Fundamental Immunology [7 ed.] 9781451117837

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Fundamental Immunology SEVENTH EDITION

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Fundamental Immunology SEVENTH EDITION EDITOR

William E. Paul, MD

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Acquisitions Editor: Julie Goolsby Product Manager: Leanne Vandetty Senior Manufacturing Manager: Benjamin Rivera Marketing Manager: Kimberly Schonberger Design Coordinator: Teresa Mallon Production Service: Absolute Service, Inc. © 2013 by LIPPINCOTT WILLIAMS & WILKINS, a WOLTERS KLUWER business Two Commerce Square 2001 Market Street Philadelphia, PA 19103 USA LWW.com 6th Edition, © 2008 by LIPPINCOTT WILLIAMS & WILKINS, a WOLTERS KLUWER business 5th Edition, © 2003 by Lippincott Williams & Wilkins 4th Edition, © 1999 by Lippincott-Raven Publishers All rights reserved. This book is protected by copyright. No part of this book may be reproduced in any form by any means, including photocopying, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the abovementioned copyright. Printed in China Library of Congress Cataloging-in-Publication Data Fundamental immunology / editor, William E. Paul. — 7th ed. p. ; cm. Includes bibliographical references and index. ISBN 978-1-4511-1783-7 (alk. paper) — ISBN 1-4511-1783-3 (alk. paper) I. Paul, William E. [DNLM: 1. Immune System Phenomena. QW 540] 616.07’9—dc23 2012035820 Care has been taken to confi rm the accuracy of the information presented and to describe generally accepted practices. However, the authors, editors, and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no warranty, expressed or implied, with respect to the currency, completeness, or accuracy of the contents of the publication. Application of the information in a particular situation remains the professional responsibility of the practitioner. The authors, editors, and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accordance with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new or infrequently employed drug. Some drugs and medical devices presented in the publication have Food and Drug Administration (FDA) clearance for limited use in restricted research settings. It is the responsibility of the health care provider to ascertain the FDA status of each drug or device planned for use in their clinical practice. To purchase additional copies of this book, call our customer service department at (800) 638-3030 or fax orders to (301) 223-2320. International customers should call (301) 223-2300. Visit Lippincott Williams & Wilkins on the Internet: at LWW.com. Lippincott Williams & Wilkins customer service representatives are available from 8:30 am to 6 pm, EST. 10 9 8 7 6 5 4 3 2 1

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For Charlotte, Gloria, Lucy, Jenna, Silvie, and Jake—and for Julien

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CHAPTER 36

CONTRIBUTING AUTHORS

Eitan M. Akirav, PhD Assistant Professor of Research Department of Medicine Stony Brook Medical School Stony Brook, New York Research Scientist Biomedical Research Core Winthrop University Hospital Mineola, New York Noelia Alonso Gonzalez, PhD Postdoctoral Fellow Department of Epidemiology and Public Health Yale University New Haven, Connecticut Yasmine Belkaid, PhD Chief, Mucosal Immunology Section Laboratory of Parasitic Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland Albert S. Bendelac, MD, PhD Professor Department of Pathology Howard Hughes Medical Institute University of Chicago Chicago, Illinois Ira Berkower, MD, PhD Lab Chief, Lab of Immunoregulation Division of Viral Products, Office of Vaccines Center for Biologics Evaluation and Research US Food and Drug Administration Bethesda, Maryland Sylvie Bertholet, PhD Unit Head Immunology Function Novartis Vaccines and Diagnostics Siena, Italy Jay A. Berzofsky, MD, PhD Branch Chief Vaccine Branch, Center for Cancer Research National Cancer Institute, National Institutes of Health Bethesda, Maryland

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Jean-Laurent Casanova, MD, PhD Professor and Head of Laboratory St. Giles Laboratory of Human Genetics of Infectious Diseases The Rockefeller University Senior Attending Physician The Rockefeller University Hospital New York, New York Marco A. Cassatella, MD Professor Pathology and Diagnostics Verona University Medical School Vice Director General Pathology Medical School Verona, Italy Ameya S. Champhekar, PhD Postdoctoral Scholar Division of Biology California Institute of Technology Pasadena, California Akanksha Chaturvedi, PhD Postdoctoral Fellow Laboratory of Immunogenetics NIH, NIAID Rockville, Maryland Sangeeta S. Chavan, PhD Associate Investigator Center for Biomedical Science The Feinstein Institute for Medical Research Manhasset, New York Yueh-hsiu Chien, PhD Professor Department of Microbiology and Immunology Stanford University Stanford, California Mary Ellen Conley, MD Professor Department of Pediatrics University of Tennessee Le Bonheur Children’s Hospital Memphis, Tennessee

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CONTRIBUTING AUTHORS

Ken T. Coppieters La Jolla Institute for Allergy and Immunology La Jolla, California Shane Crotty, PhD Associate Professor Division of Vaccine Discovery La Jolla Institute for Allergy and Immunology La Jolla, California Angel Davey, PhD Postdoctoral Fellow Laboratory of Immunogenetics NIH, NIAID Rockville, Maryland Mark M. Davis, PhD Investigator, HHMI Department of Microbiology and Immunology Stanford University Stanford, California Ennio De Gregorio, PhD Head Department of Immunology Novartis Vaccines and Diagnostics Siena, Italy Charles A. Dinarello, MD Professor of Medicine and Immunology University of Colorado Denver Aurora, Colorado Professor of Experimental Medicine Department of Medicine Radboud University Nijmegen, The Netherlands Anca Dorhoi Department of Immunology Max Planck Institute for Infection Biology Berlin, Germany Ugo D’Oro, MD, PhD Unit Head Department of Immunology Function Novartis Vaccines and Diagnostics Siena, Italy Louis Du Pasquier, PhD Professor Department of Zoology and Evolutionary Biology University of Basel Basel, Switzerland Hildegund C.J. Ertl, MD Caspar Wistar Professor in Vaccine Research Professor and Program Leader, Immunology Program Director, The Wistar Institute Vaccine Center The Wistar Institute Philadelphia, Pennsylvania

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Martin F. Flajnik, PhD Professor Department of Microbiology and Immunology University of Maryland Baltimore Baltimore, Maryland Sebastian Fugmann, PhD Investigator Laboratory of Molecular Biology and Immunology National Institute on Aging/NIH Baltimore, Maryland Christopher C. Goodnow, BVSc, BScVet, PhD Professor & NHMRC Australia Fellow Department of Immunology John Curtin School of Medical Research The Australian National University Canberra, Australian Capital Territory Siamon Gordon, FRS, MB, ChB, PhD Emeritus Professor Sir William Dunn School of Pathology University of Oxford Oxford, United Kingdom Steven Greenberg, MD Senior Principal Scientist Respiratory and Immunology Department Merck Research Laboratories Kenilworth, New Jersey Adjunct Associate Professor Department of Medicine Columbia University College of Physicians and Surgeons New York, New York Neil S. Greenspan, MD, PhD Professor Department of Pathology Case Western Reserve University Director, Histocompatibility and Immunogenetics Laboratory University Hospitals Case Medical Center Cleveland, Ohio Ted H. Hansen, PhD Professor Department of Pathology Washington University School of Medicine St. Louis, Missouri Richard R. Hardy, PhD Professor Fox Chase Cancer Center Philadelphia, Pennsylvania Dirk Homann Department of Anesthesiology Barbara Davis Center for Childhood Diabetes Aurora, Colorado University of Colorado, Denver

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CONTRIBUTING AUTHORS

Marc K. Jenkins, PhD Distinguished McKnight Professor Department of Microbiology and Center for Immunology University of Minnesota Minneapolis, Minnesota Susan M. Kaech, PhD Associate Professor and HHMI Early Career Scientist Department of Immunobiology Yale University New Haven, Connecticut Jannet Katz, DDS, PhD Professor Department of Pediatric Dentistry University of Alabama at Birmingham Birmingham, Alabama Stefan H.E. Kaufmann, PhD Director Department of Immunology Max Planck Institute for Infection Biology Professor Charite University Hospital Berlin, Germany Douglas S. Kwon, MD, PhD Instructor in Medicine Department of Infectious Disease Harvard Medical School Associate Physician Ragon Institute of MGH, MIT and Harvard Massachusetts General Hospital Boston, Massachusetts Michael J. Lenardo, MD Senior Investigator Laboratory of Immunology National Institutes of Health Bethesda, Maryland Warren J. Leonard, MD Chief, Lab of Molecular Immunology Director, Immunology Center National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland Ian Paul Lewkowich, PhD Assistant Professor Department of Cellular and Molecular Immunology Cincinnati Children’s Hospital Cincinnati, Ohio

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Judy Lieberman, MD, PhD Senior Investigator Immune Disease Institute Professor Department of Pediatrics Harvard Medical School Boston, Massachusetts Kang Liu, PhD Assistant Professor Department of Microbiology and Immunology Columbia University Medical Center New York, New York Wanli Liu, PhD Postdoctoral Fellow Laboratory of Immunogenetics NIH, NIAID Rockville, Maryland David H. Margulies, MD, PhD Chief, Molecular Biology Section Laboratory of Immunology National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland Edward E. Max, MD, PhD Associate Director for Research Office of Biotechnology Products Center for Drugs Evaluation and Research Food and Drug Administration Bethesda, Maryland James McCluskey, MD, FRACP, FRCPA, FAA Professor Department of Microbiology and Immunology University of Melbourne Melbourne, Victoria, Australia Michael G. McHeyzer-Williams, PhD Professor Department of Immunology & Microbial Science The Scripps Research Institute La Jolla, California B. Paul Morgan, MB, PhD, MRCP, FRCPath Dean and Head of School School of Medicine Cardiff University Honorary Consultant in Medical Biochemistry Department of Medical Biochemistry & Immunology University Hospital of Wales Cardiff, United Kingdom

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CONTRIBUTING AUTHORS

Philip M. Murphy, MD Chief Laboratory of Molecular Immunology National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland

Luke A.J. O’Neill, PhD Professor of Biochemistry School of Biochemistry and Immunology Trinity Biomedical Sciences Institute Trinity College Dublin, Ireland

Moon H. Nahm, MD Professor Departments of Pathology and Microbiology University of Alabama at Birmingham Birmingham, Alabama

John J. O’Shea, MD Chief, Molecular Immunology and Inflammation Branch Scientific Director, National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institutes of Health Bethesda, Maryland

Kannan Natarajan, PhD Staff Scientist Molecular Biology Section, Laboratory of Immunology National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland Mihai G. Netea, MD Professor Department of Medicine Radboud University Nijmegen Medical Center Nijmegen, The Netherlands Falk Nimmerjahn, PhD Professor for Immunology & Genetics Chair of Institute of Genetics Department of Biology University of Erlangen-Nuernberg Erlangen, Germany Luigi D. Notarangelo, MD Professor Departments of Pediatrics and Pathology Harvard Medical School Jeffrey Modell Chair of Pediatric Immunology Research Division of Immunology Children’s Hospital Boston Boston, Massachusetts Michel C. Nussenzweig, MD, PhD Sherman Fairchild Investigator and HHMI Investigator The Rockefeller University New York, New York Pamela S. Ohashi, PhD Professor Department of Medical Biophysics and Immunology University of Toronto Senior Scientist Immunotherapy Program Ontario Cancer Institute Toronto, Ontario, Canada

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William E. Paul, MD Susan K. Pierce, PhD Chief Laboratory of Immunogenetics NIH NIAID Rockville, Maryland Carlo Pucillo Department of Biomedical Science and Technology Lab of Immunology University of Udine Udine, Italy Rino Rappuoli, PhD Global Head, Vaccines Research Novartis Vaccines and Diagnostics Siena, Italy Jeffrey V. Ravetch, MD, PhD Theresa & Eugene Lang Professor Laboratory of Molecular Genetics & Immunology The Rockefeller University New York, New York Stephen T. Reece Department of Immunology Max Planck Institute for Infection Biology Berlin, Germany Eleanor M. Riley, PhD Professor of Immunology Department of Immunology and Infection London School of Hygiene and Tropical Medicine London, United Kingdom Paul A. Roche, PhD National Institutes of Health National Cancer Institute Experimental Immunology Branch Bethesda, Maryland

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CONTRIBUTING AUTHORS

Jamie Rossjohn, PhD Professor Department of Biochemistry and Molecular Biology Monash University Melbourne, Australia

Alan Sher, PhD Laboratory of Parasitic Diseases National Institutes of Allergy and Infectious Disease National Institutes of Health Bethesda, Maryland

Ellen V. Rothenberg, PhD Albert Billings Ruddock Professor of Biology Division of Biology California Institute of Technology Pasadena, California

Ethan M. Shevach, MD Chief, Cellular Immunology Section Laboratory of Immunology National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland

Nancy H. Ruddle, PhD Professor Emerita and Senior Research Scientist Department of Epidemiology and Public Health Yale University School of Medicine New Haven, Connecticut David H. Sachs, MD Paul S. Russell/Warner-Lambert Professor of Surgery Harvard Medical School Director, Transplantation Biology Research Center Department of Surgery Massachusetts General Hospital Boston, Massachusetts David L. Sacks, PhD Laboratory of Parasitic Diseases National Institutes of Allergy and Infectious Disease National Institutes of Health Bethesda, Maryland Takashi Saito, PhD Deputy Director Laboratory for Cell Signaling RIKEN Research Center for Allergy and Immunology Yokohama, Kanagawa, Japan Patrizia Scapini Department of Pathology and Diagnostics Section of General Pathology Verona, Italy Stephen P. Schoenberger, PhD Associate Professor Division of Developmental Immunology La Jolla Institute for Allergy and Immunology La Jolla, California Hans Schreiber, MD, PhD Professor Department of Pathology University of Chicago Chicago, Illinois Harry W. Schroeder, Jr., MD, PhD Professor of Medicine, Microbiology and Genetics Division of Clinical Immunology and Rheumatology University of Alabama at Birmingham Birmingham, Alabama

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Andrew L. Snow, PhD Assistant Professor Department of Pharmacology Uniformed Services University of the Health Sciences Bethesda, Maryland Hae Won Sohn, PhD Staff Scientist Laboratory of Immunogenetics NIH, NIAID Rockville, Maryland Megan Sykes, MD Michael J. Friedlander Professor of Medicine Professor of Microbiology & Immunology and Surgical Sciences (in Surgery) Columbia University Director, Columbia Center for Translational Immunology Columbia University College of Physicians and Surgeons New York, New York Nicola Tamassia Department of Pathology and Diagnostics Section of General Pathology Verona, Italy Kevin J. Tracey, MD Investigator President Head, Center for Biomedical Science Feinstein Institute for Medical Research Manhasset, New York Lucy A. Truman Postdoctoral Fellow Yale University New Haven, Connecticut Matthias G. von Herrath, MD Directory, Type 1 Diabetes Center Department of Diabetes/Immunology La Jolla Institute for Allergy & Immunology (LIAI) La Jolla, California

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CONTRIBUTING AUTHORS

David Wald, MD, PhD Assistant Professor Department of Pathology Case Western Reserve University University Hospitals Case Medical Center Cleveland, Ohio Bruce D. Walker, MD Professor Department of Infectious Disease Harvard Medical School Director Ragon Institute of MGH, MIT, and Harvard Massachusetts General Hospital Boston, Massachusetts Carl F. Ware, PhD Director and Professor Department of Infectious & Inflammatory Diseases Sanford-Burnham Medical Research Institute La Jolla, California

Kathryn J. Wood, DPhil Professor of Immunology Nuffield Department of Surgical Sciences University of Oxford Oxford, United Kingdom Thomas A. Wynn, PhD Laboratory of Parasitic Diseases National Institutes of Allergy and Infectious Disease National Institutes of Health Bethesda, Maryland Wayne M. Yokoyama, MD Investigator Howard Hughes Medical Institute Sam J. and Audrey Loew Professor of Medicine Rheumatology Division Washington University School of Medicine in St. Louis St. Louis, Missouri

Marsha Wills-Karp, PhD Rieveschl Professor Department of Pediatrics University of Cincinnati Director Division of Immunobiology Cincinnati Children’s Hospital Medical Center Cincinnati, Ohio

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PREFACE

Immunology is the quintessential medical science. Indeed, no branch of the medical sciences has improved the health of people more than the application of immunologic principles to prevention of disease. Smallpox has been eliminated from the planet as a natural infection, as has poliomyelitis from the western hemisphere. Hepatitis B vaccine has prevented more cancers than any intervention other than smoking cessation. The human papilloma virus vaccine promises to cut strikingly the toll of cervical cancer. The continued need for progress in immunology is clear. The epidemic of human immunodeficiency virus roars on. Glimmers of hope from vaccine trials have led to a redoubling of effort, and the struggle to design effective vaccines for the great infectious scourges goes on, with encouraging results but no breakthroughs yet. Highly effective therapeutic vaccines for cancers still elude us, but some immunologic therapies for cancer have met with encouraging results. We hope for much more as we marshal the tools coming from the study of the innate immune system, regulatory T cells, and lymphocyte differentiation and effector function. Understanding the basis of inflammation and the cytokine world has given us effective drugs to treat rheumatoid arthritis and a growing number of other autoinflammatory/autoimmune diseases. The value of the interventions based on this knowledge, such as the use of tumor necrosis factor, interleukin-6, and interleukin -1 blockers, is now established. The application of anti–CD20 in the treatment of autoimmune disorders shows great promise. Even more promising strategies are on the horizon. Fundamental Immunology has the goal of aiding in the education of a new generation of immunologists who can both probe more deeply into the organizing principles of the immune system and can translate this new information into effective treatments and preventatives that will extend and enlarge on the record of immunologic science in bettering the lot of human kind. Were I beginning the task of preparing a comprehensive text of immunology today, I might have titled it Immunology, Endless Fascination. Certainly that describes my own view of this science over the 30 years that I have been working on the seven editions of Fundamental Immunology. I had believed that scientific progress was marked by periods of intense creativity, during which new concepts were established, followed by longer periods of consolidation, when work that made important but anticipated advances would dominate.

Perhaps that will prove to be true of modern immunology as well when it is looked at by a disinterested observer, but for one in the midst, the pace of discovery seems to speed up with each passing year. Endless fascination certainly describes my experience of immunology. I hope that this seventh edition will convey the dynamism and creativity of modern immunology and provide the reader with a solid introduction to our field and a picture of much of the very latest that has been achieved. As with each of the previous editions, most of the chapters are entirely new and not simple reworkings of the chapter in the previous edition. In order to contain the seventh edition within one volume, a decision was made to cite references in the printed text but only to provide the detailed citations in the online version. However, the references will be linked to PubMed so cited information can be easily obtained. The electronic version can be accessed at www.fundamentalimmunology.com. As before, this edition begins with an introductory section consisting of the chapters “The Immune System” and “History of Immunology.” These give an overview of modern immunology and of its origins, and provide those new to the field with the basis to go on to the subsequent chapters. This is followed by an “expanded introduction” provided by the sections “Organization and Evolution of the Immune System,” “Immunoglobulins and B-Lymphocytes,” and “T-Lymphocytes.” These are followed by the two core “basic” immunology sections: “The Intersection of Innate and Adaptive Immunity” and “Induction, Regulation, and Effector Functions of the Immune Response.” The book concludes with sections devoted to the immune system’s role in protection against pathogenic microorganisms, “Immunity to Infectious Agents,” and to how the immune system is involved in a variety of human disorders, “Immunologic Mechanisms in Disease.” I repeat a word of caution that has been in the Preface to each edition. Immunology is moving very fast. Each of the chapters is written by an expert in the field, but in some areas there may be differences of opinion expressed by equally accomplished authors. I ask the reader to take note of the differences and to follow developments in the field. William E. Paul Washington, DC

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ACKNOWLEDGMENTS

The preparation of the seventh edition required the efforts of many individuals. I particularly wish to thank each of the authors. Their contributions, prepared in the midst of extremely busy schedules, are responsible for the value of this book. Leanne Vandetty of Lippincott Williams & Wilkins saw that the process of receiving, editing, and assembling the chapters went as smoothly as possible. Without her efforts, the completion of this edition would have been

immeasurably more difficult. Frances DeStefano’s counsel was of utmost importance in planning this edition and when she had to leave the project, Julie Goolsby took over and played a key role in making important publication decisions. I wish to gratefully acknowledge the efforts of each of the members of the editorial and production staffs of Lippincott Williams & Wilkins who participated in the preparation of this edition.

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CHAPTER 36

CONTENTS

Contributing Authors vii Preface xiii Acknowledgments xv

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Section IV. T-Lymphocytes 11 T-Cell Antigen Receptors

. . . . . . . . . . . . . . . . . . .279

Mark M. Davis, Yueh-Hsiu Chien

Section I. Introduction to Immunology 1 The Immune System . . . . . . . . . . . . . . . . . . . . . . . . . .1 William E. Paul

2 History of Immunology . . . . . . . . . . . . . . . . . . . . . . .22 Steven Greenberg

12 Mechanisms of T-Lymphocyte Signaling and Activation . . . . . . . . . . . . . . . . . . . .306 Takashi Saito

13 T-Lymphocyte Developmental Biology . . . . . . . .325 Ellen V. Rothenberg, Ameya Champhekar

14 Peripheral T-Lymphocyte Section II. Organization and Evolution of the Immune System

Responses and Function . . . . . . . . . . . . . . . . . . . .355 Marc K. Jenkins

3 Lymphoid Tissues and Organs . . . . . . . . . . . . . . . .47 Eitan M. Akirav, Noelia Alonso-Gonzalez, Lucy A. Truman, Nancy H. Ruddle

4 Evolution of the Immune System . . . . . . . . . . . . . .67 Martin F. Flajnik, Louis Du Pasquier

Section III. Immunoglobulins and B-Lymphocytes 5 Immunoglobulins: Structure and Function . . . . .129 Harry W. Schroeder Jr, David Wald, Neil S. Greenspan

6 Immunoglobulins: Molecular Genetics . . . . . . .150 Edward E. Max, Sebastian Fugmann

7 Antigen-Antibody Interactions and Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . .183 Jay A. Berzofsky, Ira J. Berkower

8 B-Lymphocyte Development and Biology . . . . .215 Richard R. Hardy

9 B-Lymphocyte Receptors, Signaling

Section V. The Intersection of Innate and Adaptive Immunity 15 The Innate Immune System . . . . . . . . . . . . . . . . . .367 Luke A. J. O’Neill

16 Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . .381 Kang Liu, Michel C. Nussenzweig

17 Natural Killer Cells . . . . . . . . . . . . . . . . . . . . . . . . .395 Wayne M. Yokoyama

18 CD1d–Restricted Natural Killer T Cells . . . . . . .432 Albert Bendelac

19 Macrophages and Phagocytosis . . . . . . . . . . . . .448 Siamon Gordon

20 Granulocytes and Mast Cells . . . . . . . . . . . . . . . .468 Patrizia Scapini, Nicola Tamassia, Carlo Pucillo, Marco A. Cassatella

21 The Major Histocompatibility

Mechanisms, and Activation . . . . . . . . . . . . . . . .246

Complex and Its Proteins . . . . . . . . . . . . . . . . . . .487

Akanksha Chaturvedi, Angel Davey, Wanli Liu, Hae Won Sohn, Susan K. Pierce

David H. Margulies, Kannan Natarajan, Jamie Rossjohn, James McCluskey

10 B-Lymphocyte Responses . . . . . . . . . . . . . . . . . . .261 Michael McHeyzer-Williams

22 Antigen Processing and Presentation . . . . . . . .524 Ted H. Hansen, Paul A. Roche

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Section VI. Induction, Regulation, and Effector Functions of the Immune Response 23 Immunogenicity and Antigen Structure . . . . . . .539

36 Complement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .863 B. Paul Morgan

37 Cell-Mediated Cytotoxicity . . . . . . . . . . . . . . . . . .891 Judy Lieberman

Jay A. Berzofsky, Ira J. Berkower

24 Fc Receptors and Their Role in Immune Regulation and Inflammation . . . . . . . . . . . . . . . .583 Jeffrey V. Ravetch, Falk Nimmerjahn

Section VII. Immunity to Infectious Agents 38 The Immune Response to Parasites . . . . . . . . . .910 Thomas A. Wynn, David L. Sacks, Alan Sher, Eleanor M. Riley

25 Type I Cytokines and Interferons, and Their Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . .601

39 Immunity to Viruses . . . . . . . . . . . . . . . . . . . . . . . .937 Hildegund C.J. Ertl

Warren J. Leonard

26 The Interleukin-1 Family . . . . . . . . . . . . . . . . . . . .639

40 Immunity to Intracellular Bacteria . . . . . . . . . . .973 Anca Dorhoi, Stephen T. Reece, Stefan H. E. Kaufmann

Charles A. Dinarello, Mihai G. Netea

27 Tumor Necrosis Factor–Related Cytokines in Immunity . . . . . . . . . . . . . . . . . . . . . .659

41 Immunity to Extracellular Bacteria . . . . . . . . . .1001 Moon H. Nahm, Jannet Katz

42 Immunology of Human Immunodeficiency

Carl F. Ware

28 Chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .681 Philip M. Murphy

Virus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . .1016 Douglas S. Kwon, Bruce D. Walker

43 Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1032

29 Helper T-Cell Differentiation

Ennio De Gregorio, Ugo D’Oro, Sylvie Bertholet, Rino Rappuoli

and Plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .708 John J. O’Shea

30 Programmed Cell Death . . . . . . . . . . . . . . . . . . . . .718 Andrew L. Snow, Michael J. Lenardo

31 Immunologic Memory

44 Autoimmunity and Autoimmune Diseases . . . .1069

. . . . . . . . . . . . . . . . . . . . . .741

Shane Crotty, Susan M. Kaech, Stephen P. Schoenberger

32 Immunologic Tolerance . . . . . . . . . . . . . . . . . . . . .765 33 Regulatory/Suppressor T Cells . . . . . . . . . . . . . . .795 . . . . . . . . . . . . . . .833

Yasmine Belkaid

35 Neurophysiologic Reflex Mechanisms in Immunology . . . . . . . . . . . . . . . . .850

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45 Immunologic Mechanisms of Allergic Disorders. . . . . . . . . . . . . . . . . . . . . . . . .1113

46 Transplantation Immunology . . . . . . . . . . . . . . .1154 Megan Sykes, Kathryn Wood, David H. Sachs

Ethan M. Shevach

Sangeeta S. Chavan, Kevin J. Tracey

Ken T. Coppieters, Matthias G. von Herrath, Dirk Homann

Marsha Wills-Karp, Ian Lewkowich

Christopher C. Goodnow, Pamela S. Ohashi

34 The Mucosal Immune System

Section VIII. Immunologic Mechanisms in Disease

47 Cancer Immunology . . . . . . . . . . . . . . . . . . . . . . .1200 Hans Schreiber

48 Inborn Errors of Immunity . . . . . . . . . . . . . . . . . .1235 Jean-Laurent Casanova, Mary Ellen Conley, Luigi D. Notarangelo

Index

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Nothing is as powerful as an idea whose time has come. Paraphrased from Victor Hugo Everything should be made as simple as possible, but not simpler. A lbert Einstein From my teachers I have learned much, from my colleagues still more, but from my students most of all. The Talmud Discovery consists of seeing what everybody has seen and thinking what nobody has thought. A lbert Szent-Gyorgyi . . . the clonal selection hypothesis . . . assumes that . . . there exist clones of mesenchymal cells, each carrying immunologically reactive sites . . . complementary . . . to one (or possibly a small number) of potential antigenic determinants. Frank Macfarlane Burnet In the fields of observation, chance favors the prepared mind. Louis Pasteur In all things of nature there is something of the marvelous. A ristotle

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SECTION

CHAPTER

I

Introduction to Immunology

1

The Immune System William E. Paul

The immune system is a remarkable defense mechanism. It makes rapid, specific, and protective responses against the myriad potentially pathogenic microorganisms that inhabit the world in which we live. The tragic examples of acquired immunodeficiency syndrome (AIDS) and the inherited severe combined immunodeficiencies graphically illustrate the consequences of a nonfunctional adaptive immune system. Patients with AIDS and children with severe combined immunodeficiency often fall victim to infections that are of little or no consequence to those with normally functioning immune systems. The immune system also has a role in the rejection of tumors and, when dysregulated, may give rise to a series of autoimmune diseases, including insulin-dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, among others. Fundamental Immunology has as its goal the authoritative presentation of the basic elements of the immune system, of the means through which the mechanisms of immunity act in a wide range of clinical conditions, including recovery from infectious diseases, rejection of tumors, transplantation of tissue and organs, autoimmunity and other immunopathologic conditions, and allergy, and how the mechanisms of immunity can be marshaled by vaccination to provide protection against microbial pathogens. The purpose of the opening chapter is to provide readers with a general introduction to our current understanding of the immune system. It should be of particular importance for those with a limited background in immunology, providing them with the preparation needed for subsequent chapters of the book. Rather than providing extensive references in this chapter, each of the subject headings will indicate the chapters that deal in detail with the topic under discussion. Those chapters will not only provide an extended treatment of the topic but will also furnish the reader with a comprehensive reference list

that can be found in the online version of Fundamental Immunology.

KEY CHARACTERISTICS OF THE IMMUNE SYSTEM Innate Immunity (Chapters 15, 17, 19, and 20) Powerful nonspecific defenses prevent or limit infections by most potentially pathogenic microorganisms. The epithelium provides both a physical barrier to the entry of microbes and produces a variety of antimicrobial factors. Agents that penetrate the epithelium are met with macrophages and related cells possessing “microbial sensors” that recognize key molecules characteristic of many microbial agents. These “pattern recognition receptors” include several families of molecules, of which the most intensively studied are the toll-like receptors (TLRs) and the nucleotide oligomerization domain–like receptors. Each TLR recognizes a distinct substance (or set of substances) associated with microbial agents; for example, TLR4 recognizes lipopolysaccharides, TLR3, doublestranded ribonucleic acid, and TLR9, unmethylated CpGcontaining DNA. Because the recognized substances are generally indispensable to the infectious agent, microbial sensors provide a highly efficient means to recognize potential pathogens. The interaction of a TLR with its ligand induces a series of intracellular signaling events, of which activation of the NFκ B system is particularly important. Macrophage activation with enhancement of the cell’s phagocytic activity and the induction of antimicrobial systems aid in the destruction of the pathogen. The induction of an inflammatory response as a result of the activation of the innate immune system recruits other cell types, including neutrophils, to the site. The innate system can provide an effective means to control or eliminate pathogens. Indeed, life forms other than vertebrates rely on the innate immune system to allow them to deal with microbial infection.

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In vertebrates, the innate immune system also acts to recruit antigen-specific immune responses, not only by attracting cells of the immune system to the site of infection, but also through the uptake of antigen by dendritic cells (DCs) and its transport by these cells to lymphoid tissues where primary immune responses are initiated. Activated DCs express cell surface costimulatory molecules and produce cytokines that can regulate the quality of the immune response so that it is most appropriate to combating the particular infectious agent, be it a virus, bacterium, or parasite.

Primary Responses (Chapters 10 and 14) Primary immune responses are initiated when a foreign antigenic substance interacts with antigen-specific lymphocytes under appropriate circumstances. The response generally consists of the production of antibody molecules specific for the antigenic determinants of the immunogen and of the expansion and differentiation of antigen-specific helper and effector T-lymphocytes. The latter include cytokine-producing cells and killer T cells, capable of lysing infected cells. Generally, the combination of the innate immune response and the primary adaptive response are sufficient to eradicate or to control the microbe. Indeed, the most effective function of the immune system is to mount a response that eliminates the infectious agent from the body, so-called sterilizing immunity.

Secondary Responses and Immunologic Memory (Chapters 10, 14, 29, and 31) As a consequence of initial encounter with antigen, the immunized individual develops a state of immunologic memory. If the same (or a closely related) microorganism is encountered again, a secondary response is made. This generally consists of an antibody response that is more rapid, greater in magnitude, and composed of antibodies that bind to the antigen with greater affinity and are more effective in clearing the microbe from the body. A more rapid and more effective T-cell response also ensues. Thus, an initial infection with a microorganism often initiates a state of immunity in which the individual is protected against a second infection. In the majority of situations, protection is provided by high-affinity antibody molecules that rapidly clear the re-introduced microbe. This is the basis of most licensed vaccines; the great power of vaccines is illustrated by the elimination of smallpox from the world and by the complete control of polio in the western hemisphere.

The Immune Response is Highly Specific and the Antigenic Universe is Vast The immune response is highly specific. Primary immunization with a given microorganism evokes antibodies and T cells that are specific for the antigenic determinants found on that microorganism but that generally fail to recognize (or recognize only poorly) antigenic determinants expressed by unrelated microbes. Indeed, the range of antigenic specificities that can be discriminated by the immune system is enormous.

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The Immune System is Tolerant of Self-Antigens (Chapters 32 and 33) One of the most important features of the immune system is its ability to discriminate between antigenic determinants expressed on foreign substances, such as pathogenic microbes, and potential antigenic determinants expressed by the tissues of the host. The failure of the system to make full-blown immune responses to self-antigens is referred to as immunologic tolerance. Tolerance is a complex process that actually involves several distinct processes. One element, perhaps the most important, is an active process involving the elimination or inactivation of cells that can recognize self-antigens. In addition, there are mechanisms through which cells that encounter antigens (such as selfantigens) in the absence of cues from the innate immune system may fail to make a response, may make a minimal response, or may be inactivated through a process referred to as anergy. Finally, a specialized set of T cells exist designated regulatory cells that actively suppress responses against self-antigens. Indeed, individuals who have mutations in the key transcription factor Foxp3 expressed by the regulatory cells develop severe multiorgan autoimmunity (Immunodysregulation polyendocrinopathy, enteropathy X-linked syndrome). The critical necessity to control selfreactivity is clearly shown by this multilayered system that involves elimination, inactivation, and suppression.

Immune Responses Against Self-Antigens can Result in Autoimmune Diseases (Chapter 44) Failure in establishing immunologic tolerance or unusual presentations of self-antigens can give rise to tissuedamaging immune responses directed against antigenic determinants on host molecules. These can result in autoimmune diseases. As has already been mentioned, a group of extremely important diseases are caused by autoimmune responses or have major autoimmune components, including systemic lupus erythematosus, rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, and inflammatory bowel disease. Efforts to treat these diseases by modulating the autoimmune response are a major theme of contemporary medicine.

Acquired Immunodeficiency Syndrome is an Example of a Disease Caused by a Virus That the Immune System Generally Fails to Eliminate (Chapter 42) Immune responses against infectious agents do not always lead to elimination of the pathogen. In some instances, a chronic infection ensues in which the immune system adopts a variety of strategies to limit damage caused by the organism or by the immune response. Indeed, herpes viruses, such as human cytomegalovirus, frequently are not eliminated by immune responses and establish a chronic infection in which the virus is controlled by immune responses. One of the most notable infectious diseases in which the immune response generally fails to eliminate the organism is AIDS, caused by the human immunodeficiency virus (HIV). In this instance, the principal infected cells are those of the immune system

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itself, leading to an eventual state in which the individual can no longer mount protective immune responses against other microbial pathogens. Indeed, under the assault of HIV, control of viruses such as cytomegalovirus is lost and they may cause major tissue damage.

Major Principles of Immunity The major principles of the immune response are: • Elimination of many microbial agents through the nonspecific protective mechanisms of the innate immune system • Cues from the innate immune system inform the cells of the adaptive immune system as to whether it is appropriate to make a response and what type of response to make • Cells of the adaptive immune system display exquisitely specific recognition of foreign antigens and mobilize potent mechanisms for elimination of microbes bearing such antigens • The immune system displays memory of its previous responses • Tolerance of self-antigens. The remainder of this introductory chapter will describe briefly the molecular and cellular basis of the system and how these central characteristics of the immune response may be explained.

CELLS OF THE IMMUNE SYSTEM AND THEIR SPECIFIC RECEPTORS AND PRODUCTS The immune system consists of several distinct cell types, each with important roles. The lymphocytes occupy central stage because they are the cells that determine the specificity of immunity. It is their response that orchestrates the effector limbs of the immune system. Cells that interact with lymphocytes play critical parts both in the presentation of antigen and in the mediation of immunologic functions. These cells include DCs and the closely related Langerhans cells, monocyte/macrophages, natural killer (NK) cells, neutrophils, mast cells, basophils, and eosinophils. In addition, a series of specialized epithelial and stromal cells provide the anatomic environment in which immunity occurs, often by secreting critical factors that regulate migration, growth and homeostasis, and gene activation in cells of the immune system. Such cells also play direct roles in the induction and effector phases of the response. The cells of the immune system are found in peripheral organized tissues, such as the spleen, lymph nodes, Peyer’s patches of the intestine, and tonsils, where primary immune responses generally occur (see Chapter 3). Many of the lymphocytes comprise a recirculating pool of cells found in the blood and lymph, as well as in the lymph nodes and spleen, providing the means to deliver immunocompetent cells to sites where they are needed and to allow immunity that is initiated locally to become generalized. Activated lymphocytes acquire the capacity to enter nonlymphoid tissues where they can express effector functions and eradicate local

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infections. Some memory lymphocytes are “on patrol” in the tissues, scanning for reintroduction of their specific antigens. Lymphocytes are also found in the central lymphoid organs, the thymus, and bone marrow, where they undergo the developmental steps that equip them to mediate the responses of the mature immune system. Individual lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens. This commitment exists before the first contact of the immune system with a given antigen. It is expressed by the presence on the lymphocyte’s surface of receptors specific for determinants (epitopes) of the antigen. Each lymphocyte possesses a population of receptors, all of which have identical combining sites (this is a slight oversimplification as occasionally T cells and less frequently B cells may express two populations of receptors). One set, or clone, of lymphocytes differs from another clone in the structure of the combining region of its receptors and thus in the epitopes that it can recognize. The ability of an organism to respond to virtually any non–self-antigen is achieved by the existence of a very large number of different lymphocytes, each bearing receptors specific for a distinct epitope. As a consequence, lymphocytes are an enormously heterogeneous group of cells. Based on reasonable assumptions as to the range of diversity that can be created in the genes encoding antigen-specific receptors, it is virtually certain that the number of distinct combining sites on lymphocyte receptors of an adult human can be measured in the millions. Lymphocytes differ from each other not only in the specificity of their receptors but also in their functions. There are two broad classes of lymphocytes: the B-lymphocytes, which are precursors of antibody-secreting cells, and the T (thymus-derived)-lymphocytes. T-lymphocytes express important helper functions, such as the ability to aid in the development of specific types of immune responses, including the production of antibody by B cells, the increase in the microbicidal activity of macrophages, and the recruitment of granulocytes to sites of infection. Other T-lymphocytes are involved in direct effector functions, such as the lysis of virus-infected cells or certain neoplastic cells. Regulatory T-lymphocytes have the capacity to suppress immune responses.

B-LYMPHOCYTES AND ANTIBODY B-Lymphocyte Development (Chapter 8) B-lymphocytes derive from lymphoid progenitor cells, which in turn are derived from hematopoietic stem cells (Fig. 1.1). A detailed picture has been obtained of the molecular mechanisms through which committed early members of the B lineage develop into mature B-lymphocytes. These events occur in the fetal liver and, in adult life, in the bone marrow. Interaction with specialized stromal cells and their products, including cytokines such as interleukin (IL)-7 and BAFF, are critical to the normal regulation of this process. The key events in B-cell development involve commitment to the B lineage and repression of the capacity to differentiate to cells of other lineages. In pro-B cells and pre-B cells, the genetic elements that encode the antigenspecific receptors are assembled. These receptors are im-

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FIG. 1.1. The patterns of gene expression, timing of gene rearrangement events, and capacity for self-replenishment and for rapid proliferation of developing B lymphocytes are indicated. (Adapted from Hardy RR, Hayakawa K. B cell development pathways. Ann Rev Immunol. 2001;19:595–621.)

munoglobulin (Ig) molecules specialized for expression on the cell surface. Igs are heterodimeric molecules consisting of heavy (H) and light (L) chains, both of which have variable (V) regions, which are responsible for the binding of antigen and that differ in sequence from one Ig molecule to another (see Chapters 5, 6, and 7) (Fig. 1.2) and constant (C) regions. The genetic elements encoding the variable portions of Ig H and L chains are not contiguous in germline DNA or in the DNA of nonlymphoid cells (see Chapter 6) (Fig. 1.3). In pro- and pre-B cells, these genetic elements are translocated to construct an expressible V-region gene. This process involves a choice among a large set of potentially usable V, diversity (D), and joining (J) elements in a combinatorial manner and depends upon the recombinating activating gene (RAG) proteins, RAG1 and RAG2. Such combinatorial translocation, together with the addition of diversity in the course of the joining process, results in the generation of a very large number of distinct H and L chains. The pairing of H and L chains in a quasirandom manner further expands the number of distinct Ig molecules that can be formed.

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The H-chain V region is initially expressed in association with the product of the μ C-region gene. Together, these elements encode the μ IgH chain, which is used in Igs of the IgM class. The successful completion of the process of Ig gene rearrangement and the expression of the resultant IgM on the cell surface marks the transition between the pre-B– and B–cell states (see Fig. 1.1). The newly differentiated B cell initially expresses surface Ig solely of the IgM class. The cell completes its maturation process by expressing on its surface a second class of Ig composed of the same L chain and the same H chain V (VDJ) region but of a different H chain C region; this second Ig H chain is designated δ, and the Ig to which it contributes is designated IgD, so that the mature naïve B cells express both IgM and IgD surface molecules that share the same V region. The differentiation process is controlled at several steps by a system of checks that determines whether prior steps have been successfully completed. These checks depend on the expression on the surface of the cell of appropriately constructed Ig or Ig-like molecules. For, example,

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B-Lymphocyte Activation (Chapter 9)

FIG. 1.2. A schematic representation of an immunoglobulin molecule indicating the means through the variable regions and the CH1 and CL regions of heavy and light chains pair with one another and how the CH2 and CH3 regions of the heavy chains pair.

in the period after a μ chain has been successfully assembled but before an L chain has been assembled, the μ chain is expressed on the cell surface in association with a surrogate L chain, consisting of VpreB and λ 5. Pre-B cells that fail to express this μ /VpreB λ 5 complex do not move forward to future differentiation states or do so very inefficiently.

A mature B cell can be activated by an encounter with antigen-expressing epitopes that are recognized by its cell surface Ig (Fig. 1.4). The activation process may be a direct one, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage–dependent B-cell activation), or an indirect one, occurring most efficiently in the context of an intimate interaction with a helper T cell, in a process often referred to as cognate help. Because each B cell bears membrane Ig molecules with identical variable regions, cross-linkage of the cell surface receptors requires that the antigen express more than one copy of an epitope complementary to the binding site of the receptor. This requirement is fulfilled by antigens with repetitive epitopes. Among these antigens are the capsular polysaccharides of many medically important microorganisms such as pneumococci, streptococci, and meningococci. Similar expression of multiple identical epitopes on a single immunogenic particle is a property of many viruses because they express multiple copies of envelope proteins on their surface. Cross-linkage–dependent B-cell activation is a major protective immune response mounted against these microbes. The binding of complement components (see Chapter 36) to antigen or antigen/antibody complexes can increase the magnitude of the cross-linkage–dependent B-cell activation due to the action of a receptor for complement, which, together with other molecules, increases the magnitude of a B-cell response to limiting amounts of antigen. Cognate help allows B cells to mount responses against antigens that cannot cross-link receptors and, at the same time, provides costimulatory signals that rescue B cells from inactivation when they are stimulated by weak cross-linkage events. Cognate help is dependent on the binding of antigen by the B cell’s membrane Ig, the endocytosis of the antigen, and its fragmentation into peptides within the endosomal/ lysosomal compartment of the cell. Some of the resultant peptides are loaded into a groove in a specialized set of cell surface proteins, the class II major histocompatibility complex (MHC) molecules (Fig. 1.5). The resultant class II/

FIG. 1.3. Organization and translocation of mouse immunoglobulin (Ig)H genes. IgH chains are encoded by four distinct genetic elements: Igh-V (V), Igh-D (D), Igh-J (J) and Igh-C. The variable (V), diversity (D), and joining (J) genetic elements together specify the variable region of the heavy chain. The Igh-C element specifies the constant (C) region. The same V region can be expressed in association with each of the C regions (μ, δ, γ3,γ1,γ2b, γ2a, ε, and α). In the germline, the V, D, and J genes are far apart and there are multiple forms of each of these genes. In the course of lymphocyte development, a VDJ gene complex is formed by translocation of individual V and D genes so that they lie next to one of the J genes, with excision of the intervening genes. This VDJ complex is initially expressed with μ and δ C genes but may be subsequently translocated so that it lies near one of the other C genes (e.g., γ1) and in that case leads to the expression of a VDJ γ1 chain.

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FIG. 1.4. Two Forms of B-Cell Activation. A: Cognate T cell/ B cell help. Resting B cells can bind antigens that bear epitopes complementary to their cell surface Ig. Even if the antigen cannot cross-link the receptor, it will be endocytosed and enter late endosomes and lysosomes where it will be degraded to peptides. Some of these peptides will be loaded into class II major histocompatibility complex molecules and brought to the cell surface, where they can be recognized by CD4positive T cells that bear receptors specific for that peptide/class II complex. This interaction allows an activation ligand on the T cells (CD40 ligand) to bind to its receptor on B cells (CD40) and to signal B-cell activation. In addition, the T cells secrete several cytokines that regulate the growth and differentiation of the stimulated B cell. B: Cross-linkage–dependent B cell activation. When B cells encounter antigens that bear multiple copies of an epitope that can bind to their surface immunoglobulin, the resultant crosslinkage stimulates biochemical signals within the cell leading to B-cell activation, growth, and differentiation. In many instances, B-cell activation events may result from both pathways of stimulation.

FIG. 1.5. Illustration of the structure of the peptide binding domain (α1 and β1) of a class II major histocompatibility complex molecule bound to an antigenic peptide from influenza hemagglutinin (adapted by D.H. Margulies from Stern LJ, Brown JH, Jardetzky TS, et al. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature. 1994;68[6468]:215–221.)

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peptide complexes are expressed on the cell surface. As will be discussed subsequently, these complexes are the ligands for the antigen-specific receptors of a set of T cells designated CD4 T cells. CD4 T cells that have receptors specific for the class II/peptide complex expressed on the B-cell surface recognize and interact with that B cell. That interaction results in the activation of the B cell through the agency of cell surface molecules expressed by the T cells (e.g., the CD40 ligand [CD154]) and cytokines produced by the T cell (see Fig. 1.4). The role of the B-cell receptor for antigen is to create the T-cell ligand on the surface of antigen-specific B cells; activation of the B cell derives largely from the action of the T cell. However, in many physiologic situations, receptor cross-linkage stimuli and cognate help synergize to yield more vigorous B-cell responses. Recently, it has been shown that the association of ligands for TLRs with antigen will strikingly enhance B cell responses.

B-Lymphocyte Differentiation (Chapters 9 and 10) Activation of B cells prepares them to divide and to differentiate either into antibody-secreting cells or into memory cells, so that there are more cells specific for the antigen used for immunization. Those cells that differentiate into antibody-secreting cells account for primary antibody responses. Some of these antibody-secreting cells migrate to the bone marrow where they may continue to produce antibody for an extended period of time and may have lifetimes very long. Memory B cells give rise to antibody-secreting cells upon rechallenge of the individual. The hallmark of the antibody response to rechallenge (a secondary response) is that it is of greater magnitude, occurs more promptly, is composed of antibodies with higher affinity for the antigen, and is dominated by Igs expressing γ, α, or ε C regions (IgG, IgA, or IgE) rather than by IgM, which is the dominant Ig of the primary response. Division and differentiation of cells into antibodysecreting cells is largely controlled by the interaction of the activated B cells with T cells expressing CD154 and by their stimulation by T-cell–derived cytokines. The differentiation of activated B cells into memory cells occurs in a specialized microenvironmental structure in the spleen and lymph nodes: the germinal center. The increase in antibody affinity also takes place within the germinal center. This process, designated affinity maturation, is dependent on somatic hypermutation. The survival of B cells within the germinal center depends on their capacity to bind antigen so that as the amount of antigen diminishes, B cells that have higher affinity receptors, either naturally or as a result of the hypermutation process, have a selective survival and growth advantage. Thus, such cells come to dominate the population. The process through which a single H-chain V region can become expressed with genes encoding C regions other than μ or δ is referred to as Ig class switching. It is dependent on a gene translocation event through which the C-region genes between the genetic elements encoding the V region and the newly expressed C gene are excised, resulting in the switched C gene being located in the position that the Cμ gene formerly occupied (see Fig. 1.3). This process also

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occurs mainly in germinal centers. Both somatic hypermutation and immunoglobulin class switching depend upon the action of activation-induced cytidine deaminase (AID) that plays an important role in the breakage and repair of DNA, which is essential for recombination events.

B1 and Marginal Zone B-Lymphocytes (Chapters 8 and 10) B lymphocytes consist of at least three distinct populations: conventional B cells, B1 B cells, and marginal zone B cells. B1 B cells were initially recognized because some express a cell-surface protein, CD5, not generally found on other B cells. In the adult mouse, B1 B cells are found in relatively high frequency in the peritoneal cavity but are present at low frequency in the spleen and lymph nodes. B1 B cells are quite numerous in fetal and perinatal life and appear to be self-renewing, in contrast to conventional B cells, in which division and memory are antigen driven. Marginal zone B cells are localized in a distinct anatomical region of the spleen (the marginal zone) that represents the major antigen filtering and scavenging area. Like B1 B cells, marginal zone B cells express a repertoire biased toward bacterial cell wall constituents and senescent self-components. Marginal zone and B1 B cells respond very rapidly to antigenic challenge, likely independently of T cells. Uniquely, among all populations of B cells, marginal zone B cells are dependent on Notch-2 signaling for their development. B1 B cells and marginal zone B cells are responsible for the secretion of the serum IgM that exists in nonimmunized mice, often referred to as natural IgM. Among the antibodies found in such natural IgM are molecules that can combine with phosphatidylcholine (a component of pneumococcal cell walls) and with lipopolysaccharide and influenza virus. B1 B cells also produce autoantibodies, although they are generally of low affinity and in most cases not pathogenic. There is evidence that B1 B cells are important in resistance to several pathogens and may have a significant role in mucosal immunity.

B-Lymphocyte Tolerance (Chapter 32) One of the central problems facing the immune system is that of being able to mount highly effective immune responses to the antigens of foreign, potentially pathogenic agents while ignoring antigens associated with the host’s own tissues. The mechanisms ensuring this failure to respond to self-antigens are complex and involve a series of strategies. Chief among them is elimination of cells capable of self-reactivity or the inactivation of such cells. The encounter of immature, naïve B cells with antigens with repetitive epitopes capable of cross-linking membrane Ig can lead to elimination of the B cells, particularly if no T-cell help is provided at the time of the encounter. This elimination of potentially self-reactive cells is often referred to as clonal elimination. Many selfreactive cells, rather than dying upon encounter with selfantigens, undergo a further round of Ig gene rearrangement. This receptor editing process allows a self-reactive cell to substitute a new receptor and therefore to avoid elimination.

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There are many self-antigens that are not encountered by the developing B-cell population or that do not have the capacity to cross-link B-cell receptors to a sufficient degree to elicit the receptor editing /clonal elimination process. Such cells, even when mature, may nonetheless be inactivated through a process that involves cross-linkage of receptors without the receipt of critical costimulatory signals. These inactivated cells may be retained in the body but are unresponsive to antigen and are referred to as anergic. When removed from the presence of the anergy-inducing stimulus, anergic cells may regain responsiveness.

Immunoglobulins Structure (Chapter 5) Igs are the antigen-specific membrane receptors and secreted products of B cells. They are members of a large family of proteins designated the Ig supergene family. Members of the Ig supergene family have sequence homology, a common gene organization, and similarities in three-dimensional structure. The latter is characterized by a structural element referred to as the Ig fold, generally consisting of a set of seven β -pleated sheets organized into two opposing layers (Fig. 1.6). Many of the cell surface proteins that participate in immunologic recognition processes, including the T-cell receptor (TCR), the CD3 complex, and signaling molecules associated with the B-cell receptor (Igα and Igβ), are members of the Ig supergene family. The Igs themselves are constructed of a unit that consists of two H chains and two L chains (see Fig. 1.2). The H and L chains are composed of a series of domains, each consisting of approximately 110 amino acids. The L chains, of which there are two types (κ and λ), consist of two domains. The carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the C region. As already discussed, the amino terminal domain varies from L chain to L chain and contributes to the binding site of antibody. Because of its variability, it is referred to as the V region. The variability of this region is largely concentrated in three segments, designated the hypervariable or complementarity-determining

regions (CDRs). The CDRs contain the amino acids that are the L chain’s contribution to the lining of the antibody’s combining site. The three CDRs are interspersed among four regions of much lower degree of variability, designated framework regions. The H chains of Ig molecules are of several classes determined by their constant regions ( μ , δ, γ [of which there are several subclasses], α and ε). An assembled Ig molecule, consisting of one or more units of two identical H and L chains, derives its name from the constant region of the H chain that it possesses. Thus, there are IgM, IgD, IgG, IgA, and IgE antibodies. The H chains each consist of a single amino terminal V region and three or four C regions. In many H chains, a hinge region separates the fi rst and second C regions and conveys flexibility to the molecule, allowing the two combining sites of a single unit to move in relation to one another so as to promote the binding of a single antibody molecule to an antigen that has more than one copy of the same epitope. Such divalent binding to a single antigenic structure results in a great gain in energy of interaction (see Chapter 7). The H-chain V region, like that of the L chain, contains three CDRs lining the combining site of the antibody and four framework regions. The C region of each H-chain class conveys unique functional attributes to the antibodies that possess it. Among the distinct biologic functions of each class of antibody are the following: • IgM antibodies are potent activators of the complement system (see Chapter 36). • IgA antibodies are secreted into a variety of bodily fluids and are principally responsible for immunity at mucosal surfaces (see Chapter 34). • IgE antibodies are bound by specific receptors (FcεRI) on basophils and mast cells. When cross-linked by antigen, these IgE/FcεRI complexes cause the cells to release a set of mediators responsible for allergic inflammatory responses (see Chapter 45). • IgD antibodies act virtually exclusively as membrane receptors for antigen.

FIG. 1.6. Schematic Drawing of the Variable and Constant Domains of an Immunoglobulin Light Chain Illustrating the “Immunoglobulin Fold.” The β strands participating in the antiparallel β-pleated sheets of each domain are represented as arrows. The β strands of the three-stranded sheets are shaded, whereas those in the four-stranded sheets are white. The intradomain disulfide bonds are represented as black bars. Selected amino acids are numbered with position 1 as the N terminus. (Reprinted with permission from Edmundson AB, Ely KR, Abola EE, Schiffer M, Panagiotopoulous N. Rotational allomerism and divergent evolution of domains in immunoglobulin light chains. Biochemistry. 1975;14:3953–3961).

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• IgG antibodies, made up of four subclasses in both humans and mice, mediate a wide range of functions including transplacental passage and opsonization of antigens through binding of antigen/antibody complexes to specialized Fc receptors on macrophages and other cell types (see Chapters 19, 20, and 24). IgD, IgG, and IgE antibodies consist of a single unit of two H and L chains. IgM antibodies are constructed of five or six such units, although they consist of a single unit when they act as membrane receptors. IgA antibodies may consist of one or more units. The antibodies that are made up of more than a single unit generally contain an additional polypeptide chain, the J chain, that appears to play a role in the polymerization process. In addition, secreted IgA expresses a chain, a secretory piece, that is derived from the receptor for polymeric IgA, which plays a role in the transport of IgA through the cells lining the lumen of the gut. Each of the distinct Igs can exist as secreted antibodies and as membrane molecules. Antibodies and cell surface receptors of the same class made by a specific cell have identical structures except for differences in their carboxy-terminal regions. Membrane Igs possess a hydrophobic region, spanning the membrane, and a short intracytoplasmic tail, both of which are lacking in the secretory form.

Immunoglobulin Genetics (Chapter 6) The components of the Ig H-chain gene have already been alluded to. To reiterate, the IgH chain gene of a mature lymphocyte is derived from a set of genetic elements that are separated from one another in the germline. The V region is composed of three types of genetic elements: VH, D, and JH. More than 100 VH elements exist; there are more than 10 D elements and a small number of JH elements (4 in the mouse). An H-chain VH DJH gene is created by the translocation of one of the D elements on a given chromosome to one of the JH elements on that chromosome, generally with the excision of the intervening DNA. This is followed by a second translocation event in which one of the VH elements is brought into apposition with the assembled DJH element to create the VH DJH (V region) gene (see Fig. 1.3). Although it is likely that the choice of the VH, D, and JH elements that are assembled is not entirely random, the combinatorial process allows the creation of a very large number of distinct H-chain V-region genes. Additional diversity is created by the imprecision of the joining events and by the deletion of nucleotides and addition of new, untemplated nucleotides between D and JH and between VH and D, forming N regions in these areas. This further increases the diversity of distinct IgH chains that can be generated from the relatively modest amount of genetic information present in the germline. The assembly of L-chain genes follows generally similar rules. However, L chains are assembled from VL and JL elements only. Although there is junctional diversity, no N regions exist for L chains. Additional diversity is provided by the existence of two classes of L chains, κ and λ. An Ig molecule is assembled by the pairing of an IgHchain polypeptide with an IgL-chain polypeptide. Although

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this process is almost certainly not completely random, it allows the formation of an exceedingly large number of distinct Ig molecules, the majority of which will have individual specificities. The rearrangement events that result in the assembly of expressible IgH and IgL chains occur in the course of B-cell development in pro-B cells and pre-B cells, respectively (see Fig. 1.1). This process is regulated by the Ig products of the rearrangement events. The formation of a μ chain signals the termination of rearrangement of H-chain gene elements and the onset of rearrangement of L-chain gene elements, with κ rearrangements generally preceding λ rearrangements. One important consequence of this is that only a single expressible μ chain will be produced in a given cell, as the fi rst expressible μ chain shuts off the possibility of producing an expressible μ chain on the alternative chromosome. Comparable mechanisms exist to ensure that only one L-chain gene is produced, leading to the phenomenon known as allelic exclusion. Thus, the product of only one of the two alternative allelic regions at both the H- and L-chain loci are expressed. The closely related phenomenon of L-chain isotype exclusion ensures the production of either κ or λ chains in an individual cell, but not both. An obvious but critical consequence of allelic exclusion is that in most cases an individual B cell makes antibodies, all of which have identical H- and L-chain V regions, a central prediction of the clonal selection theory of the immune response. During receptor editing, secondary rearrangements occur. Receptor editing is induced when the initial membrane Ig is capable of self-reactivity. As a consequence of the resultant secondary rearrangement, Ig of a different specificity is expressed, usually no longer self-reactive.

Class Switching (Chapter 6) An individual B cell continues to express the same IgHchain V region as it matures but it can switch the IgH-chain C region it uses (see Fig. 1.3). Thus, a cell that expresses receptors of the IgM and IgD classes may differentiate into a cell that expresses IgG, IgA, or IgE receptors and then into a cell secreting antibody of the same class as it expressed on the cell surface. This process allows the production of antibodies capable of mediating distinct biologic functions but that retain the same antigen-combining specificity. When linked with the process of affinity maturation of antibodies, Ig class switching provides antibodies of extremely high efficacy in preventing re-infection with microbial pathogens or in rapidly eliminating such pathogens. The associated phenomena of class switching and affinity maturation account for the high degree of effectiveness of antibodies produced in secondary immune responses. The process of class switching is known to involve a genetic recombination event between specialized switch (S) regions, containing repetitive sequences, that are located upstream of each C region genetic element (with the exception of the δ C region). Thus, the S region upstream of the μ CH region gene (Sμ) recombines with an S region upstream of a more 3′ isotype, such as Sγ1, to create a chimeric S μ /Sγ1

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FIG. 1.7. Immunoglobulin Class Switching. The process through which a given VDJ gene in a stimulated B cell may switch the constant region gene with which it is associated from μ to another, such as γ1, is illustrated. A recombination event occurs in which DNA between a cleavage point in Sμ and one in Sγ1 forms a circular episome. This results in Cγ1 being located immediately downstream of the chimeric Sμ/γ1 region, in a position such that transcription initiating upstream of VDJ results in the formation of VDJCγ1 mRNA and γ1 H-chain protein.

region and in the deletion of the intervening DNA (Fig. 1.7). The genes encoding the C regions of the various γ chains (in the human γ1, γ 2, γ 3, and γ4; in the mouse γ1, γ 2a, γ 2b, and γ 3), of the α chain, and of the ε chain are located 3′ of the Cμ and Cδ genes. The induction of the switching process is dependent on the action of a specialized set of B-cell stimulants. Of these, the most widely studied are CD154, expressed on the surface of activated T cells, and the TLR ligands such as bacterial lipopolysaccharide. The targeting of the C region that will be expressed as a result of switching is largely determined by cytokines. Thus, IL-4 determines that switch events in the human and mouse will be to the ε C region and to the γ4 (human) or γ1 (mouse) C regions. In the mouse, interferon-gamma (IFN-γ) determines switching to γ 2a and transforming growth factor-beta (TGF-β) determines switching to α . A major goal is to understand the physiologic determination of the specificity of the switching process. Because cytokines are often the key controllers of which Ig classes will represent the switched isotype, this logically translates into asking what regulates the relative amounts of particular cytokines that are produced by different modes of immunization. As already noted, both the switching process and somatic hypermutation depend upon the AID. Mice and humans that lack AID fail to undergo both immunoglobulin class switching and somatic hypermutation.

Affinity Maturation and Somatic Hypermutation (Chapters 6 and 10) The process of generation of diversity embodied in the construction of the H- and L-chain V-region genes and of the pairing of H and L chains creates a large number of distinct antibody molecules, each expressed in an individual B cell.

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This primary repertoire is sufficiently large so that most epitopes on foreign antigens will encounter B cells with complementary receptors. Thus, if adequate T-cell help can be generated, antibody responses can be made to a wide array of foreign substances. Nonetheless, the antibody that is initially produced usually has a relatively low affi nity for the antigen. This is partially compensated for by the fact that IgM, the antibody initially made, is a pentamer. Through multivalent binding, high avidities can be achieved even if individual combining sites have only modest affinity (see Chapter 7). In the course of T-cell–dependent B-cell stimulation, particularly within the germinal center, a process of somatic hypermutation is initiated that leads to a large number of mutational events, largely confined to the H-chain and L-chain V-region genes and their immediately surrounding introns. During the process of somatic hypermutation, mutational rates of 1 per 1,000 base pairs per generation may be achieved. This implies that with each cell division close to one mutation will occur in either the H- or L-chain V region of an individual cell. Such a high rate of mutation creates an enormous increase in antibody diversity. Although most of these mutations will either not affect the affinity with which the antibody binds its ligand or will lower that affinity, some will increase it. Thus, some B cells emerge that can bind antigen more avidly than the initial population of responding cells. Because there is an active process of apoptosis in the germinal center from which B cells can be rescued by the binding of antigen to their membrane receptors, cells with the most avid receptors should have an advantage over other antigen-specific B cells and should come to dominate the population of responding cells. Thus, upon rechallenge, the affinity of antibody produced will be greater than that in the initial response. As time after immunization elapses, the affinity of antibody produced will increase. This process leads to the presence in immunized individuals of high-affinity antibodies that are much more effective, on a weight basis, in protecting against microbial agents and other antigenbearing pathogens than was the antibody initially produced. Together with antibody class switching, affi nity maturation results in the increased effectiveness of antibody in preventing reinfection with agents with which the individual has had a prior encounter.

T-LYMPHOCYTES T-lymphocytes constitute the second major class of lymphocytes. They derive from precursors in hematopoietic tissue, undergo differentiation in the thymus (hence the name thymus-derived [T]-lymphocytes), and are then seeded to the peripheral lymphoid tissue and to the recirculating pool of lymphocytes (see Chapters 13 and 14). T cells are subdivided into two distinct classes based on the cell surface receptors they express. The majority of T cells express TCRs consisting of α and β chains. A second group of T cells express receptors made up of γ and δ chains. Among the α / β T cells are two important sublineages: those that express the coreceptor molecule CD4 (CD4 T cells) and those that express CD8 (CD8 T cells). These cells differ in how they recognize

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antigen and mediate different types of regulatory and effector functions. CD4 T cells are the major helper cells of the immune system (see Chapter 29). Their helper function depends both on cell surface molecules, such as CD154, induced upon these cells when they are activated and on the wide array of cytokines they secrete upon stimulation. CD4 T cells tend to differentiate, as a consequence of priming, into cells that principally secrete the cytokines IL-4, IL-13, IL-5 and IL-6 (TH2 cells) into cells that mainly produce IFN-γ and lymphotoxin (TH1 cells), or into cells that produce IL-17 and related cytokines (TH17 cells). TH2 cells are very effective in immunity to helminthic parasites, TH1 cells are effective inducers of cellular immune responses, involving enhancement in the microbicidal activity of monocytes and macrophages and consequent increased efficiency in lysing microorganisms in intracellular vesicular compartments, while TH17 cells are efficient recruiters of granulocytes and other cells of the inflammatory system and play a major role in responses to extracellular bacterial pathogens. CD4 T cells can also acquire the capacity to enter B-cell follicles and help B cells develop into antibody-producing cells and undergo immunoglobulin class switching and affinity maturation; the cells are referred to as T follicular helper (Tfh) cells. Another possible fate of naïve CD4 T cells is to differentiate into induced regulatory T cells (iTregs). However, most Tregs develop as a independent lineage of CD4 T cells. Tregs express the transcription factor Foxp3 and many express large amounts of the α chain of the IL-2 receptor (CD25). T cells mediate important effector functions. Some of these are determined by the patterns of cytokines they secrete. These powerful molecules can be directly toxic to target cells and can mobilize potent inflammatory mechanisms. In addition, T cells, particularly CD8 T cells, can

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develop into cytotoxic T-lymphocytes (CTLs) capable of efficiently lysing target cells that express antigens recognized by the CTLs.

T-Lymphocyte Antigen Recognition (Chapters 11, 21, and 22) T cells differ from B cells in their mechanism of antigen recognition. Ig, the B-cell’s receptor, binds to individual antigenic epitopes on the surface of native molecules, be they on cell surfaces or in solution. Antibody and B-cell receptors evolved to bind to and to protect against microorganisms in extracellular fluids. By contrast, T cells invariably recognize cell-associated molecules and mediate their functions by interacting with and altering the behavior of such antigen-presenting cells (APCs). Indeed, the TCR does not recognize antigenic determinants on intact, undenatured molecules. Rather, it recognizes a complex consisting of a peptide, derived by intracellular proteolysis of the antigen, bound into a specialized groove of a class II or class I MHC protein. Indeed, what differentiates a CD4 T cell from a CD8 T cell is that the CD4 T cells recognize peptide/class II complexes whereas the CD8 T cells recognize peptide/class I complexes. The TCR’s ligand (i.e., the peptide/MHC protein complex) is created within the APC. In general, class II MHC molecules bind peptides derived from proteins that have been taken up by the APC through an endocytic process (Fig. 1.8). These endocytosed proteins are fragmented by proteolytic enzymes within the endosomal/lysosomal compartment. The resulting peptides are loaded into class II MHC that traffic through this compartment. Peptide-loaded class II molecules are then expressed on the surface of the APC where they are available to be bound by CD4 T cells that have TCRs capable of recog-

FIG. 1.8. Pathways of Antigen Processing. Exogenous antigen (Ea) enters the cell via endocytosis and is transported from early endosomes into late endosomes or prelysosomes, where it is fragmented and where resulting peptides (Ea-derived peptides) may be loaded into class II major histocompatibility complex (MHC) molecules. The latter have been transported from the rough endoplasmic reticulum (RER) through the Golgi apparatus to the peptide-containing vesicles. Class II MHC molecules/Ea-derived peptide complexes are then transported to the cell surface, where they may be recognized by T-cell receptor expressed on CD4+ T cells. Cytoplasmic antigens (Ca) are degraded in the cytoplasm and then enter the RER through a peptide transporter. In the RER, Ca-derived peptides are loaded into class I MHC molecules that move through the Golgi apparatus into secretory vesicles and are then expressed on the cell surface where they may be recognized by CD8+ T cells (Reprinted with permission from Paul WE. In: Gallin JI, Goldstein, I, Snyderman, R, ed. Inflammation. New York: Raven,;1992:776.)

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nizing the expressed cell surface peptide/MHC protein complex. Thus, CD4 T cells are specialized to largely react with antigens derived from extracellular sources. In contrast, class I MHC molecules are mainly loaded with peptides derived from internally synthesized proteins, such as viral gene products. These peptides are produced from cytosolic proteins by proteolysis within the proteasome and are translocated into the rough endoplasmic reticulum. Such peptides, generally nine amino acids in length, are bound by class I MHC molecules. The complex is brought to the cell surface, where it can be recognized by CD8 T cells expressing appropriate receptors. This property gives the T-cell system, particularly CD8 T cells, the ability to detect cells expressing proteins that are different from, or produced in much larger amounts than, those of cells of the remainder of the organism (e.g., viral antigens [whether internal, envelope, or cell surface] or mutant antigens [such as active oncogene products]), even if these proteins, in their intact form, are neither expressed on the cell surface nor secreted. Although this division of class I–binding peptides being derived from internally synthesized proteins and class II– binding peptides from imported proteins is generally correct, there are important exceptions to this rule that are central for the function of the immune system. The most effective priming of naive CD8 T cells occurs in response to peptide/MHC-I complexes expressed by DCs and yet many viruses do not infect these cells but rather target other cell types. Viral antigens produced by infected cells can be taken up by specialized DCs and loaded into class I molecules in a process referred to as cross-presentation.

T-Lymphocyte Receptors (Chapter 11) The TCR is a disulfide-linked heterodimer (Fig. 1.9). Its constituent chains (α and β, or γ and δ) are Ig supergene

family members. The TCR is associated with a set of transmembrane proteins, collectively designated the CD3 complex, that play a critical role in signal transduction. The CD3 complex consists of γ, δ (note that the CD3 γ and δ chains and the TCR γ and δ chains are distinct polypeptides that, unfortunately, have similar designations) and ε chains and is associated with a homodimer of two ζ chains or a heterodimer of ζ and η chains. CD3 γ, δ, and ε consist of extracellular domains that are Ig supergene family members. The cytosolic domains of CD3 γ, δ, and ε and of ζ and η contain one or more copies of the immunoreceptor tyrosine–based activation motif (ITAM) (D/ExxYxxLxxxxxxxYxxL/I) that is found in a variety of chains associated with immune recognition receptors. This motif appears to be important in the signal transduction process and provides a site through which protein tyrosine kinases can interact with these chains to propagate signaling events. The TCR chains are organized much like Ig chains. Their N-terminal portions are variable and their C-terminal portions are constant. Furthermore, similar recombinational mechanisms are used to assemble the V-region genes of the TCR chains. Thus, the V region of the TCR β chain is encoded by a gene constructed from three distinct genetic elements (Vb , D, and Jb ) that are separated in the germline. Although the relative numbers of Vb , D, and Jb genes differ from that for the comparable Ig H variable region elements, the strategies for creation of a very large number of distinct genes by combinatorial assembly are the same. Both junctional diversity and N-region addition further diversify the genes and their encoded products. TCR β has fewer V genes than IgH but much more diversity centered on the D/J region, which encodes the equivalent of the third CDR of Igs. The α chain follows similar principles, except that it does not use a D gene. The genes for TCR γ and δ chains are assembled in a similar manner except that they have many fewer V genes from which to choose. Indeed, γ /δ T cells in certain environments, such as the skin and specific mucosal surfaces, are exceptionally homogeneous. It has been suggested that the TCRs encoded by these essentially invariant γ and δ chains may be specific for some antigen that signals microbial invasion and that activation of γ /δ T cells through this mechanism constitutes an initial response that aids the development of the more sophisticated response of α / β T cells.

T-Lymphocyte Activation (Chapter 12)

FIG. 1.9. The T-Cell Antigen Receptor. Illustrated schematically is the antigen binding subunit comprised of an αβ heterodimer and the associated invariant CD3 and ζ chains. Acidic (−) and basic (+) residues located within the plasma membrane are indicated. The open rectangular boxes indicate motifs within the cytoplasmic domains that interact with protein tyrosine kinases.

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T-cell activation is dependent on the interaction of the TCR/ CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule, on the surface of a competent APC. Through the use of chimeric cell surface molecules that possess cytosolic domains largely limited to the ITAM signaling motif alluded to previously, it is clear that cross-linkage of molecules containing such domains can generate some of the signals that result from TCR engagement. Nonetheless, the molecular events set in motion by receptor engagement are complex ones. Among the earliest steps is the activation of tyrosine kinases leading to the tyrosine phosphorylation of a set of substrates that control

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several signaling pathways. Current evidence indicates that early events in this process involve the src family tyrosine kinases p56lck, associated with the cytosolic domains of the CD4 and CD8 coreceptors, and p59fyn, and ZAP-70, a Syk family tyrosine kinase, that binds to the phosphorylated ITAMs of the ζ chain. The protein tyrosine phosphatase CD45, found on the surface of all T cells, also plays a critical role in T-cell activation. A series of important substrates are tyrosine phosphorylated as a result of the action of the kinases associated with the TCR complex. These include a 1) set of adapter proteins that link the TCR to the Ras pathway; 2) phospholipase Cγ1, the tyrosine phosphorylation of which increases its catalytic activity and engages the inositol phospholipid metabolic pathway, leading to elevation of intracellular free calcium concentration and activation protein kinase C; and 3) a series of other important enzymes that control cellular growth and differentiation. Particularly important is the phosphorylation of LAT, a molecule that acts as an organizing scaffold to which a series of signaling intermediates bind and upon which they become activated and control downstream signaling. The recognition and early activation events result in the reorganization of cell surface and cytosolic molecules on the T cell, and correspondingly, on the APC to produce a structure, the immunological synapse. The apposition of key interacting molecules involving a small segment of the membranes of the two cells concentrates these interacting molecules in a manner that both strengthens the interaction between the cells and intensifies the signaling events. It also creates a limited space into which cytokines may be secreted to influence the behavior of the interacting cells. The formation of the immunological synapse is one mechanism through which the recognition of relatively small numbers of ligands by TCRs on a specific T cell can be converted into a vigorous stimulatory process. In general, normal T cells and cloned T-cell lines that are stimulated only by TCR cross-linkage fail to give complete responses. TCR engagement by itself may often lead to a response in which the key T-cell–derived growth factor, IL-2, is not produced and in which the cells enter a state of anergy such that they are unresponsive or poorly responsive to a subsequent competent stimulus (see Chapter 32). Full responsiveness of a T cell requires, in addition to receptor engagement, an accessory cell–delivered costimulatory signal. The engagement of CD28 on the T cell by CD80 and/ or CD86 on the APC (or the engagement of comparable ligand/receptor pairs on the two cells) provides potent costimulatory activity. Inhibitors of this interaction markedly diminish antigen-specific T-cell activation in vivo and in vitro, indicating that the CD80/86–CD28 interaction is physiologically important in T-cell activation (see Chapters 12 and 14). The interaction of CD80/86 with CD28 increases cytokine production by the responding T cells. For the production of IL-2, this increase appears to be mediated both by enhancing the transcription of the IL-2 gene and by stabilizing IL-2 mRNA. These dual consequences of the CD80/86–CD28 interaction cause a striking increase in the production of IL-2 by antigen-stimulated T cells.

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CD80/86 has a second receptor on the T cell, CTLA-4, that is expressed later in the course of T-cell activation. The bulk of evidence indicates that the engagement of CTLA-4 by CD80/86 leads to a set of biochemical signals that terminate the T-cell response. Mice that are deficient in CTLA-4 expression develop fulminant autoimmune responses and anti–CTLA-4 antibodies are used as drugs to enhance antitumor immune responses.

T Lymphocyte Development (Chapter 13) Upon entry into the thymus, T-cell precursors do not express TCR chains, the CD3 complex, or the CD4 or CD8 molecules (Fig. 1.10). Because these cells lack both CD4 and CD8, they are often referred to as double-negative cells. Thymocytes develop from this double-negative pool into cells that are both CD4 + and CD8 + (double-positive cells) and express low levels of TCR and CD3 on their surface. In turn, double-positive cells further differentiate into relatively mature thymocytes that express either CD4 or CD8 (single-positive cells) and high levels of the TCR/CD3 complex. The expression of the TCR depends on complex rearrangement processes that generate TCR α and β (or γ and

FIG. 1.10. Development of `/a T Cells in the Thymus. Doublenegative T cells (4−8−) acquire CD4 and CD8 (4+8+) and then express α/β TCRs, initially at low levels. Thereafter, the degree of expression of T-cell receptiors increases and the cells differentiate into CD4 or CD8 cells and are then exported to the periphery. Once the T cells have expressed receptors, their survival depends upon the recognition of peptide/major histocompatibility complex (MHC) class I or class II molecules with an affinity above some given threshold. Cells that fail to do so undergo apoptosis. These cells have failed to be positively selected. Positive selection is associated with the differentiation of 4+8+ cells into CD4 or CD8 cells. Positive selection involving peptide/class I MHC molecules leads to the development of CD8 cells whereas positive selection involving peptide/class II MHC molecules leads to the development of CD4 cells. If a T cell recognizes a peptide/MHC complex with high affinity, it is also eliminated via apoptosis (it is negatively selected).

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δ) chains. Once TCR chains are expressed, these cells undergo two important selection processes within the thymus. One, termed negative selection, is the deletion of cells that express receptors that bind with high affinity to complexes of self-peptides with self-MHC molecules. This is a major mechanism through which the T-cell compartment develops immunologic unresponsiveness to self antigens (see Chapters 13 and 32). In addition, a second major selection process is positive selection, in which T cells with receptors with “intermediate affinity” for self-peptides bound to selfMHC molecules are selected, thus forming the basis of the T-cell repertoire for foreign peptides associated with selfMHC molecules. T cells that are not positively selected are eliminated in the thymic cortex by apoptosis. Similarly, T cells that are negatively selected as a result of high-affinity binding to self-peptide/self-MHC complexes are also deleted through apoptotic death. These two selection processes result in the development of a population of T cells that are biased toward the recognition of peptides in association with self-MHC molecules from which those cells that are potentially autoreactive (capable of high-affinity binding of selfpeptide/self-MHC complexes) have been purged. One important event in the development of T cells is their differentiation from double-positive cells into CD4 + or CD8 + single-positive cells. This process involves the interaction of double-positive thymocytes with peptide bound to class II or class I MHC molecules on accessory cells. Indeed, CD4 binds to monomorphic sites on class II molecules, whereas CD8 binds to comparable sites on class I molecules. The capacity of the TCR and CD4 (or of the TCR and CD8) to bind to a class II MHC (or a class I MHC) molecule on an accessory cell leads either to the differentiation of doublepositive thymocytes into CD4 + (or CD8+) single-positive T cells or to the selection of cells that have “stochastically” differentiated down the CD4 (or CD8) pathway. Less is understood about the differentiation of thymocytes that express TCRs composed of γ /δ chains. These cells fail to express either CD4 or CD8. However, γ /δ cells are relatively numerous early in fetal life; this, together with their limited degree of heterogeneity, suggests that they may comprise a relatively primitive T-cell compartment.

T-Lymphocyte Functions (Chapters 14, 29, and 33) T cells mediate a wide range of immunologic functions. These include the capacity to help B cells develop into antibody-producing cells, the capacity to increase the microbicidal action of monocytes/macrophages, the inhibition of certain types of immune responses, direct killing of target cells, and mobilization of the inflammatory response. In general, these effects depend on their expression of specific cellsurface molecules and the secretion of cytokines.

membrane Ig of B cells or do so inefficiently. B cells bind the antigen through their membrane Ig, and the complex undergoes endocytosis. Within the endosomal and lysosomal compartments, the antigen is fragmented into peptides by proteolytic enzymes and one or more of the generated peptides are loaded into class II MHC molecules, which traffic through this vesicular compartment. The resulting complex of class II MHC molecule and bound peptide is exported to the B-cell surface membrane. T cells with receptors specific for the peptide/class II molecular complex recognize that complex on the B cell. B-cell activation depends not only on the binding of peptide/class II MHC complexes on the B cell surface by the TCR but also on the interaction of T-cell CD154 with CD40 on the B cell. T cells do not constitutively express CD154; rather, it is induced as a result of an interaction with an activated APC that expresses a cognate antigen recognized by the TCR of the T cell. Furthermore, CD80/86 are generally expressed by activated but not resting B cells so that interactions involving resting B cells and naïve T cells generally do not lead to efficient antibody production. By contrast, a T cell already activated and expressing CD154 can interact with a resting B cell, leading to its upregulation of CD80/86 and to a more productive T-cell/B-cell interaction with the delivery of cognate help and the development of the B cell into an antibody-producing cell. Similarly, activated B cells expressing large amounts of class II molecules and CD80/86 can act as effective APCs and can participate with T cells in efficient cognate help interactions. Cross-linkage of membrane Ig on the B cell, even if inefficient, may synergize with the CD154/CD40 interaction to yield vigorous B-cell activation. The subsequent events in the B-cell response program, including proliferation, Ig secretion, and class switching, either depend on or are enhanced by the actions of T-cell–derived cytokines. Thus, B-cell proliferation and Ig secretion are enhanced by the actions of several type I cytokines including IL-2 and IL-4. Ig class switching is dependent both on the initiation of competence for switching, which can be induced by the CD154/CD40 interaction, and on the targeting of particular C regions for switching, which is determined, in many instances, by cytokines. The best studied example of this is the role of IL-4 in determining switching to IgG1 and IgE in the mouse and to IgG4 and IgE in the human. Indeed, the central role of IL-4 in the production of IgE is demonstrated by the fact that mice that lack the IL-4 gene or the gene for the IL-4 receptor α chain, as a result of gene knockouts, have a marked defect in IgE production. Similarly, IFN-γ determines switching to IgG2a in the mouse. The relationship between TFH cells that produce IL-4 and TH2 cells and those that produce IFN-γ and TH1 cells is still uncertain.

Induction of Cellular Immunity (Chapters 14 and 19) T Cells that Help Antibody Responses (Chapter 13) Helper T cells, TFH cells, can stimulate B cells to make antibody responses to proteins and other T-cell–dependent antigens. T-cell–dependent antigens are immunogens in which individual epitopes appear only once or only a limited number of times so that they are unable to cross-link the

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T cells also may act to enhance the capacity of monocytes and macrophages to destroy intracellular microorganisms. In particular, IFN-γ enhances several mechanisms through which mononuclear phagocytes destroy intracellular bacteria and parasites, including the generation of nitric oxide and induction of TNF. TH1 cells are particularly effective in

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enhancing microbicidal action because they produce IFN-γ. By contrast, three of the major cytokines produced by TH2 cells, IL-4, IL-13, and IL-10, block these activities; IL-4 and IL-13 induce an alternative gene activation program in macrophages resulting in alternatively activated macrophages, characterized (in the mouse) by the expression of arginase 1 and chitinase. Thus, TH2 cells often oppose the action of TH1 cells in inducing cellular immunity and in certain infections with microorganisms that are intracellular pathogens of macrophages; a TH2-dominated response may be associated with failure to control the infection.

Regulatory T Cells (Chapter 33) There has been a longstanding interest in the capacity of T cells to diminish as well as to help immune responses. Cells that mediate such effects are referred to as Tregs. Tregs may be identified by their expression of Foxp3 and of CD25, the IL-2 receptor alpha chain. These cells inhibit the capacity of both CD4 and CD8 T cells to respond to their cognate antigens. The mechanisms through which their suppressor function is mediated are still somewhat controversial. In some instances, it appears that cell/cell contact is essential for suppression, whereas in other circumstances production of cytokines by Tregs has been implicated in their ability to inhibit responses. Evidence has been presented for both IL-10 and TGF-β as mediators of inhibition. Tregs have been particularly studied in the context of various autoimmune conditions. In the absence of Tregs, conventional T cells cause several types of autoimmune responses, including autoimmune gastritis and inflammatory bowel disease. Tregs express cell surface receptors allowing them to recognize autoantigens; their responses to such recognition results in the suppression of responses by conventional T cells. Whether the receptor repertoire of Tregs and the conventional T cells are the same has not been fully determined, although there is increasing evidence that Tregs derive from a thymic CD4 T-cell population with relatively high affinity for selfantigen. As noted previously, iTregs can be derived in the periphery from naive CD4 T-cell populations. This is seen when naive cells are stimulated by their cognate ligands in the presence of TGF-β and IL-2.

Cytotoxic T Cells (Chapter 37) One of the most striking actions of T cells is the lysis of cells expressing specific antigens. Most cells with such cytotoxic activity are CD8 T cells that recognize peptides derived from proteins produced within the target cell and bound to class I MHC molecules expressed on the surface of the target cell. However, CD4 T cells can express CTL activity, although in such cases the antigen recognized is a peptide associated with a class II MHC molecule; often, such peptides derive from exogenous antigens. There are two major mechanisms of cytotoxicity. One involves the production by the CTL of perforin, a molecule that can insert into the membrane of target cells and promote the lysis of that cell. Perforin-mediated lysis is mediated a series of enzymes produced by activated CTLs, referred to as granzymes. Many active CTLs also express large amounts of fas ligand on their surface. The interaction of fas

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ligand on the surface of the CTL with fas on the surface of the target cell initiates apoptosis in the target cell. CTL-mediated lysis is a major mechanism for the destruction of virus-infected cells. If activated during the period in which the virus is in its eclipse phase, CTLs may be capable of eliminating virus and curing the host with relatively limited cell destruction. On the other hand, vigorous CTL activity after a virus has been widely disseminated may lead to substantial tissue injury because of the large number of cells that are killed by the action of the CTLs. Thus, in many infections, the disease is caused by the destruction of tissue by CTLs rather than by the virus itself. One example is hepatitis B, in which much of the liver damage represents the attack of hepatitis B virus–specific CTLs on infected liver cells. It is usually observed that CTLs that have been induced as a result of a viral infection or intentional immunization must be reactivated in vitro through the recognition of antigen on the target cell. This is particularly true if some interval has elapsed between the time of infection or immunization and the time of test. This has led to some question being raised as to the importance of CTL immunity in protection against re-infection and how important CTL generation is in the long-term immunity induced by protective vaccines. On the other hand, in active infections, such as seen in individuals with HIV, CTL that can kill their target cells immediately are often seen. There is much evidence to suggest that these cells play an active role in controlling the number of HIV-positive T cells.

CYTOKINES (CHAPTERS 25 TO 28) Many of the functions of cells of the immune system are mediated through the production of a set of small proteins referred to as cytokines. These proteins can now be divided into several families. They include the type I cytokines or hematopoeitins that encompass many of the interleukins (i.e., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL13, IL-15, IL-21, IL-23, and IL-27), as well as several hematopoietic growth factors; the type II cytokines, including the interferons and IL-10; the TNF-related molecules, including TNF, lymphotoxin, and Fas ligand; Ig superfamily members, including IL-1, IL-18, IL-33, IL-36, and IL-37; and the chemokines, a large family of molecules playing critical roles in a wide variety of immune and inflammatory functions. IL-17 and its congeners, including IL-25, constitute a structurally unique set of cytokines. Many of the cytokines are T-cell products; their production represents one of the means through which the wide variety of functions of T cells are mediated. Most cytokines are not constitutive products of the T cell. Rather, they are produced in response to T-cell activation, usually resulting from presentation of antigen to T cells by APCs in concert with the action of a costimulatory molecule, such as the interaction of CD80/86 with CD28. Although cytokines are produced in small quantities, they are potent, binding to their receptors with equilibrium constants of approximately 1010 M-1. In some instances, cytokines are directionally secreted into the immunological synapse formed between a T cell and an APC. In such cases, the cytokine acts in a paracrine manner. Indeed, many cytokines

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have limited action at a distance from the cell that produced them. This appears to be particularly true of many of the type I cytokines. However, other cytokines act by diffusion through extracellular fluids and blood to target cells that are distant from the producers. Among these are cytokines that have proinflammatory effects, such as IL-1, IL-6, and TNF, and the chemokines, that play important roles in regulating the migration of lymphocytes and other cell types.

encoding these molecules show an unprecedented degree of polymorphism. This, together with their critical role in antigen presentation, explains their central role as the target of the immune responses leading to the rejection of organ and tissue allografts. The MHC also includes other genes, particularly genes for certain complement components. In addition, genes for the cytokines TNF-α and lymphotoxin (also designated TNF-β) are found in the MHC.

Chemokines (Chapter 28) A large family of small proteins that are chemotactic cytokines (chemokines) have been described. While members of this family have a variety of functions, perhaps the most dramatic is their capacity to regulate leukocyte migration and thus to act as critical dynamic organizers of cell distribution in the immune and inflammatory responses. The receptors for chemokines are seven transmembrane-spanning, G-protein coupled receptors. The chemokines are subdivided based on the number and positioning of their highly conserved cysteines. Among chemokines with four conserved cysteines, the cysteines are adjacent in one large group (the CC chemokines), whereas in a second large group they are separated by one amino acid (CXC chemokines). There are also rare chemokines in which the cysteines are separated by three amino acids (CX3C) or in which there are only two conserved cysteines (C chemokines). Individual chemokines may signal through more than one chemokine receptor, and individual receptors may interact with more than one chemokine, producing a complex set of chemokine/chemokine receptor pairs and providing opportunities for exceedingly fine regulation of cellular functions.

Class I MHC Molecules (Chapter 21) Class I MHC molecules are membrane glycoproteins expressed on most cells. They consist of an α chain of approximately 45,000 daltons noncovalently associated with α2-microglobulin, a 12,000-dalton molecule (Fig. 1.11). The gene for the α chain is encoded in the MHC, whereas that for β2-microglobulin is not. Both the α chain and β2-microglobulin are Ig supergene family members. The α chain is highly polymorphic, with the polymorphisms found mainly in the regions that constitute the binding sites for antigenderived peptides and that are contact sites for the TCR. The class I α chain consists of three extracellular regions or domains, each of similar length, designated α1, α2, and α3. In addition, α chains have a membrane-spanning domain and a short carboxy-terminal cytoplasmic tail. The

THE MAJOR HISTOCOMPATIBILITY COMPLEX AND ANTIGEN PRESENTATION (CHAPTERS 21 AND 22) The MHC has already been introduced in this chapter in the discussion of T-cell recognition of antigen-derived peptides bound to specialized grooves in class I and class II MHC proteins. Indeed, the class I and class II MHC molecules are essential to the process of T-cell recognition and response. Nonetheless, they were first recognized not for this reason but because of the dominant role that MHC class I and class II proteins play in transplantation immunity (see Chapter 46). When the genetic basis of transplantation rejection between mice of distinct inbred strains was sought, it was recognized that although multiple genetic regions contributed to the rejection process, one region played a dominant role. Differences at this region alone would cause prompt graft rejection, whereas any other individual difference usually resulted in a slow rejection of foreign tissue. For this reason, the genetic region responsible for prompt graft rejection was termed the major histocompatibility complex. In all higher vertebrates that have been thoroughly studied, a comparable MHC exists. The defining features of the MHC are the transplantation antigens that it encodes. These are the class I and class II MHC molecules. The genes

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FIG. 1.11. Model of the Class I HLA-A2 Molecule. A schematic representation of the structure of the HLA-A2 class I major histocompatibility complex (MHC) molecule. The polymorphic α1 and α2 domains are at the top. They form a groove into which antigen-derived peptides fit to form the peptide/MHC class I complex that is recognized by T-cell receptors of CD8 T cells. (Reprinted from Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC. Structure of the human class II histocompatibility antigen, HLA-A2. Nature. 1987;329:506–512).

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crystal structure of class I molecules indicates that the α1 and α2 domains form a site for the binding of peptides derived from antigens. This site is defi ned by a floor consisting of β sheets and bounded by α-helical walls. The polymorphisms of the class I molecule are mainly in these areas. In the human, three loci encoding classical class I molecules have been defined; these are designated human leukocyte antigen (HLA)-A, HLA-B, and HLA-C. All display high degrees of polymorphism. A similar situation exists in the mouse. In addition, there are a series of genes that encode class I–like molecules (class Ib molecules). Some of these have been shown to have antigen-presenting activity for formylated peptides, suggesting that they may be specialized to present certain prokaryotic antigens. The class Ib molecule CD1 has been shown to have antigen-presenting function for mycobacterial lipids, providing a mechanism through which T cells specific for such molecules can be generated. CD1d, presenting certain endogenous or exogenous phospholipids, is recognized by a novel class of T cells (NK T cells) that produce large amounts of cytokines upon immediate stimulation.

Class II MHC Molecules (Chapter 21) Class II MHC molecules are heterodimeric membrane glycoproteins. Their constituent chains are designated α and β; both chains are immunoglobulin supergene family members, and both are encoded within the MHC. Each chain consists of two extracellular domains (α1 and α2; β1 and β2, respectively), a hydrophobic domain, and a short cytoplasmic segment. The overall conformation of class II MHC molecules appears to be quite similar to that of class I molecules. The peptide-binding site of the class II molecules is contributed to by the α1 and β1 domains (see Fig. 1.5); it is within these domains that the majority of the polymorphic residues of class II molecules are found. A comparison of the three-dimensional structures of class I and class II molecules indicates certain distinctive features that explain differences in the length of peptides that the two types of MHC molecules can bind. Class I molecules generally bind peptides with a mean length of nine amino acids, whereas class II molecules can bind substantially larger peptides. In the mouse, class II MHC molecules are encoded by genes within the I region of the MHC. These molecules are often referred to as I region–associated (Ia) antigens. Two sets of class II molecules exist, designated I-A and I-E, respectively. The α and β chains of the I-A molecules (Aα and Aβ) pair with one another, as do the α and β chains of I-E (Eα and Eβ). In the human, there are three major sets of class II molecules, encoded in the DR, DQ, and DP regions of the HLA complex. Class II molecules have a more restricted tissue distribution than class I molecules. Class II molecules are found on B cells, DCs, epidermal Langerhans cells, macrophages, thymic epithelial cells, and, in the human, activated T cells. Levels of class II molecule expression are regulated in many cell types by interferons and in B cells by IL-4. Indeed, interferons can

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cause expression of class II molecules on many cell types that normally lack these cell surface molecules. Interferons also can cause striking upregulation in the expression of class I MHC molecules. Thus, immunologically mediated inflammation may result in aberrant expression of class II MHC molecules and heightened expression of class I molecules. Such altered expression of MHC molecules can allow cells that do not normally function as APCs for CD4 T cells to do so and enhances the sensitivity of such cells to CD8 T cells. This has important consequences for immunopathologic responses and for autoimmunity.

Antigen Presentation (Chapter 22) As already discussed, the function of class I and class II MHC molecules is to bind and present antigen-derived peptides to T cells whose receptors can recognize the peptide/ MHC complex that is generated. There are two major types of antigen-processing pathways, specialized to deal with distinct classes of pathogens that the T cell system must confront (see Fig. 1.8). Extracellular bacteria and extracellular proteins may enter APCs by endocytosis or phagocytosis. Their antigens and the antigens of bacteria that live within endosomes or lysosomes are fragmented in these organelles, and peptides derived from the antigen are loaded into class II MHC molecules as these proteins traverse the vesicular compartments in which the peptides are found. The loading of peptide is important in stabilizing the structure of the class II MHC molecule. The acidic pH of the compartments in which loading occurs facilitates the loading process. Once the peptide-loaded class II molecules reaches neutral pH, such as at the cell surface, the peptide/MHC complex is stable. Peptide dissociation from such class II molecules is very slow, with a half-time measured in hours. The peptide/class II complex is recognized by those CD4 T cells that have complementary receptors. As already pointed out, the specialization of CD4 T cells to recognize peptide/class II complexes is partly due to the affinity of the CD4 molecule for monomorphic determinants on class II molecules. Obviously, this form of antigen processing can only apply to cells that express class II MHC molecules. Indeed, APCs for CD4 T cells principally include cells that normally express class II MHC molecules, namely DCs, B cells, and macrophages. T cells also can recognize proteins that are produced within the cell that presents the antigen. The major pathogens recognized by this means are viruses and other obligate intracellular (nonendosomal/nonlysosomal) microbes that have infected cells. In addition, proteins that are unique to tumors, such as mutant oncogenes, or are overexpressed in tumors also can be recognized by T cells. Endogenously produced proteins are fragmented in the cytosol by proteases in the proteasome. The resultant peptides are transported into the rough endoplasmic reticulum through the action of a specialized transport system. These peptides are then available for loading into class I molecules. In contrast to the loading of class II molecules, which is facilitated by the acid pH of the loading environment, the loading of class I molecules is controlled by interaction of the class I α chain

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with β2-microglobulin. Thus, the bond between peptide and class I molecule is generally weak in the absence of β2-microglobulin, and the binding of β2-microglobulin strikingly stabilizes the complex. (Similarly, the binding of β2-microglobulin to the α chain is markedly enhanced by the presence of peptide in the α chain groove.) The peptideloaded class I molecule is then brought to the cell surface. In contrast to peptide-loaded class II molecules that are recognized by CD4 T cells, peptide-loaded class I molecules are recognized by CD8 T cells. This form of antigen processing and presentation can be performed by virtually all cells because, with a few exceptions, class I MHC molecules are universally expressed. Although the specialization of class I molecules to bind and present endogenously produced peptides and of class II molecules to bind and present peptides derived from exogenous antigens is generally correct, there are exceptions, many of which have physiologic importance. Particularly important is the capacity of some DCs to load peptides from exogenous antigens into class I MHC molecules, allowing sensitization of CD8 T cells to the antigens of pathogens that infect cells other than DCs.

T-Lymphocyte Recognition of Peptide/MHC Complexes Results in MHC-Restricted Recognition (Chapter 11) Before the biochemical nature of the interaction between antigen-derived peptides and MHC molecules was recognized, it was observed that T-cell responses displayed MHC-restricted antigen recognition. Thus, if individual animals were primed to a given antigen, their T cells would be able to recognize and respond to that antigen only if the APCs that presented the antigen shared MHC molecules with the animal that had been immunized. The antigen would not be recognized when presented by APCs of an allogeneic MHC type. This can now be explained by the fact that the TCR recognizes peptide bound to an MHC molecule. MHC molecules display high degrees of polymorphism, and this polymorphism is concentrated in the regions of the class I and class II molecules that interact with the peptide and that can bind to the TCR. Differences in structure of the MHC molecules derived from different individuals (or different inbred strains of mice) profoundly affect the recognition process. Two obvious explanations exist to account for this. First, the structure of the grooves in different class I or class II MHC molecules may determine that a different range of peptides are bound or, even if the same peptide is bound, may change the conformation of the surface of the peptide presented to the TCR. Second, polymorphic sites on the walls of the α-helices that are exposed to the TCR can either enhance or diminish binding of the whole complex, depending on their structure. Thus, priming an individual with a given antigen on APCs that are syngeneic to the individual will elicit a response by T cells whose TCRs are specific for a complex consisting of a peptide derived from the antigen and the exposed polymorphic residues of the MHC molecule. When the same antigen is used with APCs of different MHC type, it is unlikely that the same peptide/MHC surface can be formed, and thus the primed T cells are not likely to bind and respond to such stimulation.

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Indeed, this process also occurs within the thymus in the generation of the T-cell repertoire, as already discussed. T cells developing within the thymus undergo a positive selection event in which those T cells capable of recognizing MHC molecules displayed within the thymus are selected (and the remainder undergo programmed cell death). This leads to the skewing of the population of T cells that emerges from the thymus so that the cells are specialized to respond to peptides on self-MHC molecules. One of the unsolved enigmas of positive selection within the thymus is how the vast array of T cells with receptors capable of reacting with a very large set of foreign peptides associated with self-MHC molecules are chosen by self-MHC molecules that can only display self-peptides. It is believed that a high degree of cross-reactivity may exist so that T cells selected to bind a given class I (or class II) molecule plus a particular self-peptide can also bind a set of other (foreign) peptides bound to the same MHC molecule. Furthermore, the affinity of an interaction required for positive selection in the thymus appears to be considerably lower that that required for full activation of peripheral T cells. Thus, thymocytes selected by a given self-peptide/ self-MHC complex will generally not mount a full response when they encounter the same peptide/MHC complex in the periphery, although they will respond to a set of foreign peptide/MHC complexes to which they bind with higher affinity. Recognition of the self-peptide/self-MHC complex in the periphery nonetheless is important in sustaining the viability of resting T-lymphocytes. Our modern understanding of T-cell recognition also aids in explaining the phenomenon of immune response gene control of specific responses. In many situations, the capacity to recognize simple antigens can be found in only some members of a species. In most such cases, the genes that determine the capacity to make these responses have been mapped to the MHC. Such immune response gene control of immune responses is based on the capacity of different class II MHC molecules (or class I MHC molecules) to bind different sets of peptides. Thus, for simple molecules, it is likely that peptides can be generated that are only capable of binding to some of the polymorphic MHC molecules of the species. Only individuals that possess those allelic forms of the MHC will be able to respond to those antigens. Based on this, some individuals are nonresponders because of the failure to generate a peptide/MHC molecule complex that can be recognized by the T-cell system. This mechanism also may explain the linkage of MHC type with susceptibility to various diseases. Many diseases show a greater incidence in individuals of a given MHC type. These include reactive arthritides, gluten-sensitive enteropathy, insulin-dependent diabetes mellitus, and rheumatoid arthritis (see Chapter 44). One explanation is that the MHC type that is associated with increased incidence may convey altered responsiveness to antigens of agents that cause or exacerbate the disease. Indeed, it appears that many of these diseases are due to enhanced or inappropriate immune responses.

Antigen-Presenting Cells (Chapter 16) T cells recognize peptide/MHC complexes on the surface of other cells. Such cells are often referred to as APCs. Although

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effector cells can mediate their functions by recognizing such complexes on virtually any cell type, naïve cells are most efficiently activated by a set of specialized APC, the DCs. DCs are a multimember family with distinctive locations and functions. Among them are the plasmacytoid DCs that are the principal source of type I interferons in viral infections. In general, in their immature form, DCs are resident in the tissues where they are efficient at capturing and endocytosing antigen. Their antigen capture activity is dependent upon expression of several surface receptors including Fc receptors, receptors for heat shock proteins, and C-type lectins. If they receive signals, such as various inflammatory stimuli, often mediated by TLRs, they downregulate the expression of these molecules but increase their expression of surface MHC molecules and various costimualtory molecules such as CD80/86. In addition, such stimulation induces expression of chemokine receptors such as CCR2 and CCR7. The latter allow stimulated DCs to follow signals from the chemokines SLC and ELC, and to migrate into the T-cell zone of lymph nodes. As part of the maturation process, they may also acquire the capacity to produce cytokines and express surface molecules that can aid in determining the polarization of T-cell priming. This includes the production of IL-12, IL-23, IL-6, and IL-10, and the expression of inducible costimulator ligand and of Notch ligands. Interaction of naïve T cells with immature DCs may induce a state of peripheral tolerance.

EFFECTOR MECHANISMS OF IMMUNITY The ultimate purpose of the immune system is to mount responses that protect the individual against infections with pathogenic microorganisms by eliminating these microbes or, where it is not possible to eliminate infection, to control their spread and virulence. In addition, the immune system may play an important role in the control of the development and spread of some malignant tumors. The responses that actually cause the destruction of the agents that initiate these pathogenic states (e.g., bacteria, viruses, parasites, tumor cells) are collectively the effector mechanisms of the immune system. Several have already been alluded to. Among them are the cytotoxic action of CTLs, which leads to the destruction of cells harboring viruses and, in some circumstances, expressing tumor antigens. In some cases, antibody can be directly protective by neutralizing determinants essential to a critical step through which the pathogen establishes or spreads an infectious process. However, in most cases, the immune system mobilizes powerful nonspecific mechanisms to mediate its effector function.

Effector Cells of the Immune Response Among the cells that mediate important functions in the immune system are cells of the monocyte/macrophage lineage, NK cells, mast cells, basophils, eosinophils, and neutrophils. It is beyond the scope of this introductory chapter to present an extended discussion of each of these important cell types. However, a brief mention of some of their actions will help in understanding their critical functions in the immune response.

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Monocytes and Macrophages (Chapter 19) Cells of the monocyte/macrophage lineage play a central role in immunity. One of the key goals of cellular immunity is to aid the macrophages in eliminating organisms that have established intracellular infections. In general, non-activated macrophages are inefficient in destroying intracellular microbes. However, the production of IFN-γ and other mediators by T cells can enhance the capacity of macrophages to eliminate such microorganisms. Several mechanisms exist for this purpose, including the development of reactive forms of oxygen, the development of nitric oxide, and the induction of a series of proteolytic enzymes, as well as the induction of cytokine production. Macrophages can act as APCs and thus can enlist the “help” of activated, cytokineproducing CD4 + T cells in regulating their function. Although macrophages function as APCs for attracting activated T cells, they do not appear to be particularly effective in the activation of naïve CD4 T cells. In instances in which they are the site of infection or have phagocytosed infectious agents or their proteins, antigens from these agents may be transferred to DCs. In such cases, the DCs would be the principal APCs that activate naïve or possibly resting memory CD4 T cells. Such activated T cells would then be available to help infected macrophages.

Natural Killer Cells (Chapter 17) NK cells play an important role in the immune system. Indeed, in mice that lack mature T and B cells due to the scid mutation, the NK system appears to be highly active and to provide these animals a substantial measure of protection against infection. NK cells are closely related to T cells. They lack conventional TCR (or Ig) but express two classes of receptors. They have a set of activating receptors that allow them to recognize features associated with virally infected cells or tumor cells. They also express receptors for MHC molecules that shut off their lytic activity. Thus, virally infected cells or tumor cells that escape the surveillance of cytotoxic T cells by downregulating or shutting off expression of MHC molecules then become targets for efficient killing by NK cells because the cytotoxic activity of the latter cells is no longer shut off by the recognition of particular alleles of MHC class I molecules. In addition, NK cells express a receptor for the Fc portion of IgG (Fc γRIII). Antibody-coated cells can be recognized by NK cells, and such cells can then be lysed. This process is referred to as antibody-dependent cellular cytotoxicity. NK cells are efficient producers of IFN-γ. A variety of stimuli, including recognition of virally infected cells and tumor cells, cross-linkage of FcγRIII, and stimulation by the cytokines IL-12 and IL-18, cause striking induction of IFN-γ production by NK cells.

Mast Cells and Basophils (Chapters 20 and 45) Mast cells and basophils play important roles in the induction of allergic inflammatory responses. They express cell surface receptors for the Fc portions of IgE (Fc εRI) and for certain classes of IgG (Fc γR). This enables them to bind antibody to

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their surfaces, and when antigens capable of reacting with that antibody are introduced, the resultant cross-linkage of Fc εRI and/or Fc γR results in the prompt release of a series of potent mediators such as histamine, serotonin, and a variety of enzymes that play critical roles in initiating allergic and anaphylactic-type responses. In addition, such stimulation also causes these cells to produce a set of cytokines, including IL-3, IL-4, IL-13, IL-5, IL-6, granulocyte-macrophage colony-stimulating factor, and TNFα , that have important late consequences in allergic inflammatory responses.

Lectin Pathway

Granulocytes (Chapter 20) Granulocytes have critical roles to play in a wide range of inflammatory situations. Rather than attempting an extended discussion of these potent cells, it may be sufficient to say that in their absence it is exceedingly difficult to clear infections with extracellular bacteria and that the immune response plays an important role in orchestrating the growth, differentiation, and mobilization of these crucial cells. Recent work indicates that TH17 cells are particularly important because of their role in recruiting granulocytes to sites of immune responses.

Eosinophils (Chapters 20 and 45) Eosinophils are bone marrow–derived myeloid cells that complete their late differentiation under the influence of IL-5. They migrate to tissue sites in response to the chemokine eotaxin and as a result of their adhesion receptors. Because TH2 cells can produce IL-5 and stimulate the production of eotaxin, eosinophil accumulation is often associated with TH2-mediated inflammation. Eosinophils store a series of proteins in their secondary granules including major basic protein, eosinophil cationic protein, and eosionphil peroxidase. When released, these proteins are responsible for much of the damage that eosinophils mediate both to helminthic parasites and to the epithelium. Eosinophils have been implicated as important in protective responses to helminths and in the tissue damage seen in allergic inflammation in conditions such as asthma.

The Complement System (Chapter 36) The complement system is a complex system of proteolytic enzymes, regulatory and inflammatory proteins and peptides, cell surface receptors, and proteins capable of causing the lysis of cells. The system can be thought of as consisting of three arrays of proteins. Two of these sets of proteins, when engaged, lead to the activation of the third component of complement (C3) (Fig. 1.12). The activation of C3 releases proteins that are critical for opsonization (preparation for phagocytosis) of bacteria and other particles, and engages the third set of proteins that insert into biologic membranes and produce cell death through osmotic lysis. In addition, fragments generated from some of the complement components (e.g., C3a and C5a) have potent inflammatory activities.

The Classical Pathway of Complement Activation The two activation systems for C3 are referred to as the classical pathway and the alternative pathway. The classical pathway is initiated by the formation of complexes of

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FIG. 1.12. The Complement System. The classical pathway of complement activation, usually initiated by the aggregation of C1 by binding to antigen/antibody complexes, resulting in the formation of an enzyme, a C3 convertase, that cleaves C3 into two fragments, C3b and C3a. The classical pathway can also be initiated by the aggregation of mannan-binding lectin as a result of binding sugars expressed in the capsules of many pathogenic microbes. The components of the lectin pathway appear to mimic the function of C1qrs. The alternative pathway of complement activation provides a potent means of activating complement without requiring antibody recognition of antigen. It results in the formation of a distinct C3 convertase. The fragments formed by cleaving C3 have important biologic activities. In addition, C3b, together with elements of the classical pathway (C4b, C2a) or the alternative pathway (Bb, properdin), form enzymes (C5 convertases) that cleave C5, the initial member of the terminal family of proteins. Cleavage of C5 leads to the formation of the membrane attack complex that can result in the osmotic lysis of cells.

antigen with IgM or IgG antibody. This leads to the binding of the first component of complement, C1, and its activation, creating the C1 esterase that can cleave the next two components of the complement system, C4 and C2. C4 is a trimeric molecule, consisting of α , β, and γ chains. C1 esterase cleaves the α chain, releasing C4b, which binds to surfaces in the immediate vicinity of the antigen/ antibody/C1 esterase complex. A single C1 esterase molecule will cause the deposition of multiple C4b molecules. C2 is a single polypeptide chain that binds to C4b and is then proteolytically cleaved by C1 esterase, releasing C2b. The resulting complex of the residual portion of C2 (C2a) with C4b (C4b2a) is a serine protease whose substrate is C3. Cleavage of C3 by C4b2a (also referred to as the classical pathway C3 convertase) results in the release of C3a and C3b. A single antigen/antibody complex and its associated C1 esterase can lead to the production of a large number of C3 convertases (i.e., C4b2a complexes) and thus to cleavage of a large number of C3 molecules. The components of the classical pathway can be activated by a distinct, non–antibody-dependent mechanism, termed the lectin pathway. The mannose-binding lectin (MBL) is

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activated by binding to (and being cross-linked by) repetitive sugar residues such as N-acetylglucosamine or mannose. The activation of MBL recruits the MBL-associated serine proteases MASP-1 and MASP-2, which cleave C4 and C2 and lead to the formation of the classical pathway C3 convertase. Because the capsules of several pathogenic microbes can be bound by MBL, the lectin pathway provides an antibody-independent mechanism through which the complement system can be activated by foreign microorganisms.

The Alternative Pathway of Complement Activation Although discovered more recently, the alternative pathway is the evolutionarily more ancient system of complement activation. Indeed, it, and the MBL activation of the classical pathway, can be regarded as important components of the innate immune system. The alternative pathway can be activated by a variety of agents such as insoluble yeast cell wall preparations and bacterial lipopolysaccharide. Antigen/antibody complexes also can activate the alternative pathway. The C3 convertase of the alternative pathway consists of a complex of C3b (itself a product of cleavage of C3) bound to the b fragment of the molecule factor B. C3bBb is produced by the action of the hydrolytic enzyme, factor D, that cleaves factor B; this cleavage only occurs when factor B has been bound by C3b. Apart from the importance of the alternative pathway in activating the complement system in response to nonspecific stimulants, it also can act to amplify the activity of the classical pathway because the C3 convertase of the classical system (C4b2a) provides a source of C3b that can strikingly enhance formation of the alternative pathway convertase (C3bBb) in the presence of factor D.

The Terminal Components of the Complement System C3b, formed from C3 by the action of the C3 convertases, possesses an internal thioester bond that can be cleaved to form a free sulfhydryl group. The latter can form a covalent bond with a variety of surface structures. C3b is recognized by receptors on various types of cells, including macrophages and B cells. The binding of C3b to antibody-coated bacteria is often an essential step for the phagocytosis of these microbes by macrophages. C3b is also essential to the engagement of the terminal components of the complement system (C5 through C9) to form the membrane attack complex that causes cellular lysis. This process is initiated by the cleavage of C5, a 200,000-dalton two-chain molecule. The C5 convertases that catalyze this reaction are C4b2a3b (the classical

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pathway C5 convertase) or a complex of C3bBb with a protein designated properdin (the alternative pathway C5 convertase). Cleaved C5, C5b, forms a complex with C6 and then with C7, C8, and C9. This C5b/C9 complex behaves as an integral membrane protein that is responsible for the formation of complement-induced lesions in cell membranes. Such lesions have a donut-like appearance, with C9 molecules forming the ring of the donut. In addition to the role of the complement system in opsonization and in cell lysis, several of the fragments of complement components formed during activation are potent mediators of inflammation. C3a, the 9,000-dalton fragment released by the action of the C3 convertases, binds to receptors on mast cells and basophils, resulting in the release of histamine and other mediators of anaphylaxis. C3a is thus termed an anaphylotoxin, as is C5a, the 11,000-dalton fragment released as a result of the action of the C5 convertases. C5a is also a chemoattractant for neutrophils and monocytes. Finally, it is important to note that the process of activation of the complement cascade is highly regulated. Several regulatory proteins (e.g., C1 esterase inhibitor, decay accelerator factor, membrane cofactor protein) exist that function to prevent uncontrolled complement activation. Abnormalities in these regulatory proteins are often associated with clinical disorders such as hereditary angioedema and paroxysmal nocturnal hemoglobinuria.

CONCLUSION This introductory chapter should provide the reader with an appreciation of the overall organization of the immune system and of the properties of its key cellular and molecular components. It should be obvious that the immune system is highly complex, that it is capable of a wide range of effector functions, and that its activities are subject to potent, but only partially understood, regulatory processes. As the most versatile and powerful defense of higher organisms, the immune system may provide the key to the development of effective means to treat and prevent a broad range of diseases. Indeed, the last two sections of this book deal with immunity to infectious agents and immunologic mechanisms in disease. The introductory material provided here should be of aid to the uninitiated reader in understanding the immunologic mechanisms brought into play in a wide range of clinical conditions in which immune processes play a major role either in pathogenesis or in recovery.

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2

History of Immunology Steven Greenberg

INTRODUCTION There comes a time during every argument at which the two opposing parties reach a critical juncture: either resolution or impasse. Variations on this essentially Socratic theme have played out in all spheres of human intellectual activity. The dialectic of science ranges from incremental and relatively harmonious shifts in key, to a few abruptly dissonant ones, taking the form of what Thomas Kuhn would refer to as “paradigm shifts.”1 Such is the case with immunology, a field distinguished by more than its fair share of paradigm shifts. Arguably, its first dialectic, between the “cellularists” and the “humoralists,” did not result in an early synthesis, but was characterized by partisan and often entrenched positions. Ultimately, the two parallel paths of cellular and chemical immunology converged, but it was not until the latter half of the 20th century that the two paths became one. How the paths were forged in the first place was an amalgam of the cultural institutions of the time, the creative output of the scientists themselves, and the imperatives of devising effective strategies to combat infection and contagion.

ANTECEDENTS TO THE GERM THEORY OF DISEASE Ancient Theories of Disease Causation Religious beliefs in ancient Greece drew contrasts between the sacred or the pure (katharos) and the polluted (miaros).2 Pollution, or miasma, was blamed for many ancient transgressions, from the petty and personal, to the gravest, most famously embodied in the Oedipus myth. To remove the stain of miasma, the transgressor must undergo rites of purification (catharsis). To the ancient Greeks in the age of Homer, these were deeply ingrained beliefs that were essentially religious in nature. Because miasma was viewed as a source of suffering, it is not surprising that miasma was implicated in disease, as described by Hippocrates in his treatise “On Air, Water, and Places” in which miasma was associated with “unhealthy vapors.”3,4 Hippocrates is credited with being the first to recognize the potential of disease to arise from the environment and not as a result of religious superstition. Mal aria, which is Old Italian for “bad air,” was one of many diseases thought to be caused by miasma. The concept that miasma was the source of disease persisted through the millennia and was a leading theory of how contagious diseases were transmitted up until the time of Pasteur. Much of what we know about the medicine of ancient Greece is codified in The Hippocratic Corpus, a collection of more than 60 volumes of text. Its authorship is disputed,

but it is generally recognized as a compilation of works by Hippocrates himself as well as his students and intellectual heirs. One of his students, as well as son-in-law, Polybus, was credited as the author of De Natura Hominis (On the Nature of Man), the earliest known text describing the ancient Greek conceptual basis for disease pathogenesis, as embodied in the four humors: black bile, yellow bile, phlegm, and blood.5 The body of man has in itself blood, phlegm, yellow bile and black bile; these make up the nature of his body, although these he feels pain or enjoys health. Now he enjoys the most perfect health when these elements are duly proportioned to one another in respect of compounding, power and bulk, and when they are perfectly mingled. Pain is felt when one of these elements is in defect or excess, or is isolated in the body without being compounded with all the others. The Greek view that disease arose from an imbalance of the four humors did not supersede the miasma theory of disease, but was rather a more general view of disease causation, compared with the subset of apparently communicable diseases best explained by miasma. The Romans developed and refined Greek concepts of disease. Marcus Terentius Varro (116 to 27 bce), referred to as “the most learned of all Romans” by the Roman rhetorician Quintilian,6 was a prolific Roman scholar, estimated to have written more than 600 volumes. During the civil war of the first century, he served as Pompey’s legate in Spain and fought at Pharsalus against Caesar but ultimately reconciled with Caesar, who appointed him director of the public library in 47 bce. Varro is perhaps best known for his only complete extant work, Rerum Rusticarum Libri Tres (On Agricultural Topics), in which he so presciently anticipated the existence of disease-causing microbes that seemed to Varro to be the immediate cause of diseases7: Precautions must also be taken in the neighbourhood of swamps, both for the reasons given, and because there are bred certain minute creatures which cannot be seen by the eyes, which float in the air and enter the body through the mouth and nose and there cause serious diseases. Thus, Varro provided a mechanistic basis for disease that was consistent with the prevailing belief of miasma as the source of illness. The Greek medical tradition was carried on for generations and was ultimately passed on to Claudius Galinus

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(Galen), the Greek expatriate who was its greatest explicator. Galen was born in Pergamum in Asia Minor in 130 ce. After beginning his medical training in Pergamum at the behest of his father, he traveled widely in pursuit of “postgraduate” medical training in Smyrna, Corinth, and Alexandria. He returned to Pergamum and practiced surgery on gladiators, which provided a unique opportunity to deepen his knowledge of human anatomy and perfect his surgical technique.8 Following an outbreak of plague among the Roman troops in Aquileia in 168 ce, he was summoned by the Emperor Marcus Aurelius and was appointed personal physician to his son, Commodus.8 Galen’s view of medicine was based on Hippocrates’ Corpus. His output was prodigious more than any other ancient author of medical texts. He distinguished symptoms from diseases and offered explanations of the former that were consistent with his interpretations of disease pathogenesis; thus, tertian fever was the result of an “imbalance of yellow bile,” quartan was caused by “too much black bile,” and quotidian by “an excess of phlegm.” Vomiting was viewed as the body’s attempt to expel poisons, and the prescription of bleeding was to rid the body of “corrupt humors.”9 Galen’s view of medicine remained the dominant one until the 17th century.

It seemed inevitable that in seeking an explanation for why some developed disease and others did not, many others would rely on a moralistic or religious view. In a remarkable passage from Galen’s On the Different Types of Fever, not only does he set forth an explanation of how disease is transmitted through the air, and using the same term as Fracastoro would, “seeds,” some 1300 years later, but he also blames a licentious lifestyle for susceptibility to the plague:

Early Concepts of Immunity

This passage has been analyzed extensively by Nutton, who questioned the extent to which “seed” is used metaphorically12 ; if so, it is particularly apt. Religious explanations for disease and immunity persisted throughout history. Particularly during the growth of Christianity during the Middle Ages, disease and sin were linked, though not inextricably; the great theologian Thomas Aquinas provided this distinction between sin and other causes of diseases13 :

The term “immunity” itself is derived from the Latin practice of “exemption” from taxes or public service that normal citizens had to discharge, a favor bestowed by the emperor to meritorious individuals or entire communities.10 However, the concept of immunity dates back at least as far as Thucydides, who described the plague of Athens of 430 bce that was responsible for the death of more than a quarter of the Athenian population11: Yet it was with those who had recovered from the disease that the sick and the dying found most compassion. These knew what I had from experience and had now no fear for themselves; the same man was never attacked twice-never at least fatally. The traditional, religious view of the plague was that it is the work of Apollo who was held responsible for earlier plagues (eg, the plague on the Greek army in Troy because the Greek general Agamemnon abducted the daughter of Apollo’s priest, Chryses). The religiously inclined could take some refuge in appealing to Apollo’s son, Asklepios, who was revered as the god of healing, as were his daughters Hygeia (Hygiene), Iaso (Remedy), Akæso (Healing), Aglæa (Healthy glow), and Panakeia (Cure-all). In contrast, Thucydides characteristically did not offer facile religious explanations for sickness or recovery. In fact, he described the futility of the Athenians’ plight in stark terms11: Neither the fear of the gods nor laws of men awed any man, not the former because they concluded it was alike to worship or not worship from seeing that alike they all perished, nor the latter because no man expected that lives would last till he received punishment of his crimes by judgment.

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Suppose, for example, that the circumambient air carries certain seeds of plague, and that of the bodies which share [breathe] it, some are full of various residues which are soon to become putrefied in themselves, while others are clean and free of such residues. Assume also that in the former there is a general blockage of their pores, a so-called plethora, and a life of ease devoted to gluttony, drink and sex, with all their necessarily concomitant digestive disorders. The others, which are clean and lack these residues, as well as being fine in themselves, have all a wholesome transpiration through pores that are neither blocked nor constricted; they take appropriate exercise and lead a temperate life. Assuming all this, which of these bodies is most likely to be affected by the rotting air they inspire?

. . . we need to consider that sin consists of a disorder of the soul, just as physical disease consists of a disorder of the body. And so sin is a disease of the soul, as it were, and pardon is for sin what healing is for disease. Yet for many, a disease as dire as the plague would continue to be viewed as divine retribution for sin; for others, it was the result of astrological phenomena, while for still others, it was the result of a “conspiracy plotted by Jews to poison wells.”14 With the exception of the latter, which at least offered a proximate physical cause of disease, regardless of the lack of evidence, there was an abstract quality to these explanations that were shrouded in belief and superstition but lacking in substance. Further theories of disease pathogenesis would have to await the 16th century.

Fracastoro’s Seeds Girolamo Fracastoro (1478 to 1553) was an Italian who would have met most definitions of a Renaissance scholar: an accomplished physician, poet, mathematician, botanist, and astronomer. Educated by his father in Verona, and later at the University of Padua, he became an instructor in logic at the University of Padua in 1501 and in anatomy in 1502. He left Padua in 1508 and returned to Verona, where he

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dedicated himself to his studies and his medical practice. In 1546, he proposed that disease was caused by seminaria (“seeds”) that could be transmitted by three ways: direct contact from one person to another; through “fomites,” or articles of clothing or dirty linen; and through the air. Although Nutton12 has pointed out that Galen and Lucretius also used the term “seeds” to describe the transmission of illness through the air (see previous discussion), Fracastoro was the first to make them the focal point for disease transmission and to describe their predilection for certain organs. Like the 10th century Arab physician Rhazes, who believed smallpox to have an affinity for blood, and specifically for the traces of menstrual blood that were believed to taint everyone in utero, as later suggested by Avicenna, Fracastoro offered the following explanation for immunity to smallpox: Following infection by smallpox seminaria, the menstrual blood would putrefy, rise to the surface, and force its way out via the smallpox pustules.15 “Hence when this process has taken place, the malady usually does not recur because the infection has already been secreted in the previous attack.” Fracastoro’s seminaria theory remained influential for nearly three centuries, in many ways serving as an early template for the germ theory of disease.

THE GERM THEORY OF DISEASE AND DEVELOPMENT OF VACCINES Until the mid-19th century, the Galenic view of disease origins was the dominant one. Not only was the etiology of diseases misunderstood but also was the origin of life itself. The theory of spontaneous generation, which arose from Aristotle, held that life originated spontaneously from inanimate matter. The first experimental evidence against spontaneous generation came from Franceso Redi, the head physician in the Medici court, who in 1668 provided early evidence against the theory.16* Nevertheless, no overarching theory was proposed to replace it. Neither the mindset nor the necessary technology were available until the latter part of the 17th century, when an apprentice in a dry goods store, Anton van Leeuwenhoek, began a lifelong obsession with grinding the perfect lens. Leeuwenhoek’s lenses were tiny but were ground with high degree of curvature, enabling him to visualize the hitherto undiscovered word of microbes. On September 17, 1683, Leeuwenhoek wrote a letter to the Royal Society, which was the first description of living, motile bacteria obtained from the plaque of his own teeth.17 Leeuwenhoek was not the fi rst to build a microscope (which was used by, among others, Redi), but his was far superior to existing multilensed or compound microscopes. Other scientists of the time, notably Robert Hooke, also observed microorganisms, and it was Hooke who was the first to publish the first image of a microorganism (the fungus Mucor) in 1665.18 Some 150 years later, Dutrochet and then later Schwann, Schleiden, and Virchow, taking advantage of *

Much later, Spallanzani and Pasteur provided key experimental evidence against spontaneous generation, a belief that some scholars still held in the late 19th century, when Pasteur, for example, showed that broth in swan-neck-bent, but not unbent or broken flasks, failed to support microbial growth.

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the microscopes of the their time, advanced the concept that “all living things are composed of cells and cell products.”19 Virchow took this one step further by declaring omnis cellula e cellula or “all cells develop only from existing cells.” Whether Virchow rejected the germ theory or not is a matter of debate, but it is more likely that Virchow’s underlying emphasis was not on external causes, but disease mechanisms, as he wrote in 1885: “First the discovery of the parasite, then the investigation of its etiology, then the question: how does it give rise to the disease.”20 Although it is hard to escape the possibility that a certain degree of professional jealousy may have played a role in Virchow’s refusal to embrace the germ theory of disease, his viewpoint is one of but many examples of apparently strict dichotomies in science that would ultimately undergo revision and later synthesis. This is a theme that was to be recapitulated many times in the history of immunology.

The Conceptual Basis for the Germ Theory of Disease Between Leeuwenhoek’s technical breakthroughs in lens design in the late 17th century and the work of Pasteur and Koch in the late 19th century, several individuals endorsed Fracastoro’s “seed” theory, which gained new relevance when bacteria were first visualized. Among these was Jacob Henle, a German pathologist who was later to become Koch’s teacher. Henle wrote a treatise that not only laid out the germ theory of disease in great detail but also arguably articulated an early version of what would later be known as “Koch’s postulates”21: “Before microscopic forms can be regarded as the cause of contagion in man, they must be constantly found in contagious material. They must be isolated from it and their strength tested.” However, Henle’s essay was a theoretical one and Henle himself never provided any experimental evidence in support of it. In the same year, decades before Pasteur and Koch would even begin to describe the germ theory of disease, Henry Holland, a Scottish-trained physician to Queen Caroline, who traveled extensively and was acquainted with the scientific luminaries of the time, including Davie, Gay Lussac, Berthollet, and Laplace, wrote a treatise in which he stated,22 The question is, what weight we may attach to the opinion that certain diseases, and especially some of epidemic and contagious kind, are derived from minute forms of animal life, existing in the atmosphere under particular circumstances; and capable, by application to the lining membranes or other parts, of acting as a virus on the human body. In a footnote, he cited others, including Kircher, and particularly Johannes Nyander, who wrote nearly a century earlier23 : . . . it may be an easy matter for very minute insects to be the causes of diverse contagious diseases,” of which he included plague, measles, smallpox, and syphilis. Nyander himself credits Lynceus Leuwenhoekius (“lynx-eyed” or “keen-eyed” Leuwenhoek) as the first to have seen such

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“animalcules.” Thus, it is fair to say that the germ theory of disease itself had been “germinating” for some time prior to its scientific proof by Pasteur and Koch.

Experimental Evidence for the Germ Theory of Disease The honor of the first experimental demonstration for the germ theory of disease may belong to two students of Francesco Redi. In 1687, soon after Redi had offered proof against spontaneous generation, two of his students, Bonomo and Cestoni, went on to observe the causative agent of scabies using the newly developed microscope and were able to transfer disease from person to person.24 Perhaps the first demonstration of the bacterial pathogenesis of diseases in animals and humans was by Casimir Davaine, a French scientist who provided essentially the same evidence that Pasteur and Koch would years later that anthrax was caused by bacteridies. In 1865, Davaine was awarded the Prix Bréant by the Académie des Science for his work.25 Davaine’s compatriot, Louis Pasteur, was the consummate experimentalist. A chemist by training, his interest in infectious disease began with his study of fermentation. He made important contributions to the science of fermentation and his work led to many practical benefits to the beer and wine industry of France. His interest in microbes began with his speculation that the same type of microbe required for fermentation was likely responsible for transmitting disease. In 1865, he was asked to investigate a disease called pébrine that affected the silk worm industry. Within a year, Pasteur had established that pébrine was caused by a microbe, which provided further proof for the germ theory of disease. Some 14 years later, his expertise was again sought out of economic interests, in this case by farmers whose poultry stocks were diminished by chicken cholera. In a famous example of scientific serendipity, his assistant, Charles Chamberland, failed to inoculate chickens with cultures of chicken cholera bacilli, as instructed by Pasteur, but instead went on vacation. Upon returning several weeks later, he inoculated chickens with the old bacterial cultures, but the chickens didn’t die as expected. Rather than disregard the experiment as a failure, “chance had favored the prepared mind” of Pasteur, who had his assistants inject fresh cholera into the same hens that had previously been injected; now none of the hens became ill. Pasteur had surmised that the bacterial cultures had become weakened by extended culture.26 Thus began the use of attenuated strains of microbes to immunize against disease, 27 and the birth of the science of vaccination. The term “vaccine,” derived from the Latin vaccus for cow, was, coined by Pasteur in honor of Jenner. Attenuation as a strategy of developing vaccines would prove to be enormously valuable, leading to development of the first rabies vaccine by Pasteur himself, a vaccine against the viral causative agent of yellow fever by Theiler,28 and the Bacillus Calmette-Guérin vaccine at Pasteur’s institute.29 Although Robert Koch is credited as the originator of “Koch’s Postulates,” it may come as a surprise that the essence of the postulates were fi rst formulated by Koch’s

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teacher, Jacob Henle (see previous discussion) and his contemporary, Edwin Klebs.30 However, neither Henle nor Klebs applied their theories to any practical benefit, which is why Koch received credit for the postulates. Koch was the fi rst to articulate then systematically apply them to prove that Bacillus anthracis was the causative agent of anthrax. Koch was a country physician living in Prussia, whose scientific career began unceremoniously with a gift of a microscope from his wife.31 His fi rst series of investigations began with observing the blood of a dead cow that succumbed to anthrax. Confi rming the observation of Davaine and others before him, he observed fi lamentous bacteria in the blood. Not content merely with the observation, he began a series of technically challenging experiments, necessitating the development of many novel techniques used in microbiology laboratories even today, such as the use of solid medium to grow individual clones or colonies of bacteria. He proved that the fi lamentous bacteria were present only in infected animals and were capable of reproducing the disease when injected into healthy animals. This was the fi rst systematic application of the eponymous postulates, which has since become the sine qua non of disease causation by infectious agents. His work was published in 1876, 32 the fi rst of many groundbreaking publications. Koch’s most profoundly important contribution to medicine was the discovery of the causative agent of tuberculosis. The lecture at which he announced his fi ndings, on March 24, 1882, described later by Ehrlich as “the most important experience of his scientific life,” is considered by many to be the single most important lecture in medical history. Koch described the invention of novel staining methods to detect the tubercle bacillus and presented tissue dissections from guinea pigs that were infected with tuberculous material from the lungs of infected apes and humans who had died from the disease. 33 For “his investigations and discoveries in regard to tuberculosis,” Koch was awarded the Nobel Prize in 1905.

The Unhealthy Rivalry Between Pasteur and Koch, and its Lasting Effects The Franco-Prusssian war of 1870, the culmination of years of tension between France and Prussia, resulted in a Prussian victory and unity among the German states under King Wilhelm of Prussia. The Treaty of Frankfurt left a unified Germany the city of Strasbourg as well as possession of Alsace and the northern part of Lorraine, which was thought to contribute to further resentment of the Germans by the French and public support for World War I. It is against this backdrop that the relationship between Pasteur and Koch must be viewed. Koch had served in the Prussian army, and Pasteur’s son was a conscript fighting for the French. Furthermore, there was intense professional rivalry between the two, especially over their work on anthrax pathogenesis. According to a letter from Charles Ruel, former privat docent at the University of Geneva, Koch was in the audience when Pasteur spoke on attenuation and vaccination at the fourth International Congress of Hygiene and Demography held in Geneva in September 1882. Pasteur spoke repeatedly about

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German collected works (recueil Allemand). According to the letter34 : Koch and his friend Prof. Lichtheim, were sitting side by side; they knew French only imperfectly and both mistook the word pride (orgueil) for collection (recueil). They felt their self-respect profoundly wounded and interpreted the words German pride as a grave insult. This is but one ironic example of the level of rancor and misunderstanding between the two great men. It is said that Pasteur and Koch underwent some form of reconciliation later in their lives. Regardless, the aftermath of their rivalry, in many ways personifying the bitter relations between France and Germany, had a lasting effect on the evolution of the nascent field of immunology. In the years to come, the intellectual heirs of Pasteur and Koch would reenact the lifelong competitive tensions that characterized their relationship.

The Germ Theory of Disease: A Summation In many ways, the “germ theory of disease” really did not begin with Pasteur and Koch, but rather by their predecessors, Henle, Klebs, and Davaine, who in turn owed credit to Fracastoro, and ultimately to Galen and Varro, some 1,600 years earlier. What enabled Pasteur and Koch to firmly establish the germ theory of disease began as a thought process that over time became distilled to something tangible: First, the idea that invisible “seeds” might propagate disease; second, the advent of an optically superior microscope, by Leuwenhoek, which enabled scientists their first glimpses at the “minute creatures” postulated by Varro; third, the growing evidence against spontaneous generation that began with Redi, thus opening the door for a new theory of disease; and finally, the inductive genius and careful experimental techniques of Pasteur and Koch.

The Long History of Vaccination: Success and the Unprepared Mind Vaccination did not originate with Pasteur; its practice had been carried out for centuries without any fundamental understanding of its basis. Probably the earliest recorded example of intentionally inducing immunity to an infectious disease was in the 10th century in China, where smallpox was endemic.35 The process of “variolation” involved exposing healthy people to material from the lesions caused by the disease, either by putting it under the skin, or, more often, inserting powdered scabs from smallpox pustules into the nose. Variolation was known and practiced frequently in the Ottoman Empire, where it had been introduced by Circassian traders in the 17th century.35 Unfortunately, because there was no standardization of the inoculum, variolation occasionally resulted in death or disfigurement from smallpox, thus limiting its acceptance. Variolation later became popular in England, mainly due to the efforts of Lady Mary Wortley Montague. Lady Montague was the wife of the British ambassador to the Ottoman court who herself had contracted a

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severe case of smallpox. While in Istanbul, Lady Montague observed the practice of variolation. Determined not to have her family suffer as she had, she directed the surgeon of the embassy to learn the technique and, in March 1718, to variolate her 5-year-old son. After her return to England, she promoted the technique and had her surgeon variolate her 4-year-old daughter in the presence of the king’s physician. The surgeon, Charles Maitland, was given leave to perform what came to be known as the “Royal Experiment,” in which he variolated six condemned prisoners who later survived. By these and other experiments, the safety of the procedure was established, and two of the king’s grandchildren were variolated on April 17, 1722. After this, the practice of variolation spread rapidly throughout England in the 1740s and then to the American colonies.35,36 It is difficult to say what influence the English country physician Edward Jenner (1749 to 1823) had on Pasteur’s later discovery of attenuation of bacterial cultures and its application to vaccination. Regardless, it is fair to say that Jenner had a major influence on public health, as he was the first to publish the development and use of a safe alternative to variolation.37 Although Jenner is rightly celebrated for his development of cowpox as a safe vaccine for smallpox, he was not the first to make use of a relatively nonpathogenic virus to induce immunity. Twenty years earlier, Benjamin Jesty, an English farmer, inoculated his wife and two sons with material taken from the cowpox lesion of the udder of a cow in his neighbor’s herd.36 In 1796, Jenner inoculated James Phipps, an 8-year-old boy, with material obtained from a cowpox lesion that appeared on the hand of a dairymaid (Fig. 2.1). Six weeks later, he inoculated the experimental subject with smallpox without producing disease. Further studies by Jenner established the efficacy of his vaccination procedure. For this feat, Jenner received a cash prize of £30,000 and election to nearly all of the learned societies throughout Europe.38

EMERGENCE OF IMMUNOLOGY AS A DISCIPLINE The Cellularists versus the Humoralists The international renown of Pasteur and Koch led to the establishment of research institutes that bore their names. Many talented young scientists were drawn to these institutes whose research missions should have been complementary, but were not, and the partisan battle that began with Pasteur and Koch would soon be reenacted on a larger scale.

Metchnikoff and the Birth of Cellular Immunology Elie Metchnikoff (1845 to 1916), an ambitious student at the University of Kharkoff (“I have zeal and ability, I am naturally talented—I am ambitious to become a distinguished investigator”),31 borrowed a professor’s microscope and began a lifelong quest to understand the cellular basis for immunity. A comparative zoologist by training, his early academic focus was on understanding the development of metazoans. Heavily influenced by Darwin’s publication of The Origin of Species in 1859,39 he viewed early embryologic development as a competition among specialized cell

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FIG. 2.1. Cowpox Pustule on the Arm of Sarah Nelmes. Reprinted with permission from Jenner37; courtesy of Dr Jenner’s House: Birthplace of Vaccination, Gloucestershire, UK.

types.40 Ontogeny was seen as a set of “interactions of cell lineages with each other to limit self-replication by any one component in favour of the interests of the organism as a whole.”40 He focused his interest on an amoeboid marker cell of the mesoderm, which was dubbed “phagocyte” (devouring cell) by a contemporary of Metchnikoff’s, the Viennese zoologist, Carl Claus. In a justly famous passage from Olga Metchnikoff’s biography of her husband, she describes the observation that became the defi ning moment of his scientific career41: One day when the whole family had gone to a circus to see some extraordinary performing apes, I remained alone with my microscope, observing the life in the mobile cells of a transparent star-fish larva, when a new thought suddenly flashed across my brain. It struck me that similar cells might serve in the defense of the organism against intruders. Feeling that there was in this something of surpassing interest, I felt so excited that I began striding up and down the room and even went to the seashore in order to collect my thoughts. I said to myself that, if my supposition was true, a splinter introduced into the body of a star-fish larva, devoid of blood-vessels or of a nervous system, should soon be surrounded by mobile cells as is to be observed in a man who runs a splinter into his finger. This was no sooner said than done. There was a small garden to our dwelling, in which we had a few days previously organised a “Christmas tree” for the children on a little tangerine tree; I fetched from it a few rose thorns and introduced them at once under the skin of some beautiful star-fish larvae as transparent as water. I was too excited to sleep that night in the expectation of the result of my experiment, and very early the next morning I ascertained that it had fully succeeded. That experiment formed the basis of the phagocyte theory, to the development of which I devoted the next twenty-five years of my life. Contrary to popular belief, Metchnikoff was not the first to visualize and describe phagocytosis, nor was he the first to suggest that it played a role in host defense. In a historical recount of the history of phagocytosis,42 Stossel argues

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that a Lutheran pastor, Johann August Ephraim Goeze, was the first to describe phagocytosis by microscopic observations of cells derived from hay infusoria in 1777. Much later, German pathologists of the mid-19th century, including Lieberkühn, Henle, and Vogel, drew connections between “pus corpuscles” of wounds and blood corpuscles.42 Others at the time, particularly the English physicians William Addison and Augustus Waller, observed leukocyte migration through capillaries in response to local injury, and Ernst Haeckel, a German marine biologist who was later to oppose Metchnikoff in an early theory of gastrulation, described molluscan leukocytes ingesting India ink particles in 1862.43 A handful of scientists of the time made the conceptual link between phagocytosis and host defense, and Metchnikoff himself cites a few: “When (the phagocytosis theory) is once firmly established, it will be time enough to determine each part taken in its foundation by workers such as Panum, Gaule, Roser, etc. . .”44 However, it seems that none of these workers seized upon this concept and appreciated its importance to the degree that Metchnikoff had; certainly, none had further developed and tested what in retrospect was the correct interpretation of the defensive function of phagocytosis. Following the key experiment in 1882, described previously, Metchnikoff performed many others to test the “phagocytosis theory,” such as the observation of infection in water fleas, which he viewed as a Darwinian struggle between pathogen and host45 : Once they have insinuated themselves into the organism’s inmost part, the spores cause an accumulation of the mobile cells round them, which correspond to the white corpuscles in human blood. A battle takes place between the two elements. Drawing the analogy with host defense in higher organisms, Metchnikoff described the killing of the spores by “mobile cells,” thus ensuring immunity for the organism. His view of phagocytosis expanded to encompass not only host defense but also organismal development, which he viewed in a teleologic context; he cited the dissolution of the tadpole’s tail by the “pervasion of phagocytes.”46 By the time he moved to Paris to become Chef de Service at the Pasteur Institute in

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1888, Metchnikoff had already formulated and tested what had become, according to his view, a cornerstone of the science of immune system. Perhaps what he had not appreciated at the time was that his move to Paris would come to signify to the Germans a complicit alliance with the French. He thus had unwittingly entered a battle that not only had been fought in the political arena but in the laboratory as well.

The Ascendance of Humoral Immunity Several related developments in the new field of immunology occurred at the end of the 19th century that would seal the fate of cellular immunology for at least 50 years: The discovery by Roux and Yersin47 that toxins alone derived from diphtheria bacilli could reproduce the symptoms of diphtheria; the discovery by von Behring and Kitasato in 189048 of humoral immunity to diphtheria and tetanus and the passive transfer of immunity to diphtheria in animals by von Behring and Wernicke in 189249 ; and the discovery of alexins (Greek for “without a name”) by Hans Buchner in 189950 and Jules Bordet at about the same time.51 Alexin was renamed “complement” by Ehrlich, as it “complemented” the activity of antibodies. Indeed, the ability of humoral components alone to lyse bacteria (the Pfeiffer phenomenon) or erythrocytes in the absence of phagocytosis51 provided strong independent evidence supporting the humoralists’ claims. The discovery of complement also had practical uses, as complement fi xation became the basis of a widely used serologic test for the diagnosis of syphilis, the so-called Wasserman test.52 The collective discoveries of the “humoralists” would lead to the successful treatment of a number of previously intractable diseases, such as diphtheria. Indeed, the first Nobel Prize in physiology of medicine was awarded to von Behring in 1901, “For his work on serum therapy, especially its application against diphtheria, by which he has opened a new road in the domain of medical science and thereby placed in the hands of the physician a victorious weapon against illness and death.” Bordet himself would later be awarded the Nobel Prize “for his studies in regard to immunity.” Among Bordet’s contributions was the development of the complement fi xation test together with his brother-in-law, Octave Gengou. This formed the basis of what Bordet termed “serodiagnosis.” Although the lines in the sand had already been drawn by the mostly Prussian humoralists led by Ehrlich on the one hand and the cellularists led by Metchnikoff on the other, it was Metchnikoff who wrote a letter to von Behring, proposing a scientific “truce”53 : I now believe . . . we can calmly work side by side. We can mutually support one another, just like the phagocytes and antitoxins, since . . . the phagocytes receive considerable assistance from the antitoxic property, just as the phagocytes . . . render great assistance to the organism or respectively its antitoxic powers, since they capture and destroy . . . bacteria. In fact, von Behring did not seem rigidly against the cellular school, and Metchnikoff viewed him as supporting the view that “active immunity requires some type of cellular

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basis.”53,54 It is difficult to know what to make of this passage; it sounds vague and seems to state the obvious, yet it does suggest a degree of flexibility that more intransigent proponents of the humoralist camp seemed to lack at the time. Regardless, scientific reconciliation would not be forthcoming until many years later, although phagocytosis theory was granted a temporary reprieve by the British physician Almoth Wright, who demonstrated the phenomenon of opsonization of bacteria. Wright was the first to describe a mechanism by which humoral and cellular components of immunity cooperate to kill bacteria.55 Wright is possibly best known in his incarnation as Sir Colenso Ridgeon in Shaw’s “The Doctor’s Dilemma.” Ridgeon defined “opsonin” as “...what you butter the disease germs with to make your white blood corpuscles eat them.”56 In what was viewed as a well-deserved but partly symbolic gesture, nevertheless, Metchnikoff and Ehrlich, two exemplars of the opposing schools of immunity, were awarded the Nobel Prize in 1908 “in recognition for their work on immunity.” It would not be until 40 years later that another cell type of the immune system would be first identified as being the source of antibody57 and 70 years later when dendritic cells (DCs) would be first identified as being the key phagocytic leukocyte responsible for initiating the immune response.58

Paul Ehrlich: The Cellularist’s Humoralist Paul Ehrlich embodies a pivotal figure in the history of immunology. Although he would make many practical discoveries in his long research career, his greatest contribution to immunology, like Jerne’s over a half century later, was a conceptual breakthrough that served to stimulate the field of immunology for years to come. Although I am tempted to consider his “side chain theory” of antibody formation a paradigm shift, that would presume that there was a preexisting paradigm to shift from, when in fact there was no paradigm of antibody specificity to begin with. Ehrlich began his research career as a medical student. One of his professors was the pathologist Wilhelm von Waldeyer, who introduced the young Ehrlich, who already showed a strong interest in chemistry, to histologic methods for staining cells and tissues. Following further training in several medical schools, he was influenced by the chemist von Bayer and the pathologists Cohnheim, Haidenhain, and Weigert (his cousin). He presented his thesis on histologic staining in Leipzig at the age of 24, in which he was the first to describe mast cells. As noted by Silverstein, the opening sentence of the thesis gave an inkling of his approach to science59 : While in the modern histological literature, directions on tintorial method are already so numerous, and still increase from day to day, yet their theoretical basis has had only a very negligible consideration. The same year, he was appointed senior physician in the Department of Internal Medicine at the Charité-Hospital in Berlin. The head of the clinic, Friedrich Frerichs, encouraged Ehrlich to continue his histochemical investigations, which led to the identification of neutrophils, eosinophils,

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and basophils as well as the diagnosis of his own case of pulmonary tuberculosis.60 It is notable that the term “side-chain” (seitenketten in German) was a chemical term in use at Ehrlich’s time meaning much the same as it does today. It is inescapable to conclude that Ehrlich’s focus and interest in chemistry would be the driving force behind his thinking about how antibody is formed, and “selected for,” by antigen. The essence of Ehrlich’s side chain theory, first proposed in 1897 (Fig. 2.2) is well articulated in Ehrlich’s Nobel lecture and is paraphrased here61: 1. “The relationship between toxin and antitoxin are strictly specific—tetanus antitoxin neutralizes exclusively tetanus toxin, diphtheria serum only diphtheria toxin . . .” 2. “For this reason it must be assumed that the (toxin and antitoxin) enter into a chemical bond . . . fitting each other ‘like lock and key.’” 3. “The group in the protoplasm, the cell receptor, must be identical with the antitoxin which is contained in solution in the serum of immunized animals.” 4. “As these receptors, which may be regarded as lateral chains (“seitenketten”) of the protoplasm...become occupied by the toxin, the relevant normal function of this group is eliminated...the deficiency is not merely exactly compensated, but made up to excess (hyperregeneration).” What Ehrlich proposed purely on theoretical grounds is a brilliant argument based on a combination of inductive and deductive reasoning. Characteristic of Ehrlich, it is

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lucid, logical, and profound. Beginning with a consideration of a simple chemical bond, Ehrlich somehow ends up with an antibody-producing cell; hence, “the cellularist’s humoralist.” Not every biologist was enamored of Ehrlich’s model, and soon after he proposed it, it came under attack, most notably by Jules Bordet. Bordet objected to Ehrlich’s insistence that the specificity of the antigen–antibody reaction required an irreversible bond, whereas Bordet, whose views seemed to be more deductively grounded in his immediate line of investigation (eg, precipitin reactions and complement fi xation) thought that adsorptive interactions between antigen and antibody were sufficient to account for specificity.15,60 Although Ehrlich’s side-chain theory provided a logically consistent mechanism to account for antibody specificity, it would later be criticized for failing to account for antibody diversity. That problem would not be conquered for another 60 years in yet another “paradigm shift” when Talmage, Burnet, and Lederberg proposed the clonal selection theory. However, the concept of clonality was not yet conceived of in 1897, and in any case, it is hard to envision how a clonal selection theory could have been developed without the prior description by Ehrlich of antibody selection itself.

IMMUNOLOGY BRANCHES OUT: BENEFICIARIES OF THE EARLY FOCUS ON HUMORAL IMMUNITY “Man built most nobly when limitations were at their greatest.” –Frank Lloyd Wright Immunology during the early part of the 20th century was heavily influenced by the early victories of the humoralists. Aside from the spectacular practical implications of the work of von Behring, Bordet, and Ehrlich, among others, the immunologist’s toolkit of the early 20th century immunologist was very limited. Advanced microscopic techniques were not yet available, and cell fractionation techniques had not yet been invented. Given these limitations, it is hard to envision how the gap between antibody and cell could have been bridged any closer than Ehrlich had managed, at least on a theoretical level. However, the focus on humoral immunity did allow for the rapid development of several fields, most notably immunochemistry as well as hematology and allergy. It would be some time before cellular immunology would catch up.

Karl Landsteiner and the Birth of Transfusion Medicine and Autoimmunity

FIG. 2.2. Ehrlich’s “Seitenkette” (Side Chains). From Ehrlich.171

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As described by Silverstein, “No single individual contributed as importantly to so many different areas of immunology as did Karl Landsteiner.”15 Landsteiner was born in Baden in 1868 and attended Vienna Medical School. He began his training in internal medicine and studied chemistry with Emil Fischer, who would receive the Nobel Prize in chemistry in 1902. He transferred to the Department of Pathological Anatomy, home to Erdheim, Billroth, Escherich, and other accomplished scientists, where he remained until

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1907.62 Landsteiner’s first major accomplishment was the discovery of the human ABO blood group antigens at the turn of the century.63,64 His motivation was succinctly described in his Nobel lecture speech65 : . . . proteins in individual animal and plant species differ and are characteristic of each species . . . The problem raised by the discovery of biochemical specificity peculiar to a species . . . was to establish whether the differentiation extends beyond the species and whether the individuals within a species show similar though smaller differences. His experiment was a simple one, in which he applied the sera of six heathy men, including himself, to red blood cells of each, and noted that the sera of the men reacted differently with each other—no serum reacted with that same individual’s red blood cells. At the end of the paper, Landsteiner noted that the results could account for the variable clinical consequences of human blood transfusion—and thus was borne the discovery of the ABO red blood cell antigens that would later become useful in blood typing prior to transfusions. Many years later, Landsteiner would discover the M, N, P isoantigens in 192766 and the Rh system in 194067). Landsteiner’s next major contribution was the codiscovery with Donath of the first autoimmune disease, paroxysmal cold hemoglobinurea,68 which challenged Ehrlich’s dictum that such a situation could not occur.69 The organism possesses certain contrivances by means of which the immunity reaction . . . is prevented from acting against the organism’s own elements and so giving rise to autotoxins . . . so that one might be justified in speaking of a “horror autotoxicus” of the organism. The nature of the contrivances was thought to be of “the greatest importance” by Ehrlich, who later stated70 : “According to our present investigations either the disappearance of receptors or the presence of autoantitoxin is foremost among these contrivances.” Depending on how this statement is interpreted, it could be viewed as Ehrlich’s formulation of either clonal deletion or anti-idiotypes. Two years after the discovery of paroxysmal cold hemoglobinurea, an Italian ophthalmologist, who observed sympathetic ophthalmia, a disease in which damage to one eye leads to inflammation of the opposite eye, speculated that this disease was due to “autocytotoxins.”71 Autoimmunity research was taken up by a few others, but perhaps owing to either a misinterpretation of Ehrlich, or possibly due to deference to his authority in the field, progress was slow. It would not be years later, until the discovery of the role of sensitization of the newly discovered Rh antigen as an etiology of erythroblastosis fetalis,72,73 that autoimmunity became an active area of research for immunologists. The fi rst time that autoimmunity was fi rst associated specifically with arthritis was 1957, when Kunkel and colleagues discovered what came to be called “rheumatoid factor,” large complexes of immunoglobulin (Ig)M directed

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against IgG in the sera of some patients with rheumatoid arthritis.74 This observation fundamentally changed the field of rheumatology.75 Landsteiner received the Nobel Prize in 1930 “for his discovery of the human blood groups.” Ironically, upon receiving the prize, it is said that he felt the prize should have been awarded for his work on haptens, which would play a great role in the development of the growing field of immunochemistry.

Discovery of Hypersensitivity: The “Other Work” In 1901 to 1902, Paul Portier and Charles Richet were attempting to raise tolerance in laboratory animals to actinotoxin, an uncharacterized toxin derived from tentacles of sea anemones. Their experiments appeared to be unsuccessful, and, in some cases, it appeared that the animals actually became sensitized to the antigen. They repeated their studies using dogs and obtained completely unanticipated results: All eight dogs collapsed and died within minutes after receiving a relatively small dose. First thinking the results were due to experimental error, they later realized that the sensitized animals had all been exposed to antigen 14 to 23 days previously.76 They proposed the name “aphylaxis” (against protection), a term later changed to “anaphylaxis.”77 Richet continued his investigations on anaphylaxis and was awarded the Nobel Prize in 1913 for his work. Soon after Portier and Richet made their seminal discovery, Maurice Arthus was able to induce a localized form of anaphylaxis (swelling and ultimately gangrene) by repeated subcutaneous injections of horse serum, considered fairly nontoxic.76 Yet, a third type of hypersensitivity was described by the pediatricians von Pirquet and Schick,78 who noted that vaccinated children occasionally developed fever, joint pains, rash, diarrhea, and hypotension. They concluded that the clinical features of what is now called “serum sickness” were not a direct result of the injection of antiserum, but the outcome of “when antigen and antibody meet.” As von Pirquet later explained,79 We are able to observe the effects of the toxic body formed when antigen and antibody meet, that is, the serum disease. We see that at the time when the antibody arises, and therefore the antigen disappears, symptoms of general disease occur. The supposed connection is that these symptoms are due to toxic bodies formed by this digestion of the allergen through the antibody. Although von Pirquet does not precisely define what he meant by “toxic body,” some have interpreted this to mean antigen–antibody complexes. It is possible that he was reluctant to be more specific as he did not actually have a way of observing or quantifying the complexes; it is also possible that he did not have a ready explanation for how such a complex, if formed, could lead to the symptoms and signs of serum sickness. It would not be until many years later that Frank Dixon and colleagues would precisely delineate the nature of the immune complexes.80 Nevertheless, it is clear that von Pirquet and Schick viewed this phenomenon as

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lying along a continuum with the normal immune response, or rather being a necessary component of it. As they later stated,81 The conception that the antibodies, which should protect against disease, are also responsible for the disease, sounds at first absurd. . . One forgets too easily that the disease represents only a stage in the development of immunity, and that the organism often attains the advantage of immunity only by means of disease. It is von Pirquet and Schick who coined the term “allergy” (from the Greek allos, other, and ergon, work).79 By highlighting the role of tissue injury in promoting immunity, they closed the loop that Metchnikoff had begun at the end of the 19th century. This theme would be revisited and expanded upon in the latter part of the 20th century when it was discovered that the inflammation that accompanied the innate immune response was a necessary prequel to the acquired immune response.

THE LONG JOURNEY FROM THE DAWN OF IMMUNOCHEMISTRY TO THE STRUCTURE OF IMMUNOGLOBULINS Challenges to Ehrlich: The Problem with the “Keys” One of the difficulties that Ehrlich faced was bridging the gap between the conceptual basis of antibody specificity and the actual basis of antibody specificity. If his side-chain theory was correct, then every cell involved in antibody production would be capable of reacting against every possible antigen it might encounter. Even without considering the cellular origins of antibodies, Ehrlich’s critics, such as the Viennese Max von Gruber, raised the question of how the astonishingly large number of different specificities of the antibody molecules themselves could be generated.15 The size of the repertoire seemed impossibly large if every “lock” had a unique “key.” Landsteiner became von Gruber’s assistant at the University of Vienna in 1896, and he inherited von Gruber’s critical view of Ehrlich’s theory. Landsteiner’s early approach to this problem was to adopt Bordet’s “colloid” explanation of the antigen–antibody interaction,15 which rejected covalent interactions in favor of multiple weaker interactions, a theme that was later taken up by Pauling, with some interesting consequences. Later, stimulated by the work by Obermeyer and Pick, who described chemical modifications of proteins, 82 Landsteiner used structurally related reactive chemicals to derivatize proteins. He showed that antisera raised against one of the chemically modified proteins would react to varying degrees with proteins modified by structurally similar, but not identical, reactive molecules. These results were interpreted as being incompatible with Ehrlich’s “lock and key” specificity but called for a more nuanced view of antigen–antibody specificity. It became immediately apparent that the size of the repertoire could be greatly enhanced if one allowed for such graded degrees of binding affi nities.

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Immunoglobulin Structure: The “Keys” to the Problem It was clear that further progress on defining the physicochemical nature of the antigen–antibody reaction required the development of specific tools that were unavailable at the turn of the century. Antibodies, whose chemical structures were unknown at the time, were considered “colloids” (from Greek kolla, glue), a suspension of particles suspended in a continuous phase of another component. In 1924, the Swedish chemist Svedberg designed a centrifuge based on a modified milk separator. The centrifuge could develop a centrifugal field of up to 5000 g and enabled Svedberg to measure the molecular mass of hemoglobin83 ; he was the first to determine the molecular mass of macroglobulins, derived from a patient with Waldenström macroglobulinemia, which we now know as IgM. A student in Svendberg’s laboratory, Arne Tiselius, developed gel electrophoresis,84 which allowed for the separation of molecules based on charge and size. These tools enabled Michael Heidelberger to establish the field of “immunochemistry.” Heidelberger devoted nearly his entire research career pursuing the implications of a simple but profound discovery he made with Oswald Avery in 1923 that type-specific antigens of pneumococcus bacteria are complex polysaccharides. Over the next three decades, this discovery enabled him to determine, for the first time, the exact weight and chemical composition of antibodies, antigens, and complement. He showed that antibodies are multivalent proteins and used these insights to devise a simple vaccine against pneumonia whose effectiveness was first proven in soldiers who fought in World War II.85

The Chemical Nature of Immunoglobulin Molecules By 1950, it was appreciated that antibodies were proteins of 150,000 molecular mass containing bivalent antigencombining sites. Based on Porter’s observation that Igs could be split into smaller products by enzymes such as papain, yielding Fab fragments that bind antigen and Fc (crystallizable) portions that do not, Edelman and Poulik further defined Ig structure by hypothesizing that Bence-Jones proteins, derived from myeloma cells, were free light chains of Ig molecules.86 As this hypothesis appeared to be correct, this provided a means of obtaining a ready supply of homogeneous Ig subunits. Reduction of disulfide bonds led to still different products, enabling them to propose that the Ig molecule consists of two light chains and two heavy chains, and that the antigen-binding site consisted of contributions from both heavy and light chains.86 Further refinement of techniques in protein chemistry allowed Edelman and Porter to work out the primary structure of Ig molecules, for which they received the Nobel Prize in 1972.

CONFRONTING THE SIZE OF THE REPERTOIRE The value of a large repertoire from which to select is appreciated by any performing musician. Yet in the 1950s, the repertoire problem was unsolved, leading to competing theories of how a large repertoire of diverse antibodies are generated. There were two mutually exclusive leading schools of thought: “instruction” theories and “selection” theories.

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Instruction Theories of Antibody Diversity 15

As noted by Silverstein, the fi rst description of antigen as template was by Bail and Tsuda, who proposed in 1909 that antigen persists after its encounter with antibody, and that by so doing, it leaves an impression on the antibody.87 This concept was further refi ned by Breinl and Haurowitz, who suggested that antigen is carried to the site of protein formation, where it would serve as a template to instruct antibody formation.88 Finally, in 1940, Linus Pauling provided a chemical explanation for antigen-directed instruction: that “antibodies differ from normal serum globulin only the way the ends of the polypeptide chain is coiled” as a result of their amino acid sequence, and that “they have accessible a very great many configurations with nearly the same stability.” Under the “influence” of antigen, they “assume configurations complementary to surface regions of the antigen,” forming two active ends, and that after “freeing one end and the liberation of the central part of the chain, it folds up to form the central part of the antibody molecule, with two oppositely directed ends able to attach themselves to two antigen molecules.” This interaction would be further stabilized by weak interactions between antigen and antibody.89 Assuming a degree of degeneracy in the initial antigen–antibody interaction, these theories were consistent with the fi ndings of Landsteiner, who showed that antigen–antibody interactions were not absolutely specific, as envisioned by Ehrlich (Fig. 2.3). In retrospect, these theories had great chemical appeal; however, a central weakness is that if the initial interactions between antigen and antibody are degenerate, that implies that the interactions cannot be of high affi nity (otherwise they would not be degenerate). Yet, if the initial interactions are weak, how would they occur in the fi rst place? At some level, there has to be a certain degree of preexisting antibody specificity, which implies a preexisting repertoire. Although the instruction theories of antibody selectivity helped explain some of the specificity of the antigen–antibody interactions, they could not account for all of them. It is notable that the underlying basic principles have been since invoked for other encounters in the immune system. For example, a “bar code model” of interactions between the T-cell receptor (TcR) and peptide-major

histocompatibility complex (MHC) (pMHC) has recently been proposed, in which the initial encounter between TcR and pMHC is governed by strong interactions, followed by “scanning” the epitope and modest changes in conformation of the TcR, leading to strengthening of the TcR–pMHC association.90

Jerne’s Natural Selection Theory of Antibody Formation Instruction theories of antibody specificity persisted through the 1950s, when they were modified to include participation of enzymes to act as intermediaries between antigen and antibody as well as the newly discovered structure of deoxyribonucleic acid (DNA). In 1955, Niels Jerne published a highly influential paper in which he proposed a new theory of antibody formation that he described as the “natural selection theory of antibody formation.”91 The antigen is solely a selective carrier of spontaneously circulating antibody to a system of cells which can reproduce this antibody. Globulin molecules are continuously being synthesized in an enormous variety of different configurations...among which... will be fractions possessing affinity toward any antigen to which the animal can respond. Jerne referred to these preexisting antibodies “natural antibodies,” and went on to state that antigens selectively attach to those globulin molecules that happen to have a complementary configuration. According to Jerne, once the interaction occurs, the antigen–antibody complexes may be engulfed by a “phagocytic cell,” at which point the antigen is eliminated. The antibody within the phagocytic cell can remain or be transferred to another cell, which is the signal for reproduction of the same specific antibodies. More antibody is released into the circulation, resulting in a larger percentage of specific circulating antibody. Jerne states that “the reproduction need not be highly faithful; copying mistakes will be harmless and may occasionally produce an improved fit.”91 These are remarkable concepts, as Jerne appeared to have invoked Metchnikoff, a Darwinian interpretation of antibody selectivity at the organismic level, as well

FIG. 2.3. Demonstration of Serologic Specificity by Landsteiner and Scheer. Reactions from immune serum for aniline with various azoproteins and with unchanged horse serum reading after 15 minutes. (1) azoprotein from chicken serum and aniline, (2) azoprotein from horse serum and aniline, (3) azoprotein from horse serum and para-toluidine, (4) azoprotein from horse serum and para-nitroaniline, (5) azoprotein from horse serum and para-aminobenzoic acid, (6) azoprotein from horse serum and para-arsanilic acid, (7) unchanged horse serum, (8) saline control. Modified from Landsteiner and van der Scheer.486

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as to have anticipated affinity maturation. It is easy to see why Jerne’s ideas were so influential. However, the central problem, the lack of an explanation for the huge existing repertoire, which hampered the instruction theories of antibody diversity, as well as Ehrlich’s side-chain theory was still unanswered. Jerne admitted this weakness and speculated that the “spontaneous production of globulin molecules of a great variety of random specificities” may reside in a “. . . specialized lymphoid tissue, such as that of the thymus.”91 Perhaps a more existential problem was that the flow of information was from the existing preformed antibody to more antibody without any genetic intermediary. What was lacking was a specific mechanism to transfer information between a specific antibody and the specific synthesis of that same antibody. Jerne admitted to this problem and nominated ribonucleic acid (RNA) as a key template; he then stated “ . . . a protein molecule may determine the order of the nucleotides in the synthesis of RNA,” citing a paper that does not actually make this assertion.91 What accounted for this conceptual block that had persisted for more than half a century? On one level, it was ignorance of the “fundamental dogma of molecular biology,” which had not yet been articulated.92 But on another level, it perhaps can be traced back to the decisive victory of the humoralists over the cellularists. So long as the focus was on the antigen–antibody interaction, there was no way to invoke a biologically plausible mechanism of generating a preexisting Ig repertoire and of selectively expanding a specific portion of that repertoire.

Development of the Clonal Selection Theory In 1957, the American immunologist David Talmage published a review whose focus was allergy; however, in the review, Talmage drew an analogy between natural selection,

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in which there is “selective multiplication of a few species out of a diverse population” and antibody production. In a very succinct but remarkably insightful passage, he lays out the essence of the clonal selection theory 93 : As a working hypothesis it is tempting to consider that one of the multiplying units in the antibody response is the cell itself. According to this hypothesis, only those cells are selected for multiplication whose synthesized product has affinity for the antigen injected. At about the same time, Burnet published his own paper outlining the key aspects of the clonal selection theory 94 (Fig. 2.4). In a later interview, Talmage discussed how he provided a preprint of his review to Burnet before he published his landmark paper on the clonal selection theory, but both he and Nossal stated that Burnet developed the theory independently. Talmage thought that the two papers were similar in substance, but that “Burnet had the good fortune to make an analogy of the idea to the clones that grow in bacteria and he gave it a very catchy name, ‘clonal selection.’”95 Among the ideas that led both immunologists to propose the clonal selection theory was the known logarithmic rise in antibody titer during the primary immune response “as if it is a product of some multiplying substance.”95 Other experiments demonstrated that lymphoid cells obtained from previously immunized rabbits that were then transferred to x-radiated recipients were sufficient to induce a recall response in the recipients.96 Although we now know that the clonal selection theory is correct, it took more than 10 years for clonal selection to be widely accepted, according to Talmage.95 Among the key pieces of evidence to prove the theory was the demonstration of antibody production from single cells, by Nossal and Lederberg,97 and the later finding that cognate antigen was capable of aggregating all surface Ig on individual cells,98

FIG. 2.4. In this diagram, antigen C (Ag.C) is recognized by clone “c,” triggering this clone, but not others, to proliferate. Antibody against Ag.C (AB.C) is indicated at the bottom. From Burnet.246 Copyright ©1959 Sir MacFarlane Burnet. Reprinted with the permission of Cambridge University Press.

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which corroborated Nossal and Lederberg’s results. Finally, in 1975, Köhler and Milstein demonstrated the production of monoclonal antibodies from single clones of immortalized plasma cells.99 Along with Jerne, they shared the Nobel Prize in 1984.

The Structural Basis of Antibody Diversity: The Dialectic Revisited By the mid-1970s, the puzzle of antibody diversity was far from solved—many of the pieces were still missing. The conceptual basis for the clonal selection theory was laid in 1957 by redirecting the focus of investigation from immunochemistry to cell biology. However, what was needed to actually prove the theory was a delineation of the molecular basis for the enormous size of the repertoire. Two lines of investigation converged to provide this evidence: the sequencing of Ig proteins followed by the sequencing of Ig genes. In 1965, Hischmann and Craig sequenced two BenceJones proteins and found that there was conservation of the amino acid sequence at the C-termini but considerable diversity at the N-termini.100 In the ensuing years, sequence data on a number of myeloma proteins became available, and in 1970, Kabat and Wu applied statistical criteria to analyze sequencing data from 77 Ig chains. They identified three regions within the 107 residues comprising the light chain variable region that demonstrated a still further degree of variability (ie, hypervariability). They hypothesized that these regions of extreme diversity represented the complementarity-determining residues and suggested by analogy with prokaryotes that they arose through episomal incorporation into the light chain locus by a recombination event.101 If this, indeed, was the underlying explanation for antibody diversity, then there would have to be a very large number of episomal elements to account for a diverse repertoire. This was a different view than the one taken by Dreyer and Bennet 5 years earlier, who proposed that variable region genes that had undergone duplication and spontaneous mutation throughout evolution underwent a “genetic scrambling” event, resulting in their juxtaposition to the constant regions of the Ig genes. They even suggested the involvement of enzymes involved in DNA repair as contributing to such an event.102 Thus, two opposing viewpoints began to emerge to account for the generation of diversity (or GOD, as playfully described by Richard Gershon): somatic mutation of a few genes, as suggested originally by Burnet,94 and supported by Weigert and Cohn’s sequencing data of the mouse λ light chain locus,103 or somatic recombination among many duplicated genes within the germline, as proposed by Dreyer and Bennet102 and later refined by Edelman and Gally104 and Hood and Talmage.105 In the years that followed, various teleologic arguments were proposed to support one theory over the other. In 1976, at least a partial resolution was provided by Hozumi and Tonegawa, who provided evidence that the Vκ and Cκ loci from embryonic DNA rearrange to form a contiguous polypeptide in mature lymphocytes.106 The advent of molecular cloning led to direct proof that the variable and constant regions of the light chain gene had undergone rearrangement at the DNA level.107,108 This

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was followed by the demonstration by Weigert et al.109 that the Ig Vκ region in the mouse is encoded by multiple V, J, and C regions joined at the DNA level during differentiation of individual lymphocytes. Finally, in 1980, Early et al.110 demonstrated how V, D, and J regions of the Ig locus could recombine to generate a virtually unlimited combination of antibody specificities. In the ensuing years, the molecular mechanisms governing VDJ recombination were uncovered. These involved recognition of conserved sequences flanking germline V, D, and J segments, introduction of double-strand breaks, potential loss or gain of nucleotides at the coding junctions, and polymerization and ligation to complete the joining process. Many talented scientists contributed to these discoveries, including Alt, Yancopoulos, Blackwell, and Gellert.111 This culminated in the isolation of the recombinase activating genes (RAG) by Baltimore’s group.112, 113 The dominant view that emerged from these studies largely favored the “germline-ists,” reinforced by Tonegawa’s receiving the Nobel Prize in 1984. However, in yet another example of a synthesis of two apparently contradictory approximations of the truth, evidence for somatic hypermutation began to emerge.114,115 Its importance was established when it was causally linked to the generation of B cells with very high affi nity antibodies,116 a phenomenon termed “affi nity maturation.”117

SPECIALIZATION WITHIN THE IMMUNE SYSTEM Division of Labor: Discovery of T and B Cells The first person to demonstrate delayed type hypersensitivity may have been Robert Koch in 1882. On his quixotic pursuit of developing a vaccination against tuberculosis, he injected himself with spent medium from cultures of human tubercle bacilli and noted a particularly severe reaction, including systemic effects.118 Although he was not successful in developing a vaccine against tuberculosis, he recognized the diagnostic potential of this procedure. It was not until 1942, and then later in 1945, that Landsteiner and Chase demonstrated that cells from guinea pigs previously immunized with Mycobacterium tuberculosis or hapten would transfer reactivity to a naïve recipient when challenged with the immunogen.119,120 This was the first demonstration that cells, rather than antibody, transmitted specific immunity, a finding that in some ways vindicated Metchnikoff’s cellular focus. The identity of the cells mediating the transferred hypersensitivity was unknown. Based on experiments performed decades earlier, as Silverstein has noted,121 James Murphy at the Rockefeller Institute argued that lymphocytes were important in the host resistance to tuberculous infection. Murphy used mice exposed to x-rays or splenectomized to manipulate lymphocyte numbers and showed that conditions that would have been predicted to deplete lymphocytes resulted in early death of the mice due to disseminated tuberculosis.122 In earlier papers, Murphy also showed more directly that lymphocytes were important in graft rejection in transplanted chick embryos. Why were these seemingly important observations ignored? Was it because the

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experiments relied to a certain extent on inference, rather than direct proof that lymphocytes were key mediators of tuberculous immunity? Most likely, it was a combination of events: The lingering vestiges of the confrontation between the cellularists and the humoralists and the fact that there was little conceptual basis for understanding how a small innocuous-appearing cell type could participate in immunity. In a completely independent line of investigation, it was known for some time that certain strains of mice had a high spontaneous rate of developing lymphocytic leukemia. In attempting to explain the finding that thymectomy of 2-month-old mice failed to develop leukemia, Jacques Miller found that neonatally thymectomized mice appeared ill and revealed a marked deficiency of lymphocytes in blood and lymphoid tissues. Furthermore, these mice failed to reject allografts or xenografts.123 However, not all areas within lymphoid organs were depleted of lymphocytes, consistent with “thymic-dependent” (paracortical areas of the lymph nodes and periarteriolar sheaths of the spleen) and “thymic-independent” regions (follicles and medullary cords). Miller concluded that the thymus was important for the development of a subset of lymphocytes important in allograft rejection. The involvement of lymphoid cells in antibody production was considered likely in the 1940s, mainly due to “guilt by association.” For example, Erich and Harris injected antigens, such as typhoid vaccine and sheep erythrocytes, into the feet of rabbits, and then compared the formation of antibody to histologic changes in the draining lymph nodes.124 Similar experiments were performed using intravenous injection of antigen, and the appearance of antibody and plasma cells in the spleen appeared to be correlated.125 However, actual proof for the involvement of B cells in antibody production was quite accidental.126 In 1952, Bruce Glick, a young doctorate student at Ohio State University, was investigating the function of an obscure avian organ, the “bursa of Fabricius.” He removed the organ, which did not result in a discernable phenotype. A fellow graduate student asked to use one of Glick’s birds to develop an antibody against Salmonella and found that the bursectomized chicken failed to make antibodies. This led to the publication that eventually appeared in Poultry Science describing the role of the organ in the generation of bursa-derived or “B” cells.127 As there was no anatomic equivalent of the bursa in mammals, an exhaustive search finally revealed the bone marrow origin of these cells. It was also found that thymusderived cells, later named “T cells,” were needed to “help” B cells produce antibody.128–130 These distinctions were further clarified when Cooper et al.131,132 showed that T cells were required for delayed-type hypersensitivity and graft versus host reaction. Thus was borne one of many central dichotomies that Mazumdar133 has argued drives the field of immunology. In 1957, Gowans134 cannulated the thoracic duct of rats and measured the rate of flow of the lymph. He suggested that “the continuous entry of living lymphocytes into the blood may be essential for maintaining the output of lymphocytes from the thoracic duct.” He later showed that intravenous transfusion of radiolabeled lymphocytes resulted

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in appearance of the radiolabeled cells into the thoracic duct, thus defining the continuous recirculation of lymphocytes from the lymphatics to the blood.135 In retrospect, these experiments were critically important in understanding how lymphocytes are constantly patrolling the lymphatics, vigilantly on the lookout for antigen.

TRANSPLANTATION BIOLOGY AND THE PURSUIT OF IMMUNOLOGIC TOLERANCE The history of transplantation began many hundreds of years ago and was vigorously pursued by surgeons and tumor researchers during the early part of the 20th century. These events are well summarized in several texts, notably Brent’s A History of Transplantation Immunology and Silverstein’s A History of Immunology. The influence of these early workers, particularly Carrel’s, on the surgical aspects of transplantation is clear, but their impact on the field of transplantation immunology was limited because the conceptual framework of immunology was still rudimentary. Analogous to the role that smallpox had in catalyzing early vaccine development, large-scale bombing campaigns in World War II resulted in many civilian and military burn victims who required skin grafting, compelling surgeons to develop better techniques to avoid homograft rejection. The British zoologist Peter Medawar was assigned to the War Wounds Committee of the Medical Research Council. In 1943, Medawar and Gibson136 published “The fate of skin homografts in man” based on a single burn victim who received multiple “pinch grafts” of skin. The authors concluded that autografts succeed, whereas allografts fail after an initial take, and that the destruction of the foreign epidermis is brought about by a mechanism of active immunization. Medawar returned to Oxford University to study homograft rejection in laboratory animals and proved that this was an immunologic phenomenon. Medawar137 concluded that the mechanism by which foreign skin is eliminated belongs to the category of “actively acquired immune reactions.” Shortly after this publication, Ray Owen138 published the provocative finding that dizygotic twin calves, who share a common circulatory system in utero, exhibit chimaerism with respect to their twin’s erythrocytes and fail to produce antibodies against each other’s erythrocytes. This led Burnet and Fenner139 to predict that introduction of a foreign antigen early enough in life would fail to elicit an immune response. Medawar reasoned that the successful exchange of skin grafts between dizygotic calves would verify this hypothesis. Together with his postdoctoral fellow Rupert Billingham, he performed a series of grafting experiments that provided direct support for this model.140 Mammals and birds never develop, or develop to only a limited degree, the power to react immunologically against foreign homologous tissue cells to which they have been exposed sufficiently early in fœtal life . . . this phenomenon is the exact inverse of “actively acquired immunity”, and we therefore propose to describe it as “actively acquired tolerance.” At the same time, Milan Hašek141 in Prague demonstrated successful parabiosis of chick embryos derived from two

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different strains. Hašek’s stated motivation to perform the experiment was to determine whether exchange of blood in the different chick strains induced a “mutual metabolic assimilation between parabionts,” rather than to induce a state of immune tolerance. It has been suggested that Hašek’s real motivation for the experiment was to advance the Lysenkoist genetic doctrine to appease the local communist regime in Czechoslovakia.142 Regardless, the result has been reinterpreted as an example of the induction of immunologic tolerance. In 1960, Medawar shared the Nobel Prize with Burnet “for the discovery of acquired immunologic tolerance.”

Mechanism versus Metaphor: “Self/Nonself” Discrimination Medawar’s experiments and Burnet’s formulation of the clonal selection theory occurred at a critical juncture in the history of the evolving field of immunology. Had their development been dyssynchronous, it is doubtful that much progress would have been made on either front. Indeed, transplantation experiments had been performed earlier in the century with little insight as to why allografts failed. The viewpoint that Burnet espoused, that the function of the immune system is to distinguish “self” from “nonself,” has proved to have enormous heuristic value ever since its formulation more than 60 years ago. Is this distinction mostly metaphorical, as suggested by Tauber,143 or does it reflect a more concrete generative reality? To begin to address this question, it is necessary to briefly review a parallel development in immunology, the discovery of the components of the immune system that define molecular self-hood.

Looking Under the Hood: The Discovery of the Major Histocompatibility Complex The clonal selection theory represented a conceptual breakthrough in the history of immunology, but it did not explain how lymphocytes actually recognize antigen. These insights would eventually come from three sources over the span of 20 years: studies of the genetics of graft rejection in inbred strains of mice by George Snell in the 1940s, studies of the agglutination of white blood cells by sera from transfused patients by Jean Dausset in the 1950s, and studies of the immune response to simple antigens in guinea pigs by Baruj Benacerraf in the 1960s. Snell was interested in identifying genes that controlled the ability of mice to resist tumor transplants. He pioneered the use of congenic mice, which are genetically identical except for a single region or locus. Snell observed that tumor grafts were accepted between inbred mice but not between mice of different strains. The same was true for normal tissues. Snell termed the underlying genes “histocompatibility” genes. In collaboration with Peter Gorer, who had prepared antisera that reacted with cells from one mouse strain but not another, Snell established that the major histocompatibility locus corresponded to a reactivity that Gorer had designated antigen II, renamed locus histocompatibility 2 or H-2.144 Two loci within this region, designated K and D, carried genes specifying antigens involved in triggering graft rejection. In

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the 1950s, Dausset observed that patients who had received many blood transfusions produced antibodies that could agglutinate white blood cells from donors but not the patient’s own cells. Several of the patients produced antibodies against the same antigen.145 Subsequent family studies indicated that a genetically determined system, named “human leukocyte antigen” (HLA) system, was found to be the ortholog of H-2 in the mouse. Dissection of the human system would take many years, during which time transplantation surgeons made use of the emerging findings to assist in tissue typing. In the 1960s, Baruj Benacerraf, an immunologist working at New York University, noticed that some outbred guinea pigs responded to simple antigens by developing delayed hypersensitivity reactions upon challenge while others did not, and that this was under genetic control. He termed these genes “immune response genes.” McDevitt and Sela observed similar genes in mice, and McDevitt showed that they are encoded in the MHC.146 In 1980, Benacerraf, Snell, and Dausset shared the Nobel Prize “for their work on genetically determined structures of the cell surface that regulated immunologic reactions.” Of course, the identification of the HLA region and the subsequent cloning of the genes encoded in this region proved to be landmarks in the history of immunology. Besides providing a molecular identity to the key orchestrators of antigen presentation to T cells, the identification of specific MHC alleles with autoimmune diseases has led to important insights into their pathogenesis. Ironically, the first recognized HLA association with human disease, HLA-B27, which was associated with the disease ankylosing spondylitis,144,147 has generated perhaps more heat than light, as there is no definitive mechanism that explains how HLA-B27 is linked to disease pathogenesis, underscoring that the HLA locus, which took so many years to uncover at the genetic level, still has much to teach us.

The Discovery of Major Histocompatibility Complex Restriction as the Molecular Basis for “Self/Nonself” Discrimination It was known for several years that cooperation between T and B cells occurred in syngeneic or H-2–compatible animals.148–150 In 1972, Kindred and Shreffler151 showed that even in nude mice, cooperation between T and B cells required H-2 compatibility; however, the exact role of that H-2 molecules played in this process and the nature of the T- and B-cell interactions remained a mystery. A valuable clue was provided by experiments by Rosenthal and Shevach,152 who demonstrated that efficient presentation of antigen by antigenpulsed macrophages to T cells also required histocompatibility matching. In 1974, Doherty and Zinkernagel sought to understand the role of T cells in the immune response to viral meningitis. They theorized that it was the strength of the immune response that caused the fatal destruction of brain cells infected with this virus. To test this theory, they mixed virusinfected mouse cells with T lymphocytes from other infected mice. The T lymphocytes did destroy the virus-infected cells, but only if the infected cells and the lymphocytes came from a genetically identical strain of mice. T lymphocytes would

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ignore virus-infected cells that had been taken from another strain of mice.153 Further experiments strongly suggested that the same TcR that recognizes viral antigen also recognizes the MHC molecule.154 The implications of the ZinkernagelDoherty experiment were profound. First, it established the principle of MHC restriction: T cells recognize antigen only in the context of MHC molecules. Second, the experiment established that cytotoxic TcR-bearing cells must recognize two separate signals on an infected cell before they can destroy it. One signal is a fragment of the invading virus that the cell displays on its surface and the other is a self-identifying tag from the cell’s MHC molecules; it was felt likely that the same TcR probably recognizes both. Thus, the experiment pointed to the identity of the molecular structure that constituted immunologic “self”—it is the MHC molecule— and therefore a virus-infected cell bearing MHC molecules was likely to constitute “altered” or “nonself.” Finally, the fact that MHC is highly polymorphic implies that any given allelic product will be capable of forming a different altered self from other MHC allelic products; thus, the specific identity of the MHC molecule itself determines the strength of the immune response. Although the Zinkernagel-Doherty experiment answered many questions, the steps between encounter of antigen by an antigen-presenting cell (APC) and presentation of that antigen to a T cell was somewhat of a “black box.” It had long been thought that intact antigen was presented to T cells, but it was not until 1981 that Ziegler and Unanue155 showed that an antigen processing event was necessary for I-region (MHC class II)-restricted antigen presentation to T cells. This appeared to require antigen processing in a lysosomal-like compartment.156 Peptide loading is a complicated affair, involving prior binding of MHC class II by an “invariant chain” to prevent premature loading of incompletely folded proteins in the endoplasmic reticulum.157,158 Peptide loading requires proteolysis of the invariant chain.159,160 Mellman and colleagues would go on to show that the actual compartment in which antigen loading occurred is uniquely specialized for antigen presentation in B cells161 and DCs.162 In a landmark paper, Unanue and colleagues purified MHC class II molecules from 1011 B cells and showed a 1:1 binding with peptide and MHC class II I-Ad, but not I-A k,163 thus demonstrating MHC restriction at the biochemical level. The processing events required for MHC class I restriction seemed more elusive, as some of the molecular components required for this were not known at the time. It was suspected that an intracellular proteolytic event was needed to process antigen, but that was not proven until 1994 when Rock et al.164 showed that proteasomal inhibitors blocked degradation of most cell proteins and subsequent generation of peptides presented on MHC class I molecules. The actual mechanism by which peptides generated in the cytosol gained entry into the secretory compartment was provided in 1990, when four groups announced the identification of a member of a family of ABC transporters, called “TAP,” which provided this function.165–168 Finally, in 1987, Bjorkman et al.169,170 demonstrated that the antigen in question was a peptide actually bound to the groove of the MHC class I molecule.

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SPECIALIZATION WITHIN THE IMMUNE SYSTEM II. Discovery of B- and T-Cell Antigen Receptors The discovery of the B-cell receptor (BcR) for antigen began with Ehrlich,171 when he proposed that cells that produced “amboceptors” expressed them at their surfaces; in fact, Ehrlich’s drawings of amboceptor-producing cells emphasized this point (see Fig. 2.2). Evidence for the existence of surface Ig was provided by indirect immunofluorescence and autoradiography, in which immunoreactivity against a single class, IgM, was observed in 1970.172–175 The function of the BcR was uncovered by Rock and Lanzavecchia, who showed that MHC class II–restricted antigen presentation by hapten-specific B cells was enhanced 103- to 104fold by specific binding, endocytosis, and loading of peptide antigen onto MHC class II molecules.176,177 In 1952, Colonel Ogden Bruton at the Walter Reed Army Hospital was caring for the 8-year-old son of a general. The boy had had recurrent pneumococcal infections, including bacteremia, but he recovered with antibiotics. Bruton noted that the boy did not mount an antibody response to pneumococcal vaccination and upon testing his serum using Tiselius apparatus, the electrophoretic pattern revealed a complete absence of γ-globulins.178 Other cases appeared, and it was soon clear that the defect was X-linked. This is often cited as the first description of a primary immunodeficiency. It was not until 1993 that the molecular defect of agammaglobulinemia was uncovered. It was due to deficient expression of a tyrosine kinase, named Bruton tyrosine kinase.179,180 This discovery highlights another function of the BcR, which is to help provide signals for B-cell maturation. Without its expression, B-cell development is blocked beyond the pre-B-cell to immature B-cell stage.181 The discovery of the TcR for antigen was likened to the hunt of the apocryphal Snark,182 according to Mak,183 except the TcR was finally captured after a hunt that lasted over 20 years or more, depending on one’s perspective. The hunt was characterized by extended periods of uncertainty, when it was not clear whether the same receptor bound to antigen and MHC simultaneously, whether two separate TcR proteins bound MHC and antigen separately (the “intimacy model”), or whether one TcR molecule bound MHC molecules altered by antigen. It was also not known what form the antigen would actually take, and many considered it likely that the binding was strictly analogous to the binding of antibody and antigen, amounting to an “IgT-antigen” interaction.184 The existence of the TcR was deemed within reach when a clone-specific monoclonal antibody (mAb) against a murine lymphoma was isolated.185 Finally, in 1984, using differential hybridization approaches, Davis and Mak independently announced the cloning of the β -subunit of the mouse and human TcR, respectively.186,187 This was soon followed by the cloning of the α-subunits of the mouse and human TcRs,188–191 and the identification of other subunits of a different type of TcR, the γδ TcR.192 Eventually, the crystal structures of pMHC combinations demonstrated that contact between TcR and pMHC was dominated by direct TcRMHC contacts, rather than TcR-peptide interactions.193–195 Beyond the initial recognition stage, signaling through the

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TcR, as in the BcR, is a highly regulated process tuned to recognize cues from pMHC that govern positive and negative selection.196 There is little doubt that had Medawar survived, he would have appreciated how the clonal selection theory has remained a driving force underlying the molecular details of antigen receptor signaling.

Discovery of Distinct Immunoglobulin Classes Ehrlich’s terminology for antibodies changed depending on the context; he sometimes referred to “amboceptors,” or immune bodies, and at other times he used the more familiar term “antikörper.” Regardless, he did not know the chemical makeup of antibodies, though he recognized that they must contain separate binding sites for antigen and complement. Elucidation of the actual structure of antibodies would have to await the seminal work of Porter and Edelman in the 1960s; however, the actual discovery of specific antibody classes, or isotypes, took place over many years. The first class of antibodies to be discovered was IgG. In 1939, Tiselius and Kabat immunized rabbits with ovalbumin, then absorbed a portion of the resulting antiserum with ovalbumin. When they applied samples of the unabsorbed and antigen-absorbed antisera to electrophoresis, they observed a marked decrease in the amount of protein that migrated in the γ region of electrophoretic mobility, farthest away from the fast-migrating albumin peak. They called the antibody that corresponded to this fraction “γ-globulin.”197 The next antibody class to be discovered was a result of an observation by the Swedish oncologist Jan Waldenström. He described two patients with oronasal bleeding, lymphadenopathy, low serum fibrinogen, and increased lymphoid cells in the bone marrow.198 With the help of a colleague in Svedberg’s laboratory, he noted that serum from these patients contained an abnormally large amount of homogeneous globulin with sedimentation coefficients corresponding to a molecular weight of more than one million. Waldenström thought this corresponded to a preformed large molecule, which became known as macroglobulin. Proof that a macroglobulin possessed antibody activity was finally provided in 1967,199 which ultimately led to its current name, IgM. IgA was discovered by Gugler et al.200 who isolated IgA from human milk, and Heremans et al.201 who isolated IgA from human serum. It was later found in high concentrations in all exocrine secretions202 and further characterized by Tomasi and Zigelbaum,203 who suggested that IgA plays an essential role in mucosal immunity. It was not until 1984 that the mechanism of secretion of IgA and IgM across epithelial barriers was uncovered; it was shown to depend on interactions of an Ig-associated polypeptide, the J chain, with a glycoprotein on the surface of epithelial cells, referred to as the “polymeric IgA receptor.”204 This receptor mediated transcytosis of secretory IgA and IgM. The discovery of IgE had a particularly long gestation period and is well summarized in a review.205 It began with a report by Prausnitz, in 1921, who injected his forearm with serum from a coworker allergic to fish (and coauthor on the ensuing paper); this was followed by a wheal and

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flare upon further injection of fish extract.206 This property was expected for a class of antibodies termed “reagins” (from German reagieren, to react). IgE was identified as a specific immunoglobulin class by Teruka and Kimishige Ishizaka in 1966207,208 and was shown to be increased in sera from asthmatics.209 In 1993, Kinet and colleagues demonstrated that its high affinity receptor, Fc εRI, was required for anaphylaxis.210 IgD is the most recent Ig subclass to be recognized. It was discovered from a patient with multiple myeloma.211 Surface IgD is coexpressed with IgM on mature B cells. The function of IgD is not well understood, although the abundance of IgM- IgD+ B cells in the human upper respiratory mucosa suggests that it is likely involved in mucosal immunity.212 Because IgA and IgE were isolated based on their predominant location (IgA) or function (IgE), their role in the immune response was relatively easy to decode, but the relationships between the different Ig classes was unknown. It was discovered in 1963 that the early immune response was initiated by the rapidly sedimenting (19S) antibody, later shown to be IgM, followed by the production of a more slowly sedimenting (7S) antibody, now known to be IgG.213,214 Although production of the different Ig classes was initially interpreted as being due to the participation of different cells, it was later shown that single clones of B cells were capable of producing multiple isotypes215–217 in a process called class switch recombination (CSR). The enzymology of CSR was worked out much later by Alt, Nussenzweig, Honjo, and others, who showed that it required DNA repair enzymes218,219 and activation-induced cytidine deaminase (AID). AID is mutated in an autosomal recessive form of the primary immunodeficiency, hyper-IgM type 2, in which afflicted patients are deficient in CSR and somatic hypermutation, consistent with an important role for AID in both processes.220–222

Discovery of Antibody Effector Functions: Fc Receptors Almoth Wright was the first to recognize the importance of antibody-mediated effector functions other than neutralization or complement-mediated lysis. His studies were ignored for a long time, and it would take many years to appreciate the importance of the “other end” of the antibody molecule. Since the 1970s, studies on the effector functions of the Fc portion of IgG made extensive use of several in vitro model systems, often involving phagocytosis.223–232 The eventual cloning of receptors for the Fc portion of IgG (FcγRs) by Ravetch and et al.233 revealed many similarities and some differences, but these early studies did not reveal how the receptors transduced signaling events, such as phagocytosis. It was not until 1989 that Michael Reth234 noted a short tyrosine-containing sequence in common with several antigen receptors, including subunits of the TcR, the BcR,235 and Fcγ and Fcε receptors236 that the signaling function of FcγRs was understood in a larger context. Following receptor engagement, tyrosine residues within this consensus sequence, subsequently named “immunoreceptor tyrosinebased activation motif” (ITAM), become phosphorylated

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by Src family tyrosine kinases and serve as docking sites for Syk tyrosine kinase,237 or ZAP-70, in the case of the TcR. The membrane-associated tyrosine kinases become activated and phosphorylate substrates that further convey downstream signals. Absence of ZAP-70 leads to a severe combined immunodeficiency disease (SCID).238 These studies uncovered the central role of nonreceptor tyrosine kinases in the immune system, which appeared to explain in large part how antigen and Fc receptors evoke calcium signaling,239 degranulation,240 phagocytosis,229,241,242 and antibody-mediated cellular cytotoxicity.243 Furthermore, the ITAM-containing γchain244 that is associated with FcγRs is required for immune complex-mediated glomerulonephritis in mice.245 Thus, it is likely that the hypersensitivity phenomena originally observed by von Pirquet at the beginning of the 20th century, and later attributed to immune complexes by Dixon et al.,80 has a similar pathophysiology, at least in part.

THE HERMENEUTICS OF LYMPHOCYTE ACTIVATION Evolution of Early Lymphocyte Activation Models By the late 1950s, it became clear that lymphocytes were highly adept at interpreting or translating environmental cues and responding by maintaining a state of activation or tolerance. The focus of immunology had shifted from identifying the relevant cell types involved in the immune response to discovering what can be viewed as the “hermeneutics” (a term derived from the Greek ε‘ ρμηνευ′ω, “translate” or “interpret”) of lymphocyte activation. Based on the clonal selection theory, Burnet246 proposed that activation or tolerance was the result of an antigen stimulating a single cell, depending on the age of the organism. This was in keeping with experiments in which tolerance was induced by exposing antigen during fetal life or shortly thereafter.138,140,141 In 1959, Lederberg247 modified this model to describe activation or tolerance as occuring depending on the age of the cell rather than the organism. If an antigen is introduced prior to the maturation of any antibody-forming cell, the hypersensitivity of such cells, while still immature, to an antigen-antibody reaction will eliminate specific cell types as the arise by mutation, thereby inducing apparent tolerance to that antigen. However, the model could not account for several observations, among which were that the dosage and specific form of the immunogen, such as a hapten, could also influence the outcome of the encounter. This led Talmage and Pearlman248 to propose an alternative model in which antigen alone would induce tolerance, but aggregated antigen, perhaps associated with complement, could induce an additional nonspecific stimulus to trigger clonal expansion. The suggestion of the need for a second “nonspecific” stimulus, and particularly complement, was remarkably prescient in light of the finding that complement can provide exactly such a stimulus to amplify immunogenicity by 103- to 104fold.249 In 1968, and later revised in 1970, Bretscher and Cohn250,251 provided a variation on this model, in which a

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thymus-derived antigen-sensitive cell-bound antigen containing at two separate sites, one for a receptor on the “antiantigen-sensitive” cell and another bound to a “carrier” antibody. If the second site on the antigen was occupied by antibody, then that would deliver a signal to the “antiantigen-sensitive” cell to induce immunity. In contrast, if the site remained unbound, the antigen would induce tolerance. In this model, two cells were involved, but they were separated in time and space. Among the problems with the model was its circularity: What would provide the stimulus for the production of the carrier antibody in the first place? Other theories were proposed to explain the phenomenon of alloreactivity. The most intriguing of these was provided by Lafferty and Cunningham,252 who proposed the existence of an “antigen bridge,” which was now provided by another cell to provide “Signal 2.” When MHC restriction was described in 1977, this model was modified somewhat to include the provision that the second signal was triggered by MHC on the stimulator cell253 (Table 2.1).

Discovery of Costimulation: T Cells as Beneficiaries By 1975, models for lymphocyte activation had matured, but many details remained sketchy, particularly the identity of the elusive Signal 2. In 1987, Jenkins and Schwartz utilized a system designed to “convert” Ag-specific T-cell clones into suppressor cells. They used a model system in which T-cell clones against a pigeon cytochrome peptide were incubated with ethylene carbodiimide-fi xed Ag-pulsed splenocytes as APCs. They obtained the surprising result that the T-cell clones became unresponsive to subsequent restimulation with untreated APCs plus peptide, although they remained viable and proliferated in response to interleukin (IL)-2.254 This showed that Ag-specific unresponsiveness could be induced in the absence of suppressor cells and that stimulation with cognate antigen alone was insufficient to induce proliferation; the missing Signal 2 was defi ned as “costimulation” and ultimately identified as cluster of differentiation (CD)28-mediated signaling by Allison and colleagues.255 Since this seminal discovery, an entire family of CD28-like molecules in T cells and their cognate ligands on APCs has been identified,256,257 which includes an inhibitory member of the family, cytotoxic T-lymphocyte antigen-4.258 This has led to the clinical development of cytotoxic T-lymphocyte antigen-4–Ig, an inhibitor of CD28,259 which was approved for the treatment of rheumatoid arthritis by the U.S. Food and Drug Administration in 2006 and by the European Medicines Agency in 2007.

Discovery of the Mechanism of B-Cell Help It was known since the 1960s that T cells were needed to “help” B cells produce antibody, but the molecular nature of that help was unknown.129,260 Experiments had suggested that T-cell help required cell-to-cell contact, implying the existence of the involvement of cell surface receptor–ligand interactions. Agonistic antibodies against CD40 drove B cells into cell cycle, raising the possibility that a cognate ligand on T helper cells participates in the mechanism of

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INTRODUCTION TO IMMUNOLOGY

Different Models of Lymphocyte Activation

Basis of Immune Recognition Self versus nonself

Signals

Components

Conditions for Tolerance

Authors

1 1 2

B cell/Ag B cell/Ag B cell/Ag–Ab + complement

2

Humoral Ag-sensitive responder cell/bivalent Ag/carrier antigen-sensitive thymusdependent cell Signal 1: Ab-bound responder cells + Ag Signal 2: Stimulator cell + Ag Signal 1: Lymphocyte + MHC/Ag Signal 2: PAMP+ APC “Signal 0”: Alarm Signal 1: (Th cell-APC–Ag; Tc-infected cells; B cell/Ag; Signal 2: Help (Th to B) or costimulation (APC to T)

Immature organism Immature Ab-forming cell Absence of bound Ab/ complement Absence of carrier Ab or lack of

Burnet, 1959230 Lederberg, 1959231 Talmage and Pearlman, 1963232 Bretscher and Cohn, 1968,234 1970235

Absence of Signal 2

Lafferty and Cunningham, 1975236

Absence of Signal 2

Janeway, 1989390

Absence of “Signal 0”

Matzinger, 1994451

2

Noninfectious self versus infected nonself Danger

2 3+

Abbreviations: Ag, antigen; Ab, antibody; APC, antigen-presenting cells; MHC, major histocompatibility complex; PAMP, pathogen-associated molecular pattern; Th, T helper.

T-cell help. Using a CD40–Ig fusion protein, murine CD40L was cloned.261,262 Use of a mAb against the human CD40L confirmed that expression was restricted to mantle and centrocytic zones of lymphoid follicles and the spleen periarteriolar lymphoid sheath in association with CD40 + B cells,263 which is the distribution that might be expected for a highly localized signal to provide T-cell help to B cells. Indeed, in 2000, the specific type of T cell that provided CD40Lmediated help to B cells, now called a T follicular helper cell, was identified by its unique anatomic location and expression of a homing receptor, CXCR5.264,265 In 1993, four groups independently announced that defects in the CD40 ligand gene are responsible for X-linked hyper-IgM syndrome in which affected adults express elevated levels of IgM and defects in class switching.266–269

T-Cell Subsets and T-Cell Signaling Paradigms The widespread use of mAbs since their discovery by Köhler and Milstein resulted in the production of many antibodies directed against T cells that had nonoverlapping patterns of expression. Much of this early work was done by Schlossman, Reinherz, and colleagues.270–275 In most cases, the antibodies themselves identified T cells with specific functions; and in 1987 and 1988, two groups found that the molecules recognized by two of these mAbs, denoted by their cluster designations, CD8 and CD4, mediated adhesion to MHC class I and II, respectively.276,277 CD4 and CD8 were shown to bind to nonpolymorphic regions of MHC molecules,277 suggesting that they might stabilize otherwise weak interactions between TcR and pMHC. Later experiments demonstrated that the cytosolic domains of these coreceptors were able to bind, and recruit, the nonreceptor Src family tyrosine kinase Lck.278,279 It was not known at the time what the relevant substrates of Lck might be, but in

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1989, at about the same time that the ITAM consensus sequence was identified, two groups showed that Lck was capable of phosphorylating subunits of the CD3 complex and the ζ subunits of the TcR.280,281 Following the discovery of another key tyrosine kinase associated with the ζ subunit of the TcR, ZAP-70, by Chan and Weiss,238 a complicated cell signaling paradigm began to emerge in which “Signal 1,” delivered by pMHC expressed on either APCs or virus-infected cells, is conveyed by a series of phosphorylation events, leading to phosphorylation of phospholipase C-γ, triggering increases in cytosolic calcium and dephosphorylation of a key transcription factor, NFAT. The elusive machinery of “store-operated calcium channels” required for calcium-based signaling282 was finally elucidated in 2006.283–286 Defects in this pathway in lymphocytes lead to a form of SCID.286 Dephosphorylated NFAT, alone or in conjunction with AP-1, translocates to the nucleus where it binds to sites on the promoters of key genes such as IL-2, culminating in T-cell proliferation.287 The initial interaction between TcRbearing T cells and pMHC-bearing APCs induces a cooperative series of protein–protein interactions in a spatially delimited fashion (the “immunologic synapse”) that facilitates signaling events.288 We now know that signal transduction by the TcR shares common elements, and in some cases identical kinases and substrates, with signaling by other ITAM-containing receptors, such as the BcR, and Fc receptors or IgG and IgE.289–291 Recent data suggest that similar mechanisms governing “immunologic synapses” in T cells may also be characteristic of “phagocytic synapses,”292 underscoring a further degree of conservation of ITAM-based pathways. It is ironic that lymphocytes, which had appeared morphologically uninteresting to so many biologists until the latter part of the 20th century, would share so many characteristics in common with their visually more intriguing cousins.

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Cell to Cell Communication: Mice Have a Tale to Tell The first cytokine to be characterized is credited to Isaacs and Lindenmann,293 who studied an influenza virus-induced factor from chick chorioallantoic membranes that blocked a second viral infection; this factor was called “interferon” (IFN). It was not purified to homogeneity until 20 years later.294,295 Type I IFNs, which include IFNα and IFN-β, and type II IFN, IFNγ, bind to distinct receptors. A major advance in our understanding of cytokine signaling occurred in 1992, when Schindler et al.296 demonstrated the tyrosine phosphorylation and nuclear translocation of a complex of four proteins, named IFNα stimulated gene factor 3. IFNα stimulated gene factor proteins were isolated from nuclear extracts derived from over 1010 cells, followed by sequencing and cloning of their genes; two of these proteins are members of what are now known as STAT proteins, which were shown to be substrates for members of the Janus-associated kinase family.297–303 The most common severe immunodeficiency attributable to defective cytokine signaling is X-linked SCID, which is due to mutation in the common γc gene that is shared by receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.304 Mutations in Janus-associated kinase 3 were found in two patients with a severe form of SCID indistinguishable from X-linked SCID.305,306 Dominant-negative mutations in STAT3, which is important in the signal transduction of IL-6 and IL-10, were found to be responsible for hyper-IgE syndrome, a disease associated with highly elevated serum IgE, recurrent staphylococcal skin infections and pneumonia, and skeletal abnormalities.307,308 Much of our knowledge of this pathway, as well as many other components of the immune system, is due to the use of gene targeting in mice. In 1985, Smithies et al.309 introduced a short DNA sequence from the human beta-globin locus into an erythroleukemia cell line and were able to detect a specific exchange of the beta-globin gene with the homologous sequence in about 1 in 103, demonstrating the feasibility of gene targeting. At the same time, Capecchi introduced DNA directly into the nucleus of a cell using a microelectrode. Capecchi noted that multiple copies of the introduced gene were integrated into the host cell’s chromosome through homologous recombination. These studies established the potential for homologous recombination in somatic cells. The next major step was based on the ability of using blastocyst-derived embryonic stem cells to introduce genes into the germline of the mouse.310,311 By injecting blastocysts with cultured embryonic stem cells that were infected with a retrovirus, Evans and colleagues generated chimeric mice in which retroviral DNA was detectable in both somatic and germ-line cells.311 Eventually, Evans, Smithies, and Capecchi refined these techniques, which led to the first knockout mouse, in which the gene encoding hypoxanthine guanine phosphoribosyl transferase was deleted. The resultant phenotypic resembled that of the Lesch-Nyhan syndrome, which is characterized by mental retardation and self-mutilation.312,313 Today, gene targeting techniques are used worldwide to study the effects of deletion or overexpression of genes important in immunity and any disease that can be reproduced in the mouse and in

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other species in which the technique has been used. In 2007, Smithies, Capecchi, and Evans shared the Nobel Prize “for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells.”

Chemokines If cytokines determine what cells do, then chemokines determine where cells go. The first chemokines purified were derived from platelets, although neither protein (platelet factor 4 [CXCL4]314,315 and β -thromboglobulin [CXCL7]316) was recognized to be chemotactic when first identified. IL-8 (CXCL8) was the first chemokine to be purified and sequenced based on its chemotactic function for neutrophils,317 and its receptors were cloned in 1991.318,319 The word “chemotaxis” was coined by Pfeffer in 1884, who observed spermatids migrating toward a pipette containing maleic acid salts.320 It is not clear who first explicitly observed chemotaxis of leukocytes, although several scientists, notably Schultze, Lieberkühn, Davaine, and Wharton Jones described amoeboid movement in leukocytes in the middle of the 19th century.321 Since then, chemotaxis has been studied in vitro using various techniques, including specialized chambers that facilitate this process.322 The effects of chemokines on cells are complex, but a major function is to orchestrate the process of diapedesis, the process of transendothelial migration first described by Dutrochet in 1824.323 In 1979, Hayward et al.324 described six infants from two families whose umbilical cords were still attached at 3 weeks of age. Five of these developed severe local and disseminated infections from which four died. The molecular defect was identified by Springer et al.325 in 1984, who showed that leukocytes from patients with the disease lacked all known β2 integrin adhesion receptors. This was eventually traced to absent or abnormal β2 subunits (CD18). Marlin and Springer326 later described the requirement of the β2 integrin, LFA-1, to bind its counter-receptor, ICAM1, on endothelial surfaces for diapedesis to occur normally. The function of chemokines is to provide chemotactic gradients as well as activate the integrins for tight adhesion prior to the active participation in endothelial cells in diapedesis.327,328 Interestingly, LFA-1 was shown to be required as an accessory molecule during interaction of T cells with their cognate targets.329 Chemokines are critical for initiating the primary immune response as APCs, such as DCs. DCs and naïve T cells both share a common chemokine receptor, CCR7. T cells migrate through high endothelial venules, which secrete the chemokine SLC (CCL21). SLC is needed for integrin-mediated adhesion of the T cells.330 Mice lacking expression of SLC have defects in lymphocyte and DC localization to the T-cell zone of secondary lymphoid organs.331 The chemokine-directed “patrolling” of naïve T cells in the lymph nodes is probably the major mechanism by which the very few lymphocytes with the relevant TcR specificity remain there to interact with the antigen-loaded APCs during an immune response. Thus, chemokines are essential components of the “clonal selection” of lymphocytes.

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T-Cell Polarization: The Power of Dichotomy In 1986, Coffman and Mosmann at DNAX Research Institute were interested in determining factors that differentiated different types of T helper cell lines. One such line was capable of inducing a 100-fold increase in IgE secretion from mouse splenocytes, which was potently blocked by IFNγ.332 When the team characterized other cloned T-cell lines, it became clear that there were very specific patterns of secretion: Some clones, which they called TH1 cells, produced IL-2 and IFNγ, while other clones, which they called TH2 cells, produced factors that stimulated B cells (B-cell– stimulating factor-1), mast cells, other T cells, and IgE and IgG1 secretion. All clones produced IL-3.333 Further work, in collaboration with Paul, who first identified B-cell– stimulating factor-1,334–336 clarified that B-cell–stimulating factor-1 was the same factor that stimulated mast cells and T cells, later renamed IL-4. Coffman and Mosmann337 defined the now well-established paradigm of polarized T helper cell secretion; they demonstrated that IL-4 and IFNγ reciprocally inhibit the outgrowth of TH1 and TH2 cells, respectively. The later identification of transcription factors that are selectively expressed in these cells (GATA-3 and T-bet, respectively) and can even reprogram the cells to transdifferentiate toward the “opposite” phenotype further substantiated this dichotomy.338,339 These findings had profound implications for immunologists, who began to classify diseases according to their predominant TH cytokine profiles. Thus, tuberculosis and most bacterial and fungal infections produced a TH1 pattern of cytokines, whereas asthma and other allergic diseases produced a TH2 pattern of cytokines. While this reductionist view of cytokine production is no doubt an oversimplification, this simple dichotomy has proved quite robust and has helped guide the development of novel therapeutics for various diseases. There is, however, a danger to oversimplification340 — sometimes it is necessary to be a “fox” rather than a “hedgehog.”341 Some diseases, such as Crohn disease, an inflammatory bowel disease that was thought to be driven by the TH1-polarizing cytokine, IL-12, is more likely driven by a related cytokine, IL-23, with which IL-12 shares a common subunit; indeed, a genome-wide association study in 2006 revealed a highly significant association of the gene encoding a subunit of the IL-23 receptor and early-onset Crohn disease.342 Ustekinumab, a mAb against the IL-12/IL-23 p40 common subunit was approved by the U.S. Food and Drug Administration for the treatment of moderate-to-severe Crohn disease in 2006 and for moderate-to-severe plaque psoriasis in 2009.

The Language of Immunoregulation: Explaining the “Contrivances” The concept of immunoregulation is implicit in the term “immunity” (exempt), and the history of how different immunologists have viewed immunoregulation, depending on their perspectives, is worthy of a chapter in itself. Jerne343 hypothesized that antibodies can act as antigens and elicit an immune response against their idiotypes, which would

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then serve to regulate the immune response. While appealing, efforts to prove the importance of this concept have achieved limited success. In 1970, Gershon and Kondo344 found that thymus-derived cells could specifically induce tolerance, and they and others spent many years trying to isolate antigen-specific T suppressor cells but were unable to do so. In the 1990s, the field shifted away from clonotypic suppressor cells toward T cells secreting specific tolerogenic factors, such as IL-10–secreting “Tr1 cells” and transforming growth factor-β –secreting “Th3 cells.”340,345 This was followed by the identification of cell surface markers of a subset of CD4 + cells that were required to suppress autoimmunity in mice. These turned out to be CD25 and CD5 in a CD4 + population of cells in the mouse, cells which were later called regulatory T cells. In 2001, the FoxP3 gene was identified as the gene that was mutated in Scurfy mice, which develop severe autoimmunity as a result of a single gene mutation.346 Similarly, mutations in the human homolog of FoxP3 were found to be associated with the disease called IPEX (immune dysregulation, polyendocrinophathy enteropathy, X-linked syndrome).347–349 Analogous to the capacity for T-bet and GATA3 to drive TH1 and TH2 differentiation, respectively, expression of FoxP3 was sufficient to drive the differentiation of T helper cells to a CD4 + CD25 + regulatory T phenotype.350,351 Central tolerance, sometimes referred to as “negative selection,” is a means of shaping the repertoire that was thought likely to exist following the discovery of the thymus as an immune organ by Miller in 1961; however, proof for this was lacking. How could one prove the existence of a phenomenon that predicted the absence of a cell, rather than its presence? It was not until 1987 that Kappler et al.352 used a mAb against a specific TcR Vβ segment to show that the mAb recognized immature thymocytes but not mature thymocytes or T cells, thus proving that negative selection occurs. Von Boehmer demonstrated a similar phenomenon using a TcR transgenic mouse model.353 Negative selection of immature B cells was also demonstrated in the classic experiments of Nossal and Pike,354 Sidman and Unanue,355 and Raff et al.356 Clearly, the field of immunoregulation is still evolving. Novel mechanisms of immunoregulation will surely be discovered, and novel regulatory interactions will be uncovered. The role of tolerogenic DCs,357,358 CD8 + T cells,359,360 and myeloid-derived suppressor cells361–363 are likely to play important roles and some of these will likely find their way to the clinic. Perhaps, however, we have made some progress in support of Ehrlich’s contention364 : The organism possesses certain contrivances by means of which the immunity reaction, so easily produced by all kinds of cells, is prevented from working against the organism’s own elements.

Immune Subversion: The Case of Tumors The idea that the immune system is capable of responding to tumors dates back to Ehrlich,365 who predicted that cancer would occur at a high frequency in the absence of

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an immune response. This theme was recapitulated by others, notably Burnet,366 who argued that T cells would be prominent in what he termed “immune surveillance.” The field enjoyed a resurgence in 1994 when Schreiber and colleagues demonstrated that tumors expressing dominantnegative IFNγ receptors demonstrated enhanced tumorigenicity in syngeneic mice.367 This was supported by studies using IFNγ receptor-deficient or STAT1-deficient mice.368 Additional evidence for an active immune response against tumors is the spontaneous development of autoimmunity in some individuals with tumors; for example, paraneoplastic cerebellar degeneration occurs in individuals with a cytotoxic T-cell response to a shared antigen on tumor cells and neuronal cells.369 The immune response to tumors is complex, with participation from cytotoxic T cells, natural killer (NK) cells, DCs, and myeloid cells.370 The “counterresponse” by the tumors, which has been termed “escape” or “evasion,” is equally complex.370 While it is beyond the scope of this chapter to discuss any of these in detail, several relevant points of historical interest should be noted. In 1976, a postdoctoral fellow named Mike Bevan was studying alloreactive cytotoxic T-cell responses and was able to demonstrate that spleen cells from an H-2b /H-2d–restricted mouse primed with H-2b cells that differed in minor histocompatibility loci contained increased numbers of both H-2b – and H-2d–restricted cytotoxic CD8 T cells.371,372 Bevan interpreted this as evidence of “cross-priming,” in which ingestion of cell-associated antigens by phagocytosis led to cytosolic entry of antigens and loading onto MHC class I molecules and then presentation to cytotoxic T cells. It was completely novel and counterintuitive, but subsequent studies validated this interpretation.373 Recent progress has been made in our understanding mechanisms by which antigens gain entry into the cytosol from phagosomes.374 These studies were based on key findings by Desjardins and colleagues who first demonstrated recruitment of endoplasmic reticulum membrane to nascent phagosomes.375,376 Mice that lack CD8α + DCs, a cell type that is adept at cross-priming, were unable to demonstrate cross-priming and were incapable of generating a cytotoxic response against West Nile virus and a highly immunogenic fibrosarcoma tumor.377 Thus, crosspriming is important in tumor immunity. Tumor cells are capable of circumventing the host immune response in various ways, including downregulation of MHC class I molecules, although this would potentially render them susceptible to NK-mediated killing, according to the “missing self” hypothesis.378 Tumors elaborate a host of cytokines and growth factors that directly inhibit various components of the immune system.370 An additional means of tumor evasion specifically bears on the meaning of “ immunologic self.” Most cells in the body express a cell surface protein, CD47, which is a ligand for a receptor present on macrophages and DCs, SIRPα .379 SIRPα interacts with tyrosine phosphatases via its cytosolic domain380 ; therefore, corecruitment of SIRPα to tyrosine kinase-coupled signaling scaffolds is predicted to inhibit kinase-mediated signaling events. Lindberg and colleagues showed in 2000 that CD47 on erythrocytes in mice prevented their phagocytosis

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until levels fell below a certain level, thus CD47 served as an aging “clock” that signified a “don’t eat me” signal.381 In 2010, in a remarkable study, expression levels of CD47 on non-Hodgkin lymphoma (NHL) cells were negatively correlated with survival. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with an anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab (anti-CD20) led to elimination of lymphoma and cure.382 In these settings, CD47, rather than MHC proteins, serve as a marker of “self.” It is somewhat counterintuitive that, at least in the case of NHL, tumor masquerading as self is one mechanism by which tumors evade the immune system. Ironically, downregulation of MHC class I molecules, the classic marker of self, is yet another mechanism of how tumor evasion of immunity.370

Immune Hijacking: The Discovery of Human Immunodeficiency Virus The first description of what would come to be known as the “acquired immunodeficiency syndrome” appeared in 1981.383 Initially described in five homosexual men, the disease soon was apparent in Haitians, transfusion recipients, infants, Africans, and female sexual contacts of infected men.384 In just 2 years, Montagnier’s team published the first paper demonstrating the presence of retroviral particles from diseased patients,385 and a year later, Gallo’s group published five papers in Science providing convincing evidence that this retrovirus was the cause of acquired immunodeficiency syndrome.386–390 Barré-Sinoussi and Montagnier won the Nobel Prize for their discovery of human immunodeficiency virus (HIV), along with zur Hausen, for demonstrating that human papillomavirus can cause cervical cancer. Many felt that Gallo should have been awarded the prize as well. Pneumonia due to Pneumocystis carinii (later re-named P. jerovici) was common in initial cases of acquired immunodeficiency syndrome, but patients soon presented with an array of opportunistic infectious diseases, malignancies (eg, Kaposi sarcoma and lymphoma), and even autoimmune diseases. In 1986, Maddon et al.391 demonstrated that CD4 is an essential receptor that mediates HIV-1 entry into lymphocytes; this was followed in 1996 by the demonstration of CXCR4 and CCR5 as coreceptors for HIV-1 entry.392 Since then, many laboratories worldwide have dedicated their efforts to uncovering HIV pathogenesis.393,394 One of the major challenges to the immune system as well as the development of an HIV-1 vaccine is the enormous plasticity of the viral sequence due to the high error rate of reverse transcription.393 Intensive efforts in academia and the pharmaceutical industry have resulted in highly active antiretroviral therapy based on targeting multiple steps of viral replication, mostly focusing on reverse transcriptase and protease. A disease that was once virtually 100% fatal is now manageable, resulting in an 80% to 90% decrease in mortality rates in the United States and Europe.393 Among the major

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challenges facing immunologists and virologists is eliminating the latent reservoir of virus in resting memory CD4 + lymphocytes, developing an effective HIV-1 vaccine, and providing treatment for the 90% of infected individuals worldwide who reside in developing countries and have poor access to antiretroviral therapy.

METCHNIKOFF’S LEGACY The Rediscovery of Innate Immunity Arguably, the most important advance in immunology in the last 15 years has been the “rediscovery” of innate immunity. The closest term to “innate immunity” that was used at the turn of the century was “natural immunity.” When the cellularists and the humoralists were debating the relative importance of cells and soluble antikörper at the turn of the century, much of the phenomena that Metchnikoff observed under the microscope represented different aspects of innate immunity. However, Metchnikoff distinguished enhanced immunity due to vaccination as a result of what we would call today an acquired immune response: “an agglutinative substance...in the...fluids of the body becomes much more developed in those of immunised animals.”395 He thought that infections could lead to an acquired immune response, but the nature of that response was to foster an enhanced response in phagocytic cells.395 In certain infective diseases terminating fatally a very marked phagocytosis is observed even in susceptible animals . . . The acquisition of immunity against microorganisms is, therefore, due not only to the change from negative to positive chemiotaxis, but also to the perfecting of the phagocytic and digestive powers of the leucocytes. In 1932, Fleming396 recounted the discovery of lysozyme, which he made in 1921, “because its importance in connection with natural immunity does not seem to be generally appreciated.” Although the discovery has been cited to represent another example of scientific serendipity, resulting from accidental dripping of nasal secretions from Fleming himself onto a culture plate, 397 Fleming wrote that “cultures of nasal mucus were made from a person suffering an acute cold” when the bacterolytic phenomenon due to lysozyme was observed.396 Fleming would go on to discover penicillin, which was a bona fide example of scientific serendipity, and receive the Nobel Prize for his discovery. Of course, lysozyme is but one of many innate immune molecules important in the early phase of host defense. Other cationic proteins include defensins, which are secreted by leukocytes and epithelial cells, and chemokines, whose tertiary but not primary structures are related to defensins. Some members of both classes have the dual role of recruiting inflammatory cells to sites of infection and killing bacteria.398–401 Other examples of components of innate immunity include “innate immune lymphocytes,” such as NK cells, NK T cells, and γδ T cells. Although it is beyond the scope of this chapter to describe these cell types except in passing, the discovery of NK cells in 1975402–404 provided a cellular

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mechanism to account for the observation that some host immune cells recognize and kill virus-infected or neoplastic cells lacking expression of MHC class I molecules. NK cells also provide an early, immediate source of cytokines, allowing them to rapidly respond to injury prior to maturation of the primary immune response.405 The “modern era” of the study of innate immunity began in 1989. In a characteristic example of his remarkable insight, Janeway406 published an article in which he described a new model for immune recognition. Janeway argued that rather than distinguish “self ” from “nonself,” the immune system had evolved to distinguish “noninfectious self from infected nonself ” (see Table 2.1). Janeway compared the innate and acquired immune systems. He emphasized that the former evolved to respond rapidly to “pathogen associated molecular patterns” by as yet unidentified receptors in a nonclonal fashion. Janeway was acutely aware of the significance of Freund’s discovery of adjuvant407 and the implications this had for the primary immune response. What was remarkable about Janeway’s hypothesis is its intuitive appeal and its Darwinian flavor, in some ways analogous to Metchnikoff ’s view of phagocytosis. In addition, it had great predictive power. In a completely independent line of investigation, Jules Hoffmann became interested in exploring inflammatory pathways in Drosophila. Hoffman was aware of Baltimore’s discovery of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κ B), a transcription factor, originally identified as a lipopolysaccharide (LPS)-inducible transcription factor in B cells in 1986.408 Mice rendered deficient in NF-κ B demonstrated multifocal defects in immune responses, including those to LPS.409,410 Based on his observation of similarities between the cytokine-induced activation cascade of NF-κ B in mammals and the activation of the morphogen dorsal in Drosophila embryos, Hoffmann demonstrated that the dorsoventral signaling pathway and an extracellular toll ligand control expression of antifungal peptide gene expression. Mutations in the toll signaling pathway dramatically reduced survival after fungal infection.411 Until that time, there was only a limited understanding of pathogen recognition in eukaryotes; the only receptor that was known to participate in the recognition of LPS was CD14, a molecule that is expressed predominantly on macrophages.412 Shortly after publication of Hoffmann’s landmark study, Janeway and Medzhitov published an article in Nature that described the cloning of a human homolog of Drosophila toll. A constitutively active mutant of human toll transfected into human cell lines induced the activation of NFκ B and the expression of the inflammatory cytokines IL-1, IL-6, and IL-8 as well as the expression of the costimulatory molecule B7.1 “which is required for the activation of naïve T cells.”413 The choice of these proteins was not accidental, and it was clear that Medzhitov and Janeway had Janeway’s hypothesis of 1989 in mind. Meanwhile, Beutler et al.414 were zeroing in on the genetic identification of a mutation in a mouse strain that was incapable of responding to LPS, a component of the cell walls of all gram-negative bacteria. Beutler and Cerami were the fi rst to show that immunization against tumor necrosis factor, the prototypic cytokine

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that is produced following administration of LPS, protected mice from lethal shock.414 Beutler demonstrated that the codominant Lpsd allele of C3H/HeJ mice corresponded to a missense mutation in the toll-like receptor-4 gene.415 Since these publications, a family of innate immune receptors has been defi ned, each responding to different ligands encountered by hosts during infection.416 Initial interactions of innate immune receptors on APCs with their ligands encounters are necessary for the upregulation of costimulatory molecules, without which tolerance would occur.417 Janeway’s prediction in 1989 was borne out, providing strong empirical evidence in support of his model for immune recognition (see Table. 2.1). In 2012, Beutler and Hoffman, together with Steinman, received the Nobel Prize. The field of innate immunity has exploded since the key observations of Hoffman, Beutler, Janeway, and Medzhitov. Many other receptors and components of the innate immune system have been discovered. These include components of the complement system,418 cell surface lectins,419 collectins such as lung surfactant proteins420 and mannosebinding proteins,421 scavenger receptors,422,423 and pentraxins.424 Most of these participate in recognition of pathogens, including fungi,425 bacteria,426 and viruses such as HIV.427 In some instances, the phagocytic cells themselves serve as the source of opsonins, such as complement.428 A variety of innate immune receptors also recognize apoptotic and necrotic cells.429,430 Secretion of opsonins, such as milk fat globule-EGF factor 8, can enhance uptake of targets such as apoptotic cells,431 and the absence of this protein has been linked to autoimmunity in mice.432 Similar results were found in other mouse strains engineered to be deficient in clearance of apoptotic cells.433,434 The conceptual basis of these experiments was built on earlier work of Savill, Henson, and others who showed that the immunologically “silent” disposal of apoptotic debris is an active process serving to divert self-antigens toward a nonphlogistic mode of phagocytosis.435–437 In the context of resolution of acute infections, a similar function is provided by the production of omega-3 polyunsaturated fatty-acid-derived “anti-inflammatory” lipids (“resolvins”), fi rst identified by Serhan et al. in 2000.438,439

Cytosolic Components of Innate Immunity: Discovery of the Nicotinamide Adenine Dinucleotide Phosphate-Oxidase In 1957, Good and colleagues described an X-linked disease in which children succumbed to chronic suppurative and granulomatous infi ltrations and chronic infections.440 Neutrophils isolated from these children showed decreased bactericidal activity, although they demonstrated normal phagocytosis.441 However, they showed decreased hydrogen peroxide production and hexose monophosphate shunt activity.442 In 1974, Curnutte et al.443 identified defective superoxide anion production in children with this syndrome who also failed to reduce the dye, nitroblue tetrazolium.444 This simple test has been widely used to diagnose what came to be called “chronic granulomatous disease.”

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In 1978, Segal et al.445,446 identified the molecular defect in X-linked chronic granulomatous disease as the absence of cytochrome b. Since then, all the components of this multiprotein enzyme complex have been identified.447–452 Of note is that the oxidase responsible for X-linked chronic granulomatous disease, now referred to as NOX2, is but one of a family of oxidases that are widely expressed and have been implicated in various disease.453 We now know that there are multiple reactive oxygen species, many of which combine with other reactive molecules, such as nitric oxide, to generate reactive nitrogen species. Nathan and colleagues demonstrated that mice deficient in inducible nitric oxide synthase proved highly susceptible Mycobacterium tuberculosis, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids.454 In addition to its role in microbial killing, NOX2 is recruited to early phagosomes in DCs and mediates the sustained production of low levels of reactive oxygen species, causing active and maintained alkalinization of the phagosomal lumen. DCs lacking NOX2 show enhanced phagosomal acidification and increased antigen degradation, resulting in impaired cross-priming.455

Cytosolic Components of Innate Immunity Signal Danger There are many additional cytosolic proteins important in innate immunity. Of particular interest is the discovery of a family of pattern recognition receptors, most commonly referred to as nucleotide oligomerization domain–like receptors.456 In 2000 and 2001, a number of genetic studies linked defects in NLR genes to inflammatory diseases, including Crohn disease,457 Blau syndrome,458 Muckle-Wells syndrome,459 and familial Mediterranean fever.460 In 2002, Tschopp and colleagues described a multiprotein complex that they called the “inflammasome,” which included members of the nucleotide oligomerization domain–like receptors family and caspase-1.461 They showed that activation of the inflammasome leads to generation of IL-1β, a cytokine that was first cloned by Dinarello and colleagues in 1984,462 representing one of the key “endogenous pyrogens” first detected in 1953.463,464 In 2006, Tschopp showed that the NLRP3 inflammasome was activated by monosodium urate crystals, implicating this pathway in gout.465 From a historical perspective, gout is among the earliest diseases to be described. It was identified by the Egyptians in 2640 bce and was later recognized by Hippocrates in the fifth century bc, who referred to it as “the unwalkable disease.” Leeuwenhoek was the first to observe urate crystals from a tophus.466 Recently, an advisory panel of the U.S. Food and Drug Administration recommended against approval of canakinumab, a humanized mAb against IL-1, for the treatment of gout; although it was effective, it was not deemed safe due to increased risk of serious infections. In 1994, Matzinger467 proposed a new model of immune recognition. She proposed that APCs are activated by danger/alarm signals from injured cells (see Table 2.1). Although this was a purely theoretical model, since then, there have been numerous instances in which injured or

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damaged components of the host have been recognized to trigger inflammation. As Matzinger explains,468 Although this may seem to be just one more step down the pathway of slowly increasingly complex cellular interaction, this small step drops us off a cliff . . . in which the “foreignness” of a pathogen is not the important feature that triggers a response, and “selfness” is no guarantee of tolerance. Thus, release of uric acid crystals can be viewed as an example of a “danger signal.” Similarly, release of intracellular stores of adenosine triphosphate from dying cells as a trigger for signaling cellular injury, predicted in 1988,469 was shown to be capable of activating the NLRP3 inflammasome.470 Although Matzinger’s model shares features in common with Janeway’s, the emphasis on endogenous danger signals rather than direct engagement of pattern recognition receptors by pathogens is novel. The fact that pathogens and “endogenous danger signals” share common receptors and signaling pathways is consistent with the idea that either exogenous or endogenous triggers of innate immune receptors accomplish the same thing: to facilitate the primary immune response.

Cytosolic Components Maintain Safety: The Role of Autophagy There are numerous examples of microbes that are ingested by phagocytosis but either remain viable within acidic organelles or escape into the host cytoplasm. Macroautophagy is an evolutionarily conserved process in which cytoplasmic components are sequestered by a double membrane sac, eventually acquiring endosomal and lysosomal characteristics.471 In 2004, Deretic and colleagues showed that IFNγ induced autophagy in macrophages, which contributed to suppression of intracellular survival of mycobacteria.472 In the same year, Nakagawa et al.473 demonstrated that autophagy was necessary for killing of group A Streptococcus, which had escaped into the host cytoplasm. Since then, many publications have confirmed the importance of autophagy in both innate and acquired immunity.471

but neither alone, were needed for an antibody response, and Mosier475 concluded that antibody production required “antigen phagocytosis by macrophages and macrophage lymphocyte interactions,” although he did not know the nature of the interactions. Further experiments established that exceedingly few adherent cells were needed for antibody production, perhaps as few as 1 in 104 adherent cells.476 In the mid-1970s, Zanvil Cohn and a postdoctorate in his laboratory at Rockefeller University, Ralph Steinman, were characterizing a novel adherent population of cells from spleen.477–479 The cells had an unusual “tree-like” morphology, prompting the name “dendritic cell.”477 These cells proved difficult to purify, necessitating Steinman and Cohn to develop a rather laborious density gradient technique for cell purification.480 In 1978, they used this technique to show that DCs could stimulate a primary mixed leukocyte reaction—an index of lymphocyte proliferation—and showed that DCs are at least 100 times more effective than B cells and macrophages for this function.58 This landmark paper was the first demonstration of the unique capability of DCs to efficiently present antigen. Since then, many DC subsets have been described with critical functions in shaping the immune response. For example, Banchereau and colleagues showed that one subtype of DC, a “plasmacytoid” DC, is the principal IFNα-secreting cell in systemic lupus erythematosus with the capacity to induce plasma cell differentiation.481,482 Other DC subsets can respond to cues from epithelial cells, which secrete the cytokine TSLP, and shape T-cell polarization toward a TH2 phenotype.483,484 This may be a critical pathway in promoting allergic inflammation in asthma.485 Since the initial discovery of DCs in 1973, Steinman and his many colleagues and collaborators have uncovered novel functions of DCs in triggering and shaping immunity as well as inducing tolerance. Many laboratories worldwide are using DCs in both capacities, and Steinman himself was the recipient of a DC-based vaccine against his own pancreatic cancer. Steinman was awarded the Nobel Prize, posthumously, in 2012—tragically, just days after he succumbed to cancer. The Nobel was a fitting tribute to a consummate cellular immunologist whose scientific curiosity and pedigree could be traced to Metchnikoff.

The Renaissance of Cellular Immunology: Discovery of Dendritic Cells

ACKNOWLEDGMENTS

Although we tend to take for granted the concept that APCs are necessary to process antigen, this was not firmly established until 1967. Mosier used a method pioneered by Mishell and Dutton for measuring in vitro antibody production with sheep erythrocytes as antigen.474 Mosier separated mouse spleen cells into an adherent fraction and a nonadherent fraction. The adherent cells, which were phagocytic, were deemed “macrophage rich,” and the nonadherent cells were deemed “lymphocyte rich.” Both populations together,

I would like to acknowledge my appreciation of my former mentor and good friend, Sam Silverstein, my former teachers, Ben Pernis and Len Chess, and to my colleagues who helped make teaching a great source of joy to me: Ned Braunstein, Vinnie Butler, Mitch Cairo, Kathryn Calame, Steve Canfield, Raph Clynes, Kathy Nickerson, Paul Rothman, Ale Pernis, Chris Schindler, and Bob Winchester. I am also indebted to Arthur Silverstein, whom I have come to know only from his scholarship, but in whom I feel a kindred spirit.

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Lymphoid Tissues and Organs Eitan M. Akirav • Noelia Alonso-Gonzalez • Lucy A. Truman • Nancy H. Ruddle

INTRODUCTION The mammalian immune system defends against invading pathogens, by both the innate and adaptive mechanisms. Although cells that can respond to pathogens are scattered in tissues throughout the body, the optimal structures for the response to antigens are organized, compartmentalized cellular aggregates that facilitate antigen concentration and presentation to a large repertoire of antigen-specific lymphocytes. The primary lymphoid organs, the fetal liver, thymus, and bone marrow, are the sites where diverse populations of naïve lymphocytes mature to disperse throughout the body to await foreign invaders. This remarkable differentiation process occurs in a foreign antigen-independent fashion. The secondary lymphoid organs, including the lymph nodes, spleen, Peyer’s patches, and other mucosal-associated lymphoid tissues (MALTs), are discrete sites in which naïve, antigen-specific Tand B-lymphocytes encounter invaders to generate an adaptive response. The lymph nodes and spleen have been considered to be somewhat static structures, while, in fact, they are responsive to environmental influences and undergo remarkable changes in the course of antigenic challenge. Precise programs control the development of the spleen, lymph nodes, Peyer’s patches, tonsils and adenoids, and (in the mouse and rat) the nasal-associated lymphoid tissue (NALT). Somewhat less anatomically restricted tissues that are even more sensitive to the environment facilitate and include accumulations of lymphoid cells are organized, but less discretely defined: the bronchus-associated lymphoid tissues (BALTs) and inducible lymphoid follicles (ILFs). Tertiary lymphoid organs, or more accurately, tertiary lymphoid tissues, are accumulations of lymphoid cells that arise ectopically in sites that are not anatomically restricted and are not regulated by developmental programs. Tertiary lymphoid tissues respond to environmental stimuli and arise during chronic inflammation subsequent to microbial infection, graft rejection, autoimmunity, or cancer by the process of lymphoid neogenesis.1,2

In this chapter, the structure, function, trafficking patterns, and developmental signals that regulate the hierarchy of lymphoid organs will be described.

PRIMARY LYMPHOID ORGANS The primary lymphoid organs are the sites where pre-B- and pre-T-lymphocytes mature into naïve B and T cells in the absence of foreign antigen. Each T cell or B cell expresses a unique receptor that can recognize and respond to exogenous antigen and, in most cases, discriminate between selfand foreign antigens. Naïve cells leave the primary lymphoid organs having received and responded to developmental cues that result in the rearrangement of their genetic material to generate a repertoire capable of recognizing and responding to a wide variety of foreign antigens. In the course of maturation in the primary lymphoid organs, the naïve lymphocytes express various chemokine receptors and adhesion molecules that direct them to secondary lymphoid organs.

Fetal Liver The earliest lymphoid cell precursors derive from self-renewing hematopoietic precursors called hematopoietic stem cells (HSCs). During ontogeny, these cells occupy several niches. In the fetal mouse, the first wave of hematopoiesis occurs in the yolk sac and aorta-gonad-mesonephros region at E10.5.3 The placenta also contains HSC activity.4–6 Cells leave these tissues and migrate to the fetal liver, and then the bone marrow and thymus and spleen under the influence of chemokines and adhesion molecules (Fig. 3.1). The cells in the fetal liver respond to CXCL12 (stromal cell derived factor) and, in contrast to those in the bone marrow, also respond to Steel factor7 (Table 3.1). The fetal liver is also the source of CD4 + CD3 − lymphoid tissue inducer cells that express lymphotoxin (LT) α (also called tumor necrosis factor [TNF] β) and LTβ. The requirement of inducer cells for the

47

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FIG. 3.1. Embryonic and Postnatal Development of T and B Cells in Mouse Primary and Secondary Lymphoid Organs. Embryonic diagram based on information summarized in Medvinsky et al.6

development of secondary lymphoid organs is described in the following. Differentiation of HSCs to B cells occurs in the fetal liver, but does not require interleukin (IL)-7. B-cell development is described in detail elsewhere in this volume. Of the several different subsets of B cells, those generated by the fetal liver HSCs are somewhat limited and are of the B-1 subset.8 They do not give rise to cluster of differentiation

TABLE

3.1

Chemokines Implicated in Lymphoid Organ Development and Maintenance

Standard Name

Common Names

Receptor

CXCL12 CXCL13 CCL17 CCL19 CCL20 CCL21 CCL25 CCL28

SDF-1 BCA-1, BLC TARC ELC, MIP-3β MIP-3α SLC, 6Ckine TECK MEC

CXCR4 CXCR5 CCR4 CCR7 CCR6 CCR7 CCR9 CCR10

BCA, B-cell chemoattractant; BLC, B lymphocyte chemoattractant; ELC, EBI-1 ligand chemokine; MEC, mucosae as-associated epithelial chemokine; MIP, macrophage inflammatory protein; SDF-1, stromal cell derived factor; SLC, secondary lymphoid tissue chemokine; TARC, thymus and activation regulatory chemokine; TECK, thymus-expressed chemokine.

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(CD)5 B cells, predominately B1-B cells, and do not express terminal deoxynucleotidyl transferase and myosin-like light chain.8,9 It is not known whether these differences are intrinsic to the cells or are due to differences in the cytokine environment of fetal liver and bone marrow. The ligands for P-selectin, E-selectin, and vascular cell adhesion molecule (VCAM)-1 are required for the cells to leave the fetal liver and home to the bone marrow.10,11

Bone Marrow Functions The bone marrow is the source of self-renewing populations of stem cells. These cells include hematopoietic precurscor cells, HSCs, and endothelial progenitor cells, which may derive from a single precursor.12 The adult bone marrow contains stem cells that can differentiate into adipocytes, chondrocytes, osteocytes, and myoblasts.13 Collectively, these cells are called hematopoietic/stem progenitor cells. In the adult, cells leave the leave the bone marrow and seed the thymus where they undergo differentiation to naïve T cells. Additional factors enable the differentiation of immature B cells from HSCs. In addition to serving as a primary lymphoid organ where B-cell differentiation and development occur, the bone marrow is also a home for antibody secreting cells.14 After B cells

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have interacted with antigen in the secondary lymphoid organs, such as the lymph nodes, spleen, and Peyer’s patches, they enter the bloodstream and travel to the marrow. Thus, this organ not only serves as a primary lymphoid organ, but also as a reservoir for fully differentiated plasma cells.

Architecture: Cellular and Functional Niches The microenvironment of the bone marrow, contained in the central cavity of bone, is a complex three-dimensional structure, with cellular niches that influence B cells during their development and later, as plasma cells, as they return to the bone marrow. The bone marrow has a rich blood supply with a nutrient artery that branches into ascending and descending arteries further dividing into cortical capillaries, periosteal capillaries, and endosteal capillaries, finally merging into a sinus.15 Previously, the prevailing understanding of B-cell differentiation in the bone marrow was that primitive HSCs were located in close contact with the endosteum near osteoblasts (the “endosteal niche”). During the course of differentiation into mature B cells, they moved into the central region of the bone marrow cavity (the “vascular niche”).16 The former niche was identified as the location of HSC; the latter, as the site of B-cell differentiation.17 This anatomic concept has been challenged, as it has been reported that HSCs are found throughout the bone marrow. More recently, a reticular niche has been described that includes CXCL12 abundant reticular cells.18 Growth factors and cytokines produced by different stromal cells influence cells at different stages in their differentiation. Thus, it is more appropriate to consider functional or cellular, rather than anatomical, niches.15,19 Once the HSCs differentiate into immature B cells expressing cell surface immunoglobulin (Ig) M, they undergo processes of negative selection and receptor editing, leave the bone marrow, and travel through the blood stream to the secondary lymphoid organs where they complete their differentiation. Several cytokines and chemokines influence B-cell differentiation in the bone marrow. Flt-3 ligand (also called Flk-2L) signals B-cell differentiation and growth and synergizes with several other hematopoietic growth factors.20 Its receptor, Flt-3 (also known as Flk-2), expressed by primitive HSCs, is a member of the class II tyrosine kinase family. In contrast to fetal liver, HSCs from adult bone marrow do not respond to Steel factor. Chemokines contribute to B-cell differentiation in the bone marrow and define the functional niches. For excellent reviews of this topic, see Nagasawa,15 Heissig et al.,18 and Mazo et al.21 Many of these factors affect other lymphoid cells, such as dendritic cells (DCs) and T cells. Several of these factors, whose functions have been identified in gene deletion studies in mice, in morphologic analysis, and in cell culture studies, play roles in multiple aspects of lymphoid organ development; their activities, though important in the bone marrow, are not limited to that organ. CXCL12, also known as stromal cell derived factor, is a chemokine that is crucial for recruitment of HSCs to the bone marrow. It is widely expressed by osteoblasts, reticular cells,19 and endothelial cells.22 In fact, the interaction of HSCs expressing CXCR4, the receptor for CXCL12, with that chemokine on the endothelial surface is the first step in the

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HSC’s exit from the circulation into the marrow.23 CXCL12 is also essential for the earliest stage of B-cell development (pre-pro-B cells). Its receptor, CXCR4, is expressed on early B cells and is downregulated in pre-B cells. CXCR4 remains at low levels in immature B cells and mature B cells in secondary lymphoid organs, but is upregulated after B cells interact with antigen and differentiate into plasma cells.14 This explains the propensity of antibody-secreting cells to return to the bone marrow. Once a pre-pro-B cell has interacted with CXCL12, it moves on to a different cell expressing IL-7. In B-cell development, IL-7 acts later than CXCL12 in a narrow window between pro-B cells and immature B cells in the scheme proposed by Nagasawa.15

Traffic In and Out: Chemokines and Adhesion Molecules The extensive vascularization of the bone marrow allows entrance of hematopoietic precursors and plasma cells and egress of mature cells. Hematopoietic progenitor recruitment to the bone marrow in the mouse is dependent on the interaction of a variety of chemokines, integrins, and selectins, and their receptors, counter receptors, and vascular cell adhesion molecules. These include α4 integrin (VLA4 or α4β1), VCAM-1,24 P-selectin glycoprotein ligand-1, E-selectin,25 α4β7, and mucosal addressin cell adhesion molecule (MAdCAM-1).26 A small subpopulation of newly formed B cells in the bone marrow that expresses L-selectin has been described,27 suggesting a mechanism for entrance into lymph nodes (see subsequent discussion). In addition to the acquisition of Ig expression that occurs in the bone marrow under the control of stromal cells, B cells express various chemokine receptors and adhesion molecules in the process of differentiation. In a study of human bone marrow B cells, it was determined that pro-B and pre-B cells migrate toward CXCL12, but not toward a wide range of other chemokines, including CCL19 and CCL21, though they do express low levels of CCR7, the receptor for those ligands. However, mature bone marrow B cells do respond to CCL 19 and CCL 21,28 CXCL13 (indicating a functional CXCR5), and CCL20 (MIP3α). On the other hand, CCR6 expression and responsiveness to CCL20 are only seen in mouse B cells after they have migrated into the periphery during the process of maturation and are in the circulating B cell pool.29

Thymus Functions The thymus is defined as a primary lymphoid organ due to its inimitable role in T-cell development. Indeed, a mutant known as the “nude” (nu/nu) mouse, which lacks a normal thymus, is completely devoid of mature T cells.30 Although some studies suggest that T cells can develop extrathymically in organs such as the “cervical thymus”31 and the gut epithelium,32 the thymus remains the main site for T-cell maturation, education, and selection. T-cell precursors represent more than 95% of total cells in the thymus and give rise to mature T cells. These cells are crucial components of the adaptive immune system in that they are highly specific in their ability to recognize a nearly infi nite number of antigens owing to their diverse repertoire.30,33

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A

B FIG. 3.2. A: Thymic structure. Low power magnification of a hematoxylin-stained frozen section of a mouse thymus. The cortex is well populated with lymphocytes; the medulla is less tightly packed and thus stains less intensely. B: Diagram of mouse thymic cellular populations. Precursor T cells enter through blood vessels at the corticomedullary junction. They progress to the medulla where they undergo differentiation from double negative to double positive (DP) cells expressing T-cell receptors. Thymic stromal cells provide growth factors. DP cells undergo positive selection, under the influence of cortical thymic epithelial cells. Single positive (SP) cluster of differentiation (CD)4 or CD8 cells migrate into the medulla where they undergo negative selection, mainly through autoimmune regulator expressing medullary thymic epithelial cells and dendritic cells. SP cells, having undergone differentiation, exit through blood vessels (not shown) to the periphery.

The T-cell repertoire is shaped during development in the thymus by the processes of positive and negative selection. Negative and positive selection are so stringent that nearly 95% of all T-cell precursors are deleted in the thymus.34 Negative selection ensures that T cells, which are capable of recognizing the organism’s self-antigens presented on major histocompatibility complex (MHC) class I or II molecules with high affinity, are eliminated prior to their export to the periphery. In contrast, positive selection, which requires a higher degree of T-cell receptor (TCR) avidity than negative selection,35 allows for T cells recognizing self-antigen with low to medium affinity to leave the thymus and eventually protect the organism against invading pathogens. While negative selection is highly efficient in eliminating the majority of self-reactive T cells, some of these cells do escape the thymus and exit to the periphery. These autoreactive cells impose a threat to various organs as their activation may result in the development of autoimmune diseases. The immune system has evolved several ways to prevent the activation of autoreactive T cells in the periphery. One mechanism that protects against autoimmunity is the generation of a T-cell population capable of suppressing activation of self-reactive T cells. These thymus-derived protective T cells, known as regulatory cells (Treg), recognize self-antigens with relatively high affinity.

Architecture The thymus in the mouse consists of two symmetric lobes located above the heart, while in humans the thymus is

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multilobed. Each lobe can be divided into three distinct regions: capsule, cortex, and medulla. The latter two regions harbor thymocytes at various maturation stages. Although maturing T cells constitute the majority of cells in the thymus, other cell types such as macrophages, DCs, B cells, and epithelial cells are also present.36 Histologic analysis of the thymus reveals a clear distinction between the thymic cortex and medulla, which are separated by a corticomedullary border. The thymic cortex appears darker and more densely populated with T-cell precursors, whereas, the medulla appears considerably lighter and contains smaller numbers of T cells relative to other cell types (Fig. 3.2A). Blood vessels and small blood capillaries are found throughout the thymus. The fact that T-cell progenitors are found in the more highly vascularized corticomedullary border suggests that blood vessels in this region facilitate the entry of T-cell progenitors into the thymic parenchyma.37 In the thymic medulla, the close association between medullary thymic epithelial cells and thymic blood vessels38 suggests that these vessels may act as organizers of the medullary thymic compartment. Lymphatic vessel distribution coincides with that of blood vessels and capillaries.39 The majority of lymphatic vessels are located in the thymic medulla, though some can also be found in the cortex. The role of lymphatic vessels in thymic function is unclear, although it has been proposed that these vessels deliver extrathymic antigens into the thymus or export mature T cells from the thymus into the circulation.

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Cellular Composition and Functions T-cell precursors at various stages of differentiation represent the majority of cells in the thymus. Antigen-presenting cells (APCs) of either hematopoietic or stromal origin mediate the education and selection of T cells in the thymus. The different stages in T-cell selection and maturation that take place in distinct regions of the thymus (Fig. 3.2B) are discussed at length elsewhere in this volume. Briefly, lymphoid progenitors enter the thymus at the corticomedullary border.37 Following their entry, these cells, identified as double negative (DN) T cells due to a lack of expression of the cell surface molecules CD4 and CD8, undergo four maturation steps termed DN1 to DN4, which are distinguished by the expression of two additional cell surface molecules: CD25 and CD44.40,41 DN3 cells migrate to the subcapsular zone while rearranging their TCRβ chain and expressing it in combination with a surrogate α chain. Those cells that have successfully rearranged the genes for α and β chains of the TCR become double positive (DP) cells, and express both CD4 and CD8 surface markers. In the cortex, DP cells undergo negative and positive selection.42,43 Positively selected DP cells further differentiate into single positive (SP) cells expressing either CD4 or CD8. Following their differentiation, SP cells relocate to the medulla where they mature and undergo further rounds of deletion. SP cells that do survive are then exported out of the thymus.44 DN precursors can give rise to an additional T-cell population expressing the γδTCR. These T cells are distinct from TCRαβ T cells in their tissue distribution and recognition of antigens. TCRαβ DP cells control the development of TCRγδ cells via the production of LTβ.45 Treg development in the human thymus occurs at the DP stage allowing for the production of CD4 and CD8 positive Tregs.46 The thymic parenchyma consists of a complex threedimensional structure supported by thymic epithelial cells (TECs). TECs in the thymic cortex and thymic medulla are phenotypically and functionally distinct and support different stages of T-cell maturation. Several cell surface markers are used to distinguish medullary TECs (mTECs) from cortical TECs (cTECs) in the mouse. Among these markers are the cytokeratins K5 and K8, the adhesion molecule Ep-CAM, and the glycoprotein Ly-51. K8 and Ly-51 are expressed by cTECs, while K5 and Ep-CAM are expressed by mTECs.47,48 TECs are unique in that they express MHC II constitutively, similarly to professional APCs. The role of TECs in T-cell selection was recently elucidated by their expression of the transcription factor, autoimmune regulator (AIRE). This transcription factor plays an important role in T-cell selection and prevention of autoimmunity, as illustrated by the fact that humans with a mutated form of the AIRE gene exhibit polyendocrine autoimmunity due to inadequate T-cell selection.49,50 It has become clear that AIRE controls the expression of certain tissue-restricted antigens in the thymus, such as insulin, a protein that is unique to the β cells of the islets of Langerhans in the pancreas.48,51 The expression of tissue-restricted antigens in the thymus facilitates the negative selection of maturing T cells, which would otherwise be allowed to leave the thymus. The thymic medulla includes distinct structures also known as Hassall

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corpuscles. Originally described by Arthur Hill Hassall in 1849, this structure consists of concentric stratified keratinizing epithelium. Hassall corpuscles are implicated in several processes of the thymus,52 including the expression of tissue-restricted antigens, such as insulin (a key antigen in type 1 diabetes),53 Igs, and fi laggrin (key antigens in rheumatoid arthritis),54,55 and serving as a prominent site for T-cell apoptosis.56 DCs are professional APCs that are found in the thymus. Thymic DCs can be divided into two distinct populations. The first population originates from a thymocyte precursor,57,58 whereas the second population is derived from partially mature peripheral DCs that continuously enter the thymus from the circulation.59 DCs do not appear to be involved in positive selection, but do contribute to negative selection.60–63 They have also been implicated in the selection of Tregs in humans,64 and recent reports in the mouse suggest that peripheral DCs can migrate to the thymus and serve as efficient inducers of Tregs.65 Activation of thymic DCs can be mediated in part by the IL-7–like cytokine, thymic stromal lymphopoietin. In the human thymus, Hassall corpuscles produce thymic stromal lymphopoietin, and thymic stromal lymphopoietin–activated DCs can alter the fate of self-reactive T cells from deletion to positive selection of Tregs.66 More specifically, thymic stromal lymphopoietin expression in a subset of DCs, plasmacytoid DCs, can induce the generation of particularly potent Tregs from CD4 + CD8 − CD25 − thymocytes.67 These findings highlight the heterogeneity of DCs and emphasize their ability to fulfi ll different roles during T-cell development in the thymus. Macrophages and B cells are additional hematopoieticderived professional APCs in the thymus. In contrast to DCs, thymic macrophages are located throughout the thymus and do not play a significant role in T-cell selection.68 B cells are detected in human and mouse thymus at relatively low numbers69,70 and are characterized by the expression of the cell surface molecule CD5. They are capable of producing antibodies of several different isotypes.71 It has been suggested that thymic B cells induce negative selection in developing thymocytes,69 although B cell–deficient mice show a limited T-cell repertoire when compared with normal mice, also suggesting a role in positive selection.72 Recent data consistent with a role for B cells in thymic negative selection have emerged. Mice whose B cells were experimentally manipulated to express a myelin oligodendrocyte glycoprotein peptide show deletion of myelin oligodendrocyte glycoprotein–specific T effector cells.73 One mechanism for the effect of B cells on T-cell selection is suggested by the observation that B cells play a role in the control of tissue-restricted antigens. Studies done in mice deficient in B cells showed fewer thymic epithelial cells with reduced expression of insulin and myelin oligodendrocyte glycoprotein, native antigens of the pancreas and brain, respectively.74

Traffic In and Out: Adhesion Molecules and Chemokines Adhesion molecules mediate the extravasation of leukocytes from blood and lymphatic vessels into the thymus. These molecules also play an important role in facilitating lymphocyte homing into various regions of the thymus. Indeed,

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thymic blood vessels express the adhesion molecules intercellular adhesion molecule (ICAM)-1, VCAM-1, CD34, peripheral node addressin (PNAd) and vascular adhesion protein-1.75 The expression of high levels of ICAM-1, VCAM-1, and vascular adhesion protein-1 on venules near the corticomedullary border suggests that these molecules may play a role in the recruitment of thymocyte progenitors. More specifically, vascular adhesion protein-1, which is restricted to the venules surrounding the sites of progenitor homing, can mediate the extravasation of leukocytes. The regional distribution of these adhesion molecules further illustrates the importance of a distinct anatomical separation between cortex and medulla and represents their individual functions. Chemokine-mediated T-cell migration and traffic to and within the thymus is crucial for normal T-cell selection. The compartmentalization of the thymus is orchestrated by a milieu of chemokines. The thymic cortex is involved in the maturation of DN cells to DP cells and by positively selecting DP T cells capable of recognizing MHC:peptide complexes and eliminating the cells that are not. The thymic medulla acts as a site of negative selection of SP T cells based on TCR recognition avidity to self-antigens and possibly positive selection of Treg. Various chemokines that are produced by the thymic cortex and medulla allow T cells expressing different chemokine receptors to home to specific regions of the thymus. This differential expression of chemokines is complemented by the fact that T cells at different maturation states express different chemokine receptors. During development, the entry of lymphoid progenitors into the thymus is highly dependent on CCL21 and CCL25, which bind the chemokine receptors CCR7 and CCR9, respectively. Mice lacking CCR7 or CCL2176,77 show a transient delay in thymus colonization by lymphocytes (day 14.5), and this delay is further extended (day 17.5) in mice lacking CCR9.78 Lymphoid progenitors enter the thymus at the corticomedullary border and commence their migration outwards toward the subcapsular region of the cortex as DN3 cells. The expression of the chemokine receptors CXCR4 and CCR7 by DN cells is important in directing cell migration.79,80 In the subcapsular region, DN thymocytes that have successfully rearranged their TCRαβ chains progress to the DP cell stage. Positively selected DP cells move inwards toward the thymic medulla for further differentiation into SP cells. The ligands for CCR7 are crucial in mediating the migration of positively selected DP cells into the medulla as illustrated by the fact that a deficiency in CCR7 or its ligands, CCL19 or CCL21, prevents DP cell relocation from the cortex to the medulla resulting in abnormal central tolerance.81,82 This abnormal tolerance is associated with a reduction in thymic B-cell numbers and reduced expression of tissue-restricted antigens.74 Interestingly, antigen itself can control T-cell migration speed from the cortex to the medulla. In the presence of a negative selection ligand, T cells slow down considerably and are limited to a confined zone 30 μ in diameter allowing for a more prolonged selection and induction of developmental arrest.83 The export of positively selected SP T cells out of the thymus is also dependent on chemokines. Chemokines involved in T-cell emigration are CXCL12 and its receptor CXCR4,

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which repel SP cells out of thymus,84 and CCL19, which promotes T-cell emigration from the thymus of newborn mice.85 The chemoattractant, sphingosine 1-phosphate (S1P), is an additional mediator of T-cell emigration. SP T cell express a S1P receptor (S1P1) and are attracted to the high levels of S1P present in the serum promoting their egress.86,87 While the role of different thymic compartments and chemokines in the maturation of naïve SP T cells has been extensively studied, the thymic regions and chemokines that control the selection of regulatory T cells remain largely unknown. It may be that chemokines produced by both cTECs and thymic DCs play a role in regulatory T-cell selection, albeit during different stages of maturation.

Development The initial development of the thymus at midgestation in the mouse is independent of vascularization or bone marrow– derived cells. In the mouse, the thymus rudiment is first evident on day 11 of gestation as it evolves from the endoderm of the third pharyngeal pouch.88 This gives rise to the thymic lobes as well as to the parathyroid gland. On day 12.5 of gestation, a separation of the primordium is observed and by day 13.5 a distinct thymus is apparent. Evidence has suggested that not only the pharyngeal pouch endoderm but also the ectoderm may also be involved in thymic development.89,90 More specifically, it was suggested that the pharyngeal pouch ectoderm contributes to the development of cTECs whereas the endoderm contributes solely to the development of mTECs. This “dual origin” model was mainly supported by histological data as well as data collected from thymi of nu/nu mice.91–93 More recently, it was shown that both thymic cortex and medulla are derived solely from the pharyngeal pouch endoderm94 ; although the endoderm and ectoderm are found in close proximity between gestational day 10.5 and 11, only the endoderm actively contributes to thymic development.95 These findings support the so-called single origin model of thymus development. The contribution of lymphocytes to the normal development of thymic cortex and medulla is well recognized. In models of T-cell deficiency, cTEC and mTEC development is halted at different stages depending on the stage of T-cell arrest. If T-cell development is arrested at the DN stage, as in the recombination activating gene–deficient mouse, the thymic medulla is greatly reduced while the thymic cortex remains unaffected.96,97 A more severe phenotype is observed in transgenic mice that overexpress the human CD3 signaling molecule. In these mice, T-cell arrest occurs earlier than in recombination activating gene knockout mice, leading to a loss of both cortex and medulla and to a shift from a three-dimensional to a two-dimensional structure of the thymic epithelia.97 Recently, LTα and LTβ have been identified as master regulators of mTEC development and expression of tissue-restricted antigens. LTα and LTβ are members of the TNF superfamily and mediate the processes of secondary lymphoid organ development and inflammation.1,98 In the absence of LT, tissue-restricted antigen and AIRE expression are reduced and certain mTEC subpopulations fail to develop.99,100 T cells, owing to their vast numbers, appear

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to be the main source for LT production in the thymus45 ; however, additional resident cells also serve as a source of LT. Recently, resident thymic B cells were identified as the highest source of LTα and LTβ on a per cell basis,74 revealing yet another aspect of thymic B cells in thymus development and function. The development of the thymus in humans closely follows the model of thymic development in the mouse and bird. Similar to the mouse, human thymic colonization by hematopoietic stem cells occurs relatively early, at week 8.2 of gestation. During this stage, the thymic medulla and cortex are organized, suggesting that thymocytes are required for normal thymic development. Between gestation week 9.5 and 10, the first signs of thymocyte negative selection are evident, and by gestation week 10 to 12.75 the gradual onset of positive selection is detected.101

SECONDARY LYMPHOID ORGANS Naïve cells express their receptors for specific antigen, leave primary lymphoid organs, circulate through the bloodstream, migrate into the tissues, and lodge in secondary lymphoid organs. The frequency of naïve cells specific for an individual antigen is quite low (estimates range from 1 in 105 to 1 in 106). Thus, the chance that an individual T or B cell will encounter its specific antigen in the circulation is rather low. Secondary lymphoid organs are strategically located in anatomically distinct sites where foreign antigen and APCs efficiently concentrate and activate rare antigen-specific lymphocytes, thus leading to the initiation of adaptive immune responses and generation of long-lived protective immunity. These organs include highly organized, compartmentalized, and mostly encapsulated tissues such as lymph nodes, spleen, appendix, tonsils, murine NALTs, and Peyer’s patches. Naïve cells are also primed in less discrete tissues throughout the body, including the BALTs, cryptopatches, and ILF.

Lymph Nodes Lymph nodes are bean-shaped structures dispersed along lymphatic vessels. The lymphatic vessel system plays important roles in tissue fluid balance, fat transport, and the immune response. In contradistinction to blood vessels, which form a closed recirculating system, lymphatic vessels comprise a blind-end, unidirectional transportation system. The absorbing lymphatic vessels, or lymphatic capillaries, remove interstitial fluid and macromolecules from extracellular spaces and transport the collected lymph through the primary collector. The collected lymph and its cellular contents are transported into the thoracic duct and returned back to blood circulation. In humans, lymph collected from the entire lower body region, the left head, and left arm region accumulates in the thoracic duct and returns to blood circulation via the left subclavian vein; lymph collected from right head and right arm region returns to blood via the right subclavian vein. Lymph nodes, usually embedded in fat, are located at vascular junctions, and are served by lymphatic vessels that bring in antigen, and connect them to other lymph

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nodes. Though most lymph nodes are classified as peripheral lymph nodes, a few (cervical, mesenteric, and sacral), termed mucosal nodes, express a slightly different complement of endothelial adhesion molecules, cooperate with the mucosal system, and are regulated somewhat differently in development (see following discussion). Although all lymph nodes are vascularized, and thus can receive antigens from the bloodstream, they are also served by a rich lymphatic vessel system and are thus particularly effective in mounting responses to antigens that are present in tissues. These antigens may be derived from foreign invaders that are transported by APCs or can be derived from self-antigens. Thus, lymph nodes extend the role of the primary lymphoid organs and discriminate between dangerous foreign antigens and benign self-antigens. This capacity relies on the APCs and their state of activation in the lymph node, and the recognition capacity of the naïve T and B cells. Lymph nodes can also function as niches for generating peripheral tolerance, an additional mechanism to minimize the effects of those self-reactive T-cells that escape central tolerance in the thymus.102 DCs constitutively sample selfantigens and migrate to draining lymph nodes even in the steady state.103–105 Because most self-antigen–bearing DCs in lymph nodes are immature106 and have low levels of costimulatory molecules, they are not effective at activating naïve cells. They regulate self-reactive T cells by inducing anergy, clonal deletion, and/or expanding Tregs.105–109 Several groups have recently described populations of cells in secondary lymphoid organs that can present tissue-restricted antigens to CD8 cells.110–114 These include two different populations of AIRE-expressing stromal cells, DCs, and AIRE-negative lymphatic endothelial cells. These cells may play a role in self-tolerance, or perhaps they can prime for autoimmunity in inflammatory conditions.

Structure and Organization A collagen capsule surrounds the highly compartmentalized lymph node (Fig. 3.3). The cortical region includes discrete clusters called primary follicles consisting of densely packed naïve B cells and follicular DCs (FDCs). After B cells encounter their cognate antigen, they are activated. They then proliferate secondary follicles and germinal centers develop. T cells and DCs distribute in the paracortex. Macrophages reside in the subcapsular zone and in the medullary area, and those in the subcapsular zone appear to be particularly adept at presenting antigen-antibody complexes to B cells.115 Follicular DCs, a population of mesenchymal origin, support B-cell follicles or germinal centers under stimulation.1 Plasma cells are also concentrated in the medulla as they prepare to leave the lymph node and circulate to the bone marrow. A network composed of reticular fibers, fibrous extracellular matrix bundles, and another mesenchymal cell population, the fibroblastic reticular cells, supports the entire lymph node.116 Compartmentalization of cells in the lymph node is orchestrated by lymphoid chemokines CCL19, CCL21, and CXCL13. Stromal cells in the paracortical region produce CCL19 and the protein is transported to the surface of high endothelial venules (HEVs).117 CCL21 is encoded by several genes118 ; CCL21-leu is expressed by lymphatic vessels

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A

B FIG. 3.3. A: Lymph node structure and functional regions. The lymph node is divided into an outer cortex and inner medulla surrounded by a capsule and lymphatic sinus. The cortex includes B cells and follicular dendritic cells. The paracortical region includes T cells and dendritic cells. Macrophages are found in the subcapsular sinus and medullary cord. Lymphocytes enter into the lymph node through an artery at the hilus region, and into the parenchyma through high endothelial venules (HEVs) expressing peripheral node addressin and CCL19 and CCL21. Stromal cells also produce these chemokines. T cells and dendritic cells (DCs) are directed to the paracortical region by CCL19 and CCL21. B cells are directed to the cortex by CXCL13. After interaction with antigen and T cells, germinal centers develop. Antigen and DCs drain into the lymph node from the tissues through afferent lymphatic vessels. Antigen continues to percolate through the node via a conduit system. Activated cells leave through efferent lymphatic vessels. Diagram of the blood network of a rat lymph node (right) is adapted from Anderson and Anderson.125 Note the arteriovenous communications, the venous sphincters, and cells leaving the HEVs into the parenchyma. B: Lymph node compartmentalization. Immunofluorescent staining of B cells (anti-B220, green) in follicles in the cortex and T cells (anti-cluster of differentiation 3, red) in the paracortical area. The medulla is unstained.

outside the lymph node119 ; CCL21-ser is made by stromal cells and HEVs in the lymph node. CCL19 and CCL21 recruit CCR7-expressing cells across the HEVs to the paracortical region. CXCL13, produced by stromal cells in the B-cell follicles, attracts CXCR5 expressing B cells.120 After naïve T and B cells encounter antigen, they undergo extensive changes in expression of chemokine receptors and adhesion molecules that result in their movement to different areas of the lymph

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node or leaving it all together.121,122 S1P1 facilitates lymphocyte egress from lymph nodes as they move toward the ligand S1P in the lymph,123,124 as discussed in more detail subsequently.

Lymph Node Vasculature: Blood Vessels and Lymphatic Vessels Soluble antigen and APCs enter into lymph nodes via afferent lymphatic vessels. After surveying antigens in the lymph

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nodes, lymphocytes leave those organs via efferent lymphatic vessels that can connect to the next lymph node in the chain and finally return to blood circulation.125–127 In this manner, HEVs and lymphatic vessels maintain lymphocyte homeostasis during the steady state. Blood endothelial cells play a crucial role in lymphocyte trafficking in the lymph node. One or two arteries enter the lymph node at the hilus. These arteries branch and pass through the medulla area, enter the cortex, and sometimes continue in the subcapsular area. Beneath the subcapsular sinus, the branching capillaries form loops and some of them become arteriovenous communications. Arteriovenous communications become HEVs in the cortex area and occasionally extend from the subcapsular sinus to the medulla. HEVs constitute a specialized postcapillary network in the lymph node, playing a critical role in lymphocyte recirculation. Each main HEV trunk receives three to five branches lined with high endothelial cells and two or three branches lined with flat endothelial cells. The luminal diameters of HEVs progressively increase from cortex to medulla. Finally, HEVs merge into segmental veins in the medulla area and join larger veins in the hilus125 (see Fig. 3.3A). Intravital microscopy has revealed that the entire venular tree consists of five branching orders with the higher orders in the paracortex and the lower orders located in the medulla and hilus areas. Only the higher order venules, located in the T-cell area, are specialized into HEVs and are recognized by the monoclonal antibody MECA-79.126,127 Recirculating naive lymphocytes leave the bloodstream via HEVs, specialized vessels with a high cuboidal endothelium, and migrate into the lymph node parenchyma. PNAd, defined by the MECA-79 antigen, is an L-selectin ligand, and is a characteristic HEV adhesion molecule. PNAd is composed of a variety of core glycoproteins, including GlyCAM-1, CD34, Sgp200, and podocalyxin; these proteins must be sialylated, sulfated, and fucosylated to become functional L-selectin ligands. The several enzymes that mediate these posttranslational modification events include FucT-IV, FucT-VII, and GlcNAc6ST2 (also called HEC-6ST, LSST, GST-3, HECGlcNAc6ST, gene name Chst4),128–132 which with the exception of a population of cells in the intestine133,134 is uniquely expressed in high endothelial cells. Together with another sulfotransferase, GlcNAC6ST1, that is expressed at sites in addition to HEVs, it sulfates glycoproteins in the Golgi apparatus135 to generate the MECA-79 epitope. PNAd, expressed on the endothelial surface, slows down (tethers) naïve L-selectinhi lymphocytes in their progress through the blood vessels. After this initial interaction, CCL19 and CCL21 on the HEVs are instrumental in activating the lymphocyte integrin lymphocyte function–associated antigen (LFA)-1. This results in tight binding of LFA-1 to ICAM-1 on the HEV, facilitating diapedesis of lymphocytes that migrate between or through the endothelial cells toward the chemokines located in the paracortical region (T cells, DCs) or cortex (B cells). Data suggest that the HEV-lymphocyte interaction is not random in different lymphoid tissues in the mouse.136,137 T-lymphocytes adhere preferentially to peripheral lymph node HEVs, B cells prefer to adhere to HEVs in Peyer’s patches, and T and B cells exhibit an intermediate pattern of

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adhesion to mesenteric lymph node HEVs.34,136,138 The selective adhesion of naïve lymphocytes to HEVs is at least partially controlled by the differentially expressed adhesion molecules in different lymphoid organs. PNAd rapidly replaces MAdCAM-1 after birth in mouse peripheral lymph nodes,139 but is expressed in mucosal lymph nodes together with MAdCAM-1, the ligand for the integrin α4β7. Lymphatic vessels also play critical roles in the immune response. The collected lymph and cell contents enter the lymph node via several afferent lymphatic vessels and fi lter through the node where they again are concentrated in the medullary sinus. Lymphatic vessels are concentrated in the subcapsular sinus and medullary area.125 Factors from afferent lymph can be either transported deep into the lymph node cortex or move via the subcapsular sinus and leave the lymph node through efferent lymphatic vessels.116 In this manner, soluble antigen and APCs from peripheral tissues are efficiently concentrated in the draining lymph node and initiate an adaptive immune response. Lymphocytes can also enter lymph nodes through afferent lymphatic vessels and leave via efferent vessels and move to the next lymph node in the chain. This is accomplished in part through a gradient of S1P, which is at a high concentration in the lymph and low concentration in the lymph node. Lymphocytes in the lymph node downregulate this receptor (S1P1) and then upregulate it as they prepare to leave and migrate toward the higher concentration in the efferent lymph.123,124 Human and murine lymphatic vessels express several characteristic markers: lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), Prox-1, podoplanin, CCL21, the vascular endothelial growth factor receptor-3 (VEGFR-3), and neuropilin-2.140

Conduit System A conduit system in the lymph nodes physically connects the lymphatic sinus with the walls of blood vessels and enables the incoming factor(s) from lymph to move rapidly deep into the paracortical area.116,125 The conduit system consists of four layers: 1) a core of type I and type III collagen bundles; 2) a microfibrillar zone composed largely of fibrillins; 3) a basement membrane abundant with laminins 8 and 10, perlecan, and type IV collagen that provides a supportive structure; and 4) fibroblastic reticular cells that embrace the entire conduit system.141,142 This conduit system enables incoming lymph to penetrate deep into the T-cell area. A special subset of immature DCs, called conduit-associated DCs, can take up and process antigens moving along the conduit.142 In this manner, the conduit system probably provides a physical support for rapid initiation of adaptive immune responses after immunization. The Intimate Relationship Between Lymph and High Endothelial Venules Incoming lymph is necessary for the maintenance of HEV phenotype and function. After afferent lymphatic vessels are severed, dramatic changes occur in HEVs. These include flattening of the endothelium, a decrease in the uptake of 35 S-sulphate143,144 (a functional marker of GlcNAC6ST-2), a reduction of lymphocyte adherence to the vessels,145–147

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and decreased expression of PNAd and the HEV genes, Glycam-1 and Fuc-TVII.146–148 An increase in MAdCAM-1 expression146 suggests that these events are not simply due to a general downregulation of blood vessel gene expression and indicates that continual accumulation of afferent lymph factor(s) in lymph nodes is necessary for HEV maintenance. The nature of such lymph factor(s) is unknown. Topographic relations between HEVs and the lymphatic sinus have been described in the rat mesenteric lymph nodes. Some HEVs are located in the medulla area and positioned closely in relation to the lymphatic sinus.125 Occasionally, HEVs are separated from an adjacent lymphatic sinus by only a thin layer of collagen bundles. The closely apposed HEV and lymphatic sinus in the medulla provide the physical support for the intimate relationship between lymph and HEV. However, most HEVs are located in the paracortical area and are separated from the lymphatic sinus by lymphocytes. The conduit system physically connects the subcapsular sinus and HEVs and allows incoming lymph factor(s) to migrate rapidly to the wall of HEVs.125,149,150 Low-molecularweight fluorescent tracers (below 70 kD) move rapidly via the conduit system and lead directly to the wall of HEVs. In this manner, low-molecular-weight tracers can migrate with lymph within minutes to HEVs and enter the HEV lumen.116 Lymph-borne chemokines likely adopt this route to regulate HEV function. IL-8 administration via afferent lymph increases lymphocyte HEV transmigration within minutes.125,151 In addition, lymph-borne chemokines, such as MIP-1α , also enter the conduit system and move rapidly to HEVs.116 These data suggest that lymph factor(s) can quickly access and regulate HEVs.

Development Despite their distinctions in the adult with regard to morphology and expressed genes, a close association of blood vessels and lymphatic vessels is seen during embryogenesis. The generation of embryonic lymphatic vessels from preexisting veins in pig embryos was first described in the early 1900s by Sabin and has recently been molecularly defined.140 A variety of transcription factors have been identified that contribute to the lymphatic specification and maintenance of the lymphatic vessels phenotype.152 As early as mouse E9, expression of Sox18 and CoupTFII is apparent in the cardinal vein. These induce Prox1 in the dorsolateral side of the vein resulting in polarization and lymphatic-biased endothelial cells. Prox1 generates a feedback signal for the further maintenance of budding and migration of endothelial cells. Targets of Prox1 include Prox1 itself, podoplanin, VEGFR3, integrin-α , and Nrp-2. Prox1 expression in blood endothelial cells also represses expression of markers of those cells. NfatC1, Foxc2, and Tbx1 are additional transcription factors that are expressed later in lymphatic vessel development and contribute to patterning, pericyte covering of lymphatic vessels, lymphatic vessel valves, and lymphatic vessel maturation. At E11.5-12.0, CCL21 is expressed in these lymphatic-biased endothelial cells, as is VEGFR-3, which is reduced in blood endothelial cells. The endothelial cells expressing LYVE-1, Prox1, VEGFR-3, and CCL21 become irreversibly committed toward a lymphatic pathway.153,154 The

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separation of lymphatic endothelial cells from venous endothelium requires a Syk/SLP-76 signal155 that is provided by platelets.156,157 Thus, during early lymphangiogenesis, some endothelial cells express both blood vessel and lymphatic vessel markers, indicating the close association of these two vascular systems. Mesenchymal lymphangioblasts may also contribute to early lymphangiogenesis.158 The lymphatic venous junction remains in adults in only limited regions but plays an essential role in connecting the function of the two vascular systems. Studies in the mouse have taken advantage of transgenic, knockout, and imaging studies to provide a mechanistic understanding of the process of lymph node development. Although lymph sacs per se do not appear to be essential for the initiation of lymph nodes, a lymphatic vessel system is necessary for the later development and cellular population of lymph nodes.159 Furthermore, an interaction between mesenchymal cells and primitive endothelial cells is apparent in the early lymph node anlage.160

Cytokines, Chemokines, and Transcription Factors in Lymphoid Organogenesis The TNF-/LT-receptor family members play key roles in secondary lymphoid organ development. LTα3, signaling through TNFRI and TNFRII, and membrane bound LTα1β2, signaling through the LTβ receptor (LTβR) have been implicated. Mice deficient in LTα lack all lymph nodes and Peyer’s patches, and exhibit a disorganized spleen and severely disorganized NALT (see subsequent discussion).161–163 Mice deficient in LTβ lack peripheral lymph nodes but retain mesenteric, sacral, and cervical lymph nodes.164–166 Ltb r- / - mice have a phenotype similar to that of Lta- / - mice.167 LTβR is also recognized by LTβ -related ligand, LIGHT, which also binds to the herpes virus entry mediator. LIGHT-LTβR signaling does not appear to play an essential role during lymphoid organogenesis, as no significant defect is observed in Light- / - mice168 ; however, mice doubly deficient in LIGHT and LTβ have fewer mesenteric lymph nodes than mice deficient in LTβ alone, indicating a cooperative effect of the two LTβR ligands. Treatment of pregnant females with an inhibitory soluble protein of LTβR (LTβR and human IgG Fc fusion protein, LTβR-Ig) inhibits most lymph nodes in the developing embryos, depending on the time of administration. Mesenteric lymph nodes are not inhibited by this treatment. These studies indicate that individual lymph nodes differ in the nature and time of cytokine signaling during development.169 Several additional cytokine and chemokinereceptor pairs are crucial for lymphoid organogenesis. Mice deficient in IL-7 or IL-7R,170,171 or TRANCE or TRANCER, also called RANKL and RANK,172,173 exhibit defects in lymph node development. CXCR5- or CXCL13-deficient mice174,175 lack some lymph nodes and almost all Peyer’s patches.98 The relative importance of the different cytokines in the development of individual lymphoid organs has been recently summarized.176 The NF-κ B signaling pathways, downstream of the TNF family receptors, play important roles in lymphoid organ development.177 The alternative pathway, characterized by NF-κ B–inducing kinase (NIK) and IKKα is particularly

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important. aly/aly mice, which have a point mutation in Nik, lack all lymph nodes and Peyer’s patches.178,179 In these mice, LTβR, but not TNFR, mediated signaling between NIK and members of the TRAF family appears to be disrupted.179–181 LTβR signaling induces gene expression via both the classical and alternative NF-κ B pathways in mouse embryo fibroblasts. The classical pathway mediated by p50:p65 heterodimers induces expression of proinflammatory genes (Vcam-1, Mip1b, Mip2). Intraperitoneal injection of an agonistic LTβR antibody induces splenic chemokines (CCL19, CCL21, and CXCL13) and requires NIK activity and subsequent p100 processing.182 IKKα is a critical component in alternative NF-κ B pathway. Mice with a mutated form of the Ikka gene have reduced HEV expression of HEC-6ST (GlcNAc6ST2) and GlyCAM-1, further confirming that the LTβR signal regulates HEVs through the alternative NFκ B pathway.183 Several other signaling pathways that contribute to lymphoid organogenesis include the helix-loop-helix transcription factor inhibitor (Id2) and retinoid acid-related orphan receptors (RORs) RORγ and RORγ t.184–186 Studies of lymph node anlage formation reveal the mechanisms by which cytokines trigger and coordinate lymphoid organogenesis. There is likely an initiating factor, though this has not been definitively identified. In the case of Peyer’s patches, a cell producing the receptor tyrosine kinase has been implicated.187,188 Recent data indicate that the earliest stages of lymph node development appear to be dependent on retinoic acid that may be produced by nerves in the vicinity of the developing node.189 Circulating CD4 + CD3 − CD45 + RORγ t + hematopoietic progenitor cells called lymphoid tissue–inducer cells, derived from fetal liver progenitors,98,185,190–192 provide crucial signals in lymph node organogenesis. These lymphoid tissue–inducer cells accumulate in the developing lymph node, forming clusters with resident stromal organizer cells, to initiate a cascade of intracellular and intercellular events that lead to the maturation of the primordial lymph node.1,98 During this early step, a positive feedback loop involves several signaling pathways, including LTαβ /LTβR, IL-7R/IL-7, CXCR5/ CXCL13, and RANK/RANKL, are expressed on the lymphoid tissue–inducer cells and the stromal organizer cells. The prolonged interaction between lymphoid tissue– inducer cells and stromal organizer cells promotes the development of HEVs, which support the entry of naïve lymphocytes.193 It is unclear how HEVs differentiate from the flat blood vessels during early lymphoid organogenesis. At birth, HEVs of all lymph nodes express MAdCAM-1, which is replaced in the first few days in peripheral lymph nodes by PNAd.194 Both MAdCAM-1 and PNAd are expressed in mucosal lymph nodes. LTα alone can induce MAdCAM-1, but PNAd requires LTαβ.2,195,196 In the remaining mesenteric lymph nodes of Ltb- / - mice, PNAd expression is impaired,196 indicating that optimal lymph node HEV PNAd expression requires LTα1β2 signaling through the LTβR and the alternative NF-κ B pathway.183 Because the maturation of HEVs is coincident with further development of the lymph node,139,197 the homing of LT-expressing lymphocytes most likely contributes to HEV maturation. Continual signaling through the LTβR is necessary for maintenance of

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HEV gene expression.198,199 Recent data suggest that the actual physical presence of lymphocytes in the close vicinity of HEVs contributes in important ways to the physical cobblestone appearance of these vessels.200

Changes in Lymph Nodes after Immunization Lymph nodes undergo dramatic changes and remodeling after immunization. Early after a variety of immunogenic exposures, such as skin painting with oxazolone, injection of ovalbumin or sheep red blood cells in adjuvant, or bacterial or viral infection, remodeling occurs. This remodeling is apparent as a complex kinetics of changes in lymph flow, lymph cell content, blood flow, HEV gene expression, and lymphatic vessels.199 Afferent lymph flow and lymph cell content increase soon after initial inflammation, and eventually return to preimmunization levels.201–205 Lymph node lymphangiogenesis occurs, which eventually resolves.199,206 Blood flow and lymphocyte migration into lymph nodes peak at 72 to 96 hours,166,204,207,208 accompanied by an increase in HEV number and dilation,209,210 accounting for the significant lymph node enlargement apparent at 72 to 96 hours.207 Efferent lymph flow also increases soon after immunization, but lymph cell content in the efferent lymph drops during the first several hours, indicating the first wave of accumulation of lymphocytes in the draining lymph node. The cell content of the efferent lymph later increases and peaks at 72 to 96 hours.211 These events, taken together, contribute to the significant enlargement of the draining lymph node at day 4 after immunization. During the early times after immunization, despite the increase in the number of HEVs, the expression of genes that contribute to L-selectin ligand, including Fuct-vii, Glycam-1, Sgp200, and Chst4, is initially downregulated followed by a recovery.148,199,212 However, despite the downregulation of chemokines CCL21 and CXCL12, some genes such as those encoding CXCL9, CCL3, and E-selectin are upregulated,213 as is MAdCAM-1,199 suggesting a reversion to an immature phenotype before the eventual recovery of the mature phenotype. In rodents, HEV maturation is coincident with the continuing development of the lymph node and population of lymphocytes after birth, indicating that the mature HEV phenotype relies on the lymph node microenvironment.139,197 Plasticity of the mature lymph node is also seen after injection of LTβR-Ig. LTβR-Ig treatment reduces lymph node cellularity, reverts the HEV phenotype to the immature state, inhibits FDC function, and disrupts immune responses to foreign antigens.198,199,214

Spleen Function The spleen is a large reddish organ located beneath the diaphragm close to the stomach and the pancreas. It is the main fi lter of the blood and integrates the innate and adaptive responses. Its structure in well-defined different compartments, red pulp and white pulp, determines a variety of functions. The red pulp is a source of hematopoiesis in the embryo that can continue in adult life under stress. Clearance of blood-borne damaged platelets, aged

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erythrocytes, and dead cells also occurs in that region. An important function, consequent to its destruction of effete erythrocytes, is its role in iron recycling. In some species, such as horses or dogs, the spleen stores erythrocytes that can be released after stress. The white pulp and marginal zone constitute the highly organized lymphoid compartment of the spleen. Due to this organized lymphoid structure and the special vasculature and circulation, the spleen is a crucial site for blood-borne antigen clearance and presentation to T and B cells. The spleen is crucial in defense against blood-borne pathogens and contributes most significantly to defense against bacterial and fungi infections.215

Architecture and Cellular Composition A fibrous capsule and trabeculae of fibrous connective tissue maintain the structure of the spleen. The general principles of splenic organization are similar across species, though specifics may differ.216–220 Classically, the spleen can

be divided into two areas: red pulp and white pulp. A transit area, the marginal zone (MZ), surrounds the white pulp (Fig. 3.4). These compartments are anatomically and functionally distinct. The red pulp, in its activities as a hematogenous organ, removes damaged cells and acts as a site for iron storage and turnover. The white pulp is an organized lymphoid structure. The complex structure of the spleen is directly related to the complexity of the vasculature of this organ. The afferent splenic artery branches into central arterioles, surrounded by white pulp areas, and end in cords in the red pulp. Blood then collects in venous sinuses that determine the engulfment of erythrocytes by red pulp macrophages. Finally, the sinuses empty into the efferent splenic vein. Some small arterial branches end in the MZ, which demarcates the red and white pulp. The MZ structure differs somewhat between humans and rodents. A perifollicular zone surrounds the human MZ, which consists of inner and outer marginal zones, whereas the rodent MZ is

FIG. 3.4. Organization of the Spleen White Pulp. Immunofluorescence staining of a white pulp unit in the mouse spleen. T cells (anti- cluster of differentiation [CD]4+anti-CD8+, red) are localized around the central arteriole. B cells (anti-immunoglobulin M, green) are localized in follicles around the T-cell area, surrounded by a layer of metalophilic macrophages (labeled with monoclonal antibody-1) and a more peripheral layer of marginal zone macrophages (labeled with monoclonal antibody ERTR9, orange). The marginal zone is located between the metalophilic macrophage and the marginal zone macrophage layers (not shown).357 From Chaplin358 with permission.

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a single structure and has no perifollicular zone. The MZ is the transition between the innate and acquired immune systems and is an important transit area for cells reaching the white pulp. It also contains a large number of resident cells. There are two specialized macrophage populations: the MZ macrophages and MZ metallophilic macrophages. MZ macrophages have a high phagocytic activity and are phenotypically different from other macrophage populations in the spleen as exemplified by the expression of SIGNR1 and macrophage-associated receptor (MARCO) with collagenous structure, molecules implicated in the recognition of pathogens. SIGNR1, a C-type lectin in the mouse that is a homologue of human DC–specific intercellular adhesion molecule,221 is also found in medullary and subcapsular macrophages in the lymph node. MZ macrophages are crucial Marco for the capture of a wide variety of pathogens, including yeast, bacteria, and viruses.222,223 MARCO, expressed constitutively on MZ macrophages,224 is a class A scavenger pattern recognition receptor. In addition to the recognition of blood-borne pathogens, MARCO interplays with MZ B cells, modulating their migration to the white pulp.225 MZ metallophilic macrophages are located at the inner border of the MZ, in contact with marginal sinus lining cells, stromal cells that express MadCAM-1.226 MZ metallophilic macrophages differ from the MZ macrophages and red pulp macrophages in that they express the sialic acid-binding Ig-like lectin sialoadhesin (Siglec-1), which binds to oligosaccharide ligands present on many cells.227 Little is known about the specific roles of this population of macrophages during the immune response; however, the phenotype of mice deficient in sialoadhesin implies a role of MZ metallophilic macrophages in T-cell activation.228,229 Recent studies confirm that MZ metallophilic macrophages are essential for cross-presentation of blood-borne adenovirus antigens to splenic CD8 DCs, activating cytotoxic T-lymphocytes.230 The MZ also contains a specialized subset of B cells that differ phenotypically and functionally from follicular B cells; they can be considered as a bridge between the innate and adaptive immune systems. They express higher levels of IgM low levels of IgD and the molecule CD1d. These B cells bind antigen in the MZ directly and/or through interactions with MZ macrophages231 and migrate to the white pulp, where they present blood-borne antigens.232 The organization of the remainder of the white pulp of the spleen is similar to that of the other secondary lymphoid organs with compartmentalized B- and T-cell areas. The white pulp consists of a central arteriole that is surrounded by T cells, also known as the periarteriolar lymphoid sheath, which is surrounded by B-cell follicles (see Fig. 3.4). T cells interact with DCs and B cells. B cells migrate to the follicles where they interact with FDCs. At the T:B-cell border, T cells, especially T follicular helper cells,233 interact with B cells. Germinal centers are the site of somatic hypermutation and Ig class switching. Plasma cells are found mainly in the red pulp. The spleen does not have an afferent lymphatic system, and initial antigen transport must occur through the blood vasculature. A conduit system has been described that allows antigens and chemokines to be transported through the white pulp in a manner similar to that described for the

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lymph node.234 This conduit differs from that in the lymph node with regard to the identity of the transported molecules by the fact that it contacts the blood rather than the lymphatic system.

Traffic In and Out: Chemokines and Adhesion Molecules Cell trafficking in the spleen is similar to that which occurs in the lymph node in some respects, and differs in others. The MZ is the main transit area for blood cells that enter the white pulp. This process seems to be controlled by marginal sinus lining cells235 and chemokines, involving signaling through G-couple protein receptors.236 Stromal cell–produced lymphoid chemokines CXCL13, CCL19, and CCL21 control the migration of lymphocytes and their organization in the various compartments in the white pulp. CXCL13 is essential to positioning of B cells in the follicles, whereas T cells respond to CCL19 and CCL21. After the immune response, germinal center B cells differentiate into plasma cells in the white pulp, then migrate to the red pulp or return the bloodstream to migrate to peripheral tissues. In the migration to the red pulp, plasma cells upregulate CXCR4, a receptor that binds the chemokine CXCL12, which is expressed in the red pulp. The role of adhesion molecules with regard to lymphocyte traffic in the spleen is not fully well understood. Lymphocytes enter into the white pulp through the marginal sinus and cells lining that structure express MAdCAM-1 and ICAM-1.226,237 MadCAM-1 expression in these cells seems to be involved in splenic structure development and therefore migration of B and T cells.235 However, treatment with anti-MAdCAM-1 or anti-α1β7 antibodies does not totally inhibit homing to the spleen, suggesting that this ligand-receptor pair is not the only receptor required for lymphocyte entrance into the white pulp. Early studies indicating that treatment with an antibody that blocks the αL-integrin chain of LFA-1 inhibits homing by only 20%, and that lymphocytes deficient in LFA-1 enter the white pulp,237 suggested that LFA-1 is not absolutely essential for entry of all cells into the white pulp. However, both α4β1 and LFA-1 integrins are necessary for B-cell retention in the MZ,238 indicating a role for VCAM-1 and ICAM-1 in cell trafficking in spleen. Once lymphocytes have encountered antigen, they most likely undergo changes in chemokine receptor and adhesion molecule expression similar to those noted in the lymph nodes, leave the white pulp and enter the red pulp (plasma cells) or the circulation. The mechanisms by which lymphocytes leave the white pulp are unclear. The observation of channels that bridge into the MZ239 suggests one route of egress of lymphocytes. In the lymph nodes, the process of egress of activated lymphocytes through the efferent lymphatic vessels is mediated by upregulation the expression of S1P1. In the spleen, S1P1 is required for B-cell localization in the MZ, and the interplay with CXCR5 regulates the constant trafficking of these cells between the MZ and the white pulp,232,240 as MZ B cells from S1P1-deficient mice are not found in the MZ but are found in the follicles. Although it is still unclear which molecules are involved in the egress of lymphocytes from the spleen, and splenic egress cannot be compared to egress in the lymph nodes,

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recent studies attribute an important role to the transcription factor Nkx2.3 in the regulation of spleen vasculature and homing of lymphocytes.241,242

Development Because the spleen has characteristics of both a hematopoietic organ and a secondary lymphoid organ, the genes that regulate its development include, in addition to LT, others that are concerned with patterning and hematopoiesis. Several genes that contribute to the development of the spleen are also crucial for normal development of other nonlymphoid organs. The first sign of splenic development in the mouse occurs at E10.5-11. Segregation of red and white pulp starts to be regulated during embryogenesis and continues after birth. Some structures, such as the MZ, develop during the 3 first weeks after birth. At E10.5-11, progenitor cells begin to condense within the dorsal mesogastrium, adjacent to the stomach and dorsal pancreas. The spleen and pancreas are so intimately associated that it is difficult to distinguish between them at these early stages. In fact, many genes that affect splenic development also contribute to pancreatic development. Several of these are homeobox genes and transcription factors that are expressed in the splenopancreatic mesenchyme at E10.5.215 The effects of just one of these genes, Hox11 (now called Tlx1215), are on the spleen. The others affect multiple organs. Tlx1 was originally described as an oncogene in T-cell childhood acute leukemias involving the (10;14) translocation breakpoint.243 Tlx1-deficient mice are asplenic,244 and the product of this gene is a cell survival factor.245 Several additional genes that are important for development of lymph nodes, NALT, and Peyer’s patches also contribute to splenic development. Mice deficient in members of the LT/TNF ligand receptor family and their downstream signaling molecules in the classical and alternative pathways exhibit defects in splenic development. However, none of these molecules is necessary for the early splenic anlagen, as mice deficient in any of the chemokines or cytokines retain a spleen. Recent studies address a role for Glce, an enzyme that modifies heparan sulfate, in early lymphoid tissue morphogenesis, as fetal spleen in Glce deficient mice exhibit a reduced size.246 The changes in lymphoid tissue organization in mice deficient in LT/TNF ligand receptor family members are due in part to a reduction or near absence of lymphoid chemokines (CXCL13, CCL19, CCL21).247 Lymphoid chemokine messenger ribonucleic acid (mRNA) expression is reduced in the spleens of mice deficient in TNFR1, TNF, LTα , or LTβ, though CXCL12 mRNA levels are normal. However, treatment with an agonistic LTβR antibody induces expression of the lymphoid chemokines and CXCL12, suggesting that signaling through both the classical and alternative NF-κ B pathways are responsible for organization and maintenance of splenic white pulp architecture. Mice deficient in LTα exhibit a disorganized white pulp with loss of T- and B-cell compartmentalization, MZ macrophages, metallophilic macrophages, MZ B cells, MAdCAM-1 sinus lining cells, and germinal centers. Ltβ - / - mice exhibit similar characteristics except that the disorganization is somewhat less pronounced. However, these lymphoid cytokines seem to be dispensable during

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fetal spleen development, when segregation between white and red pulp begins, but essential for maintenance of this structure during postnatal development of the white pulp.248 Tnf- / - and Tnfr1- / - mice show defects similar to lymphotoxin deficient mice, with the exception of MZ B cells. T cells, B cells, and CD4 + CD3 − cells produce the cytokines necessary for maintenance of splenic architecture.249,250

Plasticity after Virus Infection Though much is known regarding changes in the lymph node after immunization, the spleen has not been studied as extensively in this regard. However, after infection with cytomegalovirus, white pulp T:B compartmentalization is disrupted.251 The spleens of Lta- / - deficient mice exhibit a marked reduction in expression of CCL21-ser. This is even further reduced in cytomegalovirus infection, indicating that, in the adult, LT-independent pathways can contribute to maintenance of expression of lymphoid chemokines.

Mucosal-Associated Lymphoid Tissues General Features The MALT covers all mucosal surfaces not only in the gut, but also the oropharyngeal and lacrimal mucosae, the nasal and bronchial airways, and the genitourinary tracts. MALT protects a huge surface area and contains approximately half of the lymphocytes of the entire immune system.252 MALT is considered to be the body’s gatekeeper because it is in intimate contact with the commensal flora at the mucosal surfaces. Although the location of some mucosal lymphoid tissues like the palatine tonsils and appendix are fixed, all MALT are somewhat plastic because of their constant exposure to environmental antigens that induces them to change and remodel. Tonsils, adenoids, and their equivalent in rodents, the NALT, have a fixed location, whereas the location, number, and size of Peyer’s patches in the small intestine vary according to antigen exposure. The BALT in the lung and ILFs in the colon are the most plastic MALTs, and their number and location are subject to change by environmental influences. The mucosal epithelium that surrounds MALT is populated by a dense network of DCs, plasma cells, and intraepithelial lymphocytes that helps to maintain the epithelial barrier. These intraepithelial leukocytes provide retinoic acid and cytokines like IL-10 and TGF-β that condition the gut to become tolerant of antigens produced by the harmless commensal bacteria.253,254 They are also responsible for inducing and maintaining tolerance to food antigens and commensal bacteria.255 The large MALT structures like the tonsils, NALT, and Peyer’s patches share many features with lymph nodes. Like lymph nodes, MALT contains HEVs for the entry of naïve lymphocytes, and stromal cells that secrete chemokines that direct lymphocyte traffic in the MALT (CCL19, CCL21 and its receptor CCR7256 ; CCL25 and CCL28 and their receptors CCR9 and CCR10257; and CXCL13 and its receptor CXCR5258,259). MALT also contains separate T- and B-cell compartments with B-cell follicles, FDCs, germinal centers, and interfollicular T cells and DCs.260

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MALT differs importantly from other secondary lymphoid organs with regard to its capsules and afferent lymphatic vessels. Unlike the spleen and lymph nodes, which are surrounded by a dense fibrous capsule, MALT often has no capsule, with the exception of the tonsil that has a partial capsule that separates it from the pharyngeal constrictor muscles. Because MALT can sample antigens directly at the epithelial surface, it has no afferent lymphatics. MALT epithelium, especially that of the Peyer’s patch, is replaced by specialized lymphoepithelial cells called microfold (M) cells, which because of their high transcytotic capacity, transport antigens to the underlying lymphoid tissue. Tonsils and adenoids are covered by a squamous epithelium and have few M cells. They can sample surface antigens using epithelial DCs that push dendrites through the epithelial surface.261,262 M cells have not been characterized in human BALT.263 The proximity of MALT to the epithelium is an important point, because it helps differentiate MALT from tertiary lymphoid organs (see following discussion). MALT can be defined as organized lymphoid structures in the mucosa that are in direct contact with the epithelium. Using these criteria, a lymphoid aggregate in the submucosa that is not directly in contact with the epithelium (ie, below the lamina propria) is more correctly defined as a tertiary lymphoid organ.264

The Mucosal-Associated Lymphoid Tissue Immune Response: Inductive and Effector Sites MALT can simultaneously be a site for the induction of an immune response and an effector organ. The MALT surface containing M cells and DCs efficiently samples and transports antigens across the epithelium. Immediately below this single-cell epithelium is a dense network of DCs that can extend dendrites through the epithelium to grasp antigen to present to lymphocytes. Two distinct types of DC have been identified in MALT: CD103 + DC and CX3CR1+ DC.265 Both types of DC produce tolerogenic responses to commensal bacteria by enhancing the differentiation of Foxp3 + Tregs and inhibiting that of inflammatory Th17 cells. CD103 + DCs express αE integrin, make retinoic acid, induce Tregs, and increase T-cell expression of the two gut homing receptors CCR9 and α4β7. CD103 + DCs exit MALT via the lymphatics and present antigen in the draining lymph nodes. CX3CR1+ DCs that express the chemokine receptor for fractalkine do not migrate to the lymph node. CX3CR1+ DCs are longer-lived mucosal resident DCs that produce less retinoic acid than CD103 + DCs.265 MALT has three effector roles: local antibody secretion, systemic antibody secretion, and effector lymphocyte dispersal. MALT plays an important role in defense against pathogens by generating cells that migrate to other sites. MALT can dispatch lymphocytes to lymph nodes, the spleen, and plasma cells to the bone marrow. MALT also sends lymphocytes to other MALT effector sites including the salivary and lacrimal glands, the lactating breast,266 and the vagina. There are some differences between the effector cells made at different MALT sites. NALT-derived B cells express CCR7 and do not home back to the gut. Instead, cells induced in the NALT home to the salivary glands and the vaginal mucosa lymph nodes,267

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whereas lymphocytes leaving the Peyer’s patch express α4β7 and migrate to the gut mucosa. These “preferred pathways” for effector cells coming out of the different MALT has led to the suggestions that MALT is compartmentalized and cells circling in the BALT are in a separate compartment from the GALT. It is more likely that the nature of antigen influences the homing of effector cells. There needs to be flexibility, and there is much overlap and redundancy in the system. For example, after the removal of the tonsils and adenoids in humans, or the NALT in the mouse, the cervical lymph node can act as an inductive site.268 Likewise, the mouse BALT can mount an immune response in the absence of secondary lymphoid organs.269,270

Tonsils and Adenoids Waldeyer’s ring is a group of lymphoid tissues encircling the wall of the throat that are the fi rst defense against pathogens entering through the mouth or nose.271 Humans have several tonsils with indistinct borders: one pharyngeal tonsil (adenoid), two tubal, two palatine, and one lingual. In addition to T cells, the tonsils contain a large complement of B cells, many of which are positive for IgA.271 The tonsil epithelium makes the polymeric IgA receptor, or secretory component,272 crucial for transport of IgA dimers across the epithelium. Secreted IgA provides an early form of defense against pathogens and toxins. Overall, the adenoids produce more secreted IgA than the tonsils. The common cold virus uses ICAM-1 as receptor to invade the nasal mucosa. ICAM-1 is also an expressed HEV in tonsils.271 As noted previously, HEVs are specialized vessels that express adhesion molecules that allow naïve lymphocytes enter the tonsil. Naïve L-selectinhi lymphocytes adhere to PNAd on HEVs.258,259 L-selectinhi cells also bind to MAdCAM-1 predominantly found in the gut HEVs. Considering the “mucosal name” MAdCAM-1, it may be surprising to discover that tonsil HEVs express MAdCAM-1 only weakly or not at all.273 In mice, the HEVs also manufacture the chemokine CCL21 that can recruit naïve T cells and mature DCs that express CCR7. Human tonsil HEVs do not manufacture CCL21 but are able to display chemokines that have been made and secreted by fibroblasts.274 During tonsillitis, inflammatory chemokines, cytokines, and adhesion molecules such as CCL19,256 VCAM-1, and E- and P-selectin are upregulated.165

Nasal-Associated Lymphoid Tissue The NALTs are a pair of lymphoid organs above the soft palate in mice and rats that are considered analogous to Waldeyer’s ring.275 Despite being anatomically separate from the genitourinary tract, NALT has an important “effector” role concerning the generation of immune responses in the genitourinary tract.276 After nasal immunization with human papillomavirus 16 or ovalbumin, human papillomavirus 16–specific or ovalbumin-specific IgA is detected in vaginal washes,163,277,278 and cytotoxic T cells are found in vaginal draining lymph nodes.267 However, the NALT itself is clearly an inductive site for both humoral and cellular immune responses,279 and supports class switching to IgA.280

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The stromal cells in murine NALT produce both CCL19 and CXCL13, whereas, in contrast to lymph nodes, only HEVs transcribe CCL21 mRNA.163 NALT HEVs express high levels of luminal and abluminal PNAd and HEC-6ST.163 Similar to the tonsils, the major homing receptor-ligand pair in NALT is L-selectin and PNAd, rather than MAdCAM-1 as might have been expected of this mucosal-associated tissue.281

Development Human NALT and tonsils appear early in fetal development, are cellular, and have primary follicles. After birth, secondary follicles and germinal centers appear in response to bacterial antigens.266 By contrast, the NALT in the mouse and the rat is hypocellular at birth and undergoes dramatic changes after weaning, strongly suggesting that bacterial colonization is important for NALT development in these species. Id2 is required for initiation of the rodent NALT,282 although RORγ T, LTα , or LTβ are not required for this step.162 The expansion of rodent NALT at weaning includes changes in the expression of LTα and LTβ and lymphoid chemokines, leading to T- and B-cell compartmentalization and HEV maturation.162,163 The alternative NF-κ B pathway is required for the expression of chemokines and the HEV genes glycam-1 and chst4 in NALT.183 LTα , LTβ, IL7R, and the NIK signaling pathways are required for NALT organization and function, and mice that are deficient in these cytokines have a hypocellular NALT.163,183,283,284

Bronchus-Associated Lymphoid Tissue BALT is less organized but more responsive to environmental antigens than the NALT. The number of lymphoid aggregates varies depending on the level of microbial exposure and germ-free pigs have no BALT.252,285 BALT is found commonly in rabbits and rats, is less frequent in guinea pigs and pigs, and is absent in cats.264 BALT is not a prominent structure in the laboratory mouse, and its presence varies by strain and age.286 An inducible form of the BALT (iBALT) has been described in Lta- /- mice after infection with influenza.287 The term iBALT is misleading because these aggregates have no contact with the bronchial lumen and cannot sample inspired antigens. Therefore, iBALT should be considered as a tertiary lymphoid organ.288 Splenectomized, lethally irradiated LTα− / − mice reconstituted with normal bone marrow have no lymphoid organs. However, these mice can generate immunological memory in the iBALT.269,270 BALT is rare in normal adult human lungs, but tertiary lymphoid organs are common in chronic pulmonary diseases.283 BALT is frequent in children and is found in fetuses after infections in utero.289,290 Like NALT, the HEVs in human BALT express PNAd and not MAdCAM-1.291

Gut-Associated Lymphoid Tissue Gut-associated lymphoid tissue is the largest immune system in the body. The gut-associated lymphoid tissue in the small intestine includes the Peyer’s patches, smaller isolated lymphoid follicles, and cryptopatches. In the large intestine, there is the appendix, caecal patches, and lymphoglandular complexes.292

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Peyer’s Patches Peyer’s patches are lymphoid aggregates aligned on the antimesenteric border of the small intestine (Fig. 3.5A). They are present in most species, though their number and location vary. They are dome shaped and are covered by a specialized epithelium that lacks surface microvilli and goblet cells; they have numerous M cells. Because Peyer’s patches lack afferent lymphatic vessels, these M cells are critical transporters of antigen. Pathogens, including human immunodeficiency virus and salmonella take advantage of the M cells to facilitate their own invasion.293,294 Below the epithelium is a diffuse area, the subepithelial dome, divided into three to six parts. Peyer’s patch organization is similar to that of the lymph node (see Fig. 3.5). Peyer’s patches contain 6 to 12 basally located germinal centers, but the B-cell areas are larger with a T:B cell ratio of 0.2; CD4 + cells predominate over CD8 + cells.295 Although M cells transport antigen across the epithelial barrier, they are not believed to have a crucial role in processing or presenting antigen. Peyer’s patches are populated by several subsets of DCs that can carry out these functions.296,297 Trafficking In and Out Cells enter Peyer’s patches through HEVs. In contrast to peripheral lymph nodes and the NALT, the HEVs of Peyer’s patches express MAdCAM-1, 298 and homing depends on the interaction of MAdCAM-1 with the integrin α4β7 on lymphocytes. Luminal PNAd is rarely found in Peyer’s patch HEVs; only occasional abluminal expression is detected. This pattern is identical to that seen in lymph node HEVs in chst4- / - mice.128 However, lymphocytes from mice that lack both L-selectin ligand and α4β7 home less efficiently to Peyer’s patches than do lymphocytes from mice that lack only one or the other of the ligands, 299,300 suggesting that an L-selectin ligand does contribute to homing to Peyer’s patches. Even though P-selectin is expressed only weakly on HEVs in Peyer’s patches, cells from mice deficient in P-selectin show reduced rolling and adhesion in vivo. This suggests that P-selectin, as well as MAdCAM-1, is important for homing to Peyer’s patches. 299 Lymphoid chemokines CCL19, CCL21, and CXCL13 are found in the T- and B-cell areas301 (see Fig. 3.5B). Stromal cells make CXCL12 mRNA, but the protein is also found on HEVs, presumably transported in a manner similar to CCL19.117 The CXCL16/CXCR6 chemokine/receptor pair is important in Peyer’s patches but not in lymph nodes. Even under germ-free conditions, CXCL16 is constitutively produced by the dome epithelium.295 CD4 + and CD8 + cells that express CXCR6 are found in Peyer’s patches and require CXCL16 to home to the subepithelial dome. CCL20 and its receptor CCR6 are also important in Peyer’s patches. CCL20 is made by the intestinal epithelium and plays a crucial role in dendritic cell trafficking to Peyer’s patches.302 Peyer’s patch HEVs express CCL25, the ligand for CCR9, which has been defi ned as a mucosal homing chemokine receptor for intraepithelial lymphocytes and plasma cells.78

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B

FIG. 3.5. Organization of Peyer’s Patches. A: Photomicrograph of Peyer’s patch. Peyer’s patches are located in the intestine near intestinal villi. The follicle associated epithelium (FAE) is in contact with the gut lumen. M cells (not shown) in the FAE transport antigen into the subepithelial dome, populated by dendritic cells and T cells. The interfollicular T-cell–rich region surrounds the B-cell follicle and germinal center. Courtesy of A. Iwasaki (Yale University School of Medicine, New Haven, CT). B: In situ hybridization of chemokine messenger ribonucleic acids (mRNAs). On the left, CCL21 mRNA defines the T-cell zone and high endothelial venules; on the right, CXCL13 defines the B-cell zone.

A

Development Several cytokines and chemokines have been shown to be crucial for Peyer’s patch development. Ltα- /- and Ltβ - /mice completely lack Peyer’s patches,161,303 as do Cxcr5- / mice.174,175,301 Because mice deficient in the LTβR also lack Peyer’s patches,304 these effects are mediated in large part through LTα1β2. However, some Tnfr1- / - mice lack organized Peyer’s patches, suggesting that either LTα3 or TNFα also influences generation or later stages in maintenance of Peyer’s patches.305 Mice deficient in IL-7, RORγ t, Id2, NIK, and factors in the classical and alternative NFκ B pathways170,177,304 lack Peyer’s patches, although RANKL is not required.172 A model for the embryonic development of Peyer’s patches306–308 provided the framework for studies in that organ. The first sign of Peyer’s patch development at E15.5 in the mouse is the appearance of regions on the small intestine that stain positively with antibodies to ICAM-1, VCAM-1, and LTβR. The cells in these aggregates are called the lymphoid tissue organizer cells and express CXCL13, CCL19, CCL21, and IL-7. At E17.5, clusters of IL-7R + CD4 + CD3 + inducer cells are found. These express LTα and LTβ, CXCR5 and CCR7, Id2, and RORγ. They also express α4β1 integrin activated by CXCR5309 that allows interaction with the VCAM-1+ organizer cells. At E 18.5, mature T and B cells enter through HEVs, CCL20 is produced by the FAE, and DCs expressing CCR7 and CCR6 are found. By day 4, the typical microarchitecture is apparent, with M cells and Tand B-cell compartmentalization.310

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Lymphocytes influence the maintenance of Peyer’s patches. Mice that lack mature T and B cells have either undetectable or small Peyer’s patches that lack follicles and germinal centers.311,312 B cell–deficient mice retain some M cells, suggesting that T cells may regulate M-cell maintenance. When B cell–deficient mice are reconstituted by a membrane IgM transgene, M cells are found at levels comparable to those of normal mice,311 indicating that cells in addition to CD4 + CD3 + lymphoid tissue–inducer cells are crucial for the maintenance of mature, functioning Peyer’s patches.

Cryptopatches and Isolated Lymphoid Follicles The small intestine of a mouse contains 100 to 200 isolated lymphoid follicles and more numerous cryptopatches. The cryptopatches in the lamina propria containing lymphoid tissue–inducer cells and DCs can be precursors of ILFs. The cryptopatches are composed of lin− c-kit + cells, DCs, and VCAM-1+ stromal cells with few or no mature T and B cells. The cryptopatch cells express RORγ t, IL-7R, and CCR6,313 and their development is dependent on IL-7, CCR6, and its receptor CCL20. Cryptopatches are quite plastic, and although the LT family is necessary for their development, they can be restored by administration of wild-type bone marrow to adult Lta- / - mice. After mice are exposed to microbes or during some forms of autoimmunity, cryptopatches give rise to ILFs,314,315 which resemble small Peyer’s patches and usually only have a single

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dome. ILFs require B cells that express LT, but not T cells, for their formation.316. In contrast to lymph nodes, they also require the TNFR1.317 The location of ILFs is antigen driven, and they resolve completely after mice are treated with antibiotics or cytokine inhibitors.121,185,314,316,317

Appendix Both rabbits and humans have an appendix,318 a vestigial organ distal to the ileocecal junction that divides the small intestine from the colon. The appendix has epithelial M cells and dense lymphoid accumulations and germinal centers that are similar to Peyer’s patches. Similar to the tonsils and adenoids, the appendix involutes and regresses after puberty.

Colon Lymphoglandular Complexes The colon has the highest bacterial load of any of the MALTs, and the distribution of the MALT is more random than in the small intestine. Lymphoglandular complexes are smaller than Peyer’s patches. As their name suggests, the lymphoid cells of the lymphoglandular complexes surround the mucus glands.292

TERTIARY LYMPHOID TISSUES Similarities to Secondary Lymphoid Organs Tertiary lymphoid tissues, also termed tertiary lymphoid organs, are ectopic accumulations of lymphoid cells that arise in nonlymphoid organs during chronic inflammation through a process termed “lymphoid neogenesis” (also “lymphoid neo-organogenesis”).2 The iBALT could be considered as either a secondary or tertiary lymphoid tissue. This semantic issue epitomizes the plasticity of all lymphoid organs and the fact that lymphoid organ regulation represents a continuum from ontogeny through chronic inflammation. A notable difference between secondary lymphoid organs and tertiary lymphoid tissues is the fact that the latter can arise in almost any organ in the adult. Nonetheless, tertiary lymphoid tissues exhibit remarkable morphologic, cellular, chemokine, and vasculature similarities to secondary lymphoid organs. They exhibit Tand B-cell compartmentalization; naïve T and B cells; DCs; FDCs; germinal centers; plasma cells; lymphoid chemokines CCL19, CCL21, and CXCL131; HEVs135,196,319; and conduits.320 Lymphatic vessels have also been noted in tertiary lymphoid tissues in chronic graft rejection321,322 and mouse models of Sjögren syndrome134 (Fig. 3.6) and Type 1 diabetes.323 Although it is not completely clear whether they function as afferent and/ or efferent vessels, the fact that the tertiary lymphoid tissues respond to S1P inhibitors suggest that their lymphatic vessels function similarly to those in lymph nodes.323 Lymphoid neogenesis has been noted in humans in autoimmunity, microbial infection, and chronic allograft rejection. A few examples are shown in Table 3.2 and are described in more detail in Drayton et al.1 These accumulations also occur in atherosclerotic plaques with FDCs, organized B-cell follicles, HEVs (HECA-452), and CCL19 and CCL21 in addition to those chemokines more often associated with acute inflammation.324 Tertiary lymphoid tissues have also been noted in non–small-cell lung cancer325

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FIG. 3.6. Tertiary Lymphoid Tissue. Schematic diagram of Sjögren syndrome salivary gland. Note similarities with the organization of a lymph node.

and many other solid tumors.326 The tendency for tertiary lymphoid tissues to develop into lymphomas has also been noted in many studies.327 Ectopic lymphoid tissues are also apparent in several animal models. The pancreatic infiltrates in early stages of diabetes in the nonobese diabetic mouse express lymphoid chemokines328 and HEVs expressing MAdCAM-1,329,330 PNAd, and HEC-6ST.135 The brain in experimental autoimmune encephalomyelitis, a model of multiple sclerosis, has HEVs, CCL19, CCL21, CXCL13, and FDCs.331–333 The thyroid in the BioBreeding (BB) rat has T- and B-cell compartmentalization and DCs,334 and the gut in autoimmune gastritis in the mouse has HEVs and CXCL13.335 Atherosclerotic plaques of apoprotein-E– deficient mice exhibit a marked increase in T and B cells, expression of CCL19 and CCL21, and PNAd + HEVs.336–338 Lymphoid neogenesis also occurs in chronic mouse heart allograft rejection.339,340 Tertiary lymphoid tissues have been induced in several transgenic mouse models with the use of tissue-specific promoters driving the expression of inflammatory cytokines or lymphoid chemokines.1 These mouse models, in addition to serving as examples of human disease, have provided valuable insight into the regulation of secondary lymphoid organ development.

From Chronic Inflammation to Organized Lymphoid Microenvironments Data generated from analyzing the cellular and molecular requirements for secondary lymphoid organ development have provided a paradigm for understanding the development of tertiary lymphoid tissues in chronic inflammation. This paradigm proposes that the processes and molecules governing secondary lymphoid organs are also the basis of tertiary lymphoid tissue development,2 and informs understanding of both secondary and tertiary lymphoid tissues. The physiologic event(s) that precipitate lymphoid neogenesis remain unclear. Data obtained from experiments in knockout and transgenic

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Lymphoid Neogenesis in Human Autoimmunity, Infectious Diseases, and Graft Rejection

Disease

Affected Tissue

Characteristics

Rheumatoid arthritis

Synovial membrane

Sjögren syndrome

Salivary glands

Myasthenia gravis Hashimoto thyroiditis

Thymus Thyroid

Grave disease

Thymus

Multiple sclerosis

Brain

Ulcerative colitis Inflammatory bowel disease (Crohn disease) Psoriatic arthritis

Colon Bowel

T cells and B cells, plasma cells, GCs, FDCs, CXCL13, CCL21, HEVs (PNAd, HEC-6ST) T cells and B cells, plasma cells, GCs, FDCs, CCL21, CXCL12, CXCL13 HEVs (PNAd) T cells and B cells, GCs, FDCs T cells and B cells, GCs, FDCs CCL21, CXCL13, CXCL12, plasma cells, HEVs (PNAd) T cells and B cells, GCs, FDCs, CCL21 CXCL13, CXCL12, HEVs (PNAd) Lymphatic capillaries, B-cell follicles and centroblasts, GCs, CCL19, CCL21, CXCL12, CXCL13 CXCL13 T cell-B compartments, LVs, HECA-452+ HEV

Joint

T cells and B cells, CXCL13, CCL19, CCL21, HEVs (PNAd)

Joints Central nervous system/ cerebrospinal fluid Liver Granuloma

T and B cells, FDCs, HEVs CXCL13

Autoimmunity

Infectious Diseases

Borrelia burgdorferi/Lyme disease Borrelia burgdorferi/ neuroborreliosis Hepatitis C virus Bartonella henselae/cat scratch disease

T-cell and B-cell compartments, MAdCAM-1 CXCL13

Graft Rejection

Organ Heart Kidney

GCs GCs, LVs

FDC, follicular dendritic cell; GC, germinal center; HEV, high endothelial venule; LV, lymphatic vessel; MAdCAM-1, mucosal addressin cell adhesion molecule; PNAd, peripheral node addressin. Original references are in Drayton et al.1

mice and clinical observations indicate that cooperative activities of TNF/LT family members and the lymphoid chemokines play central roles in this process. Inflammation is a localized response to tissue injury, irritation, or infection often marked by tissue damage. Acute inflammation is an early innate immune response that is generally short-lived and self-limiting. However, in some situations, acute inflammation transitions to a chronic inflammatory response that is long-lived and self-perpetuating. Tertiary lymphoid tissues arise under conditions of constitutive cytokine and/or chemokine expression, but the precise signal(s) that initiates their development is unknown. By integrating studies of lymphoid neogenesis in human pathologies and in animal models, it is becoming clear that at least three critical events promote tertiary lymphoid tissue formation: inflammatory (eg, TNF/LT) cytokine expression, lymphoid chemokine production by stromal cells, and HEV development. It is not known if lymphoid tissue–inducer cells are necessary for lymphoid neogenesis, though such cells have been noted in the mouse models of lymphoid neogenesis,341 and lymphoid tissue–inducer-like cells have been described in several instances.

Functions of Tertiary Lymphoid Tissues Ectopic accumulation of lymphoid cells has been considered the hallmark of destructive inflammation. Indeed,

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some tertiary lymphoid tissues are accompanied by tissue damage. However, it is likely that tertiary lymphoid tissues in chronic inflammation have roles in addition to tissue destruction. In the case of microbial infection, it is likely that lymphoid neogenesis occurs as a way to sequester pathogens and to prevent their access to the other parts of the body. This may represent a primitive form of immunity. Local antigen presentation within the tertiary lymphoid tissue itself likely prevents bacteremia or viremia. Enhanced long-term survival has been noted for those individuals whose lung or breast tumors included tertiary lymphoid tissues.325,326 The propensity for tertiary lymphoid tissues to develop into lymphomas, as noted previously, the ability of lymphatic vessels to serve as conduits for tumor metastasis, and the ability of tertiary lymphoid tissues to serve as sites of prion accumulation are obvious manifestations of detrimental functions.342 Furthermore, the development of tertiary lymphoid tissues in autoimmunity may perpetuate clinical disease through epitope spreading. Data from human and mouse studies provide compelling evidence that tertiary lymphoid tissues are permissive microenvironments for the induction of antigen-specific immune responses. Extensive immunohistochemical analyses of tertiary lymphoid tissues in autoimmunity and other chronic inflammatory states have established the presence

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of germinal centers and FDC networks in these tissues; several groups have demonstrated that tertiary lymphoid tissue germinal centers can support B-cell differentiation. Microdissection of discrete lymphocytic foci and subsequent deoxyribonucleic acid sequence analysis of germinal center B cells from the inflamed synovial tissue of patients with rheumatoid arthritis revealed a restricted number of Vκ gene rearrangements, a result consistent with oligoclonal B-cell expansion in the synovial tissue.343 Somatic hypermutation is apparent in synovial germinal center B cells.344 Furthermore, synovial B cells exhibit a limited number of heavy and light chain gene rearrangements consistent with local clonal expansion of these cells. The molecular analysis of tertiary lymphoid tissue germinal centers from the salivary glands of patients with primary Sjögren syndrome or the thymus of patients with myasthenia gravis demonstrates oligoclonal B-cell proliferation in these tissues in addition to somatic hypermutation of Ig variable genes.345–347 Together, these studies indicate that tertiary lymphoid tissue germinal centers in several autoimmune pathologies can support antigen-driven clonal expansion and extensive diversification. Another important hallmark of antigen-driven B-cell responses is the terminal differentiation of activated B cells into Ig-secreting plasma cells. Plasma cells have been detected in tertiary lymphoid tissues associated with germinal centers in rheumatoid arthritis348 and in Sjögren syndrome. Mice expressing LTα under the control of the rat insulin promoter (RIPLTα) exhibit tertiary lymphoid tissue at the sites of transgene expression (pancreas, kidney, and skin). After immunization with sheep red blood cells, evidence of isotype switching is apparent in these cellular infiltrates.2 Although the presence of plasma cells in tertiary lymphoid tissues is consistent with local antigen presentation, it is occasionally unclear whether these cells develop in the tertiary lymphoid tissues themselves or migrate from canonical secondary lymphoid tissues. Nonetheless, taken together, these studies indicate that tertiary lymphoid tissues in several human pathologies and animal models support antigen-driven B-cell differentiation marked by somatic hypermutation of Ig variable genes, affinity maturation, isotype switching, and terminal differentiation into antibody-secreting plasma cells. T-cell priming occurs in tertiary lymphoid tissues as suggested by the presence of isotype switched plasma cells in the RIPLTα tertiary lymphoid tissues,2 accelerated graft rejection,349 and T-cell epitope spreading in the central nervous system during experimental allergic encephalomyelitis.350 The restricted TCR repertoire in a melanoma-associated tertiary lymphoid tissue351 further supports the concept that tertiary lymphoid tissues can act as priming sites. The demonstration of naïve T-cell proliferation in the islets of nonobese diabetic mice after surgical removal of pancreatic lymph nodes352 suggests, together with evidence noted previously, that tertiary lymphoid tissues present antigen to naïve cells at the local site and generate an immune response. Determinant or epitope spreading, a phenomenon that arises in several autoimmune diseases, occurs when epitopes other than the inducing antigen become major targets of an ongoing immune response. It is considered to occur subsequent to the tissue damage induced by the initiating

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autoreactive T cells and therefore is the result of the presentation of new antigens.353 Data generated in murine models of central nervous system inflammation support the possibility that intermolecular and intramolecular epitope spreading occur in tertiary lymphoid tissues in the central nervous system.350 More recently, it has become apparent that Tregs can populate tertiary lymphoid tissues,338 again suggesting that manipulating the immune response at the local site is an avenue of control.

Plasticity and Adaptability of Tertiary Lymphoid Tissues Tertiary lymphoid tissues are the most plastic and adaptable of the lymphoid tissues. First, lymphoid neogenesis can be induced by a variety of stimuli. Their nimbleness in this regard suggests that they might represent the most primitive tissues in the immune system. Tertiary lymphoid tissues can be “turned off” (ie, resolve) upon removal of the initial stimulus or after therapeutic intervention. The destruction of the islets of Langerhans β cells in type I diabetes mellitus is an example of a situation in which removal of the antigen stimulus is accompanied by tertiary lymphoid tissue resolution. Antibiotic treatment results in the resolution of tertiary lymphoid tissues and even MALT lymphomas.354 Treatment has been shown to resolve some established tertiary lymphoid tissues, reversing insulitis and protecting against diabetes in nonobese diabetic mice.355 Such treatment can also “turn off” established tertiary lymphoid tissues in a mouse model of collagen-induced arthritis.356 These studies are similar to those described previously regarding the plasticity of lymph nodes after mice are immunized or treated with LTβR-Ig,198,199 again emphasizing the commonality of these tissues.

CONCLUSION The immune system depends on a remarkable organization of tissues and cells. The organs have defined functions that include generation of an immune repertoire (primary lymphoid organs) and responding to antigen (secondary lymphoid organs and tertiary lymphoid tissues). Development of primary and secondary lymphoid organs depends on precisely regulated expression of cytokines, chemokines, and adhesion molecules. Similar signals regulate the transition from inflammation to tertiary lymphoid tissues. Chemokines and adhesion molecules regulate trafficking in and out of lymphoid organs. Secondary lymphoid organs are remarkably plastic in their response to antigenic assault and adapt with changes in expression of chemokines and adhesion molecules to maximize encounter of antigen with antigen-specific cells. Tertiary lymphoid tissues, characteristic of many pathologic states, may actually represent the most primitive form of lymphoid tissues in their even greater plasticity and ability to develop directly at the site of antigen exposure.

ACKNOWLEDGMENTS The authors thank Myriam Hill for assistance in figure preparation. This work was supported National Institutes of Health grants CA16885, DK57731, and HL098711 (NHR), a fellowship from Leukemia and Lymphoma (LAT) and a Richard A. Gershon Fellowship (NAG).

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4

Evolution of the Immune System Martin F. Flajnik • Louis Du Pasquier

INTRODUCTION Defense mechanisms are found in all living things, even bacteria, where they are surprisingly elaborate. Although new adaptive or adaptive-like (somatically generated) immune systems have been discovered in invertebrates and the jawless fish, adaptive immunity based upon immunoglobulin (Ig), T-cell receptors (TCRs), and the major histocompatibility complex (MHC) is only present in jawed vertebrates (gnathostomes); because of clonal selection of lymphocytes, positive and negative selection in the thymus, MHCregulated initiation of all adaptive responses, etc., the major elements of the adaptive immune system in gnathostomes are locked in a coevolving unit that arose in concert over a short period of evolutionary time.1–3 In addition, a large cast of supporting players, including a large array of cytokines and chemokines, adhesion molecules, costimulatory molecules, and well-defined primary and secondary lymphoid tissues, evolved in the jawed vertebrates as well. This scheme was superimposed onto an innate system inherited from invertebrates, from which many innate molecules and mechanisms were coopted for the initial phase of the adaptive response and others for effector mechanisms at the completion of adaptive responses. Over the last 10 years, we have learned that various components of the innate immune system are also incredibly complex and locked as well in a coevolving unit.4,5 In each group of organisms, one can detect a basic set of immune functions, and these are employed in different ways in representative species. For example, we detect that in the jawed vertebrates fine tuning, or adaptations, or even degeneration of molecules/mechanisms in each group (Taxon) are observed, and not a steady progression from fish to mammals as is documented for most other physiologic systems.6 Given that all the canonical adaptive immune system features are present in cartilaginous fish and apparently none were lost (except in particular groups of organisms that will be discussed), differential utilization of defense molecules rather than sequential installation of new elements is observed. In this chapter, there are only isolated cases of increasing complexity in immune systems, but many examples of contractions/ expansions of existing gene families; thus, “more or less of the same” rather than “more and more new features” is the rule. We observe a bush growing from a short stem rather than a tall tree with well-defined branches, and thus deducing the primitive, ancestral traits is not clear cut.

GENERAL PRINCIPLES OF IMMUNE SYSTEM EVOLUTION The Common Ancestor Hypothesis Figure 4.1 displays the extant animal phyla ranging from the single-celled protozoa to the metazoan protostome and deuterostome lineages. It is often suggested or assumed by those unfamiliar with thinking in evolutionary terms that molecules or mechanisms found in living protostomes, like the well-studied arthropod Drosophila, are ancestral to similar molecules/mechanisms in mouse and human. While this is true in some cases, one must realize that Drosophila and humans have taken just as long (over 900 million years) to evolve from a common ancestral triploblastic coelomate (an animal with three germ layers and a mesoderm-lined body cavity, features shared by protostomes and deuterostomes) that looked nothing like a fly or a human, and thus Drosophila is not our ancestor (ie, the manner by which flies and humans utilize certain families of defense molecules may be quite different and both may be disparate from the common ancestor). Thus, understanding of how model invertebrates and vertebrates perform certain immune tasks is an important first step in our understanding of a particular mechanism, but we only deduce what is primordial or derived when we have examined similar immune mechanisms/molecules in species from a wide range of phyla. We will touch upon each of the defense molecule families and will emphasize those which have been conserved evolutionarily and those that have evolved rapidly.

Rapid Evolution of Defense Mechanisms Immune systems are often compared to the Red Queen in Alice in Wonderland, (Red Queen’s Hypothesis7,8) who must continually keep moving just to avoid falling behind. Because of the perpetual conflict with pathogens, the immune system is in constant flux. This is exemplified by great differences in the immune systems of animals that are even within the same phylogenetic group (eg, mosquito [Anopheles] and fruitfly [Drosophila], both arthropods, or human and mouse, both mammals). In fact, in contrast to what was believed previously, defense mechanisms are extremely diverse throughout the invertebrate phyla, and Kepler et al. have aptly and succinctly described the situation in the title of a recent review: “not homogeneous, not simple, not well understood.”9 In the jawed vertebrate (or gnathostomes), the most rapidly evolving system is an innate system, natural killer (NK) cell recognition, governed by different classes (superfamilies) of receptors in mice and humans, and also extremely plastic even

67

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68

Jawed vertebrates (58,000)

Jawless vertebrates

900 MYA

Deuterostomes

(60)

Cephalochordates (25)

Urochordates (3,000)

Echinoderms

950 MYA

Bilateria

(7,000)

Platyhelmiths (20,000)

Annelids (9,000)

Mollusks (70,000)

Nemerteans

Protostomes

1300 MYA

(1,000)

Sipunculids (250)

Nematodes (20,000)

Arthropods (770,000)

Cnidaria (10,000)

Porifera

1600 MYA

(7,000)

Sharks

Mammals LRR, TLR, NLR Cʹ, SRCR, TNF, IL-17, IgSF, NK, lectin, AMP, IgSF V-C1,

Ig/TCR, RAG1/2, APOBEC/AID, MHC I/II, somatic DNA, Rel, TIR , PGRP

Lamprey and Hagfish

LRR, TLR, lectins, Cʹ, IgSF V-C, APOBEC/AID, somatic DNA, VLR, Rel, TIR , SRCR, IL-17, IL-8

Amphioxus

LRR, TLR, lectins, Cʹ/TEP, PPO, IgSF V, VCBP, TNF, Rel, TIR , B1-3GRP

Ciona, Botryllus

LRR, TLR, lectins, PPO, TNF, Cʹ/TEP, IgSF V-C1, NK-like, "MHC", Rel, TIR , B1-3GRP

LRR, TLR (many!), Toll, NLR (many!), Cʹ/TEP, TNF, IL17 (many!), SRCR (many!), PGRP, β1-3GRP, lectin, PPO, IgSF V, RAG1/2, 185/333, Rel, TIR , B1-3GRP

Sea urchins

Flatworms Earthworms

Hemolysins, TNF(?), NK-like, PPO, B1-3GRP

Snails, oysters

PPO, lectins, AMP, IgSF, MDM, FREP, somatic DNA, Rel, TIR , IL-17, B1-3GRP

Ribbon worms Peanut worms

NK-like

C. elegans

Toll (but not immune), IgSF, lectins, AMP, PGRP, B1-3GRP

Flies, mosquitos, shrimps

LRR, Toll, lectins, PPO, IgSF, AMP, Cʹ/TEP, PGRP, GNBP, penaeidin, DSCAM (extensive diversity via splicing), Dicer, JAK/STAT, Rel, TIR

Corals, hydra

Cʹ, PPO, “MHC”, B1-3GRP, Toll, Rel, TIR , IL-1R-like

Sponges

IL-1R-like, IgSF, lectins, PPO, “MHC”, Rel, TIR , B1-3GRP

Protozoa (50,000)

Plants (250,000)

LRR (NBS-LRR), TIR, convergent innate pathways

FIG. 4.1. Major Animal Groups and Immune Mechanisms/Molecules Described to Date in Each Group. The first box on the left in each row describes the animal taxon and the approximate number of species in that group. The next box shows specific examples of species or subgroups. The third box lists molecules/mechanisms found in each group: underlined terms indicate somatic changes to antigen receptors or secreted molecules. Figure modified from Hibino et al.48 and Flajnik and Du Pasquier.61 See Table 4.1 for definition of the acronyms.

within the same species, exemplified by the large number of killer immunoglobulin superfamily (KIR) haplotypes found in humans.10 Studies of Ig superfamily (SF) genes expressed in the nervous system and immune system showed definitively that the immune system molecules evolve at a faster rate.11 Finally, rapid evolution of immune system molecules and mechanisms is the general rule, but molecules functioning at different levels of immune defense (recognition, signaling, or effector) can evolve at widely varying rates.

Conservation of Defense Mechanisms While the immune system is the most rapidly evolving physiologic system, nevertheless there is also deep conservation

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of defense families and mechanisms. Klein11a has compared this dichotomy to the two-headed god Janus, the major idea being that certain basic mechanisms/functions are obligatory for immune systems to function, but they still must evolve rapidly to avoid pathogen subterfuge. For example, MHC class I molecules have similar structure/function/ features in all gnathostomes, but even within groups of primates class I genes are not orthologous (ie, they can be derived from totally different ancestral class I genes10). So, the idea is to preserve vital immune functions but rapidly modify the gene or pathway to outwit the pathogen. Additionally, certain features are conserved (eg, development and function of conventional αβ T cells), but a second, similar system, can be exploited in very different ways in closely related

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species (eg, the function[s] of γδ T cells). The “Janus paradigm,” therefore, can be quite useful when examining any pathway in the immune system.

Convergent Evolution Early on in the comparative study of immunity, it was assumed that the same features appearing in different taxa proved that they were present in the common ancestor as well (ie, they were submitted to divergent evolution). While this dictum still holds true and establishes one of the dogmas of comparative immunology, later we discovered, because of the aforementioned rapid evolution of immune systems, convergence of similar functions has occurred in evolution (ie, the same function or even molecular conformation has arisen independently in different organisms, sometimes in species that are relatively closely related). While we will discuss several cases of convergent evolution throughout the chapter, for frame of reference, the NK cells, which use different receptor families in primates and rodents to achieve precisely the same ends of recognizing polymorphic MHC class I molecules, is a striking example of convergence.12 Additionally, the emergence of a lymphocyte-based somatic generation of two entirely different receptor families for the same function in jawless and jawed vertebrates is another remarkable illustration of convergent evolution.13 Finally, in innate immunity, the cytosolic nucleotide-binding domain leucine-rich repeat (NLR) proteins, despite their striking similarity in deuterostomes and plants, arose (at least) twice in evolution.14

Multigene Families Genes involved in immunity are often found in clusters, with extensive contraction and expansion via so-called birth and death processes.15 It is well known that such gene clusters can change rapidly over evolutionary time due to unequal recombination crossovers and gene conversion (and not only in the immune system, but in any cis-duplicating gene family). Often, families of related immune genes— especially those involved in recognition events—are found near the telomeres of chromosomes, presumably because this further promotes gene-shuffling events. Nonclassical MHC class I loci, NK receptors, and NLRs are conspicuous examples of this phenomenon, again believed to be a consequence of the race against pathogens. We will discuss many examples of how such multigene families have been exploited in different species throughout the chapter. Gene duplication, either in the clusters mentioned above or as a consequence of en bloc duplications, certainly has been a major feature of immune system diversity and plasticity. The two types of duplications are not equivalent, the former being more taxon-specific and the latter (en bloc) having a lasting impact on the entire system. It is now universally accepted that two genome-wide duplications (the so-called 2R hypothesis2,16 ; see Fig. 4.13) occurred early in vertebrate history, tracking very well with the emergence of the Ig/TCR/MHC-based adaptive immune system.2,17 This theory forms the basis for much that will be discussed concerning the evolution of the vertebrate adaptive immune

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response as one can track the emergence of new immune mechanisms, as well as fine tuning of old ones, by examining the paralogous syntenic groups of genes. Our view is that these genomewide duplications were as crucial as the “RAG transposon”18–20 in the development of the Ig/TCR/ MHC-based adaptive immunity. In addition, the common ancestor of bony fish (teleosts) underwent a third round of genomewide duplication, which many believe to have played the major role in these fishes’ unique outlier status regarding immune system genetics and physiology.2,21

Polymorphism In addition to gene duplications, polymorphism also augments the diversity of immune recognition within a population. It can be generated any time during the history of a gene family of either receptor or effector molecules: MHC, toll-like receptors (TLRs), Ig, TCRs, NK receptors (NKRs) and related molecules, and antimicrobial peptides (AMPs) are just a few examples. Polymorphism, either within the gene itself or in its regulatory elements, provides populations with flexibility in function of the changing pathogenic environment. This subject, central to the studies on MHC, leukocyte receptor complex (LRC), and NK cell complex (NKC), is becoming well documented for immunity-related genes in insects as well. In Drosophila, polymorphism in regulatory networks is indeed expected as parasites often target their elements.22 In humans, there are two major NK cell haplotypes found in all subpopulations, which are under “balancing selection.” In such a case, the polymorphisms presumably adopt a division of labor required for the maintenance of the species: one haplotype is believed to be involved in protection from virus and the other perhaps for promoting reproduction.23

Somatic Generation of Diversity Somatic modifications can take place at multiple levels to generate immune system diversity. Long believed to be the sole domain of jawed vertebrates, modifications at the deoxyribonucleic acid (DNA) level via somatic hypermutation, gene conversion, and rearrangement (primary and secondary [eg, receptor editing]) irreversibly modify genes within an individual. The well known V(D)J joining, class switch recombination (CSR), and somatic hypermutation (SHM) are examples of this processes in the IgSF receptors of jawed vertebrates, but modifications to genomic DNA can also occur in the jawless fish and some invertebrates.7,13 The list of organisms undergoing such diversity of germline immune genes will only grow as more organisms are examined and more genome and expressed sequence tags (EST) sequencing projects are undertaken (see Fig. 4.1). Alternative splicing can be a source of tremendous diversity in some gene families encoding receptors involved in immunity in insects and crustaceans. The Down syndrome cell adhesion molecule (DSCAM) gene in several arthropods (described in detail in the following) was shown to generate enormous diversity via ribonucleic acid (RNA) processing.7,24 In the vertebrates, this mechanism is important in

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determining the function of different molecules, best known for the Igs (transmembrane [TM] versus secreted forms, as well as inflammatory versus neutralizing forms in nonmammalian vertebrates). Further diversity can be obtained by the assembly of multichain receptors in which different components are combined. The classical example in the jawed vertebrate adaptive immune system is that of Ig light (L) and heavy (H) chains of antibodies but similar combination can occur with insect peptidoglycan-recognizing proteins (PGRP), vertebrate TLRs, and many others. The study of the evolution of immunity has resulted in a fundamental appreciation of the heart of immunity, both innate and adaptive. Especially now, with studies in many plants and in both invertebrate and vertebrate animals, we can see what features have been conserved and when they arose. “Simple” genetic models such as Drosophila and Candida elegans provide a glimpse into these elemental mechanisms and also allow us to remove the clouds that surround studies of mouse and human, with so many interconnected pathways. As mentioned, examination of the well-studied mammalian models in combination with studies of invertebrates allows us to deduce the condition of the common ancestor. Interestingly, major pathways of defense known to all immunologists, such as the ones involving TLR, JAK-signal transducer and activator of transcription (STAT), NOTCH, and tumor necrosis factor (TNF) pathways, clearly arose in an early animal ancestor and have been perpetuated in derived fashions in all major taxa. We shall see how these pathways are manipulated in different animals, always drawing upon the best-known mammalian model as a foundation (whenever possible).

MAJOR GENE FAMILIES INVOLVED IN IMMUNITY Defense molecules can be composed of a very large number of protein folds, some of which are clearly some used to a large extent.25–27 Some of the most common families (Fig. 4.2) are IgSF, leucine-rich repeats (LRRs), C-type lectins, and the

TLR (LRR)

NLR (LRR)

VLR (LRR)

DD NACHT CARD

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Leucine-Rich Repeats LRRs consist of 2 to 45 motifs of 20 to 30 amino acids in length (XLXXLXLXXNXHXXHXXXXFXXLX) that fold into an arc shape (see Fig. 4.2).28 Both the concave and convex parts of the domain have been shown to interact with ligands. Molecular modeling suggests that the conserved pattern LxxLxL is sufficient to impart the characteristic horseshoe curvature to proteins with 20- to 30-residue repeats. LRRs are often flanked by cysteine-rich domains. LRRs occur in proteins ranging from viruses to eukaryotes and are found most famously in the toll/TLRs, as well as tyrosine kinase receptors, cell-adhesion molecules, resistance (R) factors in plants found at the cell surface, and in the cytosol, extracellular matrix (ECM)-binding glycoproteins (eg, peroxidasin), and are involved in a variety of protein-protein interactions: signal transduction, cell adhesion, DNA repair, recombination, transcription, RNA processing, disease resistance, apoptosis, and the immune response. LRR-containing proteins can be associated with a variety of other domains, whether they are extracellular (LRR associated with IgSF or fibronectin [FN] type III) or intracellular (caterpillar family LRR associated with a variety of effector domains; see subsequent discussion). In these chimeric molecules, the LRR moiety is involved in recognition, most likely due to its extraordinarily malleable structure. There are at least six families of LRR proteins, characterized by different lengths and consensus sequences of the repeats.29 Repeats from different LRR subfamilies never occur simultaneously and have most probably evolved independently in different organisms.

TCR (IgSF) PGRP

TIR

TNF family, and certain other domains in immune recognition (eg, scavenger receptor cysteine-rich [SRCR]). All of the domains discussed in this chapter are found in Table 4.1 and a few are displayed in Figure 4.2. As a means of introduction, two of these families, which constitute the “top two” quantitatively, will be described in the following.

B1-3GRP

C-type lectin

FIG. 4.2. Major Molecular Families Described in the Text and Representatives of Each Family: Leucine-Rich Repeats (LRRs), Immunoglobulin Superfamily (IgSF), Peptidoglycan-Recognition Protein (PGRP), a1-3 Glucan Recognition Protein (a13GRP), and C-Type Lectins. Representative structures are shown above for LRR (tolllike receptor, nucleotide-binding domain leucine-rich repeat [NLR], variable lymphocyte receptor), IgSF, PGRP, β1-3GRP, and C-type lectin. Other acronyms are defined in Table 4.1. For the NLR model, the echinoderms have N-terminal death domains, whereas all other animals have caspaserecruitment domains. Figure modified from Hibino et al.48

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TABLE

4.1

EVOLUTION OF THE IMMUNE SYSTEM

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Molecules and Abbreviations Found Throughout the Text

Acronym/Defense Molecule

Full Name

Function

AID APOBEC

Activation-induced cytidine deaminase Apolipoprotein B mRNA editing enzyme catalytic polypeptide Aorta/gonad/mesonephros Antimicrobial peptide Agnathan paired antigen receptor Avirulence protein Factor B Beta 1-3 glucan-recognizing protein Complement Caspase-recruitment domain CARD, transcription enhancer, R(purine)-binding, pyrin, lots of leucine repeats Complementarity-determining region Class switch recombination Death domain Down syndrome cell adhesion molecule

SHM/gene conversion/CSR Innate immunity (antiviral)

AGM AMP APAR AVR Bf B1-3GNP C′ CARD CATERPILLER or CLR CDR CSR DD DSCAM ECM ETI FBA FcRN FN3 FREP FuHC GALT GPI Hemolysin ICE Ig IgSF IFN IMD IRF IRG ITAM ITIM JAK

Extracellular matrix Effector-triggered immunity F box–associated domain Fc receptor neonatal Fibronectin type III repeat Fibrinogen-related protein Fusion histocompatibility Gut-associated lymphoid tissue Glycophosphatidylinositol

KIR lectins LITR LMP LRC

Killer IgSF receptor For example, galectin, C-type, S-type Leukocyte immune-type receptors Low-molecular-weight protein Leukocyte receptor complex

LRR MAC MACPF MASP MBP (or MBL) MDM MHC MIF MyD88 (also dMyD88) NITR NK cell NKC

Leucine-rich repeat Membrane-attack complex MAC-perforin domain MBP-associated serine protease Mannose-binding protein (lectin) Mollusk defense molecule Major histocompatibility complex Macrophage inhibitory factor (Drosophila) Myeloid differentiation primary response gene 88 Novel immune-type receptors Natural killer cell Natural killer cell complex

NKR

Natural killer cell receptor

Interleukin-converting enzyme Immunoglobulin Immunoglobulin superfamily Interferon Immune deficiency Interferon regulatory factor Immunity-related GTPases Immunoreceptor tyrosine-based activation motif Immunoreceptor tyrosine-based inhibitory motif Janus kinase

Intraembryonic origin of hematopoietic cells Innate immunity (eg, defensins) Similarities to Ig/TCR and NKRs Pathogen effector recognized by plant NLR Enzyme of C′ cascade Binds to gram-negative bacteria Innate/adaptive immunity Domain in intracellular defense molecules Apoptosis/immunity/inflammation Portion of Ig/TCR that binds to antigen Adaptive humoral immunity modification Cytosolic interacting domain Insect immune (adaptive?) defense and neuron specification Immunity in plants triggered by NLR Intracellular domain MHC-like FcR Domain found in many innate molecules Mollusk (adaptive?) defense Histocompatibility locus in tunicates Lipid linkage to cell membrane (eg, VLR) Cell lysis IL-1β processing Adaptive immunity Innate/adaptive immunity Innate (type I)/adaptive (type II) immunity Insect innate defense Innate (transcription factor) Innate immunity Signaling motif for NK and antigen receptors Signaling motif for NK and antigen receptors Signaling molecule associated with cytokine receptors NK cell receptor Many (eg, NKRs, selectins) Fish NK-like receptors of the IgSF Proteasome subunit Gene complex containing KIR and many IgSF molecules Innate/adaptive immunity module C′, pore-forming Potential pore former Lectin C′ pathway Lectin C′ pathway IgSF defense molecule T-cell recognition; innate immunity Innate immunity; inflammation TLR adaptor Teleost fish NK-like receptors of the IgSF Vertebrate innate cellular immunity Gene complex with many C-type lectin genes (especially NK cells) Receptor on NK cells

(continued)

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ORGANIZATION AND EVOLUTION OF THE IMMUNE SYSTEM

Molecules and Abbreviations Found Throughout the Text (Cont.)

Acronym/Defense Molecule

Full Name

Function

NALP

NACHT leucine-rich repeat and PYD-containing protein Nucleotide-binding domain LRR Nuclear factor-κB (Rel homology domain) NACHT leucine-rich repeat protein Nucleotide oligomerization domain protein Nitric oxide synthase Pathogen-associated molecular pattern Programmed cell death

Intracellular PRR

NBD-LRR NFκB NLR NOD NOS PAMP PCD Penaedins PGRP

Peptidoglycan-recognition protein

PPO PRR PMSB Polμ PPO PYD RAG RFP-Y RFX RIG

Propolyphenol oxidase Pattern-recognition receptor Proteasome subunit beta subunit DNA polymerase μ Prophenoloxidase Pyrin domain Recombination-activating gene Restriction fragment polymorphism-Y Regulatory factor X Retinoic acid-inducible gene

RSS

Recombination signal sequence

RXR SHM SPE SRCR TAK

Retinoid X receptor Somatic hypermutation Spaezle-processing enzyme Scavenger receptor cysteine-rich TGF-β activated kinase

TAP (and TAP-L) TAPBP TCR TdT TEP TGF TNF UPD

Transporter associated with antigen processing TAP-binding protein T-cell receptor Terminal deoxynucleotidyl transferase Thioester-containing protein Transforming growth factor Tumor necrosis factor Unpaired

TLR

Toll-like receptor

TM TNF TRIM

Transmembrane Tumor necrosis factor Tripartite motif-containing proteins

V-, C1-, C2-, I-

Variable, constant 1 and 2, intermediate IgSF domain Guanine exchange factor, the “onc F” proto-oncogene Variable domain chitin binding Variable lymphocyte receptor

VAV VCBP VLR WKRY XMIV XNC 185/333

Xenopus MHC-linked IgSF V region Xenopus nonclassical Sea urchin defense molecule

Motif of intracellular defense molecules Evolutionarily conserved transcription factor Intracellular PRR Intracellular PRR Intracellular killing innate defense molecule Conserved target epitopes on pathogens Many pathways Defense molecule in shrimp Gram-positive bacteria defense family; receptor and effector Plant/invertebrate defense (melanization) Recognize PAMP, innate/adaptive immunity Proteolytic member of 20S proteasome Error-prone polymerase (related to TdT) Invertebrate defense molecule Domain in intracellular defense molecules Ig/TCR rearrangement Chicken nonclassical MHC gene cluster Transcription factor, class I regulation Intracellular double-stranded RNA recognition DNA element next to Ig/TCR gene segments necessary for RAG-mediated rearrangement Transcription factor encoded in MHC Adaptive humoral immunity Insect defense molecule in toll cascade Innate immunity recognition molecule ubiquitin-dependent kinase of innate pathways Rransports peptides from cytosol to ER lumen Tethers TAP to class I Adaptive defense Involved in Ig/TCR rearrangement Opsonization (like C3) Immunosuppressive cytokine Protostome cytokine induced by viral infection Innate receptor on the cell surface or in endosomes Proinflammatory cytokine (and family) Large family of cytosolic innate defense molecules IgSF domain types Encoded in MHC, involved in adaptive signaling pathways Amphioxus defense molecule Agnathan adaptive defense molecule Plant transcription factor used to upregulate defense genes (analog of NF-κB) Xenopus MHC-linked NKR-like genes Xenopus class Ib cluster (Adaptive?) Defense

DNA, deoxyribonucleic acid; ER, endoplasmic reticulim; IL, interleukin; mRNA, messenger ribonucleic acid; RNA, ribonucleic acid.

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LRR-containing proteins are involved in immunity from plants to animals. The functions in the immune systems range from control of motility of hemocytes and lymphocytes30 to specific recognition of antigens via a novel system of gene rearrangement (the variable lymphocyte receptors [VLR] described in the following; see Fig. 4.9). LRRs can occur in soluble forms, the ECM, in the cytosol, or as TM forms, either integral membrane proteins or glycophosphatidylinositol (GPI)-anchored. The bottom line is that because of its basic structure and malleability, the LRR module was locked in early in evolution as an ideal motif for recognition of essentially any ligand.

Immunoglobulin Superfamily IgSF domains are encountered in a very large number of molecules in the animal kingdom (see Fig. 4.2).31 They are found intracellularly (eg, connectin) or as cell adhesion molecules, many of which are in the nervous system (eg, the neural cell adhesion molecule, NCAM), coreceptors and costimulatory molecules of the immune system (eg, cluster of differentiation [CD]79, CD80), molecules involved in antigen presentation to lymphocytes (eg, class I molecules), certain classes cytokine receptors (eg, interleukin [IL]-1R), and of course Ig (and TCR), where they were first characterized and were bestowed with their name (Ig). They can be associated with other domains such as FN (eg, titin and FREP) and LRRs,32 or they can be the sole constitutive elements of the polypeptide chain often associated to a transmembrane segment and a cytoplasmic tail (or GPI-linked). The β barrel IgSF structure was adopted independently in other families such as cadherins, calycins, lipocalin, etc., and the super (or über) family has hundreds of members and has been selected for several different functions. These functions are somehow related, almost all involved in protein-ligand interactions. The vertebrate lymphocyte surface can express 30 different IgSF members simultaneously. IgSF domains are commonly classified according to different domain constitution in their β strands and loops.31,33 All conform to the stable shape of a β barrel consisting of two interfacing β sheets, usually linked by a disulfide bridge. There are three types of domains: variable (V), and two types of constant (C1 and C2); the so-called I set domain is intermediate between the C1 and C2. The V domain is most complex with more strands (C′ and C″), which make up complementarity determining region (CDR) 2 in conventional Igs and TCRs. C1 domains lack these strands entirely, and C2/I domains have varying sizes in the C′/C″ region. V domains, either alone (eg, the new antigen receptor [NAR]) or in association with another V domain (eg, Ig H/L), recognize the antigenic epitope and are therefore the most important elements for recognition. Domains with the typical V fold, whether belonging to the true V-set or the I-set, have been found from sponges to insects (eg, amalgam, lachesinm and fascicilin) and even in bacteria. The mollusk fibrinogenrelated proteins (FREPs, described in the following) have one or two V-like domains at their distal end, associated with a fibrinogen-like domain. For V domains, the interface between dimers is the beta strand bearing the C, C′, C″, F,

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and G strands, so that in Igs the CDR3 are in the center of the binding site; for C domains, the other beta strand bearing the A, B, D, and E strands forms the interface. The binding capacities of V domains in molecules besides Ig/TCR can reside in different areas of the molecule (interstrand loops, A-A′ strand, F strand), while in Ig/TCR, CD8, and certain NKR, these regions are the targets for variation in shape and charge. The binding capacities can be modulated whether one domain acts as a single receptor unit (eg, IgNAR) or whether it is associated with a contiguous domain (eg, KIR, variable domain chitin-binding protein [VCBP]) or with another polypeptide chain (eg, TCR, Ig). In the case of a dimer, the binding capacity can again be modulated by the presence or the absence in the G strand of a diglycine bulge, which can modify the space between the faces of the Ig domain. In several cases, the sites responsible for binding are known (KIR, Ig, TCR); in many other cases, they are not known but inferred from crystal structures and/or variability plots (leukocyte immune-type receptor [LITR], chicken Ig-like receptor [CHIR], triggering receptor expressed on myeloid cells [TREMs], DSCAM, hemolin).

HEMATOPOIEIS AND CELL TYPES IN THE INVERTEBRATES Invertebrate Cell Types Examples of conservation of fundamental mechanisms of genetic control of developmental pathway between protostomes and deuterostomes, even in the absence of homology of the cells or organ considered, are accumulating: the organization and expression of the homeotic gene clusters and eye formation through the function of a complex of proteins including Pax-6.34 The cell types involved, besides direct interaction with the external layer of cells on the skin, or external teguments, have been specialized cells of mesodermal origin devoted to defense. This is true for all coelomates where effector cells have been identified, but recent data have shown that cnidarian diploblastic organisms that lack mesoderm also have many of the same genetic systems as the coelomates35 (see Fig. 4.1). The cells can be circulating or sessile, and often are found associated with the gut. Several morphologically distinct hemocyte types in insects cooperate in immune responses: they attach to invading organisms and isolate them, trapping larger organisms in nodules or forming large multicellular capsules around them. Indirect evidence for the role of hemocytes in immune responses can be derived by contrasting properties of such cells in healthy and parasitized animals (ie, modifications in adherence and opsonic activity). All animals show heterogeneity of the free circulating cells, generically called hemocytes (arthropods), coelomocytes, amebocytes (annelids, mollusks, and echinoderms), or leukocytes (sipunculids). However, the repertoire of insect “blood cells” is clearly less heterogeneous than that of vertebrates. Basically, three or four types of cell lineages can be identified in Drosophila (Fig. 4.336): plasmatacyte, crystal cells, and lamellocytes, and an equivalent number in Lepidoptera (butterflies). The functional roles they play consist of immune defense, disposing of apoptotic and other debris, contributing to the ECM, and modeling of the

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Cytokines (UPD)

PGRP-LC

Lymph glands

Serine proteases/ serpins Hemolymph PRRs (PGRP/ β1-3GRP)

ENCAPSULATION

Plasmatocyte

PHAGOCYTOSIS

COAGULATION

Imd JAK/STAT

dMyd88

Fat body

Prohemocyte

Lamellocyte

Domeless

Toll

Crystal cell

MELANIZATION

– AMP – Serine proteases – TEPs – Clotting factors – DSCAM

FIG. 4.3. Types of Immune Responses and Cells in Insects, with Drosophila as the Prototype. Secreted defense molecules are made by fat body cells in response to pathogens, which either act as direct effector molecules or feed back on hemocytes to stimulate their defense functions. Unpaired (UPD) is produced after virus infection, stimulating defense molecule upregulation via the JAK/signal transducer and activator of transcription pathway. Not shown is the RNAi pathway, also induced upon virus infection. Details on stimulation of the toll and immune deficiency pathways are found in Figure 4.4. This figure was modified from Lemaitre and Hoffmann.36

nervous system. The immunity role encompasses phagocytosis, encapsulation, and sometimes production of effector molecules (see Fig. 4.336). These roles all require recognition of pathogen-associated molecular patterns (PAMPs) or self-derived defense molecules (ie, opsonization) at the cell surface.37–40 Only in a few organisms has the characterization of hemocyte lineages gone beyond morphologic or basic physiologic functions. Among these free circulating cells are always one or more types that can undergo phagocytosis. Different cells participate in encapsulation, pinocytosis, and nodule formation, and can upon stimulation produce a great variety (within an individual and among species) of soluble effector molecules that may eliminate the pathogen. In an attempt to integrate all of the data available in invertebrates, Hartenstein has proposed a unified nomenclature of four basic types: prohemocytes, hyaline hemocytes (plasmatocytes or monocytes), granular hemocytes (granulocytes), and eleocytes (chloragocytes).37 These designations will be found in the following description of the blood cell types. Earthworm (annelid) coelom-tropic coelomocytes are called eleocytes. They contain glycogen and lipid and are considered of the same lineage as the chloragocytes involved in the production of immune effector molecules such as fetidin or lysenin. The phagocytic cells of annelids are apparently granular “leukocytes” derived from the somatopleura

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and involved in wound healing, whereas the ones derived from the splanchnopleura participate in immunity. Heterogeneity of annelid coelomocytes is not encountered in primitive oligochaetes or in hirudinae (leeches). Phagocytic coelomocytes show an acid phosphatase activity and a beta glucoronidase activity.41 The large coelomocytes and free chloragocytes (eleocytes) in the typhlosole of Eisenia foetida appear to produce the bacteriolytic and cytolytic factor lysenin.42 From electron microscopy studies, macrophagelike cells seem to be involved in graft rejection. In the closely related sipunculid phylum, two main cell types can be identified in the blood: erythrocytes (a rare occurrence in invertebrates) and granular leukocytes. The latter are capable of cytotoxicity and even have dense granules reminiscent of vertebrate “NK cells.”43 Two developmental series have been described in mollusks, the hyaline and granular cells, but cephalopods seem to have only one lineage. They participate in encapsulation, with hemocytes adhering around the foreign body like Drosophila lamellocytes. Phagocytosis is carried out by the wandering granular cells, which resemble vertebrate monocytes/macrophages. In oysters, electron microscopy revealed different types of circulating hemocytes, including granular hemocytes resembling the granulocytes of sipunculus mentioned previously.44,45 In crustaceans, the situation is similar to that in mollusks, with three main populations identified based again on the presence of granules in the cytoplasm.

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The hyaline cells are involved in the clotting process and the granular cells in phagocytosis, encapsulation, and the prophenoloxidase (PPO) pathway. The hematopoietic organ is located on the dorsal and dorsolateral regions of the stomach.39 Crustacean hemocytes can now be cultured and their response to virus can be examined,46 and markers of the three hematopoietic lineages are available.47 In insects, the so-called prohemocytes are believed to be stem cells. They are only found in the embryonic head mesoderm and the larval lymph glands but not in the hemolymph. However, prohemocytes are frequent in both the hemolymph and hematopoietic organs of the lepidopteran Bombyx (silkmoth). Plasmatocytes of Drosophila have a phagocytic function. This type of hemocyte is equivalent to the granulocytes of Bombyx, which play a key role in phagocytosis in larvae. Lamellocytes seem to be unique to Drosophila, but they are probably the equivalent of the lepidopteran plasmacytoid cells. Their precursors reside in the larval lymph gland, where they differentiate in response to macroscopic pathogens, following a brief phase of mitosis linked to the presence of the pathogens and under hormonal control via ecdysone. The transcription factors (GATA, Friend-of-GATA, and Runx family proteins) and signal transduction pathways (toll/NF-κ B, Serrate/Notch, and JAK/STAT) that are required for specification and proliferation of blood cells during normal hematopoiesis, as well as during hematopoietic proliferation that accompanies immune challenge, have been conserved throughout evolution. The specific differentiation of lamellocytes requires the transcription factor Collier. The mammalian early B-cell factor, an ortholog of Collier, is involved in B-cell differentiation in mice. The Drosophila crystal cells are responsible for melanization through the PPO system (see subsequent discussion). In silkworm oenocytoids, crystallike inclusions are also found, but they disappear later after bleeding.36,37,40 Echinoderm coelomocytes express a diversity of effector functions, but no studies of lineages have been performed. In echinoderms, the number of different coelomocytes may vary according to the particular family. The sea urchin is endowed with at least four cell types, only one of which only is phagocytic and corresponds to the bladder or fi liform forms. Another type is described as the round vibrating cell involved in clotting. Pigment cells (red spherule cells) have been detected ingesting bacteria; the morphology of phagocytic cells can vary enormously, precluding any easy classification.48 In tunicates, amoeboid cells circulate in the blood and are involved in a large number of processes, such as clotting, excretion, nutrition budding, and immunity. Large numbers of blood cells are present (average of 107 per mL) in the blood of ascidians such as Ciona. Hemoblasts are considered to be undifferentiated cells, perhaps the equivalent of the prohemocytes of arthropods or the neoblasts of annelids. Blood cells in ascidians proliferate in the connective tissue next to the atrium. The pharyngeal hematopoietic nodule of this animal contains a large number of hyaline and granular cells called “leukocytes” with supposed intermediary forms of differentiation between blast and granular mature types.

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The granular form is likely to be involved in postphagocytic activity, like in earthworms.49 Adoptive transfer of alloimmunity in the solitary tunicate Styela can be achieved via lymphocyte-like cells. In Amphioxus, cells with phagocytic capacity have been identified in the coelom with a morphology resembling more the phagocytic echinoderm cells than urochordate blood cells, a fact that is consistent with the new systematic positions of amphioxus and echinoderms.50 Both free cells and the lining of the perivisceral coelom are able to phagocytose bacteria. Cells with the morphologic appearance of lymphocytes and expression of lymphocyte-specific genes were detected in this species, the earliest identification of such cells in phylogeny.51

Hematopoiesis in the Invertebrates The history of the hemocytes is associated with that of the mesoderm among triploblastic organisms. The bilaterian ancestor was most likely a small acoelomate or pseudocoelomate worm similar to extant platyhelminths (flatworms) (see Fig. 4.1). A specialized vascular system or respiratory system was probably lacking, although cells specialized for transport and excretions were likely present because they exist in most extant bilaterian phyla. One can further assume that groups of mesoderm cells in the bilaterian ancestor could have formed epithelial structures lining internal tubules or cavities (splanchnopleura). In coelomates, the mesoderm transforms into an epithelial sac, the walls of which attach to the ectoderm (somatopleura) and the inner organs (splanchnopleura). Blood vessels are formed by tubular clefts bounded by the splanchnopleura. Excretory nephrocytes are integrated into those vascular walls, which also gives rise to blood cells circulating within the blood vessels (the pronephros of anurans and head kidney of teleost fish are important hematopoietic organs in vertebrates). Thus, further evolutionary changes separated the three systems, but there was a close original connection between them. The origin of hemocytes has been investigated mainly in arthropods. When examining principles that govern hematopoietic pathways, similarities have been observed with vertebrates, raising interesting evolutionary issues.37,40 In jawed vertebrates, the yolk sac or its equivalent gives rise to blood precursors that are primarily erythroid in nature (but see the following: recent data suggest that B1 cells and macrophages are also derived from this embryonic tissue). In succession, definitive hematopoiesis occurs in the aorta/ gonad/mesonephros (AGM) region of the embryo, encompassing all of the different cell types and multipotent progenitors (although this is controversial). Like in the vertebrates, hematopoiesis in insects is biphasic. One phase occurs in the embryo and the other during larval development. Additionally, these waves occur in distinct locations of the embryonic head mesoderm and the larval lymph gland. In the early embryo expression of the GATA factor, serpent (Srp) can be detected in the head mesoderm. This GATA family of zinc-finger transcription factors is conserved from yeast to vertebrates where they are involved in various aspects of hematopoiesis. Blood cell formation in the head

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follows Srp expression, whereas in the lymph gland there is a long delay between Srp expression and the appearance of the lymph gland–derived hemocytes.38 Hematopoiesis in the head mesoderm and yolk sac may be related evolutionarily. A further similarity occurs at the AGM/lymph gland level in Drosophila. The lymph gland develops from a part of lateral mesoderm that also gives rise to vascular and excretory cells, much like the vertebrate AGM. The conserved relationship between blood precursors and vascular and excretory systems is intriguing.

Hematopoiesis and Transcription Factors in the Vertebrates As mentioned previously, transcription factors of the family PAX 2/5/8; GATA 1, 2, 3; ets/erg; and runt domain– containing factors have been cloned in several invertebrates. One plausible model to explain the genesis of true lymphocytes in vertebrates is that closely related members of transcription factor families are the result of a relatively late divergence in lineage pathways followed by specialization of duplicated genes.52 These duplications could be those that apparently occurred during the history of chordates (see MHC and “Origins” section2). Within deuterostomes, the generation of true GATA 2 and 3 probably occurred after echinoderms diverged from the chordate branch and the GATA, ets, early B-cell factor, and Pax5-dependent pathways of T-/B-cell differentiation are thus specific to vertebrates. It is already known that lampreys express a member of the purine box 1/spleen focus–forming virus integration-B gene family that is critically and specifically involved in jawed vertebrate lymphocyte differentiation. Expression has been detected in the gut, which may be related to the fundamental nature of “gut-associated lymphoid tissue (GALT)” as a lymphoid cell–producing organ. In vertebrates, the generation of T-, B-, and NK lymphocyte lineages from pluripotent hematopoietic stem cells depends on the early and tissue-specific expression of Ikaros (and related loci), which by means of alternative splicing produces a variety of zinc-finger DNA-binding transcription factors. The orthologs of Ikaros, Aiolos, Helios, and Eos have been identified in the skate Raja eglanteria, where two of the four Ikaros family members are expressed in their specialized hematopoietic tissues (epigonal and Leydig’s organs; see subsequent discussion) like in mammals.52 In lower deuterostomes, single genes that seem to be related to the ancestor of the Ikaros and Ets family of transcription factors exist, further suggesting that the division of labor between the family members in the jawed vertebrates was a result of en bloc duplications.52,53 The conservation of Ikaros structure and expression reinforces its role as a master switch of hematopoiesis. We discuss this topic further in the lymphoid tissues section.

Responses of Hemocytes In this section, we simply touch on classical and specific responses in the invertebrates, responses that are more universal are found in the innate immunity section. Proliferation

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of hemocytes upon stimulation is an unresolved issue in the invertebrates; clearly, clonal selection resulting in extensive proliferation is not the rule. The turnover of cell populations has been the object of numerous, often unconvincing experiments. Still, new data have emerged, and it is clear that in several invertebrates, proliferation occurs in certain cell types following encounters with pathogens. Very little cell proliferation occurs in the circulation of crayfish, but cells in the hematopoietic tissue divide after an injection of the PAMP β1-3-glucan. New cells in the circulation developed into functional synthetic germinal centers (GCs) and GCs expressing the proPO transcript. RUNT protein expression was upregulated prior to release of hemocytes. In contrast, proPO was expressed in these cells only after their release into the circulation.54 By contrast to the study of transcription factors that regulate hematopoiesis, relatively little is known about cytokines that drive hematopoiesis among invertebrates. It was reported that differentiation and growth of hematopoietic stem cells in vitro from crayfish required the factor astakine, which contains a prokineticin domain55 ; prokineticins are involved in vertebrate hematopoiesis, another case of conservation during the evolution of growth factors and blood cell development. Parasitization of Drosophila by the wasp Leptopilina boulardi leads to an increase in the number of both lamellocytes and crystal cells in the Drosophila larval lymph gland. This is partially due to a limited burst of mitosis, suggesting that both cell division and differentiation of lymph gland hemocytes are required for encapsulation. In genetic backgrounds where ecdysone levels are low (ecdysoneless), the encapsulation response is compromised and mitotic amplification is absent. This ecdysone-dependent regulation of hematopoiesis is similar to the role of mammalian steroid hormones such as glucocorticoids that regulate transcription and influence proliferation and differentiation of hematopoietic cells.56

Phagocytosis To obtain phagocytosis at the site of microorganism invasion implies recruitment of cells via chemoattraction. In vertebrates, this can be done by several categories of molecules such as proinflammatory chemokines/cytokines or the complement fragments C3a and C5a (as mentioned in the following section, C3a fragments as we know from mammals may be found in tunicates but not other nonvertebrates; yet, C3 may be cleaved in different ways in the invertebrates). C3b, mannose-binding lectin (MBL), and many other lectins can function as opsonins, and recent studies of the PGRPs, thioester-containing proteins (TEPs), DSCAMs, and eater have added to this repertoire.36 Ingestion follows phagocytosis, and then killing occurs by an oxidative mechanism with the production of reactive oxygen radicals and nitric oxide. These mechanisms are conserved in phylogeny, and other basic mechanisms are being examined in more detail now in protozoan models.57 Signaling pathways in common between vertebrates and the protozoon Dictyostelium include involvement of cyclic AMPs, integrins, and perhaps mitogen-activated protein (MAP) kinase cascades. Unique to all jawed vertebrates studied to date, the activation of

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phagocytes also leads to upregulation of the antigen processing machinery, costimulatory molecules, and proinflammatory cytokines that can enhance adaptive immunity.

INNATE IMMUNE RESPONSES Immune responses are often subdivided into recognition, signaling, and effector phases, which are subjected to different pressures, defined by whether orthology is maintained and the relative divergence rates of the genes responsible for the various phases. Recognition molecules are from evolutionarily conserved families, but as described previously, their genes are subjected to rapid duplication/ deletion so that orthology is rarely preserved. By contrast, signaling pathways can be conserved (see Fig. 4.5), despite the fact that the genes are often divergent in sequence. Effector molecules can either be extremely conserved (eg, reactive oxygen intermediates) or extremely divergent to the point of being species-specific (eg, AMP). Here, we break the immune response down into these three phases, beginning with the recognition phase. Initiation of an immune reaction can theoretically involve either the recognition of nonself, altered self, or the absence of self. Nonself-recognition can take place with receptors (pattern recognition receptors [PRRs]) that detect PAMPs, which were originally defined by Janeway and colleagues as evolutionarily conserved epitopes displayed by pathogens but not host cells.58,59 The second mode, altered self, is typified by molecules that are induced in self-cells during infections and recognized by conserved defense molecules, similar to the SOS systems mentioned in the MHC section, or by peptide presentation on MHC molecules. A third mechanism, “am I still myself,” depends upon recognition of self-tags and their changes in expression60 (eg, NK recognition of self-MHC molecules through KIR and C-type lectins). These latter two mechanisms have not been described in the invertebrates for immune defense against pathogens, but it would not be surprising if they were revealed in the future, considering the new features of invertebrate immune systems that have been discovered recently and the usage of this mode of recognition in many invertebrate histocompatibility systems. Whether the invader is related to its host (cells from individuals of the same species or cells from a parasitoid) or are very distant from the host (fungi and bacteria in metazoa), there are different principles of recognition. Yet PAMP determinants have been identified on very different organisms—sugars such as β1-3 glucan of fungi, lipopolysaccharide (LPS) and peptidoglycans of bacteria, phosphoglycan of some parasites, and especially nucleic acids of bacteria and viruses—and they can trigger similar cascades of events. The foreign ligand can be bound by a molecule in solution that initiates an effector proteolytic cascade (eg, clotting or the complement cascade). On the other hand, a proteolytic cascade can be initiated and result in the production of a self-ligand that interacts with a cell surface or endosomic or cytosolic receptor. In this way, there need not be a great diversity of cell surface receptors, especially in the absence of clonal selection.

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Of the over 1 million described species of animals (see Fig. 4.1), approximately 95% are invertebrates representing 33 phyla, some with one species (Placozoa, Cycliophora) and others with over 1 million (Arthropoda). Because they have major differences in body plans, development, size, habitat, etc., wildly different types of immune systems in diverse species should be expected. Early studies of invertebrate immunology reached no consensus of how immunity should be examined, but because vertebrate cellular adaptive immunity was often defined (indeed, was discovered for T cells) through transplantation reactions, attempts to reveal specific memory by allograft rejection were often used. After many unsuccessful attempts to demonstrate memory of such responses (see the following) and after extensive molecular studies, a consensus was reached that an invertebrate adaptive immune system involving somatic generation of antigen receptors and their clonal expression was highly unlikely. However, the term “innate” is rigid and masks the possibility of other somatic alterations of invertebrate immune system molecules, as will be discussed.61,62 We will categorize the molecules based on their location within the cell.

Intracellular Recognition Nucleotide-Binding Domain Leucine-Rich Repeat (NLR) One major group of intracellular sensors in animals and plants is the NLR family (see Figs. 4.2 and 4.4).14,63 Each of the family members has a central NB/NACHT (nucleotidebinding domain) and C-terminal LRR used for recognition, and a unique N-terminal domain. The subfamilies are defined by their N-terminal domains, coiled-coil and toll-IL-1 receptor (TIR) in plants, and baculoviral inhibitory repeat, caspase-recruitment domain (CARD), pyrin domain (PYD), and activation domain in animals (see Fig. 4.4). Thus far, the NLRs have been found in deuterostomes but not protostomes (see Fig. 4.1), which is surprising considering that plants have intracellular defense proteins with a similar structure, and seem to have been derived via convergent evolution.14

PLANT NLR CC TIR

NB-ARC

ANIMAL NLR

LRR

• Detection of plant-specific affectors (Avr) • Disease resistance or cell death

CARD PYD BIR NACHT

LRR

• Cooperation with TLR to upregulate proinflammatory cytokines of the IL-1 family • Pyroptosis (cell death) • Upregulation of Class I (NLRCS) and Class II (CIITA)

FIG. 4.4. Structure and Major Functions of Nucleotide-Binding Domain Leucine-Rich Repeats in Plants and Animals.14 Although the structures are quite similar, and recognition can be analogous, these families seem to have arisen via convergent evolution (see text for more details).

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The specificity of plant NLR depends principally on the LRRs, and these are targets for diversifying selection, as described previously for multigene families. Plant NLR can recognize pathogen effectors (pathogen-derived avirulence factors), or viral and fungal PAMPs directly via the LRR domains, or via modifications of a host target that interacts with the N-terminal domains, altered self if you will (see Fig. 4.4). This type of activation in plants is termed effector-triggered immunity, which is specific of the NLRs (see Fig. 4.4). A host of downstream effectors are generated, some involved in defense but others activating cell death pathways.14,64 Best described in mammals, the nucleotide oligomerization domain (NOD)/NLR recognizes PAMPs such as peptidoglycan and induces an autophagy-mediated destruction of intracellular pathogens as well as production of proinflammatory cytokines; however, it remains controversial whether there is direct or indirect recognition of the PAMPs (similar to some responses in plants). Polymorphisms in the NOD proteins are associated with inflammatory bowel diseases. The NLRP and NAIP NLRs are activated by in ways that are not well understood by various PAMPs or dangerassociated molecular patterns and form inflammasomes, best known for the activation of caspase 1 and the processing of pro–IL-1beta (or mature IL-18) for release from cells.14,65 The founding member of the family, CIITA, has long been known to upregulate class II genes (and associated genes, such as cathepsins and invariant chain), and its function is somewhat outside the norm. Another member, NLRC5, has been shown to upregulate MHC class I expression, but the mechanism is unknown.66 While the shuttling of the vertebrate NLRs CIITA and NLRC5 to the nucleus seems to be a derived characteristic, movement of plant NLRs into the nucleus to activate transcription occurs, either directly or after recruitment of transcription factors like WRKY described in the following. NLRs are expressed by echinoderm coelomocytes, again representing a highly diversified family48 (> 200 members, similar to the TLRs and SRCRs). As mentioned, it is surprising that these genes do not seem to be represented in protostomes, and thus the emergence of the family in plants and deuterostomes occurred through convergent evolution.67 On the contrary, in the vertebrates a search of the Danio rerio (and other teleosts) database have yielded a large number of NLR sequences, more similar to the situation in plants.63 In humans, most NLR genes are encoded in clusters on chromosomes 11p15, 16p12, and 19q13, where six sequences are found in a single telomeric region.

Rig-I-Like Receptors (RLR) The retinoic acid-inducible gene (RIG)-I is an intracellular defense molecule that is unrelated to the NOD proteins, with N-terminal CARD and C-terminal helicase domains.68 With the helicase domain, RIG-1 binds to an uncapped 5′ phosphate group, which is diagnostic of viral RNAs. RIG-I also recognizes short double-stranded RNAs, while a second member of this family MDA5 recognizes long double-stranded RNAs. These molecules contain two CARD domains at the N-terminus, a DEXDc domain, a he-

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licase domain, and a regulatory domain. Ligands bind to the regulatory domain, inducing a conformational change leading to interaction with the adaptor protein MAVS (or IPS-1) and ultimately to the induction of type I interferons (IFNs). A third member of the RLR family is LPG2, which lacks the CARD domain; this molecule was originally believed to be a negative regulator of RIG-I/MDA5-induced signaling, but that has been called into question. This family is found in all of the vertebrates and in lower deuterostomes, such as amphioxus and echinoderms.63 Somewhat surprisingly, the RLR family is only mildly expanded in sea urchins (12 members). While there is no report of bonafide RLR family members in protostomes but RLR activity is present,69 the cnidarian sea anemone has been reported to have a RLR homologue,70 again showing the importance of studying this taxon for the emergence of immune-related molecules.

Cytosolic Deoxyribonucelic Acid (DNA) Sensors There are four mechanisms of cytosolic DNA sensing, three of them, the DNA-dependent activator of IFN-regulatory factor, IFI16, and RNA polymerase III (which converts viral DNA into RNA recognized by RIG-I), induce type I IFN production through the intermediate STING, a protein associated with the endoplasmic reticulum (ER).71 In addition to being an intermediate in IFN upregulation (through IFN regulatory factor-3), STING is also a PRR in its own right, responding to the PAMP cyclic dinucleotides produced by intracellular bacteria like Listeria ; this suggests that STING was originally a PRR, and then was co-opted by several other PRR sensors to induce effector functions.72 IFI16 is part of the AIM2-like receptor family; the founding member, AIM2, like the inflammasome, activates caspase-1 to process pro–IL-1beta. These new molecules/mechanisms have so far only been studied in mammals, but it would be surprising if they were not operative (at least) in other vertebrates as a way to combat DNA viruses. To date, they have not been found in the sea urchin or jawless fish databases.

Tripartite Motifs Tripartite motif (TRIM) proteins belong to a family induced by type I and II IFNs, with 68 members in the human genome. TRIMs are involved in resistance against pathogens in mammals, especially lentivirus (eg, human Trim 5α is a retroviral restriction factor with activity against human immunodeficiency virus).73,74 The activity of proteasomes, responsible for cytosolic protein degradation, has been implicated in the TRIM5α-dependent attenuation of retroviral reverse transcription. TRIMs contain an N-terminal moiety composed of three modules: RING (with an E3 ubiquitinase activity)-Bbox-coiled xoil motif followed by different C-terminal domains. TRIMs fit into two major categories by the function of their C-terminal domain: Category 1 with a PHD, MATH, ARF, FNIII, exoII, or NHL domains, and Category 2 with a B 30-2 domain shared with butyrophilins and other proteins and essential for ligand binding.75 The tertiary structure of TRIM21 revealed two binding pockets in the B30.2 domain formed by six variable loops.76

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Despite reports to the contrary, the TRIM family is ancient.77 The family has been greatly diversified in vertebrates and in a taxon-specific manner, as observed for many multigenic immune families.77 The zebrafish genome harbors a striking diversity of a subset of Category 2 TRIMs not encountered in mammals, called finTRIM, with 84 genes distributed in clusters on different chromosomes. This subset, specific of teleosts, is overexpressed after virus infection in the trout. In the B30.2 domain, residues under positive selection are concentrated within a viral recognition motif first recognized in mammalian Trim 5α .78 Finally, trim genes encoding Category 2 proteins are preferentially located in the vicinity of MHC or MHC gene paralogs both in fish and human, suggesting that they may have been part of the ancestral MHC.79 The B30.2 domains most closely related to finTRIM are found among NLRs, indicating that the evolution of TRIMs and NLRs was intertwined by exon shuffling.80 Exon shuffling was likely responsible for the presence of the B30.2 domain in butyrophilin and TRIM genes where it was perhaps favored by the proximity of gene in the MHC. It has been argued that during evolution the combination of SPRY and PRY motifs that build up the B30.2 domain were selected and maintained for immune defense.81

P47 GTPases Among IFN-inducible immunity-related genes with an interesting evolutionary history, immunity-related GTPases (IRG/p47 in mouse) function as cell-autonomous resistance factors by disrupting the vacuolar membrane surrounding parasites (eg, toxoplasma).82 The IRG system studied primarily in mice (absent in humans83) is present throughout mammals but the number, type, and diversity of genes differ greatly even between closely related species, one of the common themes in immunity described previously. Concerning the evolutionary origin of the IRGs, the homologs of zebrafish and pufferfish seem to form two teleost-specific groups, another common theme in this chapter. Their putative promoter regions suggest an expression regulated by an IFN. Homology searches failed to find any convincing ancestral form to the vertebrate IRG proteins in the genomes of invertebrates, but in phylogenetic trees vertebrate IRGs clusters with some families of bacterial GTPases. Thus, IRGs may be derived from a prokaryotic GTPase acquired by a horizontal transfer subsequent to the appearance of eukaryotes.82

Integral Membrane (and Sometimes Secreted) Proteins C-Type Lectins Lectins were originally defined by their ability to bind carbohydrates in a calcium-dependent manner (how C-type lectins got their name84) and some have been described previously (and throughout the chapter). They are found in many phyla in both the deuterostome and protostome lineages in both membrane and/or secreted forms (eg, MBL described in the following). A large number of C-type lectins have been uncovered in the mosquito genome, and

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some are involved in bacterial defense through direct binding and others through the melanization reaction.85 Some C-type lectins are encoded in the NKC, including the Ly49 and NKG2 families, as well as CD94 and several other members of the family are central to NK-cell function in mammals. A molecule resembling CD94 but unlikely to be an ortholog (see the following) has been detected on a subset of hemocytes in Botryllus and Ciona, the functions of which are unknown.86 Another large gene family that is implicated in the response of the sea urchin to immune challenge includes 100 small C-type lectins,48 consistent with the enormous expansion of several immune defense families in this animal. We describe other functions of C-type lectins in the NK cell sections.

Scavenger Receptors The SRCR superfamily is an ancient (from sponges to chordates) and highly conserved group of cell surface and/or secreted proteins, some of which are involved in the development of the immune system as well as the regulation of both innate and adaptive immune responses; they are especially well known for their function in macrophages.87 Group B SRCR domains usually contain eight regularly spaced cysteines that allow the formation of a well-defined intradomain disulfide-bond pattern. Scavenger receptors are best known for their housekeeping function of taking up lipids modified by oxidation or acetylation, but they have many other functions as well, such as uptake of apoptotic bodies (eg, croquemort in Drosophila of the CD36 subfamily88). SRCRs have been studied mainly in the coelomocytes of echinoderms. Within a few hours after bacterial injection, sea urchin coelomocytes upregulate a variety of genes including an extremely diverse family of SRCRs.48,89 A very large number of SRCR domains are present (approximately 1,200), but each individual may express different groups of SRCR genes at different levels (and even with differential splicing). To assume that they are all involved in defense is premature, as SRCR genes can be both up- and downregulated after infection with bacteria. As mentioned, this high level of gene duplication is a general rule in the echinoderms. In mammals, the SRCR family as a whole is also poorly defined but is involved in endocytosis, phagocytosis, and adhesion, and some members acts as PRRs that bind to LPS or other bacterial components. SRCRs are widespread in the human genome and participate as domains in the structure of numerous receptors (eg, S4D-SRCRB, CD6, CD5-L, CD163), but without showing the high level of duplication seen in the echinoderm families.87 Down Syndrome Cell Adhesion Molecule DSCAM in Drosophila and other arthropods was described originally by neurobiologists as an axon-guidance protein, dependent upon a large number of isoforms (> 30,000) generated by alternative splicing for the IgSF domains and the transmembrane segment. DSCAM is also involved in insect immunity, expressed in cells of the hematopoietic lineage, and clearly capable of binding to bacteria; like in the nervous system, a large number of splice variants are generated,

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clearly different from the ones expressed in neurons.24 In Drosophila, the DSCAM gene is composed of 115 exons, 95 of which encode alternative possibilities for splicing of exons 4, 6, and 9. The molecule consists of 10 IgSF domains and 6 FN domains, and present as either a membrane or soluble form, presumably generated by proteolysis of the membrane form. Each cell expresses only a fraction of the isoform repertoire. Knock out (RNAi) and anti-DSCAM treatment significantly suppresses phagocytosis, at least in Drosophila. Soluble DSCAM constructs with different exon combinations were found to have differential pathogen-binding properties.90 In addition, suppression of DSCAM in mosquitoes results in an impaired immunity to Plasmodium ; exposure of hemocytes to different pathogens in culture gives rise to specific modifications and selection of alternative splicing patterns. A similar finding was made in crustaceans, in which particular DSCAM isoforms were induced in response to different pathogens in one species91 and epitope II was under selection in a study in Daphnia.92 The diversification of DSCAM seems to be specific of arthropods as neither flatworm nor sea urchin nor vertebrate DSCAM are diversified. The vertebrate DSCAM has only two forms, using two alternate TM exons. The cytoplasmic tail can also be modified by alternative splicing that could change its signaling properties by modulation of tyrosine-based motifs.93 Human DSCAM is duplicated on chromosomes 21 and 11, but does not appear to be involved in immunity.

A

VERTEBRATES

INSECTS Gram+ Fungi Spaezle

PAMP DAMP

PGRP β1-3GRP SPE (proteolytic enzyme) Toll

TLR MyD88 IRAKs (others)

NF-kB Ikβ p50 p65 (REL)

PLANTS

Pelle

Peptodoglycan-Recognizing Protein and a1-3 Glucan Receptors PGRPs are found in a wide range of organisms but have been best studied in insects, where they are classified into short (S) and long (L) forms. S forms are soluble and found in the hemolymph, cuticle, and fat-body cells.94 L forms are mainly expressed in hemocytes as integral membrane proteins where their fi nal structure depends on combinatorial association of different isoforms, modulated by alternative splicing. We provide a short description here, but delve more deeply in the discussion of the insect toll and immune deficiency (IMD) pathways subsequently (Fig. 4.5).36 The expression of insect PGRPs is often upregulated by exposure to bacteria. PGRPs can activate the toll or IMD signal transduction pathways (see the following) or induce proteolytic cascades that generate AMPs, melanization, or induce phagocytosis. PGRPs directly kill bacteria by inducing a suicide mechanism, fi rst demonstrated to be activated by a type of unfolded protein (stress) response in prokaryotes.95 Besides their defense functions, insect PGRPs expressed in the gut are believed to promote homeostasis with commensal bacteria (also discussed briefly in the following). Both soluble and transmembrane forms are present in sea urchins, some with potential catalytic function.48 In vertebrates, PGRPs are all secreted and have direct microbicidal activity. Best studied in zebrafish, PGRPs are

B

VERTEBRATES

PAMP DAMP

INSECTS Gram (–)

TNF

PGR LRR (not homologous to Toll/TLR)

PGRP-LC

TNFR TRADD RIP1

dMyD88

?

Kinase

Tube

MAP kinase (dicots)

FADD

MAP kinase

Gene WRKY transcription factors

Caspase 8

JNK

Cactus DIF

(REL)

DAP-type PGN

Apoptosis Gene regulation

Gene regulation

Gene regulation

–Proinflammatory cytokines –MHC, costimulation

–Drosomycin (fungi) –Defensin (gram+)

–Multiple defense genes

AP1

TAK1/TAB1 NEMO K63 Ikkβ NF-κB Ikβ p50 p65 (REL)

Gene Gene regulation regulation –cJUN –Proinflammatory cytokines

dTAK1/dTAB2 Kenny K63 IRD5 Ankyrin RELISH

REL

Gene regulation –Diptericin –Cercropin

FIG. 4.5. Comparison of Immune Response Induction, Intracellular Pathways, and Immune Outcome in Insect (eg, Drosophila), Vertebrates (eg, human), and Plants (a Composite of Pathways in Monocots and Dicots). Note that the initiation of the response and the outcome(s) in insects and vertebrates are quite different for both the toll/toll-like receptor pathway (left, A) and the immune deficiency/tumor necrosis factor pathways (right, B), but the intracellular signaling pathways are well conserved evolutionarily in insect and vertebrate (details in the text). Note as well that plants use similar molecules for recognition (leucine-rich repeat–containing molecules) and have similar intracellular pathways with kinase cascades, but all of the molecules of recognition, signaling, and effector are derived by convergent evolution as compared to animals. This figure was modified from Beutler et al.152

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expressed in many tissues such as gills, skin, and intestine, providing immune defense. They are expressed before the development of adaptive immunity and likely provide an important protective role.96 The human PGRP genes are found on the MHC chromosomal paralogs 1q21 and 19q13/ p13. All detected splice-variant isoforms bind to bacteria and peptidoglycan. Like the fish molecules, mammalian PGRPs are also positioned at epithelial surfaces and promote intestinal homeostasis by discriminating somewhat between commensal (eg, lactobacilli) and pathogenic bacteria. Knockout mice have increased pathogenic bacteria on mucosal surfaces that induce colitis after injury in the dextran sulphate sodium autoimmune assay.97 β1-3 glucan receptor proteins (β1-3GRPs, formerly known as gram-negative binding proteins (GNBPs) are related to bacterial β1-3 glucanases.98 They are found in insects and other arthropods where they bind bacteria, fungal β-1, 3-glucans, LPS, and/or bacterial lipoteichoic acid (without necessarily showing glucanase activity) . An ortholog is present in the sea urchins, but not in vertebrates to date. Drosophila GNBP1 together with PGRP-SA are required to activate the toll pathway in response to infection.36

Toll and Toll-like Receptors The toll receptors were originally described in Drosophila as genes involved in early development, specifically in dorsoventral patterning. Later, they were also shown to be essential sensors of infection, initiating antimicrobial responses.36,99 This family was then revealed to be a major force in innate immunity in the vertebrates as well.100,101 As mentioned previously, across the metazoa structurally closely related members of the toll family range from not being involved in immunity (in C. elegans and apparently in the horseshoe crab), to being the equivalent of a cytokine receptor (in Drosophila), to being PRR in the vertebrates and invertebrates.102 Six spaetzle-like and eight toll-like molecules have been identified in Drosophila, but only one or two of them are clearly immunity-related.36,102 In jawed vertebrates, they belong to a multigene family of PRR specific for diverse PAMPs and exhibiting different tissue distributions and subcellular locations.27 In humans, many are on chromosome 4p and q (TLR 2, 1, 6, 10) but the others are distributed on chromosomes 9, 1, 3, and X. Ectodomains of TLRs comprise 19 to 25 tandem repeats of LRR motifs made of 20 to 29 aa capped by characteristic N- and C-terminal sequences. All of the toll receptors are homologous and appear similar in domain constitution among all animals. They also share the TIR domain, which is the intracellular segment shared with the IL-1/-18/-33 receptors of vertebrates, as well as other molecules in plants. TIR domains associate with Myd88 to initiate signaling cascades culminating in the activation of NFκ B/Rel (see the following) (see Fig. 4.5). In Drosophila, the toll dimer is triggered by an interaction with the unique ligand spaetzle, which is the product of a series of proteolytic cascades, with the most critical enzyme identified (spaetzle-processing enzyme). Activation of the cascades triggers the production of antimicrobial peptides (see Fig. 4.5). The specificity of recognition is not

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achieved at this receptor level but rather in solution via other intermediates (see the following). C. elegans has only one toll receptor, and rather than being antimicrobial responses, it promotes avoidance of a flatworm pathogen upon engagement; the signaling mechanism for the C. elegans TOL (toll) is not evolutionarily conserved (it clearly does not induce the NF-κ B pathway) and is under investigation.67 A toll/TLR gene is present in the sea anemone, a cnidarian, but not in other cnidarians such as hydra or coral, which nevertheless have TIR domains associated with other molecules.35 A TIR domain of the toll-receptor types was detected in sponges, but, like IL-1R in vertebrates, it is associated with a receptor with three IgSF domains.103 Plants do not have toll/TLR per se, but do have LRR-containing transmembrane sensors that function in a similar fashion102 (see Fig. 4.5). In summary, toll/TIR arose before the split of protostomes and deuterostomes, but has been lost in some invertebrate groups and has been recruited to perform multiple functions. The arsenal of TLRs in vertebrates is endowed with specific and diverse capacities. Each vertebrate TLR has its range of specificities and, in addition, combinations of different TLR can create different binding specificities (eg, the association of TLR2 with TLR6 or TLR1 and 2104 or even TLR2 homodimers in regulatory T cells105). This divergence in recognition function is well illustrated by the phylogenetic analysis of the toll and toll-related receptors in different phyla such as arthropods and vertebrates. Toll and related proteins from insects and mammals cluster separately in the analysis, indicating independent generation of the major families in protostomes and vertebrates.102 Consistent with the expansion of SRCR and NLR genes in sea urchins, hundreds of TLRs also were found in this species.48 TLRs of the protostome type are in small numbers (3 members) while the vertebrate type has been enormously amplified, all within a single family (222 members) and most without introns. Vertebrate TLRs do not diverge rapidly and evolve at about the same rate, and while there have been some duplications in amphibians and fish, they are not greatly expanded like in the echinoderms (no more than approximately 20 genes in any species).

Signaling Through Innate Surface Recognition Molecules Four pathways of innate immunity triggering have conserved elements in eukaryotes: the toll/TLRs, the TNF-α / IMD receptors, the intracellular NOD, and the JAK/STAT. Although toll receptors have been found in almost all triploblastic coelomates, most of the work and the elucidation of pathways have been accomplished in Drosophila and Anopheles.36 The diversity of AMPs that can be produced via the toll/IMD pathways is substantial, and as described previously is classified in several categories depending upon the type of pathogen recognized (eg, gram (+), drosocin, gram (−), diptericin; fungal, drosomycin) with different effector functions (see Fig. 4.5). Insect antimicrobial molecules were originally discovered by the late Hans G. Boman and colleagues in 1981, a seminal finding that

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heralded the molecular analyses of innate immunity in the invertebrates.106

Toll and Immune Deficiency Pathways As described, invertebrate toll receptors are homologous to the vertebrate TLR, in the sense that they are integral membrane LRR-containing proteins (see Fig. 4.5). Drosophila toll is activated after it binds spaetzle, the product of a proteolytic cascade activated in solution after the interaction of molecules produced by fungi or gram-positive bacteria with GNBP and PGRP.107 The TIR cytoplasmic domain of the toll receptor then interacts with MyD88 (itself having a TIR domain) followed by Tube and Pelle, leading to activation of the homologous NF-κ B system (Cactus or Diff) that then induces transcription of various defense peptides.36,99,108 This is remarkably similar to the cascade of events following activation of mammalian TLRs where after their interaction with PAMPs at the cell surface, a cascade is induced through TLR including MyD88, IRAK, TRAF, TAK1, to NF-κ B via the IKK signalosome. Thus, infection-induced toll activation in Drosophila and TLR-dependent activation in mammals reveal a common ancestry in primitive coelomates (or previous), in which defense genes under the control of a common signaling pathway lead to activation of Rel family transactivators. The Drosophila IMD pathway is employed in responses to gram-negative bacteria109 (see Fig. 4.5). After interaction with the cell surface receptor PGRP-LC mentioned previously, in a cascade similar to the mammalian TNF-αR signaling pathway, Drosophila tak1, an IKK signalosome, and a Relish-mediated (instead of Diff) NF-κ B step, results in transcription of antibacterial peptides like diptericin. The Drosophila intracellular pathway is similar to the mammalian TNF-α receptor cascade, which also progresses via a death domain Mekk3, the signalosome, and NF-κ B resulting in cytokine production. In both cases, a link to pathways leading to programmed cell death is possible; overexpression of Drosophila IMD leads to apoptosis. When the activation of either the fly toll or IMD pathway is considered, they are analogous to a mammalian cytokine/cytokine receptor system (eg, TNF-α) in which a soluble self-molecule activates cells via a surface receptor. Fitting with the paradigm put forward on recognition, signaling, and effector phases of the immune response, the diversity of external recognition systems is not matched by an equivalent diversity of intracellular signaling pathways.22 There are conserved signaling cascades coupled to the receptors, giving the impression of conservation of the innate immunity pathways; yet, these pathways are also used in development, so which is primordial remains an open question. Plants do not have toll/TLR, but do have transmembrane LRR-containing microbial sensors, of which FLS2 that binds to flagellin, is best characterized27 (see Fig. 4.5). These molecules do not have an intracellular TIR domain (note that TIR domains exist in plants, but not associated with the TM sensors), but do recruit a kinase of a similar nature to the toll/TLR kinases (the so-called non-RD kinases) to activate downstream mitogen-activated protein kinase cascades. However, as mentioned previously, the NFκ B transcriptional

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system arose early in the animal kingdom110 ; plants employ a different system of transcriptional activators, the WRKY molecules, which are activated directly by the mitogen-activated protein kinase cascades, similar to transcription factor found in animals, AP1.

Extracellular Soluble Receptor with Effector Cascade Proteolytic cascades are initiated immediately following interaction of foreign material bound by preformed proteins in solution, and this principle is conserved throughout evolution. Indeed, the proteolytic cascade upstream of production of the toll ligand spaetzle resembles the complement or clotting cascades. The PPO cascade of arthropods leading to melanization and the genesis of antibacterial products described in the following is another example in which peptidoglycans on microbial surfaces initiate the cascade resulting in the degranulation of hemocytes.

The Complement System The best-studied immune proteolytic cascade that is surprisingly well conserved in the animal kingdom is complement111,112 (Fig. 4.6). In contrast to the other defense molecules that we have discussed, orthologous complement genes can be detected in all of the deuterostomes without a great deal of expansion/contractions of the gene family. The three major functions of complement in jawed vertebrates are 1) coating of pathogens to promote uptake by phagocytes (opsonization); 2) initiation of inflammatory responses by stimulating smooth muscle contraction, vasodilatation, and chemoattraction of leukocytes; and 3) lysis of pathogens via membrane disruption. Additionally, in the vertebrates, C′ is vital for the removal of immune complexes as well as elicitation of adaptive humoral immunity. The focal point of complement is C3, which lies at the intersection of the alternative, classical, and lectin pathways of complement activation. It is the only known immune recognition molecule (besides its homologue C4) that makes a covalent bond with biologic surfaces via a thioester linkage. C3 has a nonspecific recognition function, and it interacts with many other proteins, including proteases, opsonic receptors, complement activators, and inhibitors. In the alternative pathway, C3 exposes its thioester bond in solution, and in the presence of cell surfaces lacking regulatory proteins that block C3 activation (by cleaving it into iC3b), it associates with the protease factor B (B or Bf). After binding to C3, B becomes susceptible to cleavage by the spontaneously active factor D, resulting in formation of the active protease Bb that in combination with the covalently attached C3 cleaves many molecules of C3 in an amplification step. Another nonadaptive recognition system, the lectin pathway, starts with the MBL (or the lectin ficolin), which is a PRR of the collectin family that binds mannose residues on the surface of pathogens and can act as an opsonin. MBL is analogous to C1q with its high-avidity binding to surfaces by multiple interaction sites through globular C-terminal domains, but apparently it is not homologous to C1q. Like C1q, which associates with the serine proteases C1r and C1s, the MBL-associated serine proteases (MASPs) physically interact with MBL and not only activate

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CLASSICAL

LECTIN

ALTERNATIVE

Ab-Ag

MBL (Ficolin)

C3

|

83

(IgM or IgG/Y)

C1qrs

MBL-MASP

[C3-H2O]B

Thioester

(C1r cleaves C1s)

(D cleaves B)

C3, C4, (a2m, TEP) C3: From Diploblastic C4: Jawed vertebrate a2m: All metazoa TEP: Protostomes

C4 (Cleaved by C1s or MASP2)

[C3-H2O]Bb (Bb cleaves C3 in plasma)

Inflammation

Function

C5a > C3a > C4a Jawed vertebrates

Opsonization C3b: From Diploblastic TEP: Protostomes

C4b C2

C3

(C1s or MASP2 cleaves C2)

(Cleaved by C2b or Bb)

Microbial surface; properdin (factor P) stabilizes

C3a

C2a

Cell Lysis MAC (C5b-9): Jawed vertebrates MACPF: All animals

C4b C2b

Homologous members

C3, C4, C5, a2m, TEP, CD109 C3, a2m: Diploblastic C4, C5, CD109: Jawed vertebrates TEP: Arthropods

C2, B

C3b

(C3 convertase)

C3b Bb (C3 convertase) (Factors H, I and CR4 inhibit)

(CR1 inhibits)

C4b C2b C3b

C5

C3b Bb C3b

(C5 convertase)

(Cleaved by C2b or Bb)

(C5 convertase)

C2: Amphibian, mammal B: Diploblastic

C5a

C1r, C1s, MASP

C5b

C1r, C1s: Jawed vertebrates MASP: Deuterostomes

C1q, MBL C1q: Jawed vertebrates MBL: Deuterostomes

MAC: C5b C6 C7 C8 (C9)n (CD59 inhibits)

FIG. 4.6. Evolution of the Complement System. The general pathways and appearance of the various components in the phylogenetic tree are emphasized.

the classical pathway of complement by splitting of C4 and C2 (the same function as C1s; MASP2 appears to be the active protease), but also can activate the alternative pathway in ways that are not understood and thus completely bypass the classical pathway. Indeed, MASP-1 and -2 are homologs of C1r and C1s (see Fig. 4.6). Both C1q and MBL can promote the uptake of apoptotic bodies by phagocytes, via collectin receptors. Another lectin, ficolin, can also initiate the MASP pathway,113 and it would not be surprising if other activators were discovered in the future (eg, the ancient molecule C-reactive protein is also capable of activating C′). Finally, the classical pathway, which is dependent upon antibody molecules bound to a surface, results in the same potential effector outcomes described previously for the alternative pathway. Novel molecules initiating this pathway are C1q, C1r, C1s, C4, and C2, as well as specific negative regulatory proteins such as C4-binding protein.

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C3 and MBL (and ficolin) are vital players in the immediate innate immune response in vertebrates, and both have been described in nonvertebrate deuterostomes.48,112 Thus far, the best-studied invertebrate systems for investigation of C3 evolution are the sea urchin and the ascidians Halocynthia and Ciona, in which C3 and B molecules and genes have been analyzed in some detail. In contrast to the very high levels of C3 found in the plasma of jawed vertebrates, sea urchin C3 is not expressed at high levels but is induced in response to infection in coelomocytes.114 The C3 opsonic function clearly has been identified, but so far initiation of inflammatory or lytic responses (if they exist) has not been obvious. Receptors involved in the opsonization in echinoderms have not been identified, but in the ascidian gene fragments related to the C3 integrin receptor CR3 were identified, and antisera raised to one of the receptors inhibited C3-dependent opsonization.115

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Hagfish and lamprey C3-like genes were thought to be ancestral C3/C4 genes because the sequence predicts two processing sites (leading to a three-chain molecule), like C4, but a C3-like properdin-binding site is clearly present.116 However, like C3 in other animals the hagfish protein is composed of only two chains of 115 and 72 kDa, and sea urchin and ascidian C3 sequences predict only two chains (one proteolytic processing site). The lamprey, but not sea urchin C3, has a recognizable C3a fragment known from gnathostomes to be involved in inflammation, so the role of complement in inflammation may be a vertebrate invention (but see the following). TEPs have been isolated from Drosophila and the mosquito Anopheles, as well as several other arthropods.117,118 While the insect molecules function in a C3-like fashion (opsonization), phylogenetic analysis shows them to be more related to α2-macroglobulin (note that a few insects actually have molecules more related to C3). TEPs in insects function as opsonins, binding to parasites and promoting their phagocytosis or encapsulation. The evolution multimember TEP families in Drosophila and mosquito followed independent evolutionary paths, perhaps as a result of specific adaptation to distinct ecological environments as described in the introduction. The Drosophila genome encodes six TEPs (whereas there are 15 genes in Anopheles, again consistent with the major expansion of many immune gene families in the mosquito), three of which are upregulated after an immune challenge. Mosquito TEPs are involved in killing of parasites, and the reaction is regulated by LRRcontaining molecules to avoid destruction of self-tissues; thus, full-blown complement-like systems complete with inhibitors have arisen independently in protostomes and deuterostomes.119,120 C3-like genes are present in cnidarians121,122 and in the horseshoe crab Limulus.123 Good phylogenetic support was obtained for their relationship to C3, as compared to other members of the thioester-containing family like the TEPs. Thus the emergence of C3 as a defense molecule predates the split between protostomes and deuterostomes. A gene resembling the proteolytic enzyme Bf was discovered in these protostomes as well (and in sea anemones), suggesting that the fundamental system was in place a billion years ago (see Fig. 4.1). The lack of C3 in many other protostomes suggests that the ancestral gene was lost and replaced by the TEPs.117,118 In jawed vertebrates and some lower deuterostomes, certain species express more than one C3 gene, suggesting that the innate system might compensate in animals that do not optimally make use of their adaptive immune system.124 Changes in the amino acid composition of the C3-binding site are found that may somehow regulate the types of surfaces bound by the different isotypes.125 Likewise, in lower chordates such as Ciona, C3 and other complement components can be duplicated.115 Diversification of the carbohydrate recognition domains has been observed also in the Ciona MBP family (nine members). Like Ig/TCR/MHC, the classical pathway and the terminal pathway membrane-attack complex (MAC) appears fi rst in cartilaginous fish.112 However, as MBL can activate

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the classical pathway in mammals, it is possible that some portion of this pathway exists in prejawed vertebrates. Nevertheless, C4 and C2 genes have not been detected to date in jawless fish or invertebrates. A bonafide C2 homologue has only been identified to the level of amphibians, although duplicate B genes were isolated from cartilaginous fish and teleost fish that may function both in the classical and alternative pathways. The lytic or MAC pathway, which is initiated by the cleavage of C5 into C5a and C5b, also has not been described in taxa older than cartilaginous fish. Thus, opsonization and perhaps the induction of inflammatory responses were the primordial functions of the lectin/complement pathways. However, a complementary DNA clone for CD59, a molecule that inhibits MAC formation in self-cells, was identified from a hagfish library, and some of the terminal components of the pathway have been detected in lower deuterostomes with no described functions.116 Interestingly, proteins with the MAC/perforin domain have been detected throughout the animal kingdom,48 and some are even involved in cytotoxic reactions; however, it seems that only vertebrates have bonafide terminal C′ components that are highly evolved for targeted destruction of cell membranes. The perforin gene itself also seems to have arisen in gnathostomes, from an ancient MAC/perforin domain-containing gene, macrophage-expressed gene 1 protein, which dates back to sponges; thus, cellular cytotoxic reactions in the invertebrates described in the following must use novel cytotoxic effector molecules.126 C3, C4, C5, and α2m (and TEP) are members of the same small family. A cell-surface–expressed (GPI-linked) member of this family, CD109, has been shown to associate with the transforming growth factor (TGF)-β receptor and modulate its expression.127 The protease inhibitor, α2m, clearly present in invertebrates (protostomes and deuterostomes) and vertebrates, is thought to be the oldest, but obviously this must be viewed with caution considering the data in cnidarians. Along with its ability to bind to and inactivate proteases of all known specificities through a “bait region,” it also has been shown to be opsonic in some situations. α2m, C3, C4, and CD109 (as well as the TEPs) have internal thioester sites, so this feature is primordial; C5 subsequently lost the site. The first divergence probably occurred between α2m and C3, with C5 and then C4 emerging later in the jawed vertebrates.128 Consistent with Ohno’s vertebrate polyploidization scheme is the fact that C3, C4, and C5 genes are located on three of the four previously described paralogous clusters in mammals, and this is also fits with the absence of classical (no antibody) and lytic (no MAC) pathways in phyla older than cartilaginous fish.2 α2m is encoded at the border of the NKC in mice and human, and there are similarities between these regions and the other MHC paralogs (see Fig. 4.13). The C3a and C5a receptors that promote the inflammatory responses upon complement activation have been identified in several vertebrates and (perhaps) some lower deuterostomes; like the chemokine receptors they are G-protein coupled receptors whose genes may also be found on the ohnologs (C3aR, chr 12p13; C5aR, chr 19q13). If indeed such receptors are found in the prejawed vertebrates

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as suggested by recent pioneering experiments in Ciona and Styela, it will be interesting to determine whether they are involved in some type of inflammation, thought to be the domain of the vertebrates.129

Melanization (Prophenoloxidase Cascade) A major defense system in invertebrates is the melanization of pathogens and damaged tissues,130 popularized by poor Gregor in Kafka’s Metamorphosis, when the cockroach Gregor undergoes a melanization reaction from an apple thrown into his thorax by his father. The process is controlled by the circulating enzymes PPO and phenol oxidase. The system is activated by β1-3GRP, PGRP, LPS-binding proteins, and other proteins that can bind to various PAMPs (see Fig. 4.3). The complexes launch a cascade of serine protease activities resulting in cleavage of the pro-form of a prophenoloxidase-activating enzyme into the active form that in turn activates the PPO into phenol oxidase. This leads to the production of quinones and finally melanin. Melanization can completely inhibit parasite growth, whereas concomitant with PPO activation, many other immune reactions are initiated, such as the generation of factors with antimicrobial-, cytotoxic-, opsonic-, or encapsulation-promoting activities. The presence of specific proteinase inhibitors (of the serpin family) prevents unnecessary activation of the cascade and overproduction of toxic products. Phenoloxidase is the key enzyme responsible for the catalysis of melanization. It is a marker of the PPO activating system, and it can be an immune effector by itself as demonstrated in ascidians. It is therefore interesting to assess its conservation within all metazoa. A survey of the different organisms revealed the presence of phenoloxidase in many deuterostome and protostome phyla, and related molecules are also present in sponges. In arthropods, several PPO genes are present in the genome (nine in Drosophila and Aedes). Some may have different “immune” functions such as injury repair. Several components that would maintain the role of melanization in immunity may be lacking in different phyla even if many elements are conserved, and so far the best examples of melanization associated with immunity are still found almost exclusively among arthropods and to a lesser extent in annelids. Despite the presence of molecules involved the pathway, the PPO cascade per se does not exist in vertebrates.

Effector Molecules Peroxidasin Among molecules containing LRR motifs, peroxidasin occupies a special place because of its involvement in hemocyte biology in insects and because of its homology to the LRR motifs in the agnathan VLR and Ig domains similar to Ig itself. Drosophila peroxidasin is an assembly of a cysteinerich motif, six LRR, and four IgSF domains.131 The molecule is conserved in vertebrates, although a role in immunity has not been reported. Another molecule called peroxinectin, with similarity at the level of the peroxidase region, has been described in crustaceans and shown to be associated with immunity via the PPO cascade.132 Its involvement in

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immunity is unlike any other effector so far described but illustrates the utility of LRR in many different types of molecules and processes. Pathogens bound by AMPs can be phagocytosed or walled off by a barrier of flattened hemocytes and ECM. The ECM forms a basement membrane that becomes stabilized partly through peroxidases that generate tyrosine-tyrosine bonds. The combination of LRR and Ig structures suggests that peroxidasin may precisely mediate adhesion of cells to the ECM. As mentioned, a large number of LRR-Ig–containing proteins has been discovered, most of them playing roles in embryologic development.32 Many LRR-Ig proteins are encoded in paralogous regions in the vicinity of immune genes, showing an ancient direct connection between the families (see the following).

Fibrinogen-Related Proteins FREPs are proteins that were first discovered in the hemolymph of snails with an IgSF moiety (one or two V-like domains) and a fibrinogen domain. The fibrinogen domain is found in a large number of defense molecules throughout the animal kingdom (eg, the ficolins). FREP gene expression is upregulated following exposure to the mulluscan parasites such as schistosomes133 ; a snail strain resistant to schistosomes shows an upregulation of the FREP 2 and 4 genes of up to 50-fold. The original discovery of FREPs followed the recovery of snail proteins that bound to worm antigens, and thus this is one case in which the correlation between an invertebrate receptor and its ligand is clear. However, it is not known whether the IgSF or fibrinogen domain (or both) bind to the antigen or which effector functions are induced after FREP binding. FREP diversity is remarkable in that there are many polymorphic genes as well as alternate messenger RNA splicing to generate the diversity. In addition, based on the number of genes and alleles in individual snails, there appears to be a somatic diversification mechanism that modifies FREP genes, either via mutation or gene conversion in the region that encodes the IgSF domains.134 Over 300 unique sequences were found in 22 snails, consistent with a somatic diversification mechanism. Currently, there is no mechanism to account for the mutations, but data accumulate for somatic modifications and the use of FREPs in critical defense against pathogens.135 FREPs are also present in arthropods where they lack the Ig domains,136 once again with an expansion of genes in the mosquito Anopheles gambiae as compared to Drosophila. RNAi studies have shown that subsets of FREP genes are vital for defense against malarial parasites, and different FREPs bind to bacteria with different affinities.137 Homoand heterodimers can form between different FREPs, and multimers can be fashioned that likely increase the avidity of binding. In summary, this ancient family of defense molecules has all of the attributes described in the introduction: rapidly evolving multigene family, conservation in divergent protostomic invertebrate phyla, somatic diversification via alternative splicing, and perhaps an unknown mutational/ gene conversion mechanism, as well as heterodimeric (and multimeric) association.

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185/333 There are a number of additional expanded gene families in the sea urchin genome that encode proteins with immune-related functions. The 185/333 genes were first noted because they are highly upregulated in coelomocytes after exposure to LPS, constituting up to 7% of the messenger RNAs in such cells.138 Subsequently, transcripts were shown to be upregulated by many different types of PAMPs. The encoded proteins have no detectable similarity to any other gene family, but they are highly diversified and are produced (at least) by a subset of coelomocytes. There are estimated to be at least 50 185/333 genes in the sea urchin genome.139 The gene is composed of two exons, one encoding the leader and the other encoding the mature protein. The second exon is made up of so-called elements and repetitive sequences that are quite different from gene to gene, which can to a large extent explain the diversity of expressed loci. However, there are hints of RNA editing, a unique form of alternative splicing, somatic mutation (perhaps targeting cytosine residues), and other (perhaps) novel mechanisms of diversity to explain the incredible number of different isoforms; additionally, like many other defense molecules, there is evidence of protein multimerization. Whatever the mechanism of diversity generation, it would be surprising if this family were not vital for defense in echinoids. Furthermore, the presence of this unique multigene family in sea urchins is consistent with the great expansion of other immune gene families in this group, including TLRs, SRCRs, and NLRs.7

Variable Domain Chitin-Binding Proteins VCBP, fi rst discovered in Amphioxus but present in Ciona as well, consist of two Ig domains of the V type but with a different folding motif when compared to Ig or TCR V domains140 followed by a chitin-binding domain. The chitin-binding domain resembles chitinases found throughout the animal kingdom, and like dedicated chitinases VCBP is usually expressed in the gut. Apparently, there are no cell-surface–expressed forms and thus all VCBPs are likely to be secreted, effector molecules. In Amphioxus, their diversity is enormous, apparently entirely because of polymorphism and polygeny, and not somatic alterations. Each individual can carry up to five genes per haplotype, and in limited studies (11 individuals), no identical haplotype has been encountered.141,142 The general structure of the V domain is like that of the vertebrate rearranging antigen receptors, but with some unusual properties, including packing in a “head-to-tail” dimeric fashion, totally unlike Ig and TCR. VCBP diversity does not reside in the Ig/TCR CDR resides, but rather in the A, A′, and B strands, like in DSCAM. By contrast to the Amphioxus VCBP, there are only a few nonpolymorphic Ciona VCBP genes, which are expressed by gut epithelium and amebocytes. Soluble VCBPs bind to bacteria and induce opsonization in amebocytes. It is hypothesized that these molecules may perform a function similar to mucosal IgA in vertebrates, which provides a “firewall” protecting from invasion of intestinal bacteria and promoting homeostasis.143 If true, this would provide

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a link to the regulation of commensals in early deuterostomes. It should be noted here, however, that recent data suggest that “tolerance” of commensals occurs in the protostomic invertebrates as well.

Antimicrobial Peptides (Defensins) Each metazoan taxon produces a variety of molecules with intrinsic antimicrobial activity,144,145 the majority of which fall into three major categories: defensins, catelicidins, and histatins. Even in species with very small genomes (such as the tunicate Oikopleura [60 Mb genome]), selection pressures have been strong enough to lead to expansion of the Phospholipase a2 family, with 128 members.146 Some families are evolutionarily conserved but generally they diverge rapidly and orthologous relationships are not apparent. The best studied group of AMPs is the defensins, which are amphipathic cationic proteins; their positively charged surface allows them to associate with negatively charged membranes (more common in pathogens), and a hydrophobic surface that allows them to disrupt the membranes, either by disordering lipids or actually forming pores. Most of the molecules are proteins, but an antimicrobial lipid called squalamine, which also is modeled to have hydrophobic and positively charged surfaces, is found at very high levels in dogfish and lamprey body f luids.147 Defensins can either be constitutively expressed (eg, in respiratory epithelia in mammals) or inducible (eg, see the following for Drosophila and see Fig. 4.5). Certain responses that seem systemic, like the production of Drosophila defensins, can also take place locally in the damaged tissues themselves; otherwise, a systemic response is initiated in organs distant from the site of infection such as the fat body in Drosophila where induction of bactericidal peptide expression occurs.36 Defensins are the focus of great attention in commercially bred species such as oysters, mussels, and crustaceans. Besides their direct defense functions, in mammals defensins play other roles, such as chemotaxis and immune regulation. Penaedins. One set of diverse AMPs is the penaedins, present in crustaceans (shrimp). Penaedins are small antimicrobial peptides (5 to 7 kda) that bind to bacteria and fungi, and consist of a conserved leader peptide followed by an N-terminal proline-rich domain and a C-terminal cysteine-rich domain.7,148 Most of the diversity is found in the proline-rich domain,149 suggesting that it is most important for recognition, but both domains are required for recognition of bacteria and fungi. Four classes of penaedins, PEN2 to 5, are expressed by shrimp hemocytes. A great diversity of isoforms is generated, with substitutions and deletions within the proline-rich domain, suggesting that this domain recognizes ligand; nevertheless, both domains seem to be required for function. Like the VCBPs, each penaeidin class seems to be encoded by a unique gene and isoform diversity is generated by polymorphism. Multiple copies of penaedin genes are present in different species, and there is rapid expansion and contraction even within closely related organisms.

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Responses to Viruses in the Invertebrates Compared to immunity to extracellular pathogens in the invertebrates, the study of responses to intracellular pathogens like viruses is in its infancy.150 First discovered in plants and in C. elegans, the RNAi pathway of defense against viruses is also operative in Drosophila and Anopheles.151 Doublestranded RNAs (viral or otherwise) are recognized by the enzyme Dicer 2, generating small interfering RNAs that can associate with complementary RNAs and induce their degradation. Viruses also induce an “IFN-like response” through a cytokine receptor (domeless) that is homologous to the IL-6 receptor and signals through the JAK/STAT pathway.152 After viral infection, unknown signals (RNA, perhaps) induce the production of cytokines of the unpaired family that bind to domeless on neighboring cells and upregulate a large number of genes involved in defense (see Fig. 4.3). Nothing is known about the effector pathways of these responses, but mutants of one of the induced genes results in increased viral load. It should be noted that this pathway is not cell autonomous, inconsistent with the IFN pathway in vertebrates. In a paradigm-changing paper, foreign antigen was shown to directly interact with Drosophila toll7, resulting in the induction of antiviral autophagy and inhibition of viral replication.153 Toll-7 interacted with the glycoprotein from vesicular stomatitis virus at the cell surface to initiate the response. Thus, the dual paradigm of indirect and direct recognition by toll and TLR, respectively, must now be modified. There are several other tolls without functions in insects that might be involved in immunity, perhaps via such mechanisms. In summary, arthropods (and presumably other invertebrates) use an RNAi pathway as well as a signaling pathway to combat viruses.150,152 As opposed to the systemic plant RNAi response, the same pathway in protostomes is rather cell autonomous. This response was lost in the vertebrates, presumably because 1) viruses have been able to effectively counter this response and render it ineffective; 2) there have been remarkable evolutionary innovations in the vertebrate innate and adaptive immune systems to combat viruses; and 3) vertebrates use a Dicer pathway extensively to regulate expression of their own genes. The discovery of a viral immune response quite like a type I IFN response in vertebrates demonstrates that the three major signaling pathways of defense in Drosophila, toll, IMD, and JAK/STAT, are similar to the vertebrate TLR/IL-1R, TNF, and IL-6/IFN pathways, respectively (the fi rst two homologous). Finally, other insect tolls may interact directly with PAMPs to promote antiviral defense such as autophagy or apoptosis. This is a field in which rapid progress will be made in the near future.

Natural Killing Activity Across Metazoa The word “cytotoxicity” encompasses vastly different protocols of cell killing by of different cell types. It can be an effector function of cells of the adaptive (cytotoxic T-lymphocyte [CTL] or NKT cells) or of the innate arm

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(bonafide NK, see the following) of the jawed vertebrate immune system. Similarly the term “NK cells” covers different cell types and different functions. NK cells of vertebrates can “recognize” missing self-MHC class I, but also ligands induced on stressed cells following virus infection, transformation or stress. They can also have an immunoregulatory role by interactions with antigen-presenting cells (APCs). Many of these features obviously profit from a comparative approach. In natural killing, the common denominator is a spontaneous reaction (ie, it does not require any [known] antigenic priming but only cell contact). Some form of natural killing can be observed from the earliest metazoans onwards. Some marine sponge and corals avoid fusion with one another by mechanism of cytotoxic cells or induce apoptosis at the level of the teguments.154 Phenomena more similar to vertebrate NK killing are observed in sipunculid worms where allorecognition among populations was shown to result in killing of allogeneic erythrocytes by lymphocyte-like cells.155 Similar cases can be also encountered in annelids and mollusks,156 The role of IgSF and lectin receptors known to be involved as NK cell receptors in vertebrates has not been examined in these invertebrates, even though some candidate homologs have been identified. Note that the mechanism(s) cannot be perforin-mediated, based on the recent bioinformatics analysis on MAC/perforin domains described previously in the complement section.126 When comparative morphology or function is not informative, searching for conservation of transcription factors or cell surface markers may be useful. Survey of IgSF genes across databases have not yielded any promising candidates to date. Despite the presence of polymorphic IgSF members of the receptor tyrosine kinase family in sponges, their role in allorecognition or killing has not been demonstrated.157 As mentioned, similarities were found among the lectin families, especially in prochordates where cytotoxicity has been reported and associated with a discrete population of hemocytes the granular amoebocytes. The urochordate genome (Botryllus, Halocynthia, Ciona) encodes many lectins with or without typical carbohydrate recognition signatures, among them a putative CD94 homolog has been cloned and its expression followed in Botryllus158 ; its predicted sequence does not match well to vertebrate CD94. This gene is differentially regulated during allorecognition in Botryllus, and a subpopulation of blood cells has the receptor on their surface in both Botryllus and Ciona. Phagocytosis is inhibited by an antiserum recognizing the Ciona homologue.159 Other C-type lection homologs of CD209 and CD69 are linked to the CD94/L gene on Ciona chromosome 1 (L.D.P., personal observation). Could they be part of a “pre-NK complex”? Interestingly, all the human homologs of those lectin genes are present either in the NK complex on 12 p13 (CD94, CD69) or on an MHC paralog 19p13 (CD209). Taken together with studies of the chicken MHC which encodes some C-type lectins (see the following), the data suggest that a conserved MHC-linked region containing several lectin genes was present before the emergence of MHC class I and II genes.2

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In addition, a number of genes encoding membrane proteins with extracellular C-type lectin or immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motif s (ITAMs) (plus their associated signal transduction molecules) were identified in Ciona, which suggests that activating and inhibitory receptors have an MHC class I– and II–independent function and an early evolutionary origin.160 The ligands of these Ciona molecules are of great interest to uncover.

Natural Killer and Natural Killer–Like Cells NK cells express both activating and inhibitory cell surface receptors; in fact, the paradigm for positive and negative signaling via such receptors began with these cells60,161; however, activating and inhibitory receptors (often paired) are conserved throughout vertebrates and invertebrate deuterostomes, and are expressed in hematopoietic cells of all types. In NK cells, stimulation of the activating receptors, which associate with proteins having an intracellular ITAM (CD3ζ, DAP12, or DAP10 conserved at least to the level of bony fish162), results in killing of target cells. Inhibitory signaling receptors all possess cytoplasmic ITIMs, which recruit phosphatases and generally are dominant over the activating receptors. These receptors fall in two categories IgSF and C-type lectin group V(II). In general, NKRs recognize MHC class I molecules of either the classical or nonclassical type, the latter sometimes encoded by viruses.163 General Evolution of Natural Killer Receptor Families. As mentioned, NK cells in mammals can use different types of receptors, even encoded by different gene families, IgSF (KIR) in primates and C-type lectins (Ly49) in rodents. A few receptors are conserved but most others are highly variable. Very few families show conservation of domains throughout the jawed vertebrates.163 When dealing with the origin of these genes in invertebrates, one has to imagine under what pressures they evolved. The question remains as to whether NK cells, or NK-like cells, preceded the emergence of T- and B-lymphocytes. As described in the introduction, NKRs are the most rapidly evolving molecular component of the gnathostome immune system. Most ligands for these diverse NKRs are MHC class I molecules, or molecules of host or pathogen origin related to MHC class I. The KIR families are divergent, as very few genes are conserved even between chimpanzees and humans, and there are different numbers of genes in KIR haplotypes within a species. Humans have two KIR haplotypes A and B, one encoding a large number of activating receptors and the other very few.23 It is speculated that the haplotypes are under balancing selection within the population both for defense against virus and for maternal/ fetal interactions. By contrast, CD94 and NKG2 receptors are conserved throughout mammals. So whereas receptors for polymorphic class I molecules are divergent, those for nonpolymorphic or stress-induced class I molecules are relatively conserved (despite the fact that their ligands Qa1 and HLA-E are not orthologous). NK cells play other important roles in other innate immune responses, for example

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in antiviral immunity. NK cell recognition of virus-infected cells engages the activating KIR and Ly49 receptors and NKG2D in this process. Thus, viruses are hypothesized to supply the evolutionary pressure on diversification of NKRs. In fact, it has been shown in mice that inhibitory receptors can rapidly mutate into activating receptors when viral “decoy” class I molecules evolve to engage inhibitory receptors.164 Generally speaking, inhibitory receptors are older and more conserved, whereas activating receptors evolve more rapidly and can be derived from inhibitory receptors via mutations that result in loss of the ITIM.165 Comparative Studies of Natural Killer Function. NK cells were detected in Xenopus by in vitro 51Cr-release assays. Splenocyte effectors from early thymectomized frogs spontaneously lyse allogeneic thymus tumor cell lines that lack MHC antigen expression.166 This activity is increased after the injection of tumor cells or after treating the splenocytes in vitro with mitogens, suggesting lymphokine activation of the killers. Splenocytes isolated with an anti-NK monoclonal antibody (mAb) revealed large lymphoid cells with distinct pseudopodia. Immunohistology indicated that each anti-NK mAb routinely labeled cells within the gut epithelium but NK cells were difficult to visualize in spleen sections.167 In amphibians, NK cell studies are especially interesting because of natural experiments done by nature (ie, the absence or low levels of MHC classical class I during larval life of some species like Xenopus).168 They are bonafide NK cells, distinct from T cells, as they fail to express TCR Vβ transcripts. NK emerge in late larval life, 7 weeks postfertilization, which is about 2 weeks after the time when cell surface class I can be detected. The proportion of splenic NK cells remains very low until 3 to 4 months of age, but by 1 year there is a sizeable population. Therefore, NK cells fail to develop prior to MHC class I protein normal expression (at least NK cells of the type that can be measured with these assays and with NK cell–specific mAbs) and do not contribute to the larval immune system, whereas they do provide an important backup for T cells in the adult frog by contributing to antitumor immunity. NK cells have also been described in a number of teleost fish with the most in-depth studies in catfish, in which there are clonal likes of cytotoxic cells,167 some that clearly lack TCR expression.169 A subset of the fish NK cell bears a highaffinity FcR that can be utilized for antibody-dependent cellular cytotoxicity.170 Other subsets of NK cells spontaneously kill allogeneic targets. Further study of these cloned lines may provide much needed information on NK function in phylogeny.

Phylogeny of Natural Killer Lectins Besides the well-described Ly49 family of receptors in rodents (and horse) and the CD94 and NKG2 families in all mammals, other mammalian NKRs are of interest. Studies in mammals have shown that some NKC-encoded lectin-like receptors in the Nkrp-l family can recognize other lectin-like molecules, termed Clr, also encoded in the NKC.171 Having linked loci encoding receptor-ligand pairs suggests a genetic

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strategy to preserve this interaction; perhaps the CD94 homologs of invertebrates are genetically linked to genes encoding their ligands. In addition, as described in the following, the close genetic linkage of receptor and ligand genes is a common theme in “histocompatibility reactions” throughout the animal and plant kingdoms described below. In chickens, a single gene similar to CD94/NKG2 is encoded in a region syntenic with the mammalian NKC.172 It is linked to CD69, another C-type lectin also encoded in the NKC of mouse/human. Chickens and quail MHC encode two C-type lectin NKR B-lec and B-NK, the latter being most similar to NKPR1.173 Other C-type lectins are found in the RFP-y locus, one that is also most similar to NKRP1.174 These linkages give credence to the idea that the NKC and MHC were syntenic in early jawed vertebrates (see the following). While C-type lectin genes with some similarity to mammalian NKR have been detected in ectothermic vertebrates, no convincing orthology or synteny to the NKC has been found to date. In mammals including marsupials, NKG2D is conserved, and CD94/NKG2 is found in mammals and birds (as well as NKPR1).163 If such C-type lectin NKR are found in the future in cold-blooded vertebrates, they will have to be studied in functional assays. Given the apparent lack of MHC class I and class II in agnathans and their convergently acquired adaptive immune system (see the following), it is difficult to envisage how NK cells with receptors of any type might function in these animals. It should be mentioned, however, that sequence similarity might be difficult to detect for an MHC peptide-binding region (PBR), given the rapid rate of evolution of this gene family. Furthermore, it would not be shocking if there were NK cells with ligands encoded by other gene families—in mammals, ligands for some activating NKRs have not been identified. It would be of interest to study the non-VLR–expressing lymphocytes in agnathans (if such cells exist) for their killing potential or gene expression.

Phylogeny of Immunoglobulin Superfamily Natural Killer Receptors IgSF-activating receptors have been recognized from cartilaginous fish onwards with a convincing activating NKp44 homolog first found in carp (called NILT), but with no functional data.175 Subsequently, this family was found in other bony and cartilaginous fish and definitive orthology to mammalian NKp44 was shown. The activating receptor NKp30 is also conserved, with orthologs found to the level of cartilaginous fish176 ; in addition, as described in the MHC section, there are V(J) genes within the frog MHC called XMIV that are ancient homologues of NKp30 and may be NKRs of both activating and inhibitory types.177 The ligand for NKp30 has recently been uncovered, the stress-induced molecule B7H6.178 Interestingly, there is a perfect correlation between the presence or absence of NKp30 and B7H6 in the vertebrate line, with both genes lacking in birds and bony fish but present in all other gnathostome classes.176 Furthermore, phylogenetic trees suggest a close relationship between NKp30 and NKp44, consistent with their ancient origins within the vertebrate line.

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There are other bony fish–specific IgSF NKR families. One family, the novel immune-type receptors (NITRs),179 have one or two Ig domains with a charged residue in the TM and could therefore be associated (by analogy) to an ITAM DAP12 equivalent, and indeed was shown to interact with mammalian DAP12.162 Like NKp30, the NITR N-terminal V domain is also of the VJ type. NITRs were originally believed to be part of the LRC, but this was shown to be unlikely upon further analyses. NITRs can be expressed by cells of the hematopoietic lineage, presumably lymphocytes. NITRs have been found in all bony fish, with rapid contraction and expansion of the gene family, with the majority of proteins predicted to have inhibitory ITIMs in their cytoplasmic tails. In zebrafish, there are many NITR genes that group into 12 distinct families.180 An extreme level of allelic polymorphism is apparent, along with haplotype variation and family-specific isoform complexity. By contrast, only 11 related genes encoding distinct structural forms have been identified in the channel catfish, and the relatively small number of genes allowed functional studies to be performed. Additionally, taking advantage of the ability to grow clonal lines of catfish hematopoietic cells, one granular cell line lacking all markers of B/T cells was shown to express several NITRs. Expressed NITRs were fused to an ITAM-containing motif and transfected into a T-cell hybridomas line with a nuclear factor of activated T cells (NFAT) promoter, and the specificity for particular catfish MHC alleles was maintained.181 Subsequent crystal structure analysis showed the NITR V domains to form dimers, much the same as Ig/TCR heterodimeric V regions. Thus, the sequence analysis (ITAM/ITIM), signaling properties, involvement in cytotoxicity, and recognition of MHC molecules identify the NITRs as excellent candidates for NKRs in bony fish. Furthermore, the work serves as a paradigm for study of potential NKRs when homology and/or conserved synteny are lacking or ambiguous. IgSF inhibitory receptors usually form larger families of molecules in comparison to activating receptors. This function can be devoted to two distinct families of receptors, giving another example of the extremely rapid evolution of these molecules. There are many ITIM-carrying IgSF integral membrane receptors across the classes of vertebrates, and they seem to have had independent histories as it is difficult to convincingly detect orthologous genes between species. This is especially true of multigene families in fish, with members equipped with possible ITIMs, including the teleost NITR and LITR, and bird CHIR and CD300L.163 Several members of these families can be expressed on NK cells, but expression studies are in their infancy in fish. It is sometimes difficult to distinguish FcR families from NK KIR-like domains, and both FcR and KIR seem to stem from a same lineage. Given the role of the FcR binding to bonafide antibodies and conferring specificities to cells of the innate arms of the immune system, it is likely that these molecules will be restricted to jawed vertebrates. The KIR activity that can incorporate pathogen and virus recognition may be more primitive, but the ancestry of KIR is not well understood, and the bonafide KIR family seems to be restricted to primates.163

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Genes encoding the classical FcRs on the long arm of human chromosome 1 (1q21–23) are linked to other FcR-like genes. A large multigene family, which includes genes encoding the Fc γR and the NK cell Ig-like receptors, is located in the LRC (human chromosome 19q13). This region could in fact be paralogous to 1q23 and may even have been originally associated with the MHC (see the following). These families belong to a larger class of activating or inhibitory receptors. Their phylogenetic conservation in birds, amphibians, and bony fish suggests a biologic importance even though the size of the families, their expression pattern, and the specific nature of the receptors vary greatly among species.182 In several cases, a commitment to a task in the immune system may not be conserved among homologous members, and the evolutionary fate of the family will be probably affected. Comparison of key residues in the domains may suggest a possible common involvement in MHC recognition for the two families recently discovered in birds (CHIR)183 and the teleosts (LITR).184 Other families were generated within a single class or even within a single order of vertebrates (eg, the KIR described previously). The relationships of KIR with many other multigene families such as IpLITR or NITR remain to be explored. What was the scenario that led to the present mammalian situation? If the fish observation on potential MHC binding holds true, the IgSF type of receptor seems to be the most primitive NKR.163

Other Immunoglobulin Superfamily Families to Explore Further In more primitive vertebrates, the physical or genetic linkage of relatively large IgSF families is well documented in the teleost NITR but not yet elucidated in the case of other interesting families in prochordates like the VCBP. In the sea urchin genome, many IgSF await a complete analysis and will certainly contribute to a better understanding of the evolution and origin of Ig/TCR.48 Large families of LRR-IgSF in amphioxus185 could perhaps represent interesting intermediaries in the genesis of either VLR in agnathans or Ig/TCR in gnathostomes (see Fig. 4.10). In hagfish, the discovery of leukocyte expressed receptors agnathan-paired receptors (APARs) revealed what might have been a precursor of Ig or TCR.186 APARs resemble Ag receptors and are expressed in leukocytes and predicted to encode a group of membrane glycoproteins with organizations characteristic of paired Ig-like receptors. Based on their transmembrane regions, APAR-A molecules are likely to associate with an adaptor molecule with an ITAM and function as activating receptors. In contrast, APAR-B molecules with an ITIM are likely to function as inhibitory receptors Thus, the APAR gene family has features characteristic of paired Ig-like receptors. APAR V domains have a J region and are more closely related to those of TCR/B-cell receptor (BCR) than any other V-type domain identified to date outside of jawed vertebrates. Thus, the extracellular domain of APAR may be descended from a VJ-type domain postulated to have acquired recombination signal sequences (RSS) in a jawed vertebrate lineage (see Fig. 4.10). In jawed vertebrates, three such receptor families with VJ-type domains have been identified: a small family of mammalian proteins known as signal-regulatory proteins,

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a large family of the previously described teleost NITR, and the MHC-linked XMIV in Xenopus. These molecules are examined in more detail in the conclusion. Many IgSF proteins expressed in the immune systems are also expressed in nervous systems where the signaling cacscades may be conserved. This selection of IgSF domains, in two different systems in which homologies are found in molecules with quite different functions, may reveal adaptation capacities and constraints exerted on surface receptors.187

VERTEBRATE ADAPTIVE IMMUNITY Not long ago, it was believed that only jawed vertebrates had a true adaptive immune system. From the previous discussion of invertebrate immune responses, clearly mechanisms exist to generate high levels of immune diversity—even at the somatic level—one of the hallmarks of an adaptive response.7 As described in the introduction, many in the field agree that the boundary between innate and adaptive immunity is artificial, and it may not be a useful dichotomy when studying immune responses in diverse organisms.61,62 Despite this reluctance to exclusively classify systems as innate or adaptive, some features clearly fall into the latter category, such as clonal expansion of uncommitted lymphocytes and specific memory. These conditions are not fulfi lled for the DSCAM, FREP, 185/333, or any other invertebrate systems described previously, but of course we must be open to new mechanisms besides the conventional outlook of adaptive immunity. Additionally, such adaptive immunity arose in concert with the emergence of lymphocytes in the lower chordates, as these cells clearly are the major players; we will discuss this in the conclusion.

Immunoglobulins A typical Ig molecule is composed of four polypeptide chains (two heavy [H] and two light [L]) joined into a macromolecular complex via several disulfide bonds (Fig. 4.7). Each chain is composed of a linear combination of IgSF domains, and almost all molecules studied to date can be expressed in secreted or transmembrane forms.

Immunoglobulin Heavy Chain Isotypes Like all other building blocks of the adaptive immune system, Ig is present in all jawed vertebrates (see Fig. 4.7). Consistent with studies of most molecules of the immune system, the sequences of IgH chain C region genes are not well conserved in evolution and insertions and deletions in loop segments occur more often in C than in V domains. As a consequence, relationships among non-μ isotypes (and even μ isotypes among divergent taxa) are difficult to establish.188 Despite these obstacles, the field has developed a working evolutionary tree among all of the isotypes.

Immunoglobulin M IgM is present in all jawed vertebrates and has been assumed to be the primordial Ig isotype. It is also the isotype expressed earliest in development in all tetrapods; until recently, it was believed to be the case in fish as well, but

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MAMMALIAN Ig ISOTYPES

IgM –Primordial Ig –Transmembrane form defines B cells –Pentamer/hexamer in most vertebrates –Tetramer in teleosts –Secreted monomer in cartilaginous fish (associated with affinity increase) –Associated with J chain in all but teleosts and shark monomeric IgM

B

IgD

IgG

–Primordial Ig –Highly plastic in evolution –Lost in several species –Known as ‘IgW’ in several fish –Secreted form associated with granulocytes and inflammation in fish and human –H chain lacks V region in bony fish

IgM

–Derived from ‘IgY-like’ ancestor –First appeared in amphibians –TM region conserved in evolution; involved in memory

IgD (IgW)

MAMMALS

IgE

IgA

–Derived from ‘IgY-like’ ancestor

IgG (IgY) IgE

IgA

–First found in reptiles –Dimer in secretions, monomer in sera –Mucosal Igs in other vertebrate via convergence (e.g. IgX in frogs)

Camelid IgG/IgNAR

Camelid IgG –Derived form of bona fide IgG –’Single-chain V’ convergent with a similar form in cartilaginous fish IgNAR

IgZ/T

or (IgY)

(IgY)

BIRDS or (IgY)

REPTILES (IgY)

(IgYʹ, IgF)

IgX1

or

AMPHIBIANS

2

(IgZ/T)4

3

BONY FISH

5

6

7

or

CARTILAGINOUS FISH

(IgM1gj)8

100

IgMgj

400

IgA

400

IgM/IgD/W IgX

350

400

300

IgE

IgY 100

IgG

Camelid IgG

IgNAR

IgZ/T

FIG. 4.7. Immunoglobulin (Ig) Isotypes in the Jawed Certebrates. A: Mammalian isotypes and their relationships to Igs in vertebrates from other classes. Each oval represents an Ig superfamily C1 domain. IgD is shown in two forms, mouse (left) and human (right).2 B: Major Ig isotypes in all vertebrates. The bottom panel displays the approximate divergence times of all isotypes. IgM/D/W was found at the inception of adaptive immunity. 1, IgX is in the IgA column because it is preferentially expressed in the intestine, and IgA seems to have been derived from an IgX ancestor; IgX seems to have been derived from both IgM and IgY ancestors; 2, secreted IgM in teleost fish is a tetramer, and the transmembrane (TM) form only has three C domains; 3, the teleost fish IgD H chains incorporate the μC1 domain via alternative splicing in the TM form, and the secretory form does not have a V region and does not associate with L chains; 4, the new bony fish isotype, IgZ/T, may not be found in all fish species; 5, the secreted form of shark/skate IgM is present as a pentamer and monomer at approximately equal levels; 6, the TM form of IgW has four C domains; 7, a major TM form of IgNAR has three C domains, and IgNAR is related to camelid IgG by convergent evolution; 8, no TM form has been found (to date) for IgM1gj. The bottom panel displays the approximate divergence times of all isotypes. IgM/D/W was found at the inception of adaptive immunity.

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this view has changed (see the following). The secretory μ H chain is found in all vertebrates, usually consists of one V and four C1 domains, and is heavily glycosylated. H chains associate with each other and with L chains through disulfide bridges in most species, and IgM subunits form pentamers or hexamers in all vertebrate classes except teleost fish, which form tetramers.189 The μ CH4 domain is most evolutionarily conserved, especially in its C-terminal region, whereas the CH2 domain evolves at the fastest rate.188 There are several μ-specific residues in each of the four CH domains among vertebrates suggesting a continuous line of evolution, which is supported by phylogenetic analyses. Like TCR TM regions, μ TM regions are also well conserved among sharks, mammals, and amphibians, but the process by which the Ig TM messenger RNA is assembled varies in different species. In all vertebrate classes except teleosts, the μ TM region is encoded by separate exons that are spliced to a site on μ messenger RNA located approximately 30 basepairs from the end of the CH4-encoding exon. By contrast, splicing of teleost fish μ messenger RNA takes place at the end of CH3 exon.190 In holostean fish (gar and sturgeon), cryptic splice donor sites are found in the CH4 sequence that could lead to conventional splicing, but in the bowfin there is another cryptic splice donor site in CH3.191 The TM region itself is interesting as it is the only one that does not contain a residue capable of making an ionic bond with the ITAM-containing molecule (in this case, Ig-α and Ig-β.) As mentioned, some modifications apparently related to the particular environment were noticed in the Antarctic fish Trematomus bernacchii. There are two remarkable insertions, one at the V H-CH1 boundary and another at the CH2-CH3 boundary; the latter insertion results in a very long CH2-CH3 hinge region. Rates of nonsynonymous substitutions were high in the modified regions, suggesting strong selection for these modifications. These unusual features (also unique glycosylation sites) may permit flexibility of this IgM at very low temperatures.192 It has been known for a long time that in all elasmobranchs, IgM is present at very high amounts in the plasma of cartilaginous fish and that it is found in two forms: multimeric (19S) and monomeric (7S).193 It is unlikely that the two forms are encoded by different gene clusters because 1) peptide maps are identical; 2) early work by Clem found the sequences of the cysteine-containing tail of 19S and 7S H chains to be identical; and 3) all identified germline VH families are represented for the 19S form.194 Although most studies (but not all) reported that 19S and 7S are not differentially regulated during an immune response, in a recent study, the 19S response wanes over time and a stable 7S titer is maintained for periods of up to 2 years after immunization.195 In addition, antigen-specific 7S antibodies observed late in the response have a higher binding strength than those found early, suggesting a maturation of the response, also generally at odds with the previous literature. Finally, when specific antibody titers were allowed to drop, a memory response was observed that was exclusively of the 7S class. This work has shown that a “switch” indeed occurs in the course of an immune response; whether the “switch” is due to an induction of the 19S-producing cells to

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become 7S producers or whether there are lineages of 19Sand 7S-producing B cells is an open question. One working hypothesis is that J chain expression is important for regulating whether a B cell makes 19S or 7S Ig, but of course that could be at the lineage level or the switch level (see the following).

Immunoglobulin M1gj Nurse shark Ginglymostoma cirratum expresses an IgM subclass in neonates.196 The V H gene underwent V-D-J rearrangement in germ cells (“germline-joined” or “gj,” see the following). Expression of H1gj is detected in primary and secondary lymphoid tissues early in life, but in adults only in the primary lymphoid tissue, the epigonal organ (see the following). H1gj associates covalently with L chains and is most similar in sequence to IgM H chains, but like mammalian IgG it has three rather than the typical four IgM constant domains; deletion of the ancestral IgM second domain thus defines both IgG and IgM1gj. Because sharks are in the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system. It is suggested that this isotype interacts either with a common determinant on pathogens or a self–waste product.

Immunoglobulin New Antigen Receptor and New Antigen Receptor-T-Cell Receptor A dimer found in the serum of nurse sharks and so far restricted to elasmobranchs, IgNAR is composed of two H chains each containing a V domain generated by rearrangement and five constant C1 domains.197 IgNAR was originally found in sera, but TM forms exist as ccomplementary DNA and cell-surface staining is detected with specific mAbs. The single V resembles a fraction of camel/llama (camelid) IgG that binds to antigen in a monovalent fashion with a single V region, but it clearly was derived by convergent evolution. In phylogenetic trees, NAR V domains cluster with TCR and L chain V domains rather with that V H. A molecule with similar characteristics has also been reported in ratfish, although it was independently derived from an ancestral Ig like the camelid molecule emerged from bonafide IgG.198 IgNAR V region genes accumulate a high frequency of somatic mutations (see the following). The crystal structure of a Type I IgNAR V regions showed that, in contrast to typical V regions, they lacked CDR2 and had a connection between the two IgSF sheets much like an IgSF C domain.199 The domain wraps around its antigen (hen egg lysozyme [HEL]), with the CDR3 penetrating into the active site of the enzyme. The structure of a Type II V region has a disulfide bond between CDR1 and CDR3 that forces the most diverse regions of the molecule to form raised loop, similar to what has been described for camelid V domains. In total, the differential placement of disulfide bonds forces major changes in the orientations of CDR1 and CDR3, and provides two major conformations for antigen binding.200 The structure of a Type II NAR bound to HEL showed that it also interacted with the active site of the enzyme.

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While analyzing the TCRVδ repertoire in nurse sharks, an entirely new form of this chain, which encodes three domains, V-V-C, was detected.201 The C is encoded by the single-copy Cδ gene, and the membrane-proximal V is encoded by a Vδ gene that rearranges to the DJδ elements. The membrane distal V domain is encoded by a gene in the NAR family, found in a rearranging VDJ cluster typical of all cartilaginous fish Ig clusters. The NAR-TCR V genes, unlike IgNAR V genes which have three D segments, only have a single D region in each cluster. The particular Vδ loci linked to each NAR-TCR gene—called NAR-TCR– supporting Vδ —encode a cysteine in CDR1 that likely makes a disulfide bridge with the NAR-TCR V domain. The J segment of the rearranged NAR-TCR V gene splices at the RNA level directly to the supporting Vδ segment, which has lost its leader exon (Fig. 4.8). This organization likely arose from an IgNAR V cluster that translocated to the TCRδ locus upstream of a Vδ gene segment. After modifications of the supporting Vδ genes, this entire V-V gene set duplicated and diverged several times in different species of sharks. About 25% of the expressed nurse shark TCR δ repertoire is composed of this TCR (encoded by 15 to 20 V-V genes in this species), and have proposed that the typical γ /δ TCR acts as a scaffold upon which sits the single chain NAR V. Our interpretation is that, true to the proposal that γ /δ TCRs interact with free antigen, the NAR V is providing a binding site that can interact with antigen in a different way than conventional heterodimeric Vs. Thus, this is the first case in which a particular V region family has been shown to be associated with a BCR and TCR; in the case of the BCR, the function likely resides within the Fc portion of IgNAR and for the TCR the function (cytokine secretion, killing) lies within the T cell itself. Interestingly, a second TCRδ chain locus has also been described in marsupials and

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monotremes with properties similar to NAR-TCR.202 In this case, there are also two V domains, but in marsupials one (proximal to the membrane) is germline-joined, and only the membrane-distal domain undergoes rearrangement. In monotremes, both of the V regions undergo rearrangement, like for the NAR-TCR. This new TCRδ locus is preferentially expressed early in development. This type of TCR is described in more detail in the TCR section.

Immunoglobulin R/Immunoglobulin New Antigen Receptor/Immunoglobulin W/Immunoglobulin X/Immunoglobulin D All elasmobranchs studied to date have another isotype called IgW. It was probably discovered long ago in skates as a non-IgM secreted isotype called IgR, but no protein sequence of this molecule has been obtained for confirmation.203 Subsequently, an Ig gene was discovered in skates encoding a three-domain molecule with an unusual secretory tail that was named IgX (not to be confused with another isotype with that name in amphibians).204,205 A high molecular weight (MW) species detected by northern blotting with an IgW probe suggested that there might be a longer form of this isotype, subsequently shown to be true in the sandbar shark (IgW), nurse shark (IgNARC—it was so-named because the C domains had highest similarity to IgNAR C domains), and skate.206,207 It was originally believed that sharks only expressed the long (seven-domain) IgW form, but they were later shown to have both secretory forms; the reason for the discrepancy was shark-to-shark variation in expression of the short form, for unknown reasons. The major IgW TM form, like IgM, is composed of five domains, but variants with three domains—like the secretory forms—were also detected.208 Very little is known about the function of this isotype, as IgW-specific mAbs have not been generated as they have for IgM and IgNAR.

Vn

Dn

Jn

Cμ

Vn

Dτn

Jτn

Cτ

Cγ

C others TRANSLOCON

IgH bony fish

(

IgH cartilaginous fish IgL bony fish

Mammal

(

IgL bony, cartilaginous fish

V

Vλ, κ, σ

Jλ, κ, σ

Cλ, κ, σ

Dn

TCRγ cartilaginous fish

Mammal

Vn

TCRα/δ cartilaginous fish

Mammal

Vαn D

J

V

D

J

V

n

Cμ D

D

Cδ D

J

CNAR

)

CLUSTER n

TRANSLOCON

)( n

Vσ’

Jσ’

Cσ’

)

CLUSTER: Some germ-line-joined

n

C

TRANSLOCON

Jn

C

TRANSLOCON OR CLUSTER

Dδ1–3

)

Jμn

Jn

Vδn

V

)(

Cμ/x Cλ, κ, σ

Vn

NAR-TCR cartilaginous fish (form of δ)

J

Jλ, κ, σn

Mammal

( (

D

Vλ, κ, σn

TCRβ cartilaginous fish

TCRμ marsupial (form of δ)

D

Dμn

Jδn

V

n

Vδ

)

Cδ D J

n

Vn

Jαmany

Cα

TRANSLOCON DOUBLE V (ONE REARRANGING): Cluster + germline-joined

Cμ D

Jn

Cδ

DOUBLE V (TWO REARRANGING): Cluster + translocon

FIG. 4.8. Organization of Immunoglobulin (Ig) Heavy and Light (L) Chain and T-Cell Receptor (TCR) Genes in All Jawed Vertebrates. All coldblooded gnathostomes except bony fish have three L chain isotypes, κ, λ, and σ; mammals have κ and λ; and birds have only λ. RSS are found at the 3′ end of V segments, 5′ end of J segments, and on both sides of D segments. Marsupial μ TCR is related to Ig in its V regions and TCRδ in its C region (and is not related to the μ of IgM).

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IgW was thought to be a dead-end isotype in the cartilaginous fish, but a homologue was found in the lungfish.209 It also is present in two secreted forms, one with eight domains and the other, like in the elasmobranchs, with three domains (unfortunately the secretory tail was not sequenced, and the TM form was not studied.) An Ig isotype was found in Xenopus tropicalis most related to the lungfish IgW.210,211 Computer searches of databases for the X. tropicalis genome project uncovered a new isotype flanked by the IgM and IgX genes at the IgH locus. The deduced amino acid sequence obtained from the exons on the genomic scaffold suggests a nine-domain molecule. The N-terminal C domains and the TM regions are most similar to mouse and human IgD regions, and its genomic location also suggest that it is an IgD equivalent. Thus, these new data reveal that like IgM, IgW/D is an isotype that was present at the emergence of all extant vertebrate taxa. Catfish IgD was the first member of this Ig class found outside of mammals,212 but this was not well accepted until the isotype was found in all vertebrate classes. It is found attached to FcR on a subset of myeloid cells, and the secreted form does not contain a V region as the leader is spliced to the CH2 domain in plasma cell RNA.213 This strongly suggests that the IgD is acting as a PRR, perhaps interacting with a conserved region of a pathogen leading to innate immunity. The work on catfish prompted a reappraisal on the role of secreted IgD in humans.214 Like the fish IgD, the human IgD is bound to a subset of myeloid cells, which are activated upon IgD cross-linking. The receptor on human cells has not been identified, but this new work argues for strong evolutionary conservation of the function of secreted IgD. An interesting feature of the IgD/W locus is its high plasticity in evolution, both in terms of the number of domains in different fish species and the plethora of splice variants found, at least in cartilaginous fish.194,215 In sharks, two of the C domains were derived approximately 250 million years ago by a tandem duplication event, and there was a Xenopus-specific, two-domain tandem duplication event as well. Within teleost fish, the number of C exons for this isotype is different in various species and the secreted and TM forms are encoded by different loci in the catfish. In addition to the splice variants previously described in the cartilaginous fish and lungfish, in teleosts the IgM C1 domain exon is spliced into the IgD transmembrane transcript.212 Even in mammals, there are different numbers of C domains in different species, and even exons that have emerged quite recently in evolution (see Fig. 4.7). It is our impression that this is the Ig locus that evolution “plays with,” perhaps using it for different functions in vertebrate taxa. The beginning of understanding the function of the secreted form of IgD is a triumph for comparative immunology; likely the evolutionary approach will reveal the function of the TM form as well.

Immunoglobulin Z/T A third, novel bony fish isotype was uncovered in screens of the EST and genomic databases called IgZ in zebrafish 216 and IgT in trout.217 Its genomic organization parallels the TCR α /δ locus in that the IgZ/T D, J, and C elements are

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found between the VH and Cζ /C μ exons (see Fig. 4.8). IgZ/T is a five-domain H chain that associates with L chains (see Fig. 4.7). The authors of the zebrafish paper proposed that lymphocytes bearing IgZ may be B1-cell equivalents, but a preliminary VH repertoire analysis did not suggest that unique sets of V regions are expressed on IgZ compared to IgM. IgT is not preferentially expressed over IgM early in trout development. Recent work has shown that IgT is the mucosal Ig of trout, with preferential expression in the intestine and skin.218 Mucosal immunization with the parasite Toxoplasma gondii showed a preferential induction of specific IgT. While all teleosts have lost the J chain in evolution,219 biochemical analysis of IgT showed that it contained a secretory piece derived from the poly-Ig receptor (pIgR). Furthermore, like mammalian dimeric IgA, IgT coats trout commensal bacteria, presumably acting as a “firewall” preventing commensals from breeching the blood–brain barrier. In sum, IgT, while displaying unique evolutionary features, has attained almost all of the characteristics of IgA via convergent evolution.

Other Isotypes Related to Immunoglobulin G, Immunoglobulin E, Immunoglobulin A, and the Class Switch Other isotypes consist of four C domains in nonmammalian vertebrates including Xenopus IgY and IgX, non-μ isotypes of Rana, IgY of axolotl, and IgA and IgY of birds.194,220 In Xenopus, IgY is thymus-dependent; IgM and IgX are not, although thymectomy impacts specific IgM antibody production (ie, antigen-specific IgM can be produced, but there is neither an increase in affi nity after immunization nor elicitation of plaque-forming cells). IgM and IgX plasma cells are abundant in the gut,221 whereas IgY is expressed primarily in spleen. Axolotl IgM is present in the serum early during development and represents the bulk of specific antibody synthesis after antigenic challenge.222 In contrast to Xenopus IgY, the axolotl ortholog appears late in development and is relatively insensitive to immunization. From 1 to 7 months posthatching, axolotl IgY is present in the gut epithelium associated with a secretory component.223 IgY progressively disappears from the gut and is undetectable in the serum of 9-month-old animals. Thus, axolotl IgY, like Xenopus IgX and trout IgT, may be analogous to mammalian IgA. Xenopus IgX seemed to be most similar to IgM, but as more sequences have become available it does seem to be orthologous to IgA, obviously consistent with its assumed function (Criscitiello, personal communication). In addition, IgX was formerly believed to be derived from IgM, but recent data suggest that the N-terminal C-domains were derived from an IgY ancestor and the C-terminal domains from IgM.224 The TM and cytoplasmic domains of Xenopus TM IgY share residues with avian IgY and mammalian IgG and IgE, suggesting that mammalian/avian isotypes share a common ancestry with amphibian IgY.225 This homology is especially interesting because studies in mice suggested that the IgG cytoplasmic tail is the central molecular element promoting rapid memory responses of plasmablasts.226,227 The motif in

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the IgY/G tail is also found in other receptors and seems to recruit PI3kinase, which amplifies the signal derived from Ig-α and -β. Finally, another Xenopus isotype was discovered from the databases, called IgF or IgY′ (because it is closely related to IgY, probably duplicating within the Xenopus lineage). Like other isotypes described in this section, it only has two C domains, but rather than alternative splicing, the gene is organized in such a manner.211 There has been no biochemical identification of IgF. CSR results from fusing switch regions upstream of the μ gene and another 3′ isotype gene, accompanied by the deletion of the intervening sequences. Among vertebrates, mammalian and bird class switch regions are GC-rich and contain tandem repeats in which certain motifs, such as TGGGG, GGGGT, AGCT, and GGCT, are abundant. Because of the GC richness of these regions, transcription generates stable R loops, which provide single-stranded substrates for activation-induced deaminase (AID) activity. The fi rst comparative studies on switch were done in the amphibian Xenopus where the switch regions are not GC-rich but adenine-thymine (AT)-rich and cannot form R loops.228 Replacement in mouse of the switch region with a Xenopus switch region showed that it mediated efficient CSR and that the junctions were associated with the short palindromic AGCT motifs, already recognized as the main component in Xenopus switch.229 As predicted from the absence of R loops, the Xenopus switch region supported recombination in both orientations. The breakpoints were located in the AGCT palindrome-rich region of the switch box. Other motifs have been identified in the other switch regions, such as in IgX and chicken isotypes; all of these correspond to the DGYW hotspot consensus. AIDmediated deamination in the context of these motifs may be the conserved major event in the initiation of CSR. As described in the following, until recently it was believed that CSR fi rst appeared in amphibians, but recent data have uncovered a precursor to switch in cartilaginous fish (but not teleosts). Thus, we should add another conserved feature of adaptive immunity that appears to have arisen in the early gnathostomes.230 As mentioned, a single gene can encode different Ig forms, like for duck IgY, cartilaginous fish IgW, and camelid IgG loci. It has been suggested that the two avian IgY short and long forms could be the functional equivalents of both IgE and IgG, respectively; the same may be true of the cartilaginous fish IgW short and long forms with two and six domains, respectively. Also as mentioned, IgF or IgY′ also falls into this category but not through alternative splicing.194,211

Immunoglobulin Light Chains L chains can be classified phylogenetically not only by their sequence similarity, but also by the orientation of their V and J RSS, which differ for mammalian λ and κ. There has been much debate regarding the affi liations of L chains in various vertebrates, but as sequences have accumulated, we have a grasp on their phylogenetic emergence. Contrary to what was believed, κ and λ L chains emerged early in vertebrate evolution, in the cartilaginous fish or placoderms. In

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addition, a third L chain, σ, originally described in Xenopus as a dead-end isotype, is in the ancestral clade and is present in all cold-blooded vertebrates.231 Elasmobranchs have four L chain isotypes (Type I, II, III, and IV), and the combined data suggest that they are present in all chodricthyans.231 Type IV is the ortholog of the unusual Xenopus σ L chain, and Type I is a dead-end variant of this isotype called σ′ or σ-cart (for cartilaginous fish). Type II L chains are of the λ isotype and have been cloned from all groups of cartilaginous fish; all genes of this isotype are “germline-joined.” The type III is clearly κ-like, at least in the V region and RSS orientation (κ is the only antigen receptor gene in which the V is associated with a 12mer RSS). These relationships are more noticeable in the V sequences, which have defining characteristics such as CDR length; although, the C sequences also fall into the same clusters, they do so with much less phylogenetic support. Different elasmobranch species express the L chain isotypes preferentially (eg, κ in nurse sharks, σ in horned sharks, and λ in sandbar sharks); this pattern of expression may be due to expansions/contractions of the different isotype genes in various elasmobranch lineages. Almost all of the L chains in bony fish can be categorized as either κ or σ, despite the large number of gene expansions and contractions in this group.232 As expected, recent data have shown that λ exists in some species, consistent with the work in sharks.233 Genes in different species can be found either in the cluster type organization, translocon, or some intermediate type, and this organization is especially well suited to allow for receptor editing, either to generate the repertoire or as a consequence of binding to self-ligand.234,235 mAb studies suggested the existence of three Xenopus L chain isotypes of 25, 27, and 29 kD with heterogeneous two-dimensional gel patterns and preferential association of some L chain isotypes with IgY H chains.236 Indeed, three Xenopus L chains genes have been isolated: ρ (now κ), σ, and λ. Only one C gene is present in the ρ locus, and it encodes the most abundant L chain. The V and J RSS are of the κ-type and the five identified J segments are nearly identical. The locus is deleted, like mammalian κ, when the other isotype genes are rearranged. Southern hybridizations with genomic DNA from different animals showed V L sequences to be both diverse and polymorphic. The λ Xenopus laevis L chain isotype predicted from the biochemical studies consists of six distinct V L families. In the σ locus, the J segment has an unusual replacement of the diglycine bulge by two serines. The Rana major L chain type has an unusual intrachain disulfide bridge that is seemingly precludes covalent association of its H and L chains.237 Two L chain types were identified in reptiles. Chickens and turkeys only express one L chain (λ) with a single functional V and J gene, and the manner by which diversity is generated is likely responsible for this unusual evolutionarily derived arrangement (see the following). Nonproductive rearrangements are not detected on the unexpressed L chain allele, and thus there is a strong pressure to generate functional joints (see the following). Such a system probably rendered a second (or third) L chain locus

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superfluous.236 Within mammals (marsupial Monodelphis domestica), the Vλ repertoire is comprised of at least three diverse families related to distinct placental families, suggesting the divergence of these genes before the separation of metatherians and eutherians more than 100 million years ago. Opossum λJC sequences are phylogenetically clustered, as if these gene duplications were recent and the complexity of the λ locus seems greater than that found at the H chain locus.238 In summary, all vertebrate groups except the birds have two to four L chain isotypes, all of which can be categorized as κ, λ , or σ (and in cartilaginous fish σ-cart).231 However, we are still at a loss to understand the significance of possessing multiple isotypes as there is scant evidence that L chains have any effector functions. It has been suggested that different isotypes may provide distinct CDR conformations in association with H chains, or there may be L chain/H chain preferences that provide some advantage that is not obvious.239,240 At least the differential CDR sizes for σ as compared to κ /λ suggest that the former rationale is plausible. The preferential association of certain L chains with particular IgH isotypes is suggestive of different functions as well, but there is no evidence for such functions to date.

VH Evolution Diversity of the immune repertoire depends on the variety of V segments inherited in the germline and upon the further diversification by rearrangement (CDR3 only) and SHM (all CDR). Early in life, the repertoire depends chiefly on the inheritable genes as one finds little N-region diversity and somatic mutation (with exceptions; see the following). A central question is how antibody germline V genes diversify CDR during evolution while they are subject to homogenizing forces operating in most multigene families. Perhaps environmental antigens have played a major role in shaping the germline repertoire and have selected some V H/V L germline sequences used by neonates. VH families arose in a bony fish lineage and have been conserved for hundreds of millions of years.241 Conserved regions defining families are found on solvent-exposed faces of the V H, at some distance from the antibody-combining site. Phylogenetic analyses show clustering of V H into groups A, B, C, D, and E. All cartilaginous fish V H belong to the monophyletic group E; bony fish V H genes to cluster into all Groups (one group [D] unique only to them). By contrast, group C includes bony fish sequences as well as V H from all other classes except cartilaginous fish. Another phylogenetic analysis classifies mammalian V H genes in three “clans” (I, II, and III), which have coexisted in the genome for > 400 million years. Only in cartilaginous fish does it appear that V H gene families have been subjected to concerted evolution that homogenized member genes (except for the IgM1gj V region described previously). It has been debated whether Ig V genes could be under direct positive selection or not because these genes hypermutate somatically. However, several features (eg, codon bias) and discovery of high replacement/silent ratios in germline gene CDR codons indeed argue for positive selection during evolution.242,243

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In summary, much of the V H germline repertoire has been conserved over extremely long periods of vertebrate evolution. The birds and some mammalian species that rely on GALT to generate Ig diversity (see the following) are exceptions with a reduced germline repertoire (at least expressed repertoire), but as will be seen in the following, gene conversion and SHM compensate for this situation in formation of the primary repertoire. Even in the cartilaginous fish where there is a single V H family, there is nevertheless great heterogeneity in CDR1 and CDR2 sequences (as well as hypermutation) that must boost diversity in the expressed repertoire.

The J Chain The joining (J) chain is a small polypeptide, expressed by plasma cells, that regulates polymer formation of IgA and IgM.244 J-chain incorporation into dimeric IgA and pentameric IgM endows these antibodies with the ability to be transported across epithelial cell barriers. J chain facilitates creation of the binding site for pIgR (secretory component in the Ig polymers), not only by regulating the polymeric structure but apparently also by interacting directly with the receptor. Therefore, both the J chain and the pIgR/secretory component are key proteins in secretory immunity. J chain complementary DNAs have been reported in all jawed vertebrates except the teleost fish, which have lost it.245 The existence of Xenopus J chain suggests that, unlike mouse IgM, Xenopus IgM forms hexamers with J chain; alternatively, the previous electron microscopy studies identified IgX as the hexameric isotype (the ξ chain has a stop codon before the Cys of CH4 domain and thus cannot make a covalent attachment to J chain). The highest level of J chain expression was detected in frog and bird intestine, correlating well with a role for J chain in mucosal immunity (although obviously not for IgX secretion). Elasmobranch J chain shows high similarity to the N-terminal half of J chains from other vertebrates, but is divergent or even absent in the other regions. This result suggests that the function of J chain may be solely for IgM polymerization in elasmobranchs, and the transporting function arose later in evolution; consistent with this idea, Xenopus, but not shark, J chain is capable if interacting with human IgA and pIgR.245 As mentioned previously, the loss of J chain in bony fish does not preclude the interaction of IgT with pIgR and transport across epithelia, implying a strong pressure to maintain a mucosalspecific Ig.218 There was a claim for the presence of J chain in many protostomes because a homologue was cloned in earthworms, but no J chain sequences have appeared in the numerous protostomes or deuterostome invertebrate databases.246

T-Cell Receptors Ig, TCRs, and MHC class I and class II are all composed of IgSF domains. The membrane-proximal domains of each Ig/TCR/ MHC chain are IgSF C1-set domains, while the N-terminal domains of Ig and TCR proteins are V-set domains encoded by genes generated via rearrangement of two or three gene segments during ontogeny (the membrane-distal domains of

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MHC are a special case; see the following). In all vertebrates studied to date, TCRs are membrane-bound and never secreted, while almost all Ig proteins have transmembrane and secreted forms.247

`/a Constant Domains

Genes encoding the two types of TCR, α / β (which accounts for all known MHC-restricted regulatory and effector functions) and γ /δ (which to the best of our knowledge recognizes antigens in an Ig-like manner and may play immunoregulatory or homeostatic roles during certain infections), existed in the earliest jawed vertebrates. The α / β TCR is considered rather “boring” evolutionarily, as all gnathostomes from the basal cartilaginous fish appear to have this MHC restricted form.248 As mentioned, although many IgSF members exist in the invertebrates, thus far no bonafide Ig/TCR sequences (ie, IgSF genes generated by somatic rearrangements) have been isolated from jawless vertebrates or invertebrates, although a renewed search in jawless vertebrates may be fruitful (see the following). While TCR genes have been cloned from representatives of most vertebrate classes, few biochemical data are available, except in birds where α / β and γ /δ TCR have been identified with mAbs.249 In amphibians, the Xenopus α / β TCR was coimmunoprecipitated with cross-reactive antibodies raised against human CD3ε chains250 (note that this same antibody indentifies CD3 epsilon from many divergent species, which is quite unusual, 251 in turtles). α chains from diverse vertebrates are poorly conserved and the structure of the C α IgSF domain itself is unusual: only strands A, B, C, E, and F can be identified, although strands E and F are shorter than those of mammals and strand D is absent; this modification has an important role in TCR dimerization and subsequent signaling.252 The lack of conservation in this extracellular domain as well as deletions found in bird and teleost fish TCR (especially in the connecting peptide) suggest that the coreceptor may be structurally distinct from mammalian CD3 complex components. Pre-Tα , which associates with TCR β chains during thymocyte development, has been identified only in warmblooded vertebrates.253 The pre-Tα protein has no V domain, and its interaction with the TCRβ chain is unique, based on recent structural studies.254 Unlike all of the other TCR chains, pre-Tα has a long cytoplasmic tail, which seems to be important for T-cell differentiation. Interestingly, the pre-Tα gene is linked to the MHC in mammals, and more phylogenetic studies should be performed to determine whether this linkage group is ancient (see the following). The TM region and cytoplasmic tail of Cα are the most conserved parts of the molecule. Cα and Cβ TM segments in all species have the so-called conserved antigen receptor transmembrane motif (CART) motif, in which conserved amino acids form an interacting surface with the CD3 complex.255 Besides CART, the opposite TM face with conserved residues Ile-Lys-Leu interacts with other CD3 components. The cytoplasmic region is remarkably conserved among all vertebrates. TCR β genes have been sequenced from several species of cartilaginous and bony fish and two species of

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amphibians (axolotl and Xenopus). In addition to the typical IgSF domain features, there are several conserved regions among vertebrate TCRβ chains, especially at positions 81 to 86, probably involved in TCR dimerization. There are also remarkable differences: the solvent-exposed segment 98 to 120 in mammals is absent in all nonmammalian vertebrates. This loop has been shown in mouse TCR to be important for negative selection events in the thymus; perhaps the absence of this region in nonmammalian vertebrates results in subtle differences in tolerance induction as compared to mouse/human.256 The number of Cβ genes varies in different species. Like Cα , Cβ sequences are not well conserved in evolution (eg, the X. laevis Cβ gene does not cross-hybridize with X. tropicalis genomic DNA, and catfish Cβ has only 41% to 42% identity with other teleost Cβ and 26% identity with horned shark Cβ). Two different catfish Cβ complementary DNA sequences were identified, suggesting the existence of either two loci or allotypes, as is found in mammals.257 Indeed, the damselfish Cβ was shown to be encoded by two polymorphic genes, and this feature seems to extend to other teleosts. As the polymorphic sites are believed to interact with the associated CD3-signaling molecules, the authors suggested that signals might be transduced to T cells in different ways depending on the particular expressed Cβ allele. The damselfish Cα gene seems to be encoded by polymorphic alleles as well.258

`/a T-Cell Receptor Variable Domains Because T-cell recognition is MHC-restricted, TCR V regions have been evolutionarily selected for different properties as compared to Ig; indeed, TCR V regions are much less similar to each other than are Ig V regions from other antigen receptor loci.257 Furthermore, TCR Vs, unlike IgV H, have conserved CDR3 lengths, suggesting that there is a restricted size for recognition of MHC-peptide complexes.259 α loci in all vertebrates examined have many J segments, and consistent with the mammalian paradigm, the absence of D and the large numbers of J segments favors the potential for receptor editing during thymic positive selection.260 A number of Vβ gene families are another evolutionarily conserved feature. There are 12 families in nurse sharks and 19 in Xenopus. In axolotls Vβs are classified into nine categories each with 75% or more nucleotide identity; as only 35 genes were cloned, there are probably more families, and several are related to mammalian Vβ genes (human Vβ13 and Vβ20257). There is evidence that the TCR has coevolved with MHC, displayed by the canonical types of interactions seen in crystal structures.261 Vβ sequences from many different vertebrates share residues in CDR2 that interact with class II and CD1 MHC molecules (Y-46 and Y-48, especially).262 It will be of interest to search for other conserved interaction sites as crystal structures become available. Studies of several teleost α /δ loci suggests an organization similar to mouse/human, but with more rearrangement by inversion and more gene segments than are found in mammals.263 Pulsed field gel analysis suggested that the horned shark α and δ TCR loci are closely linked.264 There

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has been a recent, major modification in our thinking of the α /δ locus that will be described in the following.

The f/c TCR Complementary DNA sequences from the skate have significant identity with prototypic mammalian γ and δ TCR genes with extensive V region diversity, putative D segments in δ, and varying degrees of junctional diversity.248 In the nurse shark, there is a great diversity of the δ V regions, the highest among the TCR chains; consistent with this diversity is the presence of the NAR-TCRδ, which is found in approximately 25% of the δ Vs.257 The NAR-TCR has been found in all cartilaginous fish to date, including holocephalins, whereas NAR and IgW have only been detected in elasmobranchs. Thus, it is possible that NAR (especially the V region) arose as a TCR and was transferred to an Ig cluster sometime in evolution. In axolotls, Vδ diversity was diminished in thymectomized animals and TCR δ chains are expressed by cells in lymphoid organs, skin, and intestine.265 Chicken γ /δ T cells were identified long ago. Expression is found in thymus, spleen, and a γ /δ T-cell line, but not in B cells or α / β T-cell lines. Three V subfamilies, three J gene segments, and one C gene were identified at the TCR γ locus. All Vγ subfamilies participate in rearrangement during the first wave of thymocyte development, and the γ repertoire diversifies from embryonic day 10 onwards with random V-J recombination, nuclease activity, and P- and N-nucleotide addition.249 In ruminants and chickens (so-called GALT species; see the following), the γ /δ repertoires are quite diverse, and there seems to be ligand-mediated selection of γ /δ cells during ontogeny. In sheep, where γ /δ TCR diversity is thymus-dependent and follows a developmentally regulated progression, no invariant γ /δ TCRs are found.266 The degree of γ /δ expression is correlated with the evolution of the TCR V families in warm-blooded vertebrates. Indeed, mammals can be classified into “γ /δ low” (humans and mice, in which γ /δ T cells constitute limited portion of the T-cell population) and “γ /δ high” (chicken, sheep, cattle, and rabbits, in which such γ /δ cells comprise up to 60% of T cells). TCR V genes form subgroups in phylogenetic analyses, and humans and mice have representative loci in most subgroups whereas the other species appear to have lost some.267 Thus, γ /δ -low species have a high degree of TCR-V gene diversity, while γ /δ -high species have limited diversity. Interestingly, this pattern is similar to that found for IgV H genes. Recent work in Xenopus has shown that the presence of prototypic VH at the TCRδ locus, in addition to typical α and δ V domains.268 Furthermore, consistent with the presence of VH, the αδ locus is closely linked to IgH and IgL λ in the frog genome. These data suggest that these heterodimeric loci arose via a cis duplication early in vertebrate history.263 The TCRμ locus in monotremes and marsupials202,269 can now be extended to the chicken as well,270 although like Xenopus there are chicken V H with a three-domain molecule as is found in monotremes, marsupials, and sharks. In total, all of the nonplacental mammals except bony fish show some sort of chimeric Ig/TCR locus, with TCR δ V domains that are more Iglike than TCR-like. Generally speaking, comparative studies

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have revealed that the γ /δ TCR can either be used for innate recognition (eg, the cells in the skin of mice or the blood of humans) or adaptively with an enormous repertoire in ways we do not understand—this will be discussed in more detail in the conclusion. Consistent with the potential of γδ TCR to interact with nominal antigen, SHM has been detected in the γ genes of the sandbar shark, although it is not known whether the mutations generate the repertoire or appear in mature T cells after immunization.271,272

Immunoglobulin Gene Organization VH Regions A rearranged V H gene consists of a leader segment, encoded by a canonical split exon, followed by four framework regions and three CDRs (see Fig. 4.8). Canonical V H CDR1 nucleotide sequences are conserved in all jawed vertebrates and serve as targets for SHM.242,243 A major germline difference is the lack of conserved octamers and TATA box in the 5′ region of shark Vs. In all species, functional V genes are assembled by rearrangement and joining of germline V, D, and J elements. Cartilaginous fish H chains are encoded by large numbers of clusters (> 100 in horned shark and dogfish; approximately 15 in the nurse shark). For IgNAR, there are only four V genes/haploid genome and only a few IgW V genes are detected in nurse sharks, but a large number in skates and dogfish.273 There are widely varying numbers of V genes in different species; importantly, the V H complexity does not seem to limit diversity of the antibody repertoire in any ectothermic vertebrate studied to date. There are actually fewer functional human V H (44 functional, 79 pseudogenes that fall into seven families) than in many ectotherms. Dynamic reorganization of the H chain V regions seems to have occurred at least eight times between 133 and 10 million years ago.241 Perhaps species that utilize somatic mutation/selection “optimally” rely less on germline diversity and therefore fewer functional genes are required. Only approximately 10% of Xenopus V H are pseudogenes in the three families (V H1–3) that have been exhaustively studied; thus, Xenopus with fewer lymphocytes has a greater number of functional V H genes than humans.

Chondrichthyan Germline-Joined Genes In all vertebrate species, functional Ig genes are assembled by rearranging DNA segments scattered on the chromosome. However, in cartilaginous fish some V genes are the products of V(D)J rearrangement in eggs/sperm.196,274,275 Type I L chain (σ) genes are all germline-joined in skates but split in horned sharks, and the piecemeal germline joins (eg, VD, VDD, VDDJ) found in many horned shark H chain gene clusters and in nurse shark L chain Type I (σ) clusters strongly suggest that the germline-joining is a derived feature. Definitive proof came from a study of a germline-joined nurse shark Type III (κ) L chain gene, shown by phylogenetic analysis to have been joined within the last 10 million years236 ; this was followed by the identification of the nurse shark germline-joined IgM1gj described previously.196 When there is a mixture of joined and conventional genes, the split genes are expressed in adults, while the joined genes are

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expressed at significant levels only early in ontogeny. When all of the genes in a particular family are joined (eg, skate Type I L chain genes and Type II [λ] L chains in all elasmobranchs), they continue to be expressed into adult life at high levels. In mammals, what may appear like germlinerearranged V genes are in most cases processed pseudogenes (eg, pseudo Vκ on chromosome 22 in human or in mouse). However, it is possible that the surrogate L chain gene VpreB is the product of a germline-joining event in the line leading to mammals.276 Additionally, as described previously, there is a marsupial germline-joined V that serves as the membrane-proximal domain of TCRμ.202

Organization of Rearranging Genes As mentioned, shark IgH-chain genes are structured into tens to hundreds of clusters, each consisting of V, D, J, and C elements277; all evidence from studies in horned shark, nurse shark, skate, and sandbar shark (and holocephalin ratfish) suggest that V, D, and J genes rearrange only within one cluster (but, as detailed in the following, switch may occur between clusters, most likely as a consequence of antigen stimulation). While there is extensive N-region diversity and sometimes usage of two D segments (three Ds in IgNAR), and there are V H subfamilies having substantial CDR1/2 heterogeneity, diversity of the primary repertoire is lower than in other vertebrates as there is no (or infrequent) rearrangement between clusters. The special constitution of the shark H and L chain loci suggests an exclusion mechanism similar to that of mouse TCR γ loci, also found in clusters. It appears that only one V H transcript is expressed in each lymphocyte, consistent with isotypic exclusion, despite the many clusters (see the following).278,279 Bony fish (teleosts and chondrosteans like the sturgeon), frogs, reptiles, and mammals have very similar architectures of their H chain locus—the so-called translocon configuration. As described previously, multiple families of V H genes, each consisting of many apparently functional elements (1 to 30 per family), are separated from a smaller number of genomic D and J elements. The possibility of combinatorial rearrangement enables more diversification than is possible with the cartilaginous fish clusters for a given number of segments. In birds, the organization is similar but all V genes except those most 3′ to the D elements are pseudogenes (see the following). One exception is the coelacanth (Latimeria), in which V genes are immediately followed by D segments, and then multiple J segments as are found in all tetrapods.280 L chain gene organization is variable. In elasmobranchs, the organization is the same (ie, in clusters) as the H-chain locus without the D segments. The prototypic horned shark Type I (σ) L chain has a cluster organization in which V, J, and C segments are closely linked. As mentioned previously, bony fish L-chain genes have the shark cluster-type organization, but some species have multiple V genes in some clusters, demonstrating that there is rapid evolution of not only sequence but gene organization as well in this taxon.232 In Xenopus, there are multiple Vκ (ρ) presumably derived from one family: five J and a single C gene segment. In cartilaginous fish and birds there has been coevolution of Ig gene architecture for H and L loci, but the teleosts have shown

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that this is not a rule. As mentioned previously, two groups have suggested that the teleost gene organization seems to promote receptor editing.234,281

D Segments D segments are always present in one of the two loci encoding an Ig/TCR heterodimer (IgH and TCR β,δ), and the pressure maintaining this asymmetry is unknown. Cartilaginous fish usually have two D genes/H chain cluster, and there are only minor variations among the clusters; one RSS follows the 12-12 paradigm seen in tetrapods and the other is like the TCR 12-23. In teleosts, amphibians, and reptiles where the organization of the H chain locus is similar to humans, the number of Ds deduced from complementary DNAs ranges from 10 to 16. Two germline D segments have been identified in Xenopus, and their RSS follow the rules defined in mammals. In birds, there are 15 very similar DH. There are several reasons why D segments may have been preserved throughout evolution. Incorporation of D segments augments CDR3 diversity and size, obviously directly influencing the combining site.259 Three different Ds contribute to IgNAR CDR3, and besides generating great diversity, CDR3 length and amino acid composition fulfi lls special tertiary structure requirements: D-encoded cysteine residues bond with cysteine(s) in the body of the V domain, thereby stabilizing a loop involved in the antigen-binding of this unusual monomeric receptor.197,200 A similar situation has been reached by convergence in the monomeric variant of camel IgG.198 Finally, rearrangement of one locus “locks it in” and allows the second locus to undergo receptor editing, as is the case for negative selection of the B-cell repertoire and positive selection of the T-cell repertoire. Finally, in mice, one of the TCRδ D segments encodes a section that interacts with the ligand, the nonclassical class I molecule T lymphocyte antigen (TLA).282

Agnathan Variable Lymphocyte Receptor Structure and Function In the 1960s, jawless vertebrates (hagfish and lampreys; see Fig. 4.1) were reported to mount humoral responses to foreign antigens. However, for anti–group A streptococcal antigens, the hagfish “antibodies” (at least a proportion of them) were actually the complement component C3283! Lamprey and hagfish were long known, however, to possess cells resembling lymphocytes and plasma cells,284 with expression of lymphocyte- or at least leukocyte-specific genes,285 but the quest for RAG or bonafide Ig/TCR/MHC genes was a complete failure. Reports of specific memory in allograft rejection and other specific humoral responses were difficult to reconcile with absence of the RAG-based rearranging machinery and the possibility of generating specific lymphocyte clones. Our view of the jawless fish immune system was radically transformed when Pancer et al. prepared complementary DNA libraries of naïve lymphocyte RNA subtracted from lymphocyte RNA derived from lamprey larvae (ammocoetes) that had been immunized to a bacterial/PAMP mixture, and found a highly diverse set of LRR sequences enriched in the

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immunized animals.286 The clones were not only diverse in sequence, but also in the number of LRR “cassettes” found between invariant 5′ (LRR NT) and 3′ (LRR CT) cassettes. Unlike what has been discussed previously for immune genes many species (especially sea urchins), there were not a great number of the germline NT and CT cassettes, suggesting that a somatic recombination process, convergent with Ig/TCR rearrangement, occurred in the developing lamprey lymphocytes (Figs. 4.9 and 4.10). The genomic organization of germline VLR (the original gene discovered was VLRB) have 5′ and 3′ LRR cassettes separated by an intron. Upstream and downstream of the invariant exons are a large number of LRRs, which become inserted between the 5′ and 3′ cassettes during lymphocyte differentiation. Individual lymphocytes apparently express a uniquely rearranged VLR gene in monoallelic fashion. The potential VLR repertoire can be as great as 1014, vastly outnumbering the lymphocytes within an individual.287,288 Fragments of homology between the LRR cassettes allow for joining and then priming of synthesis of a copy of the particular transferred cassette.289 Because these small regions of homology are found throughout the cassettes, “hybrid” LRR can also be formed, further enhancing the diversity over a simple insertion of cassettes. The insertion of LRRs can occur at either end of the somatic recombinant, but it is always 5′-to-3′ on the growing strand. This type of genomic modification resembles the initial stage of gene conversion, but because there is not a complete transfer of genetic material between two homologous gene segments, but actually an addition of sequence, the assembly of the mature VLR is more similar to a recombinatiorial mechanism described in yeast called “copy choice.” The enzymology of “copy choice” has not been examined; because a gene conversion-like process occurs, it is likely that an APOBEC family member is involved. Indeed, APOBEC family members were detected

LRR

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in the lamprey genome project and are expressed in a lymphocyte-specific fashion288 ; in fact, the two members that have been discovered, CDA1 and CDA2, are differentially expressed in agnathan lymphocyte subsets (see the following). Because APOBEC family members are involved in repertoire building in jawless and jawed vertebrates, mutation/gene conversion may have predated RAG-mediated repertoire building of the repertoire (see the following). VLR homologs were found in two additional lamprey species and in hagfish, the only other order of living jawless vertebrates reviewed in Boehm et al.13 As in the sea lamprey, the incomplete hagfish germline VLR generate somatically highly diverse repertoires. Interestingly, the Amphioxus genome harbors a large number of intronless VLR-like sequences that could represent an alternative germline VLR diversity akin to the echinoderm gene families (TLR, SRCR) described previously; one could even speculate that they are related to the ancestral VLR before invasion of its analogous “RAG transposon” (see the following). There are three VLR types: A, B, and C. VLR-B cell-surface receptors and secreted molecules are analogous to the jawed vertebrate membrane and secreted BCR, and it was proposed that cell activation was similar to T-independent pathways in jawed vertebrates (ie, either direct stimulation through the surface VLR or surface VLR stimulation in combination with a PRR/PAMP interaction). It was suggested that the jawless vertebrate adaptive system might be dedicated exclusively to humoral immunity, with the PAMPs providing the second signal to activate lymphocytes.290 Thus, it was proposed that the lamprey system was “B cell-centric” and therefore focused on humoral immunity. This model has been disproved as subsequent work demonstrated that the VLRA and VLRB were differentially expressed (see Fig. 4.10). The former receptor could not be found in plasma and was expressed exclusively as

CT STALK

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Insertion of flanking LRR modules LRR LRR1 LRR NT LRR-1

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Lamprey lymphocyte VLR GPI-linked

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FIG. 4.9. Genetics, Generation of Diversity, and Speculative Cell Biology of the Hagfish Variable Lymphocyte Receptor (VLR) System.13 The top line shows an incomplete VLR gene (NT and CT, N-terminal and C-terminal cassettes, respectively). During lymphocyte ontogeny, upstream and downstream LRR cassettes are inserted between the NT and CT gene segments, resulting in an intronless, mature VLR gene (second line). VLR proteins are attached to the lymphocyte surface via a glycophosphatidylinositol linkage, and may be released into the blood upon antigenic stimulation (also see Fig. 4.10). Boxed is a hagfish VLR protein, the first structure to be elucidated by Kasahara and colleagues.499 Note the loop at the C-terminal leucinerich repeat, which is mentioned in the text as a prominent region inserting into antigens, like single-domain antibodies.293,294

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Cytokines (IL-17) Cytotoxicity?

Cytokines Cytotoxicity

Antigen

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Ig

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2R RAG IgSF IgSF

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Differential recruitment of receptors

VJ

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1R GPIb-α (LRR)

IgSF IgSF (VJ-type) (C1-type)

B-cell like

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APOBEC diversification?

FIG. 4.10. Emergence of Variable Lymphocyte Receptor (VLR) and Immunoglobulin (Ig) Early in Vertebrate Evolution. T and B cells likely preceded the divergence of the leucine-rich repeat and Ig superfamily antigen receptors. The VLR is most similar to a molecule called GPIb-α288 present in Amphioxus, and Ig superfamily antigen receptors are derived from molecules with so-called VJ and C1 domains, probably in the proto–major histocompatibility complex (see text for candidates). Both precursor genes were present in basal chordates. The RAG transposon and 2R likely ignited the appearance of the Ig superfamily receptors (Ig/T-cell receptor) (see text for full explanation). This figure also shows B and T cells today in jawless and jawed vertebrates, with secretion of IgM from gnathostome B cells and a VLR tetramer of dimers from VLRB-secreting lamprey B cells. Figure modified from Flajnik and Kasahara.2

a cell-surface receptor, while, as described, the latter was present as both a lymphocyte receptor and a secreted molecule.291 When microarray expression analysis was performed on VLRA- and VLRB-positive cells, the patterns were consistent with expression profi les in gnathostome T cells and B cells, respectively. The lamprey adaptive scheme, therefore, parallels the situation in jawed vertebrates where Ig is found both as a cell-surface receptor on B cells and as a secreted effector molecule in the serum, whereas the TCR is present only as a cell-surface receptor on T cells; effector functions in T cells, such as cytokine secretion or production of

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cytotoxic mediators, are properties of the T cells themselves. It is not clear how VLRC fits into this scheme at the moment. As expected for members of this family, the framework, or backbone, of the LRR is very similar between the cassettes. Diversity between cassettes is concentrated in the concave surface, presumably the region coming in contact with antigen. The crystal structures of two VLR-B molecules have been determined, one specific for HEL292 and the other for H-trisaccharide.293 In both cases, the concave surface makes contacts as well as a loop in the LRR-CT, which in the case of HEL inserts into the active site, similar to

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what has been found for single-domain camelid and shark Vs.198 One VLR-A crystal structure was determined, also specific for HEL.294 This VLRA was derived from a HELhyperimmunized adult lamprey, which apparently underwent affinity maturation with induced molecules having affinities in the picomolar range. The antigen is bound in the region of the molecule that is most diverse among all VLRAs; surprisingly, the LLRNT among all VLRA are not diverse, suggesting that this region might bind to a self– “restricting element” and the rest of the binding site to the true antigen. These are early days, but the comparisons to date suggest that VLRA and VLRB recognize antigen in different ways. Like gnathostome BCR, VLRB is expressed at the cell surface and is secreted as a pentamer of dimers, much like IgM (see Fig. 4.10). The affinities detected to date from immunized animals are in the micromolar range, again similar to low-affinity pentameric IgM in gnathostomes. By contrast, VLRA has never been detected in the sera or secreted in culture, fitting with its similarity to TCR. The affinity of VLRA for soluble antigen was high (in the nanomolar range) for the HEL-immunized animal, which appeared to undergo affinity maturation.294 Combining the information with the conserved and diverse regions of the VLRA biding site, perhaps VLRA recognizes antigen (budding virus?) associated with a self-protein (MHC analogue?) at the cell surface. It remains to be seen whether VLRA clones will be found from other immunized animals, and the HEL experiment was an artifact of the immunization schedule and not physiologic. The independent development of two different strategies for receptor somatic diversification at the dawn of vertebrate evolution approximately 500 million years ago reveals the magnitude of the selective pressure applied on to the immune system and the emergence of individualized adaptive responses (see Fig. 4.10). Are VLR responsible for the graft rejection results seen in the old experiments? If so, this is most likely a “convergent MHC” coopted for the VLR system. Skin grafting experiments (or other tests of alloreactivity) should be repeated to study potential VLR involvement. The VLR system is discussed below as well in the lymphoid tissues section and the conclusion.

Adaptive Immune Responses in Gnathostomes The quality of T-cell and B-cell responses depends on the heterogeneity and the diversity of the antigen receptor repertoires, and the ability to select cells in secondary lymphoid tissues. Because of the indefinite number of potential Ig/TCR V region sequences in most taxa, potential diversity exceeds the number of available lymphocytes; furthermore, all jawed vertebrates are capable of AID-dependent SHM or gene conversion after antigen stimulation. Yet, while potential repertoires are diverse in all vertebrate classes, and polymorphic MHC class I and II and TCR genes have been isolated from all classes, antibody diversity in nonmammalian vertebrates is relatively low.295–297 The expressed repertoire has been studied via structural studies, affinity measurements during the maturation of the

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immune response, enumeration of antigen-binding Igs by isoelectrofocusing (IEF), and idiotypic analysis. Sequences of Ig and TCR genes expressed over the course of a response help to estimate diversity at another level, allowing studies of V genes diversified by gene conversion and/or somatic mutation during a response in a precise way. In the following survey, we describe studies of specific antibody synthesis, T-cell responsiveness (T-B collaboration, MHC restriction).

Cartilaginous Fish Natural antibodies binding many antigens have been detected at surprisingly high levels in chondrichthyans and in some teleosts. In older experiments, after immunization the horned shark mounted a low-affinity 19S (pentameric) IgM antibody response, which varied little among individuals and did not increase in affinity after prolonged immunization.298 The relative homogeneity and large number of V genes hindered SHM studies until a single unique reference horned shark IgM V H gene was found. Mutations in this gene were slightly more frequent than those in Xenopus (see the following). This first study proved that SHM preceded diversity obtained by combinatorial association of gene segments in evolution.299 In contrast to mutations in the horned shark IgM V H genes, unusual patterns of somatic mutation were detected in nurse shark IgNAR (see the following) and Type II (lambda) germline-joined L chains. Half of the mutations (338/631) occur in tandem without the GC bias seen in Xenopus or horned shark H chain V genes. Tandem mutations and point mutations that take place simultaneously were not generated by gene conversion as there are no repeated patterns or potential donor genes.300,301 The germline-joined L chain genes can only diversify through SHM, perhaps like the hypothetical prototypic V region gene prior to RAG-mediated rearrangement (ie, SHM may have preceded gene rearrangement as the primordial somatic diversification mechanism)300 (see the following). Lastly, a reappraisal of mutation at H chain and other L chain loci in the nurse shark showed that the mutations were not so different from the L chains; the differences from the previous work were the different shark species and the analysis of all H chain loci in the species rather than only one unusual locus.302 As mentioned, the small number of IgNAR genes also made it possible to analyze SHM, and in the first experiments, random complementary DNAs were examined.197,296 The mutation frequency was about 10 times that of Xenopus and horned shark IgM, and even higher than in most studies in mammals. lt was difficult to establish a pattern for the mutations due to their high frequency and because they are often contiguous, like in the L chain gene study described previously. Mutations even in randomly isolated clones appeared to be under positive selection in IgNAR secretory but not TM clones, strongly suggesting that mutations do not generate the primary repertoire like in sheep but arise only after antigenic stimulation.303,304 In total, the shark mutations seem quite mammalian-like but with unusual features. Analysis of the mutations in noncoding DNA suggests

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an AID-dependent process coupled with an error-prone polymerase.305 Affinity maturation and memory generation can be detected in sharks.195 Soon after immunization with HEL, an IgM response can be detected, primarily of the pentameric class. Over time, 7S IgM and IgNAR responses develop, and the 7S antibodies have a higher binding strength than those of the 19S class. When titers were permitted to drop to baseline (or close to baseline), a memory response was induced by immunization of antigen without adjuvant. However, unlike responses in higher vertebrates, the titers do not increase over those in the primary response, suggesting a unique type of regulation of antigen-specific IgM and IgNAR. Nevertheless, these data strongly suggest that the hallmarks of an adaptive response occur in sharks. This type of response is reminiscent of IgA responses of intestinal lymphocytes in mice.306 In another study, a family of HEL-specific IgNAR clones was followed over time after immunization, and a 10-fold increase in the affinity of an already high-affinity germline clone (10 −9 M) was observed.307 These results suggest that affinity maturation, memory, and “switch” to the monomeric IgM isotype occurs, but it takes much longer to attain these adaptive hallmarks compared to mammals, perhaps a paradigm for ectotherms (see the following). Recent work showed that isotype switch can occur in sharks, despite many reports to the contrary.230 Previously, studies of nurse and horned sharks complementary DNAs showed that the V and C regions were derived from the same gene clusters. By contrast, in the new work, immunized animals showed switching between IgM clusters and even between IgM and IgW clusters. It is not known whether there is any functional significance to the switch, but one might predict the switch could dictate a change from 19S to 7S antibodies (see the following), or a modification of effector class in IgM and IgW. None of the classic hotspots (RGYW) upstream of constant region genes in tetrapods vital to switch appear to be targeted to initiate the switch in sharks, but other repetitive elements were detected that might play a role. The role of T cells in shark immune responses has not been studied in detail. No thymectomy experiments have been performed, and T cells have not been monitored during an immune response. Shark mixed lymphocyte responses (MLR) and graft rejection have been attempted, MLR with little success (probably for technical reasons) and grafts with the demonstration of a chronic type of rejection for which the genetics has not been analyzed. However, from the MHC and TCR studies, it is clear that all of the molecular components are available for proper antigen presentation in sharks and skates, and studies of splenic architecture suggestive class II + dendritic cells in the white pulps argue for a prominent T-cell regulatory role in adaptive immunity.308 Furthermore, an increase in binding strength and memory response, as well as mutation and now switch, also strongly suggests a T-cell involvement in humoral immunity. Finally, recent studies of the thymus have shown that the architecture and expression of well-known markers are wholly consistent with a typical T-cell regulation of

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adaptive immunity.257 In summary, it appears that all of the components of adaptive immunity in mammals occur in the cartilaginous fish.

Bony Fish There are high levels of low-affinity natural antibody (up to 11% of total Ig) to nitrophenylacetate in some bony fish. Natural antibodies in catfish have been correlated with resistance to virus infection or furonculosis. As a rule, and similar to cartilaginous fish, little affinity maturation has been detected in fish, although some changes in fi ne specificities were noticed in the trout with a sensitive enzyme-linked immunosorbant assay–based test.309 The mild increase in trout antibody affinity (similar to that found in Xenopus and shark IgNAR) is attributed to selection of either minor preexisting B-cell populations or somatic mutants. In partially-inbred self-fertilized or gynogenetic trout, variability of specific responses is even more restricted. Affinity measured by equilibrium dialysis was of the order of 2.0 × 10 −6 M for trinitrophenol (TNP)-specific antibodies. A large literature deals with vaccination attempts in teleost fish, due to their economic importance. The availability of catfish B-cell, macrophage, and T-cell lines have been instrumental in analyses of antibody production.310 There are puzzling differences in responses from different teleost groups, much like differences between urodeles and anurans (amphibians). Cod, for example, do not respond well to specific antigen and have very high levels of “natural antibodies”; recent studies showing that cod have lost class II genes provides an explanation for the poor humoral responses (see the following). Like the sharks, isolation of TCR genes and the existence of a polymorphic class I and class II molecules suggest that antigen presentation is operative teleosts, but unlike sharks, functional experiments examining mammalian-like T-APC interactions have been performed. TCR messenger RNAs are selectively expressed, and specific TCR rearrangements have been detected in catfish clonal cell lines, which produce factor(s) with leukocyte growth–promoting activity reviewed in Miller et al.310 Modifications of the trout T-cell repertoire during an acute viral infection (rhabdovirus) have also been followed.311,312 In nonintentionally immunized trout, adaptation of the spectratyping technique for TCRβ CDR3 length revealed a polyclonal naïve T-cell repertoire. After an acute infection with viral hemorrhagic septicemia virus, CDR3 size profi les were skewed for several Vβ /Jβ combinations, corresponding to T-cell clonal expansions. Both “public” and “private” T-cell expansions were detected in the infected genetically identical individuals. The “public” response resulted in expansion of Vβ4/Jβ1-positive T cells that appeared fi rst in the primary response and were boosted during the secondary response. Further work examined fi ne specificity of the viral T-cell response, which is a model for studies in cold-blooded animals. Recent results suggest that, despite the fact that IgM is the major Ig expressed in an immune response, high- and lowaffinity IgM appears to be a function of the degree of polymerization of the tetramer.309 It is suggested that high-affinity interactions of TM IgM on B cells modifies the enzymes

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involved in disulfide-bond formation, resulting in modifications of secreted IgM.313 In addition, expression of transcription factors in different subpopulations of trout B cells suggests that the typical populations of naïve, memory, and long- and short-lived plasma cells exist in bony fish; these populations have been followed in blood, spleen, and head kidney by measuring proliferation and B cell–specific transcription factors.314–316 Such studies should prove very useful for vaccination of large populations of teleosts. Studies of SHM in teleost fish have been hampered by the lack of good reference genes, but it has been clear that AIDtargeting motifs are found in Ig V genes.317,318 Furthermore, AID is expressed in the spleen during immune responses, again consistent with its role in SHM.319 A recent study examined mutations in a reference zebrafish L chain gene and detected the typical mutational pattern described previously (without the tandems seen in sharks).320 AID from several bony fish species is capable of inducing CSR in mammalian B cells, despite the fact that teleosts do not undergo CSR 318 ; perhaps the new results that demonstrate switching in sharks may shed light on this conundrum.230 As mentioned, species living in extreme cold develop adaptive structural differences in their Igs.192 At the level of global immune response, temperature exerts a great influence in ectothermic vertebrates in general, low temperature generally being immunosuppressive. Lowering the water temperature from 23° to 11°C over a 24-hour period suppresses both B- and T-cell functions of catfish for 3 to 5 weeks as assessed by in vitro responses.321 Virgin T cells are most sensitive to this cold-induced suppression, a property shared with mammals when tested appropriately. Fish have developed ways to adapt to the lack of fluidity of their B-cell membranes by altering the composition of fatty acid by using more oleic acid at low temperatures. After appropriate in vivo acclimation, catfish T cells are better able to cap cell surface molecules at low assay temperatures than are B cells, suggesting that capping is not the low temperature-sensitive step involved in T-cell immunosuppression in catfish. In the NK section, we briefly discussed fish cytotoxic cells.169 In vitro studies have now shown that leukocytes from immunized fish specifically kill a variety of target cells (allogeneic erythrocytes and lymphocytes, hapten-coupled autologous cells); fish CTL of the αβ (and perhaps γδ) lineages as well as NK cells were found (see previous discussion). Naïve catfish leukocytes spontaneously kill allogeneic cells and virally infected autologous cells without sensitization, and allogeneic cytotoxic responses were greatly enhanced by in vitro alloantigen stimulation.322 Cloned cytotoxic cells contain granules and likely induce apoptosis in sensitive targets via a putative perforin/granzyme or Fas/FasL-like interactions. All catfish cytotoxic cell lines express a signaltransduction molecule with homology to the Fc γ chain of mammals; this chain with an ITAM is an accessory molecule for several activating receptors on mammalian NK cells.161 Importantly, these cytotoxic cells do not express a marker for catfish nonspecific cytotoxic cells. As described previously, nonspecific cytotoxic cells have been found in other fish species, including trout, carp, damselfish, and

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tilapia, and they spontaneously kill a variety of xenogeneic targets, including certain fish parasites and traditional mammalian NK cell targets.

Amphibians Differences in immune system features between urodele (axolotl) and anuran (Xenopus) amphibians, already discussed for MHC and Ig complexity, are also seen in immune responses. Rarely is such divergence seen within one vertebrate class (although the two groups diverged over 250 million years ago!). Urodeles express a very restricted antibody repertoire in response to specific antigen that peaks at 40 days postimmunization, and is entirely of the IgM class, even though the serum also contains IgY.323 They do not respond well to thymus-dependent antigens, which may be due to lack of T-cell help, yet their expressed TCR diversity looks normal. A population of axolotl B cells proliferates specifically in response to LPS and also secretes both IgM and IgY. Moreover, a distinct lymphocyte subpopulation proliferates significantly in response to the T cell mitogens Con A. T cells from young axolotls (before 10 months) do not have this functional ability. Axolotl T cells also can be stimulated with to SEA/SEB, known from mammalian studies to be superantigens.324 Anuran larvae can respond specifically (with only 106 lymphocytes) to many antigens, with a modest affinity maturation of the IgM anti-dinitrophenol (DNP) response.325 In adults, the number of different anti-DNP antibodies does not exceed 40, versus 500 in mammals. In secondary responses, the peak of the response is about 10-fold higher and is reached in 2 weeks; there are no major changes in affinity over this initial rise. Isogeneic Xenopus produce homogenous antibodies to DNP, xenogeneic red blood cells, or phosphorylcholine with identical or similar IEF spectrotypes and idiotypes, while outbred individuals differ.326 Both IEF spectrotypes and idiotypes are inheritable, suggesting that diversity is a reflection of the germline repertoire without a major contribution from somatic mutations. Thus, somatic mutations were followed during the course of an antigenspecific immune response at the peak of the modest affinity maturation.327 The V H genes, like their mammalian homologues, contain the sequence motifs that target hypermutation, as described previously. Of the 32 members of the V H1 family involved in the anti-DNP response, expression of only 5 was detected, indicating that immunization was being monitored. Few mutations were detected (average: 1.6 mutations per gene; range: 1 to 5), and there was not a strong preference for mutations in CDR1 and 2 and virtually none in CDR3. Like in the horn shark IgM study noted previously (but not IgNAR or Type II L chains), the mutations were targeted to GC bases, and such a pattern has been suggested to be the first phase of the SHM phase in mouse/ human; perhaps Xenopus has lost the second phase of the process that results in an evening of mutation frequency for all bases. While the mutation frequency was lower than in mammalian B cells, the rates were only four- to sevenfold less in Xenopus. Thus, there is no shortage of variants, and the reasons for the low heterogeneity and poor affinity mat-

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uration may be due to less than optimal selection of the mutants. Indeed, because of a relatively low ratio of replacement to silent mutations in the CDRs, it was argued that there is no effective mechanism for selecting mutants, which in turn might be related to the absence of GC in Xenopus. In summary, the data from hypermutation, complementary DNA heterogeneity, and spectratype dominance suggests that in the absence of refined modes of selection in late-developing clones, B cells producing somatic mutants may be outcompeted by antibodies generated earlier in the response. Essential T-cell functions in anurans have been demonstrated with in vitro assays for T-B collaboration and MHC restriction, demonstrating the similarity of the role of MHC in Xenopus and mammals.328 Regulatory T cells have been shown indirectly in hematopoeitic/thymic chimaeras for control of CTL generation and in antibody responses. Ig synthesis can be enhanced following late thymectomy in axolotl or Xenopus, again implying a role for thymicdependent regulatory cells. Thymectomy early in life totally prevents CSR from IgM to IgY, but not IgX synthesis; thus, T cells are absolutely required for the switch to the “IgG-like isotype” and for high-affinity IgM responses, but switch to the mucosal Ig can be T-independent. Switching can also be induced in tadpoles, although one must hyperimmunize animals for this response, due to a paucity of T cells in larvae. The switch is also temperature-dependent, and as described previously for channel catfish, ectotherm T cells are quite temperature-sensitive. AID is expressed in lymphocytes in the spleen as well as in secretory cells.329 However, consistent with many previous studies, there is no evidence for a typical GC response to date in amphibians or any other ectothermic species. The continued expression of AID in plasma cells (and, presumably, early in embryogenesis) is of interest and deserves further study. Similar to studies in mammals, the chaperone gp96 has been shown to shuttle peptides into cells making them targets for MHC-restricted CTL lysis.330 Immunization of frogs with gp96 from a thymic tumor results in the elicitation of CTL that display antitumor activity. Elegant experiments with gp96 vaccination have also shown that CTL activity against minor histocompatibility antigens is MHCrestricted. As mentioned previously, NK cells have been characterized in Xenopus with mAbs that recognize non-B/T cells. Those cells kill MHC class I–negative target tumor cells but not class I–positive lymphocytes, and after thymectomy these cells are enriched in the spleen.331 CD8 + cells expressing TCR were isolated with the same mAb, suggesting the existence of amphibian NKT cells; expression of the mAb epitope on cells is induced by phorbol 12-myristate 13-acetate (PMA)/ionomycin, and is also detected in CTL when MHCdependent cytotoxicity is reduced.332 Robert and colleagues have developed one of the best-defined model of innate and adaptive immunity to viral infection in cold-blooded vertebrates, the ranavirus FV3 in Xenopus.70,333 Involvement of CD8 cytotoxic cells and humoral responses have been studied over the course of primary and secondary infections with this virus.334,335 The system is now primed for the study of other cell type involvement, ontogeny, repertoire, etc.

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Reptiles Lack of an increase in affinity and homogeneity of IEF spectrotypes suggest low-antibody heterogeneity in reptiles. In the turtle Pseudemys scripta, a number of genomic VH sequences, representing possibly four families, were isolated, as was a genomic Cμ, all shown to be encoded at a single locus. In northern hybridizations, the Cμ4 probe detected two transcripts; of the four VH groups, only one was expressed, and multiple bands indicated the presence of at least two non-μ transcripts. Among 32 unique VDJ rearrangements from one animal, there were 22 sequence variants in FR4, suggesting either a large number of J segments or somatic modification.336 The latter interpretation is supported by point mutations found in FR3 and CDR3. For T cells, there are no data on T effector function, but there are studies on the behavior of T-cell population changes due to seasonal and hormonal variations. Thymocytes from the turtle Mauremys caspica proliferate in response to phytohemagglutinin (PHA) and ConA, and can kill tumor target cells by both antibody-dependent cellular cytotoxicity–mediated and NK-mediated cytotoxicity. Proliferative responses to PHA and Con A were higher for both sexes in spring and for females in winter than in the other seasons.337 Birds and Mammals The poor increase in affinity of chicken anti-DNP and antifluorescein antibodies again indicates lower heterogeneity in chickens. Few changes occur after immunization, even if one waits 1 year after several injections. Perhaps similar to the trout study described previously, a restricted population of high-affinity antibodies was found only after immunization in complete Freund's adjuvant (CFA).338 Hyperconversion and somatic mutation in Ig genes have been found in splenic GC B cells after immunization.339 The relatively poor affinity maturation of the chicken response may be due to a balance between gene conversion and somatic mutation. Indeed, modification of V genes with segments of DNA is not an optimal strategy for fine-tuning antibody responses.340 In the rabbit, there is also conversion/mutation by B cells in GCs after immunization. Within mammals, large variations are found from marsupials with no obvious secondary response, to mouse with 1,000-fold increases in affinity, but the basis for the relatively poor responses has not been established. In conclusion, although all vertebrates have a very large potential for generating diverse antibodies after immunization, only some mammals studied to date make the most of this potential. Perhaps pressures on the immune system of cold-blooded vertebrates have been less intense due to a stronger innate immunity, and architecture of their lymphoid system is not optimal for selecting somatic mutants, or the great rises in affinity detected in antihapten responses are not physiologically relevant.297 An immune system using somatic diversification at its “best” is well adapted to species where the value of single individuals is important (ie, species with small progenies); has that been the condition for the creation and selection of somatic rearrangement and of the optimal usage of somatic mutations? If this explanation provides a rationale for the utilization of somatic mechanisms

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in generating a repertoire and improving it, it does not tell us why it works so well in certain species and not in others. Perhaps one key is the organization of secondary lymphoid organs. Likely a combination of factors (eg, quantitative and qualitative effects such as endothermy, secondary lymphoid tissues, mutation versus conversion, the hypermutation mechanism itself, rates of proliferation thogen and lymphocyte], etc.) are at work in the regulation of antibody responses.296,297 Finally, significant differences have been detected in the mutational mechanism and targeting in ectotherms (eg, GC-richness in amphibians, tandem mutations in sharks, TCR loci mutation in sharks, etc.) that may enrich our understanding of the general mutational mechanism, including the enzymes involved and the targeting to V genes.318

Lymphoid Tissues In addition to the molecules and functions characteristic of adaptive immunity, primary (lymphocyte-generating) and secondary (immune response–generating) lymphoid tissues also define the specific immune system341 (Fig. 4.11). The thymus is present in all jawed vertebrates, and recent evidence suggests its origin at the dawn of vertebrate emergence.342 All animals have hematopoietic cell–generating tissues, and outside of the so-called GALT species, B cells develop in such bone marrow equivalents in all jawed vertebrates. With the advent of clonal selection, the accumulation and segregation of T and B cells in specialized organs for antigen presentation became necessary and indeed the spleen as such an organ is found in all jawed vertebrates, but not in agnathans or invertebrates. All jawed vertebrate species rearrange their antigen receptor genes somatically (except the case mentioned previously for some shark germline-joined genes276). Besides rearrangement, with combinatorial joining of gene segments and imprecision of the joins, there are two other sources of diversity

AGNATHAN

CARTILAGINOUS FISH

to generate the repertoires: the terminal deoxynucleotidyl transferase (TdT) enzyme that modifies boundaries of rearranging gene segments, and somatic mutations, found exclusively in B cells usually introduced during immune responses. However, progression of rearrangement during Band T-cell development and diversification follow different rules in different vertebrates.220 It is conceivable that species hatching early with just a few lymphocytes are under pressures to develop a rapid response and may not use the same mechanisms as species protected by the mother’s uterine environment. It is also possible that immune systems of species with few offspring are under stronger pressures than species that have many offspring, and this could be reflected in the manner diversity is generated. Studies of B- and T-cell differentiation have been performed in many vertebrates. RAG and TdT genes have been cloned in representatives of all vertebrate classes, probes that allow the monitoring of lymphocyte development (see the following). Reagents have become available permitting a monitoring of T-cell appearance in the lymphoid organs of ectotherms (crossreactive anti-CD3 sera mentioned previously or TCR probes), as well as mAbs and gene probes specific for Ig H/L chains that allow examination of B cells. As a rule, the thymus is the first organ to become lymphoid during development. Another emerging rule is that development of the thymus-dependent MHC-restricted T-cell repertoire is similar in all species, and this is reflected in the evolution of TCR gene organization described previously as well as a core set of molecules that are required for thymocyte migration and differentiation343; in contrast, B-cell repertoire generation differs dramatically among different species, at times even within the same class of vertebrates.220

Cartilaginous Fish Like all other major adaptive immune system components, cartilaginous fish are the first in evolution to possess a prototypic thymus originating from pharyngeal pouches. As

BONY FISH

AMPHIBIAN

BIRD

Thymus Thymoid Fat column

Thymus

Epigonal organ

MAMMAL Thymus

Spleen

Head kidney Spleen

Thymus

Peyer’s patches

Bursa Typhlosole

Leydig’s gland

Bone marrow

Spleen Thymus

Spleen

Spleen

Bone marrow

Bone marrow

GALT

GALT

GALT

GALT

GALTA

1° Thymoid

1° Epigonal 1° Leydig’s 1° Thymus 2° Spleen

1° Head kidney 1° Thymus 2° Spleen

1° Bone marrow 1° Thymus 2° Spleen

1° Bone marrow 1° Bursa 1° Thymus 2° Spleen 2° Nodes (?) Germinal centers

GALTA 1° Bone marrow 1° Thymus 2° Spleen 2° Lymph nodes 2° Peyer’s patches Germinal centers

FIG. 4.11. Evolution of Lymphoid Tissues in the Vertebrates. All jawed vertebrates have a thymus and a spleen with demarcated T- and B-cell zones. Fish have different bone marrow equivalents (epigonal, Leydig’s gland, head kidney), and amphibians are the oldest group with lymphopoietic bone marrow (also the first to have a typical immunoglobulin [Ig]H chain class switch, although sharks seem to have switch as well, despite the IgH cluster organization). Germinal centers are found only in warm-blooded vertebrates. A thymus equivalent (thymoid) has recently been discovered in agnathans.375

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in mammals, it has a distinct cortex/medulla structure, and TdT expression was detected in thymocytes with crossreactive antisera and more recently by northern blotting and in situ hybridization, where it is found throughout the cortex.257 Interestingly, unlike most other vertebrates, age is not an indicator as to the size of the cartilaginous fish thymus; it can be small or large at any stage of development. GALT is also found in elasmobranchs, but lymphoid tissue in the spiral valve (intestine) clearly does not have typical secondary lymphoid tissue structure; the spleen is the only tissue with compartmentalization of cells into discrete T-cell and B-cell zones.308 The Leydig’s and epigonal organs (associated with the gonads) are lymphopoietic and erythropoietic, producing mainly granulocytes and lymphocytes, and there is high RAG expression in these tissues (see the following). Lymphocytes form nodules in the epigonal organ, probably indicative of differentiative events. In addition, many plasma cells are found peppered throughout the epigonal, fitting with the bone marrow connection. At hatching, when dogfish embryos are exposed to waterborne antigens, structural development of the lymphomyeloid tissues is well advanced.344 In the nurse shark, neonatal spleen white pulp consists entirely of class II–negative B cells; by 5 months after birth, T-cell zones appear adjacent to the B-cell zones. Both the B-cell and T-cell zones are vascularized, and no detectable marginal zone separates red pulp from white pulp. Class II–positive dendritic-like cells are found throughout the white pulp.345 In the skate Raja eglanteria, Ig and TCR expression is sharply upregulated relatively late in development (8 weeks) by quantitative polymerase chain reaction. IgM expression is first detected in the spleen of young skates but IgW is expressed first in gonad, liver, Leydig’s organ, and thymus.346 In adults, Leydig’s organ and spleen are sites of the highest IgM and IgW expression. In nurse sharks, IgM1gj and 19S IgM appears in the serum before 7S IgM and IgNAR, and this profi le is reflected in the lack of IgNAR + cells in the spleen until 2 months after birth. RAG and TdT expression in the thymus and epigonal organ of the nurse shark suggests that lymphopoiesis is ongoing in adult life. In contrast to most other vertebrates, N-region diversity is detected (albeit reduced by approximately 50%) in skate and nurse shark IgM and IgNAR CDR3 from the earliest stages analyzed, suggesting that a diverse repertoire is important for young elasmobranchs.308 As mentioned previously, a subset of Ig genes is prerearranged in the germline of chondrichthyans, and many of those germline-joined genes are transcribed in the embryo and hatchling, but not in the adult. This pattern fits with the expression of the nurse shark IgM1gj with its germline-joined V region, and suggests that some germlinejoined genes “take advantage” of their early transcriptional edge and thus some clusters can be selected for specialized tasks in early development. With many gene clusters, it is not known how “clusteric exclusion” is achieved at the molecular level (and why the germline-joined gene expression is extinguished in adult life), but as mentioned previously, studies in two cartilaginous fish species suggest that only one H chain cluster is expressed in each lymphocyte.278,347

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It was suggested, based on the paucity of rearranged gene segments at the nonexpressed IgH loci, that there are limiting factors regulating accessibility to RAG proteins and a short time window for rearrangement of accessible loci in B cells.347 The architecture of cartilaginous fish Ig loci allows greatest diversity only in CDR3 because the CDR2 and CDR1 are always encoded in the germline and V segments do not combine with (D)J segments from other clusters. Yet the number of possible CDR3 is essentially limitless, and the number of germline clusters is also high (at least 15 genes in each species and as many as 100 genes; and usually three rearrangement events take place because two D segments are in each cluster). Thus, the potential diversity is greater than the number of lymphocytes, the general rule for generation of diversity in the vertebrates.

Bony Fish In all teleosts examined, the thymus is the primary organ for T-lymphocyte generation and head kidney the primary organ for B-cell development. The teleost thymus gland originates from the pharyngeal pouches and can be uni-, bi-, or trilobed, depending on the species,348 and it is the first organ to become lymphoid. The cortex/medulla architecture is not as precise in other vertebrate species, but the duality of the compartment is apparent and varies from species to species.349 The spleen contains the basic elements seen in other vertebrates—blood vessels, red pulp, and white pulp—but the distinction between red and white pulp is less obvious (the white pulp being poorly developed). In spleen, the ellipsoids, which are actually terminal capillaries, have a thin endothelial layer surrounded by fibrous reticulum and an accumulation of cells, mainly macrophages. Lymphocyte accumulations are often seen in their vicinity, especially during immune responses, which have been suggested to be primitive GCs, but they are not homologous; as mentioned, AID expression is found in cells319 during immune responses. Red pulp is rich in melanomacrophage centers, groups of pigment-containing cells at bifurcations of large blood vessels, which may regulate immune responses. The other main lymphoid organ is head kidney, believed to function as mammalian bone marrow. The transparent zebrafish is being developed as a new model to study T-cell differentiation.343 In the sea bass Dicentrarchus labrax, a mAb detects differentiating T cells (perhaps pre-T cells) as well as mature T cells as evidenced by the presence of TCR messenger RNA in the sorted populations. Cells seem to migrate from surrounding mesenchyme and subsequently mature in the thymus like in all vertebrates studied so far. T cells appear earlier in ontogeny (between 5 to 12 days after hatching) than cytoplasmic Ig + pre-B cells, which are detected only at 52 days posthatching. Adult levels of T and B cells are reached between 137 to 145 days after hatching, which is quite a long time compared to young amphibians.350,351 Teleost RAG1 differs from mammalian RAG1 genes by the presence of an intron of 666 base pairs (an intron is also found in the sea urchin RAG1 gene in a similar position352).

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Compared with other RAG1 sequences, trout RAG1 has a minimum of 78% similarity for the complete sequence and 89% similarity in the conserved region (amino acid 417-1042). RAG1 transcripts are detected starting at day 20 after fertilization. Trout TdT is highly expressed within the thymus and to a lesser extent in the pronephros beginning at 20 days postfertilization, which correlates with the appearance of lymphocytes in these two tissues.353 Because the H chain cluster is in the translocon configuration and there are many V H families, it is assumed that diversity is generated in the mouse/human mode. As described previously, studies of mature B-cell activation and homing have been done in trout. Immunization of animals results in the production of short-term Ig-secreting cells in the blood and spleen and long-lived plasma cells in the head kidney. Further analysis with B cell–specific transcription factors like PAX5 and BLIMP1 reinforced the functional studies and showed that the blood contains primarily “resting” B cells and the head kidney both plasma cells and B-cell precursors.314,354 These last findings appear to be true of the cartilaginous fish as well, with the epigonal organ as the head kidney primary lymphoid tissue equivalent. Interestingly, recent studies have shown trout B cells to be quite efficient at phagocytosis, raising questions about myeloid/lymphoid lineage commitment in the vertebrates.355 It is predicted that B cells from all ectothermic vertebrates are capable of phagocytosis (shown for Xenopus), and the work was recently extended to B1 cells in mice.356 It certainly will be of interest to study these innate characteristics of B-cell subsets in the future, considering the early appearance of B1 cells and macrophages differentiating from the yolk sac of mouse embryos.328,357

Amphibians In anurans, the thymus develops from the dorsal epithelium of the visceral pouches (the number of pouches varies with species) and is the first tissue to become lymphopoietic. It is colonized from days 6/7 onward by precursors derived from lateral plate and ventral mesoderm through the head mesenchyme. Precursors proliferate in situ as the epithelium begins to express MHC class II molecules but not classical class I molecules. By day 8, thymic cortex/medulla architecture resembles that of other vertebrates.328 Amphibians possess a spleen with red and white pulp, GALT with no organized secondary lymphoid tissue, and many nodules (but no lymph nodes), with lymphopoietic activity in the kidney, liver, mesentery, and gills. The general morphology of lymphoid organs varies greatly according to species and changes with the season. In Xenopus, splenic white pulp is delineated by a boundary layer, and the central arteriole of the white pulp follicle terminates in the red pulp perifollicular area, a T-dependent zone. Anurans, like all ectothermic vertebrates, lack GCs. In Bufo calamita, colloidal carbon particles injected via the lymph sac are trapped by red-pulp macrophages, which then move through the marginal zone to the white pulp.358 Giant, ramified, nonphagocytic cells found in both white and red pulp have been proposed to be dendritic cells. Xenopus bone marrow does not appear to be a major

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lymphoid organ from histologic observation, but high RAG expression in this tissue suggests lymphopoietic activity.359 The maintenance of RAG expression throughout adult life suggests that lymphocytes are continually produced. Thymectomy decreases or abolishes allograft rejection capacity, MLR and PHA responsiveness, IgY antibody synthesis, and all antibody responses that increase in affinity to classic thymus-dependent antigens.331,360 MLR reactivity matures before the ability to mount IgY responses in primary responses. Thymectomy at 7 days of age delays allograft rejection and abrogates specific IgY responses, whereas later in life it only abrogates antibody responses. Thymectomy performed later greatly affects the pool of peripheral T cells, as monitored with mAbs specific for molecules such as CD8. Early thymectomy results in the complete absence of T cells, but lymphocytes with T-cell markers, perhaps corresponding to NKT cells, can still be detected. In Xenopus, thymocytes induce weak graft-versus-host reactions, whereas splenic T cells are good helpers and strong graft-versus-host inducers. The thymus contains some IgM-producing B cells and memory cells poised to switch to IgY synthesis, and in vitro responses are downregulated by naïve thymus cells. Xenopus B cells respond in vitro to low doses of LPS not by proliferation, but rather by Ig synthesis, and also respond to PMA. Old reports of B-cell proliferation can be attributed to contaminants in LPS preparations.328 Urodele embryos initially produce five pairs of thymic buds, the first two of which disappear.284 This results in a three-lobe thymus in Ambystoma, but in Pleurodeles and Triturus it forms one lobe. No cortex-medulla boundary is present, and the thymus generally resembles a canonical cortex. There are at least three types of stromal epithelial cells. There is no lymphopoietic activity in axolotl bone marrow, and hematopoiesis takes place in the spleen and in the peripheral layer of the liver. The spleen is not clearly divided into white and red pulp. About 40% of TCR-β VDJ junctions in 2.5-month-old Ambystoma larvae have N-additions, compared to about 73% in 10- to 25-month-old animals.247 These VDJ junctions had approximately 30% defective rearrangements at all stages of development, which could be due to the slow rate of cell division in the axolotl lymphoid organs and the large genome in this urodele. As mentioned previously, many axolotl CDRβ3 sequences, deduced from in-frame VDJ rearrangements, are the same in animals of different origins. In contrast, in Xenopus, rearrangement starts on day 5 after fertilization for the V H locus, and within 9 days all V H families are used. V H1 rearranges first followed by V H3, and by day 9/10 V H 2, 6, 9, and 10 begin being rearranged and then V H 5, 7, 8, 11 on day 13. For VL, the κ locus is the first to rearrange on day 7 (2 days after V H), a situation similar to that found in mammals. During this early phase, B cells are present in the liver, where their number increases to approximately 500 cells.325 Later in larval life, rearrangement resumes at metamorphosis, as suggested by the low incidence of pre-B cells and by the reexpression of RAG during the second histogenesis of the lymphoid system. T cells show a similar type of RAG expression/cell renewal during

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ontogeny as the B cells, and the larval and adult Vβ T-cell repertoires differ significantly. Even early in development, tadpoles express a highly variable TCR-β repertoire despite the small number of lymphocytes (8,000 to 10,000 splenic T cells); little redundancy in TCR complementary DNA recovered from young larvae implies that clone sizes must be extremely small, unlike in axolotls. In Xenopus, no lymphoid organ apart from the thymus is detectable until day 12 when the spleen appears and with it the ability to respond to antigen. For B cells, until this time no selection occurs as suggested by the random ratio of productive/non-productive VDJ rearrangements (2:1). After day 12, this ratio becomes 1:1 (ie, the rearrangements have been selected). Complementary DNA sequences on days 10 to 12 (when the number of B cells increases from 80 to 500) are not redundant as if each sequence was represented by one cell.325 RAG expression together with the detection of DNA rearrangement circles in the bone marrow suggests that rearrangement is ongoing throughout life and is not restricted to an early period, like in birds and rabbits. Tadpole rearrangements are characterized by a lack of N-region diversity, like in mammals but not axolotls or shark/skate (see previous discussion), and thus very short CDR3.361 During ontogeny, TdT appears in significant amounts in thymus of tadpoles at metamorphic climax, but little expression is detected at earlier stages, which correlates well with the paucity of N-region addition in larval IgH chain sequences.362 Studies of the ontogeny of the Xenopus immune system have revealed a less efficient tadpole immune response (skin graft rejection and Ig heterogeneity and affinity); the absence of TdT expression during tadpole life fits well with the findings of lower larval Ig (and perhaps TCR) diversity.

Reptiles In all reptiles studied, the thymic cortex and medulla are clearly separated. The spleen has well-defined white and red pulp regions, but T- and B-cell zones have not been delineated with precision.348 In Chrysemys scripta, white pulp is composed of two lymphoid compartments: lymphoid tissue surrounds both central arterioles and thick layers of reticular tissue called ellipsoids. Even after paratyphoid vaccine injection, splenic GCs are not formed, as in fish and amphibians. Splenic red pulp is composed of a system of venous sinuses and cords. In Python reticulatus, dendritic cells involved in immune complex trapping have been identified and may be related to mammalian follicular dendritic cells. GALT develops later than spleen during development, and it appears to be a secondary lymphoid organ (but does not seem to contain the equivalent of the bursa of Fabricius). Lymph node–like structures, especially in snakes (Elaphe) and lizards (Gehyra), have been reported. Reptiles, the evolutionary precursors of both birds and mammals, are a pivotal group, but unfortunately the functional heterogeneity of reptile lymphocytes is poorly documented. There seems to be T-/B-cell heterogeneity because an antithymocyte antiserum altered some T cell–dependent functions in the viviparous lizard Chalcides ocellatus. Embryonic thymocytes responded in MLR at all stages, but ConA responsiveness

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increased gradually during successive stages and declined at birth. In the alligator (Alligator mississippiensis), like in mammals after glass-wool filtration, nonadherent peripheral blood leukocytes (PBL) responded to PHA and not to LPS, whereas adherent cells were stimulated by LPS.

Birds The thymus, which develops in chickens from the third and fourth pharyngeal pouches, consists of two sets of seven lobes each with definitive cortex/medulla. The thymus becomes lymphoid around day 11 of incubation. Splenic architecture is less differentiated than in mammals. It is not lymphopoietic during embryogenesis as RAG-positive cells are found mainly in yolk sac and blood. Birds are the first vertebrate group where follicular GCs and T-dependent areas comprising the periarteriolar lymphatic sheath are encountered. Plasma cells are located in the red pulp. γ /δ TCR+ T-lymphocytes are chiefly concentrated in sinusoids, whereas α / β T cells fi ll the periarteriolar lymphatic sheath.249 Lymph nodes seem to be present in water and shore birds but not in chickens and related fowl. The bursa of Fabricius is a primary lymphoid organ unique to birds in which B cells are produced.363 It arises at day 5 of development and involutes 4 weeks later. T-B heterogeneity is obviously well defined in birds (indeed, the “B” in B cell stands for bursa.) The effects of thymectomy—T- and B-cell collaboration and generation of MHC-restricted helper and killer cells—are very similar to mammals, the other class of warm-blooded vertebrates. During the embryonic period, chicken stem cells found in yolk sac and blood rearrange their IgH and L V genes simultaneously over a very restricted period of time, and very few cells colonize each bursal follicle (about 104 follicles).364 Three weeks after hatching, these cells have differentiated in the bursa and then seed the secondary lymphoid tissues, after which time B cells are no longer be generated from multipotent stem cells; thus, only approximately 2 × 104 productive Ig rearrangements occur in the life of the chicken. When an antiserum to chicken IgM was administered in ovo to block this early bursal immigration, there were no stem cells arising later in development that can colonize the bursa, and these chickens lacked B cells for their entire lives.365 Although the general Ig locus architecture is similar to that of frogs and mammals, only one rearrangement is possible as there is only one functional V L or V H on each allele.366,367 Diversity is created during bursal ontogeny by a hyperconversion mechanism in which a pool of pseudogenes (25 ψL and approximately 80 ψH) act as donors and the unique rearranged gene acts as an acceptor during a proliferative phase in bursal follicles. For H chains, the situation is more complex as there are multiple D elements. During ontogeny, selection of productive rearrangements parallels the selection of a single D reading frame, suggesting that the many D segments favor D-D joins to provide junctions that are diversified by gene conversion; the hyperconversion mechanism can also modify Ds because most donor pseudogenes are fused VD segments. The gene conversion process requires AID, which is also required for SHM and CSR.368

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Because diversification by gene conversion occurs after Ig rearrangement and cellular entry into bursal follicles, and there is only a single germline V H and V L expressed on all developing B cells, it was tempting to implicate a bursal ligand binding to cell surface IgM to initiate and sustain cellular proliferation and gene conversion. However, surface expression of IgM devoid of V regions permitted the typical B-cell developmental progression, demonstrating that such receptor/ligand interactions are not required.369 Thus, currently we know little of how cells enter the bursa, which signals induce them to proliferate/convert, and how cells arrest their development and seed the periphery.364

Generation of Diversity in Mammals Mechanisms leading to the generation of repertoire diversity vary among mammalian species.220 Categories can be made depending upon the mode of B-cell development: rabbits, cattle, swine, and chickens, unlike fish, amphibians, reptiles, and primates/rodents, use a single V H family, of which only a few members (sometimes only one) are functional. To diversify their antibody repertoire, this group uses gene conversion or hypermutation in hindgut follicles of GALT early in life (rather than bone marrow throughout life) and is therefore known as the “GALT group.” At the rabbit H locus, as in the chicken, a single V H is expressed in most peripheral B cells. During development, B cells that have rearranged this particular V in the bone marrow (and other sites) migrate to the appendix where this rearranged gene is diversified by gene conversion using upstream donor V segments.370,371 This development of B cells in rabbits is dependent on the intestinal microflora, and efforts are being made to define the potential bacterial superantigens involved, as well as binding sites on IgM necessary for the differentiation.372 In ruminants, the ileal Peyer patches are the bursa-like primary B cell–generating tissues.373 Although bursa, appendix, and sheep ileal Peyer patches show morphologic similarities, the mechanisms generating diversity are different: conversion in the chicken and hypermutation in sheep, and both in the rabbit. As described previously, most of the “GALT group” also appears to lack IgD; thus, IgD might serve some purpose in repertoire development in some groups of mammals and not others. In summary, the organization of the lymphoid tissues is perhaps the only element of the immune system that shows increasing complexity that can be superimposed on the vertebrate phylogenetic tree. The absence of primary and secondary lymphoid tissues (thymus and spleen) is correlated with the absence of a rearranging and/or hypermutating receptor family in other animals, with the exception of the agnathan VLR in which the relevant lymphoid tissues are being defined (see the following). While all jawed vertebrates have a true secondary lymphoid tissue (spleen), ectotherms lack lymph nodes and organized GALT. In addition, while ectotherms clearly have B-cell zones resembling follicles, and despite the clear ability for ectothermic B cells to undergo SMH and at least some degree of affinity maturation, GCs with follicular dendritic cells are not formed after immunization; clearly, this was a major advance in the evolution of the vertebrate immune system.

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The potential repertoire of Ig and TCR as well as VLRcombining sites is enormous in all vertebrates. The potential antigen receptor repertoire in all species for both T and B cells is far greater than could ever be expressed in an animal because of cell number limitations. Not all species or all gene families use combinatorial joining for repertoire building, but all species assemble V, (D), and J gene segments to generate their functional Ig genes during B-cell ontogeny, and the imprecision of this assembly creates great somatic diversity. Thus, from this survey in various species, one could not predict that there would be major differences in immune responses in representatives of different vertebrate classes, and yet as mentioned previously the mouse/human antibody responses are superior to those in many taxa.

More on Evolution of the Thymus The discovery of a second (cervical) functional thymus in mice has raised ontogenetic and immunologic questions.342,374 Is this second thymus the result of an atavism, or does it correspond to what is seen in human when a cervical thymus can form under certain pathogenic conditions during the migration of the thymus to its final mediastinal location? There are examples of cervical thymi in primitive mammals such as marsupials but also in some prosimians. An “extra” thymus also is reminiscent of the multiple thymi encountered quite frequently in cold-blooded vertebrates. As described, the thymus is promiscuous with regard to its precise developmental origin.284 The thymus arises from pouches two to six in cartilaginous fish, from the second pouch in frogs, the second and third in reptiles, and from the third and/or fourth in bony fish, birds, and mammals. The final number of thymi can also be variable. It ranges from five pairs of organs in sharks, to four in caecilian amphibians, to three in urodeles, and finally to one in many teleost fish species, anurans, and many mammals. The thymus of reptiles varies in term of location and number of lobes, reflecting variation in embryonic origin. The adult thymus may be found anywhere from the base of the heart to the neck. For example, in lizards and snakes, there are two lobes on each side of the neck with no subdivision in lobules. The crocodilian thymus is an elongated chain-like structure, not unlike that of birds. In turtles, the thymus is a pair of lobes divided in lobules at the bifurcation of the common carotid associated with the parathyroids. The multilobed thymus in birds is not the equivalent of the multiple thymi found in sharks because the subdivision is secondary to primary organogenesis. Regardless of the underlying ontogenetic mechanism leading to the development the cervical thymus in mice, the result is suggestive of the secondary “thymus spreading” in birds, but it differs with regard to an uneven final size distribution. All marsupials, except koalas and some species of wombats that only have a cervical thymus, have a thoracic thymus similar to that of placentals. Some marsupials (kangaroos and possums) also have a cervical thymus; their thoracic thymus derives from the third and fourth pharyngeal pouches, whereas the cervical thymus arises mainly from the ectoderm of the cervical sinus with some participation from the second and third pouch (like in reptiles).

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The second mouse thymus seems to show primitive characteristics such as the existence of a single lobe as is found in amphibians, a superficial location, and a position compatible with an origin involving another pharyngeal pouch (presumably the second) that would be a marsupial and therefore perhaps a reptilian character. Embryology helps in determining the possible scenarios: During mouse ontogeny, the canonical thymus anlage can be recognized, beginning on day 11.5 in development, as a group of Foxn1-expressing cells located ventrally in the third pharyngeal pouch. If the cervical thymus were derived from an independent anlage, one would rather expect to detect Foxn1+ cells in the endoderm outside the third pouch. This argues for a common origin of thoracic and cervical thymi and against a second thymus anlage outside the third pouch, and, hence, against the above hypothesis that the cervical thymus represents an atavistic organ. A Thymus in Jawless Fish?. Until recently, it was believed that the thymus appeared in evolution with the emergence of adaptive immunity in the extinct placoderm lineage approximately 500 million years ago. There has never been any controversy concerning the presence of a thymus in all living jawed vertebrates, and as described, its requirement for T-cell differentiation is universal. The lack of a thymus in the jawless fish was consistent with the non-Ig/TCR adaptive immunity in these animals, but this has changed in a new study.375 The finding that lampreys have two types of lymphocytes heralded a renewed search for a thymus equivalent in these animals.291 Indeed, there was an extensive literature on this topic over the past century identifying accumulations of cells in various cranial regions, suggesting that lymphocytes could be differentiating in these areas. However, it has always been clear that there is no specialized tissue with a defined cortex and medulla in lampreys or hagfish, as is seen in all gnathostomes. This conclusion was not well supported because there were no molecular markers for either lymphocytes or thymic epithelium in jawless fish. Indeed, consistent with almost all other accumulated data, a recent study concluded that there was “no evidence for a thymus in lampreys.”343(p189) In that study, the major transcription factor involved in the development of T cells described previously, foxn1, was expressed by the pharyngeal epithelium, but the lack of expression of any known lymphocyte markers made it unlikely that this region was truly the thymus equivalent; a similar transcription profi le was seen in the gill epithelium of the model basal chordate Amphioxus, which truly seems to lack an adaptive immune system. The discovery of “T cells” in lampreys opened a new panorama with the expression of T cell–specific genes besides the antigen receptors. As mentioned, Pancer and colleagues discovered that lamprey lymphocytes express two APOBEC family member genes, CDA1 and CDA2, and suggested that they were involved in the rearrangement (and perhaps mutational) events in the VLR genes.288 In the T/B split paper described previously, these APOBEC family members seemed to be expressed specifically in either VLRA (CDA1) or VLRB (CDA2) cells. In the new study, CDA1 was shown

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to be expressed by lymphocytes in close proximity to the foxn1-positive pharyngeal epithelial cells.375 Furthermore, only in these “developing” lymphocytes, but not in mature VLRA-positive cells, could a high percentage (approximately 25%) of out-of-frame VLRA genes be detected, implying that cells were differentiating in this region. In summary, this tissue in lampreys, which was christened the “thymoid,” 1) is derived from the pharyngeal epithelium, 2) expresses classical thymic epithelial markers such as foxn1 and notch ligands, and 3) is associated with developing VLRA cells, based on expression of the APOBEC family member CDA1, out-of-frame VLRA gene sequences, and failure to respond to activation signals (such as the T cell mitogen PHA) that stimulate mature lymphocytes. In addition, consistent with the high percentage of cells with a nonfunctional receptor, many lymphocytes undergo apoptosis in the thymoid, which is also comparable to the situation in jawed vertebrates. Much more work is necessary to understand this system, but the basic finding is extraordinary. Because the thymoid is expressed at the tips of all of the gill filaments,375 thymectomy will not be possible in jawless fish. Perhaps procedures will be developed to block the interactions between the VLRA cells and the pharyngeal epithelium, or the development of the thymoid itself can be disrupted. Assuming that this tissue indeed is the thymic equivalent in lampreys, what is the significance of having the VLRA cells develop in a unique organ? In gnathostomes, T cells recognize antigen in the form of peptides in association with MHC class I or class II molecules. Because of the high levels of MHC polymorphism, as mentioned previously, T cells are positively selected in the thymus for cells that recognize antigen in association with the thymic MHC. Despite major effort, neither MHC molecules nor the specialized proteins associated with antigen processing have been detected in the jawless fish, and thus if there is positive selection it must be orchestrated by a convergent system of antigen processing/presentation. In the same vein, perhaps there is an AIRE equivalent expressed by the thymoid that ensures deletion of self-reactive clones376 ; if so, it would also imply that a convergent antigen presentation system will be discovered in lampreys. It will be of interest to reexamine differentiation of lymphocytes in the pharyngeal epithelium of basal chordates; perhaps this will lead us to an understanding of the origins of adaptive immunity in the vertebrates. Finally, what is the significance of T cells developing in association with the pharyngeal epithelium? Is it because this area in the gill region is evolutionarily plastic or is there some relevance to exposure of the thymoid to the external environment? Discovery of a New Proteasome Element Expressed in Thymus. Recently, a new form of the proteasome only expressed in the thymus was discovered in mammals, called β5t. The β5t gene was generated via a cis duplication from LMPX (β5), and in its absence cytotoxic T cells are impaired.377 Like many other components of adaptive immunity we have discussed, β5t is found in all gnathostomes, beginning with the cartilaginous fish. As we will see in the following, the LMPX equivalent of the immunoproteasome, β5i or LMP7, is polymorphic in amphibians and bony fish,

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suggesting that it is a lynchpin in formation of the immunoproteasome.378 It is interesting as well that both β5t and β5i (and all of the immunoproteasome elements) have been lost in birds (see the following).

The Major Histocompatibility Complex T cells distinguish self from nonself through the presentation of small peptides bound to MHC class I and class II molecules (ie, MHC restriction). The genetic restriction of T cell–APC collaboration, processing of antigen by professional APCs, and T-cell education in the thymus described in mice hold true (or is assumed) for most jawed vertebrate classes. For technical reasons, no MHC-regulated T-cell responses have been documented in cartilaginous fish, but the identification of polymorphic class I and II and rearranging TCRα / β genes and segregated T- and B-cell zones in spleens (see previous discussion) strongly suggest that functional analyses will reveal MHC restriction of adaptive responses.257 By contrast, urodele amphibians and teleost cod are notorious for their poor immune responses (see the following), but biochemical and molecular evidence suggests that class II polymorphism is low in the axolotl and cod have lost the entire class II–based immune system. Recent results in agnathans and other vertebrates have opened our eyes to new functions in MHC.

Class I/II Structure Through Evolution The three-dimensional organizations of class I and class II are remarkably similar: the two membrane-distal domains of both molecules form a PBR composed of two antiparallel α helices resting on a floor of eight β strands, and the two membrane-proximal domains are IgSF C1. Although sequence identity among class I and class II genes in vertebrates is low (like most other immune genes), the four extracellular domain organization and other conserved features are likely to be found in the ancestral class I/II gene.379 An intrachain disulfide bridge exists within the class I PBR α2 and class IIβ1 domains, but not the class I/II PBR α1 domains, and phylogenetic trees show that these respective domains are most similar. Bony fish class II α1 domains, like class II DMα molecules, do have a disulfide bridge. The exon/intron structure of class I and class II extracellular domains is also well conserved, but some teleosts have acquired an intron in the exon encoding the IgSF β2 domain. Other conserved features of class I genes include a glycosylation site on the loop between the α1 and α2 domains important in biosynthesis (shared with class II β chains), a Tyr and one to three Ser in the cytoplasmic regions that can be phosphorylated in mammals, as well as several stabilizing ionic bonds. Class II with its two TM regions differs from class I with only one; conserved residues in the class II α and β TM/cytoplasmic regions facilitate dimerization. In summary, because sequence similarity is very low among MHC genes in different taxa, these conserved features are important for function, biosynthesis, and maintenance of structure. β2m was the second IgSF molecule (C1 type) ever to be identified, originally found at high levels in the urine of patients with kidney disease. It associates with most class I

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molecules (see the following). Besides mammals, β2m genes have been cloned from representatives of all jawed vertebrate classes. The β2m gene is outside the MHC in all tetrapods and bony fish tested, and is a single copy gene in all species except cod and trout, in which it has undergone multiple duplications. Based on the levels of similarity between the various domains of class I and class II, it was predicted that β2m was originally encoded in the MHC; indeed, recent work has shown that it is linked to the MHC in the nurse shark, adjacent to the ring3 gene.380 Like TCR/Ig/class I/II, β2m has not been found in jawless vertebrates, so its origin remains mysterious.

Classical and Neoclassical Class I and Class II Class Ia (classical) and class Ib (nonclassical) genes are found in all of the major groups of jawed vertebrates. Class Ia genes are defined by their ubiquitous expression, their presence in the MHC proper, and by high polymorphism (in most species). In addition, class Ia proteins almost always have eight conserved residues at both ends of the PBR that interact with “mainchain” atoms of bound peptides and constrain their size to eight or nine residues; this feature often distinguishes class Ia from class Ib (see the following). Thus, tight binding of peptides, a likely source of conformational changes in class I allowing transport through the endoplasmic reticulum and cell surface expression, is an evolutionarily conserved trait.381 In nonmammalian vertebrates, one of these residues at the C-terminus is lysine rather than tyrosine, the functional significance of which is unclear.379 The class Ia/Ib distinction holds in most taxa: one to three polymorphic class Ia genes are expressed ubiquitously in most species, whereas other minimally polymorphic or monomorphic class Ib genes are expressed in a tissue-specific fashion. The class Ib genes can be split into two major groups: one set that is most related to the class Ia genes within a taxon and thus recently derived, and one group that is ancient and emerged early in evolution.381 In mouse and human, the set most closely related to class Ia genes are closely linked within the MHC. In nonmammalian vertebrates, however, this first set is found in gene clusters on the same chromosome as the MHC proper but far enough away to segregate independently from MHC (eg, chicken [Rfpy] and Xenopus [XNC ]). One Xenopus class Ib gene is expressed specifically in the lung and thus likely has a specialized function, and another gene (XNC10) is expressed by T cells and may serve as a ligand for unconventional subsets of T cells.382,383 Chicken Rfpy is associated with resistance to pathogens, and the recent structure of one of these class Ib genes has shown an unusual hydrophobic structure in the groove.384 Class Ib genes related to but unlinked to the classical class I have also been found in bony and cartilaginous fish, and a lineage of class II–linked class I genes was discovered in bony fish.385 Thus, class Ib genes that arise in each taxon seem to have true class I–like functions, but perhaps have become specialized (sometimes the distinction between class Ia and class Ib is blurry; see the following). The second set of older class Ib genes that predates divergence of taxa can have very different functions.381 For example, the neonatal Fc receptor is involved in binding

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and transport of IgG molecules across epithelia as well as protecting them from degradation (the Brambell receptor). Furthermore, molecules only described so far in mammals are composed only of a PBR without IgSF domains; these unusual class I molecules do not bind peptides but rather are important for the regulation of NK- and T-cell function during infection. The paradigm for these SOS responses is the MHC class I-related protein (MIC) and UL16-binding protein (ULBP) class Ib molecules, which clearly do not bind peptides. Some teleost class Ib genes that fall outside the major cluster of fish class I genes may fit into this category.385,386 Finally, molecules like CD1 bind nonpeptidic antigens for presentation to innate-like NKT cells. The phylogenetic analysis predicts that CD1 and FcRN are old class I genes (see the following on CD1); the age revealed by the phylogenetic tree also correlates well with the hypothesis that ancient duplication events predating the emergence of jawed vertebrates resulted in the appearance of CD1, FcRN, and MHC-linked class I genes (see the following). Was the original function of class I linked to antigen presentation (peptidic or otherwise), induction of an SOS response, or to housekeeping functions? We do not have the answer because class Ia and class Ib molecules are present in the oldest living gnathostomes. The discovery of class I–like genes in animals derived from ancestors predating adaptive immunity or in the jawless fish would help resolve this question, but to date no recognizable MHC molecules or their kin have been detected in prejawed vertebrates; this topic is especially of interest since the dichotomy of lymphocytes as well as a thymus candidate in jawless fish were discovered.291,375 Class II molecules also have nearly invariant and evolutionarily conserved residues that bind to main-chain atoms of peptides, but these are in the center of the groove.379 Thus, tight binding to main-chain peptide atoms occurs in the center of the class II PBR, and peptides are free to protrude from both ends. The only nonclassical class II molecules so far identified are the previously mentioned DM molecules that lack these residues and DO proteins. DM molecules so far have been cloned only from tetrapods177 and have not been detected in any fish species despite the large genomic and EST databases for the bony fish. Thus, they either had not emerged at the time fish arose or were lost in the bony fish lineage; phylogenetic trees suggest the latter, and would be consistent with the rapid rate of genome evolution in the teleosts. DO class II molecules, believed to modulate DM function, have only been detected in placental mammals.387 The invariant chain is another protein found only in gnathostomes388 ; its recent role in class I cross-presentation suggests that it may have been important for biosynthesis of both class I and class II.389

Class I/II Expression In Xenopus species, immunocompetent larvae express high levels of class II on APCs such as B cells, but express only very low levels of class Ia molecules on all hematopoietic cells until metamorphosis.328 Expression of the immunoproteasome element lmp7 and all identified class Ib isotypes is also very low.390 Larval skin and gut, organs with epithelia in contact with the environment, appear to coexpress class I (transcripts) and class II. Such expression may provide

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immune protection during larval life; perhaps expression of class Ia is limited to organs that undergo massive destruction and remodeling at metamorphosis. Class II molecules also change their distribution after metamorphosis and are highly expressed by unstimulated T cells.391 Axolotl class II molecules are also regulated differentially during ontogeny, expressed in young animals on B cells, and then expanded to all hematopoietic cells, including erythrocytes, later in life.392 Changes in MHC expression are not correlated with cryptic metamorphosis in axolotls, but class II expression by erythrocytes is correlated to the switch from larval to adult globins. Unlike Xenopus, class I transcripts isolated so far are expressed early in ontogeny, from hatching onwards. Carp class I and class II transcripts are detected in embryos 1 day after fertilization and reach a plateau at day 14. However, the suspected class Ia protein does not appear until week 13, whereas β2m can be detected several weeks earlier.393 It was suggested that another class I molecule is expressed during early development of the carp hematopoietic system, perhaps one of the unusual nonclassical molecules that groups outside the teleost cluster. Interestingly, class I is expressed in the brain of young, but not adult fish, suggesting that class I molecules may play a role in neurogenesis.394 As mentioned previously, the cod was recently shown to have lost class II genes, as well as the invariant chain and CD4 genes.395 The apparent lack of the entire class II system of antigen recognition correlates well with the inability of cod to make antigen-specific antibody responses.396 The authors speculate that the large numbers of class I molecules in cod may somehow compensate for the loss of a class II system. Axolotls, which have very low class II polymorphism,397 also have a highly expanded class I system.398 It is possible that in both cases there has been a “use it or lose it” scenario, in which a major arm of the immune system was lost due to a low pathogen load.

Major Histocompatibility Complex Gene Organization As class I and class II proteins are structurally similar, it is no surprise that their genes are linked, a primordial trait subsequently lost only in bony fish (Fig. 4.12).2 But why are structurally unrelated class I processing genes, including the immune proteasome components lmp2 and lmp7 and the TAP and TAPASIN genes, also found in the MHC? There are two possible scenarios: primordial linkage of ancestral processing and presenting genes in the MHC, or later recruitment of either the processing/presenting genes into a primordial MHC. Based on the presence of similar clusters of MHC genes on paralogous chromosomal regions in humans and mice, Kasahara et al.17,399 proposed that ancestors of class I, class II, proteasome, transporter, and class III genes were already linked before the emergence of the adaptive immune system. Genomewide duplications around the time of the origin of vertebrates (the so-called 2-Round or 2R hypothesis), as proposed by Ohno et al.,16 may have provided the raw material from which the immune system genes was assembled (see the following; Fig. 4.13). As for all other adaptive immune genes described so far, neither class I/II nor immunoproteasome/transporter associated with antigen processing (TAP) have been isolated from hagfish nor

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Class I/II

LMP7χ* CL Ib

Bf/C2 (2 genes)*

CL Ib

CL IIαβ

NURSE SHARK

CLIa TAP 1 TAP 2 LMP 2* LMP 7* MECL1* β2m RING3* NOTCH4* PBX2* PPT2* TNX* C4* Bf* HSPA1* BAT2* TNFSF* B7-like* NKR*

CL IIα/β

ZNF297* COLL11A2* RXRB* TAPBP (IgVC1)* FABGL*

HYPOTHETICAL JAWED VERTEBRATE ‘UR’ MHC

C4*

SECTION II

CL Ia

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TAP 1 TAP 2 LMP 2* LMP 7* LMP 7/X* β2m RING3*

114

– Class I, II, III genes linked – True class I region

Class III

C4*

Bf*

CL II β

CL II β

CL II αβ

TAP 1

PBX2* SKII2W CSK2B BAT2* DSP PPT2* HKE4 SACM2L RPS18

KNSL2* ZNF297* DAXX TAPBP* CL Ia LMP* RXRB-L* CL Ia COL11A2* RING3* FABGL* TAP 2 LMP 2* LMP 2/7* MECL1* LMP 7* CL Ia

TELEOST

– Loss of class II gene linkage to class I by translocation or differential silencing after polyploidization – Loss of class III immune genes – True class I region

Class Ib (XNC) (Many loci)

HSPA1 (At least 2 loci)

Ly6 (2 loci)

C5NK2B*

IKBL

TNFSF (3 loci)*

RING3* DMα DMβ CL Ia LMP 2* LMP 7* TAP 1 TAP 2 NOTCH* PBX2* RAGE (IgV) LPAAT C4* MECL1* TNXB* PPT2 C2* Bf* NEU AIF1 BAT2*

CL IIαβ (At least 4 loci)

FABGL* TAPBP* ZNF297* DAXX* KNSL2* RxRB* COL11A2*

CTX (IgSFV)

“Adaptive MHC”

XMIV (IgSFVJ) (At least 6 loci)

XENOPUS

– Highly conserved MHC compared with humans – Class II, class I, TAP, proteasome genes tightly linked (Adaptive MHC) and true class I region – MECL1 in class III region – IgSF(VJ) NK-like receptors in class III region

Bf*

CL αβ

R-fpy* CL Ib

BG (>10 loci IgV)

CD1 (2 loci)

BG (IgV) NKR LECTIN CL IIβ TAPBP** CL IIβ RING3* DMα DMβ DMB CL Ia TAP 1 TAP 2 CL Ia C4*

CL IIa

CHICKEN

– Compaction of genome including MHC; loss of “non-essential” genes especially LMP from the genome – Expansion of non-classical class I/II far from MHC proper – CD1 genes linked to MHC – C type lectin NK receptor genes linked to MHC – TAP/Class I co-evolution demonstrated

Extended class II

Class II

Class II

MOG (IgV)

CL Ib (ABC-like)

CL Ia (A,B,C)

CL Ib (MIC)

KBL

TNFSF (3 loci)*

CSK28 BAT2* AIF1 NKP30 (IgV) LST1

G6 (3 loci)

HSPAI (3 loci)*

SKII2W Bf(Cʹ)* C2 (Cʹ)∗ NEU (sialidase)

C4 (2 loci Cʹ)*

BTL II (IgVC1) NOTCH4* PBX2* RAGE (IgVo) LPAAT PPT2* TNXB*

CL II (DR) αβ

CL II (DQ) αβ

LMP 2* TAP 1 LMP 7* TAP 2 CL II (DO) β

CL II (DM) αβ

CL II (DO) α RING3*

CL II (DP) αβ

KNSL2* DAXX* ZNF297* TAPBP* RPS18 SACM2L FABGL HKE4 RXRB* COL11A2*

“Inflammatory region”

BTN (8 loci, IgVC1)

HUMAN

Class I

– Translocation of class I and subsequent expansion – Translocation of MECL1 to chromosome 16 – IgSF NK Receptor (NKp30) in class III region

FIG. 4.12. Major Histocompatibility Complex (MHC) Evolution. White indicates that the gene is found in at least one other species besides human; black designates a gene with known or inferred immune function; asterisk indicates an ancestral gene found on at least two paralogous MHC regions in mammals; large box indicates at least two genes in the particular region; double slash indicates linkage far away on the same chromosome; space between lines indicates that the genes are on different linkage groups. The teleost MHC is a composite of the zebrafish, trout, Fugu, and medakafish maps. Extensively modified from Flajnik and Kasahara.498

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B2M

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(IgSF) (C-type lectin)

(cathepsin)

(proteasome)

CHAPTER 4

Proto-MHC RXR PSMB TAP TAPBP TRIM RFX CTS NOTCH VAV TNFSF JAK MHCI/II NKR BRD B7 chromosome Two rounds whole genome duplication

• LRC (Chr 19q13) KIR, IgSF, ‘leukocyte’ • NKC (Chr 12p11) NKG2, CD4, LAG3, A2M (C3-like), TAPBP-L, TAP-L • (Chr 14q11) CTS, PSMB5, 11, 5t • (Chr 11q12) CTSD, CTSC

• (Chr1p11-32) NOTCH2, JAK1, VAV3, JUN, B7H4, RXRG, CTSK/S, TNFSF4, 6, 18, (TRIM)n • MHC (Chr5p21) TAPBP, TAP, PSMB8/9, C4, TNSF1–3, NOTCH4, RXRB, (TRIM)n • (Chr19p13) NOTCH3, C3, JAK3, VAV1, TNSF4/7/9, PIAS4 • (Chr9q32) JAK2, CTSL, CTSL2, NOTCH1, DXRA, PSMB7, C5, VAV-2, TNSF8, TRIM14, TRIM32

FIG. 4.13. Genomewide Duplication (2R) Model of Major Histocompatibility Complex (MHC) Evolution. Displayed are the MHC paralogous regions and their human chromosomal designations, first identified by Kasahara and modified over the years.2,17 Note that such paralogous regions have also been studied for chemokine receptors,477 tumor necrosis factor and tumor necrosis factor receptors,459 immunoglobulin superfamily molecules involved in cell-cell interactions,442 and others. Note that the natural killer cell complex and leukocyte receptor complex, respectively, are likely to have been in the Ur MHC based upon linkages seen today in various taxa. The box on the right indicates the original MHC paralogous regions identified by Kasahara on chromosomes 1, 6, 9, and 19.17 The box on the left shows paralogous regions uncovered in further analyses.500 Modified from Flajnik and Kasahara.2

lampreys, and all of these genes as well as other genes involved in immunity could have emerged as a consequence of the duplications. Because class I genes are found on two or three of the clusters, class I–like molecules may have predated class II in evolution. Indeed, NK-like recognition of a class I or class I–like molecule encoded in an ancestral linkage group may have been at the origin of the adaptive immune system (see the following). TAP is an interesting case in that the MHC-linked TAP 1/2 genes were clearly not part of the Ur MHC, but rather they were “recruited” to the MHC early in evolution. TAPs are members of the very large ABC transporter family, and are most closely related to bacterial ABC transporters; this suggests that TAP1/2 were derived from a bacterial, or more likely a mitochondrial gene, via horizontal transfer.400 Furthermore, a close homologue of TAP1/2, TAP-L (TAPlike), unlike all of the other Ig/TCR/MHC genes we have discussed, is present in jawless fish.401 The function of TAP-L is not known, but it may be involved in cross-presentation402 or “typical” presentation in jawless fish. In summary, the TAP genes are not following any of the “rules” that seem to hold true for the other genes involved in adaptive immunity, and this story will be exciting to monitor, especially in the context of the agnathan VLR system. In all nonmammalian vertebrates, and even in marsupials, the immunoproteasome and TAP genes are closely linked to class I genes, not to class II, in a true “class I region.”2 This result is most striking in bony fish (Fugu, zebrafish, medaka, trout) because class I/lmp/TAP/TAPBP and class II are found on different chromosomes.403,404 The class III region, historically defined by the innate immune genes such as Bf/C2, and C4 are also present in the Xenopus and elasmobranch MHC, showing that the class III association of class I/II with such genes is ancient.177 If Kasahara’s interpretation is correct

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(ie, MHC syntenic groups found on different mammalian chromosomes resulted from ancient block duplications), it is expected that the physical association of ancestral class I, II, and III genes predated the emergence of jawed vertebrates, and such syntenies in ectothermic vertebrates are not surprising. Indeed, linkage studies in nonvertebrates Amphioxus and Ciona do support an ancient linkage of class I, II, and III genes.405 Taken together, the data reveal that lack of synteny of class I, class II, and class III genes in teleosts is a derived character. Independent assortment of class I and class II may allow these genes to evolve at different rates: in some teleosts, class Ia alleles form ancient, slowly evolving lineages, whereas class II genes evolve at similar rates as mammalian MHC alleles.406,407 The chicken MHC, the B complex, is on a microchromosome, and intron sizes and intergenic distances are both quite small so that the entire complex is only a few hundred kb as compared to over 4,000 kb in humans and Xenopus.408 Class Ia (BF), class IIβ (BL and DM), and TAP genes are in the MHC, but there is no evidence for immunoproteasome genes, and almost all class III genes have been deleted except for C4. The quail MHC is similar, although is somewhat expanded, especially in genes related to the C-type lectin NK receptors.409 Although most class III genes are found on other chromosomes, lmp2/7 and MECL1 genes are actually absent from the genome; indeed, peptides bound to chicken class I molecules sometimes have C-terminal glutamic acid or aspartic acid, which are rare after proteolysis by mammalian proteasomes containing lmp2 and lmp7. Indeed, the recent crystal structure of a chicken class I molecule shows that the alleles can interact with a broader array of peptides as compared to mammalian class I alleles; the PBR is “broader” and one of the conserved peptide-binding residues is noncanonical.410 To explain the correlation of

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diseases with particular haplotypes, Kaufman proposed that the chicken has a minimal essential MHC composed of only those genes absolutely required to remain in the complex. This concept has been reinforced recently by an analysis of resistance to viral infection (Rous sarcoma virus) governed by classical class I molecules.411,412 Additionally, the presence of polymorphic TAP alleles closely linked to particular class I alleles has demonstrated coevolution of the transporters and peptide-binding molecules.413 Surprisingly, CD1 genes are closely linked to the chicken MHC.414,415 As mentioned previously, CD1 genes in mammals seem to be on one of the MHC paralogous regions and it was suggested that it arose as a consequence of the en bloc duplication. While this is still possible (ie, there was differential silencing of CD1 and other class I genes on the two paralogous chromosomes), the more likely scenario is that CD1 arose by gene duplication within the MHC itself in an ancestor of warm-blooded vertebrates; no bonafide CD1 genes have been detected to date in any fish or amphibians, so the entire NKT system may be specific of warm-blooded vertebrates. In summary, in all animals except placental mammals, classical class I genes map closely to the TAP, lmp, and tapasin genes, suggesting that the processing, transport, and presenting genes were in an original “class I region.”416 The tight linkage of the functionally, but not structurally, related genes strongly suggests that such genes coevolve within particular MHC haplotypes. Indeed, in Xenopus there are biallelic lineages of class Ia, LMP7, and TAP, which are always found as a set in wild-caught animals.417 Although teleosts underwent an explosive adaptive radiation 100 million years ago and primordial syntenies have been lost in many cases, there are deep lineages of class Ia genes in many species, also found for Xenopus and cartilaginous fish class Ia genes. A study in medaka suggests that divergent noncoding regions between the class I–processing and –presenting genes do not permit recombination between lineages, hence preserving the linkage disequilibrium.418 Nonaka et al. proposed that these deep proteasome/class I lineages emerged at the dawn of vertebrate immunity and may even have been maintained perhaps by convergent evolution in certain groups.419 In (most) eutherian mammals, the class I region is not closely linked to lmp/TAP and is very unstable, with rapid duplications/ deletions expected in a multigene complex (see Fig. 4.12); the same class I instability extends to the non–MHC-linked class Ib genes in Xenopus species.420

EVOLUTION OF ALLORECOGNITION Histocompatibility Reactions in Invertebrates Scrutinizing graft rejection within a species (allograft) across the animal kingdom demonstrated that allorecognition was almost universal among metazoa. Scrutinizing specific memory in the same systems was the major tool to establish the universality or not of adaptive immunity. Moreover, it fueled speculations the origins of vertebrate MHC. Most of these studies led to inconclusive results

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because of the poor immunogenetic aspects of the reactions. However, the genetic regions and mechanisms responsible and molecular underpinnings of such alloreactions are now becoming known, and no (or little) resemblance to the vertebrate MHC has been found even though regions homologous of the extended vertebrate MHC have been identified in some organisms but without functional correlation.405,421 Colonies of Porifera, Cnidaria, Bryozoa, and Tunicata (see Fig. 4.1) often compete for space and may develop histocompatibility reactions in the zone of contact. In addition, cell-lineage parasitism, in which the somatic and/or germ cell lineage of one partner replaces that of the other, may ensue if colonies fuse into a chimera. Thus, effector functions following allorecognition also protect the genetic integrity of the individual. In some species, fusion is apparently restricted to tissues of the same individual (complete matching), in other species such as bryozoans, fusion also occurs between genetically distinct individuals if they share kinship (partial matching). Two divergent invertebrate phyla have been studied in detail: Cnidaria and Tunicata.

Porifera Sponges (Porifera) and placoza are the phylogenetically oldest extant metazoan phyla. Sponges, whether marine or freshwater species, possess a sophisticated histocompatibility system.422 Elements of the sponge immune system involved in these reactions have been analyzed at the molecular level. Sponge cells associate in a species-specific process through multivalent calcium-dependent interactions of carbohydrate structures on a 200 kd extracellular membrane-bound proteoglycan called “aggregation factor,” well studied in Microciona and Geodia .422 The glycan moiety is involved in cell adhesion and exhibits differences in size and epitope content among individuals, suggesting the existence of allelic variants. Therefore, strong carbohydrate-based cell adhesion evolved at the very start of Metazoan history. Other genes involved in these reactions include one that is similar to the vertebrate MHC-linked allograft infl ammatory factor and another to the T-cell transcription factor. AIF-1 and T-cell transcription factor genes are upregulated in vivo after tissue transplantation, and in vitro in mixed sponge cell reaction.423 Polymorphic IgSF molecules are found on the surface of sponge cells, but their relationship to allorecognition events (if one exists) is not clear.424 Allogeneic recognition in vitro led to apoptotic cell death in one partner and survival in the other.425 The process is controlled by a differential expression of the proapoptotic and prosurvival proteins that are characteristic for the initiation of apoptosis (caspase, MA3, ALG-2 protein) and the prevention of programmed cell death (2 Bcl-2 homology proteins, FAIM-related polypeptide, and DAD-1–related protein). In an apoptotic mixed cell combination, characteristic apoptotic genes were expressed, while in the nonapoptotic aggregates the cell-survival genes are upregulated.423 In another species, Microciona, allogeneic interactions also

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induce cellular reactions involving gray cells (sponge immunocytes) and finally apoptosis. Analogous (but most likely not homologous) to T-cell responses, the response is inhibited by cyclosporin A.426 From observation of 50 pairs of larval grafts within one F1 progeny of the marine sponge Crambe, 75% could fuse, a proportion suggesting that the genetic control depends on one locus and sharing of one haplotype results in fusion; a 100% fusion between mother and offspring is consistent with this interpretation. Unfortunately, the issue is complicated as individuals from a given mother may not have the same father. Still, the few data available are consistent with a single or at least major histocompatibility locus (or region), the “rule” in other invertebrate phyla described in the following.

Cnidaria The existence of highly polymorphic histocompatibility loci was demonstrated long ago in various corals where apoptosis induction was responsible for the death of partners in incompatible combinations,154 whereas in sea anemone nematocyte discharge was induced between incompatible individuals.427 The colonial cnidarian Hydractinia was the only species to provide a model to analyze genetic control of such reactions. In 1950, Hauenschild noticed that allorecognition seemed to be under the control of a single genetic region with multiple alleles.428 Colonies of Hydractinia encrust the shells of hermit crabs, where they grow by elongation and branching of stolons. Embryos and larvae fuse indiscriminately. However, when two or more larvae are recruited to the same substratum, colonial forms may come into contact through their stolons. If the two colonies are histocompatible, stolon tips adhere and fuse, establishing gastrovascular connections and a permanent genetic chimera. If tips of incompatible colonies fail to adhere, they swell (hyperplastic stolons) with the migration of nematocysts, which discharge and damage the tissues. In addition, transitory fusions can also occur in a few cases. Similar to the genetics of the sponge alloreactions previously mentioned (and in other invertebrates and plants), colonies fuse if they share one or two haplotypes, reject if they share no haplotypes, and display transitory fusion if they share only one allele at one haplotype and no alleles at the other. Examination of the polymorphic locus governing this reaction (alr) revealed the involvement of two closely-linked polymorphic loci 1.7 cm apart (likely encoding receptor and ligand), alr1 and alr2.429 Alr1 encodes a three IgSF domain surface molecule, not unlike other metazoan nectin-like molecules, with a cytoplasmic tail equipped with multiple signaling motifs (including a ITAM-like motif). In the first domain, the molecule shows a high level of variability at particular residues, suggesting positive selection. Alr1 is embedded in a genetic region consisting of multiple IgSF members. Alr2 encodes another highly polymorphic gene with three IgSF domain IgSF with the distal V-like domain being the most variable, and an ITIM-like motif in the cytoplasmic tail adjacent to various phosphorylation motifs. Like the corals studied by Theodor,430 a high level of allele diversity was found at the

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alr2 level as nearly all sampled alleles encoded unique gene products.431

Urochordates Compared to Cnidaria, tunicates shared a recent common ancestor with the vertebrates (see Fig. 4.1), and thus might be expected to have a histocompatibility system more related to MHC. Allorecognition has been studied in both colonial and solitary ascidians. In Botryllus, a colonial ascidian, extra attention was afforded this system because the locus controlling histocompatibility was linked to (or was the same locus as) the locus controlling fertilization by preventing self-fertilization. For fusion, at least one histocompatibility locus must be shared between the colonies and for fertilization the sperm must be mismatched from the egg.432 Metamorphosis in Botryllus is followed by budding that eventually gives rise to a large colony of asexually derived genetically identical individuals (zooids), united through a vascular network. At the periphery of the colony, the vasculature ends in ampullae, which are the sites of interaction when two colonies meet during their expansion. The interaction results in either fusion of the two ampullae to form a single chimeric colony sharing a common blood supply or a rejection reaction during which the interacting ampullae are destroyed, thus preventing vascular fusion. Hemocytes (morula cells) are involved in the reaction.433 Fusion or rejection is governed by a single highly polymorphic (tens to hundreds of alleles) locus called the FuHC (for fusion/histocompatibility; 10 wild-type individuals collected from around the Monterey Bay area yielded 18 cFuHC alleles). As mentioned, when two colonies share one or both FuHC alleles, they will fuse; rejection occurs if no alleles are in common. However, like in Hydractinia, intermediary pathways have been reported in the past and have not been entirely elucidated. The C-terminal region of the FuHC molecule consists of a nectin-like segment with three Ig domains, showing most similarity to chicken Igsf4 and related members conserved in all vertebrates as well as to one of the Ciona nectins.434 Those members are found in a tetrad of paralogs in vertebrates but not linked to the tetrad of MHC paralogs (see the following). The N-terminal region contains an epidermal growth factor (EGF) domain and some other unrecognizable regions not conserved among ascidians. Any two FuHC alleles differ by an average of 4% at the nucleotide level. Unlike MHC class I and class II, polymorphic residues in FuHC alleles are not concentrated in particular regions, and alternative splicing can generate a fragment devoid of the IgSF moiety. Some 200 kb away from FuHC is a second polymorphic (and polygenic) locus, fester, which is inherited in distinct haplotypes.435 Diversified through extensive alternative splicing, with each individual expressing a unique repertoire of splice forms (each individual expressing up to three splice variants in addition to the regular full-length product), it potentially exists as both membrane-bound and secreted forms, all expressed in tissues intimately associated with histocompatibility. After fester knockdown, the histocompatibility reaction is blocked at the stage of initiation as

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if the colonies ignored each other. By contrast, when Fester was blocked with specific mAbs, fusion reactions were unaffected, but rejection reactions were turned into fusions in an allele-dependent manner. These data combined with its genetic location suggest that Fester encodes the FuHC ligand. Fester contains a short consensus repeat (or sushi domain) often found in vertebrate complement receptors. Beside these two polymorphic products, another Botryllus cell surface–expressed nonpolymorphic molecule related to fester, uncle fester, is required for incompatibility reactions, while fester seems to be required for allele discrimination and inhibition of killing.436 So, several independent pathways seem to control the final outcome of the interaction between individuals.437 It should be mentioned that a recent study, based essentially on the incongruence between histocompatibiity profi les and FuHC polymorphism, questions the validity of these reports.438

Solitary Ascidians In Halocynthia roretzi, a “polymorphism of color” has been observed and histocompatibility reset by a mixed hemocyte technique in vitro resulting in a melanization reaction likely to involve the PPO cascade.439 Depending on the strains, the percentage of positive reaction varied from approximately 55% to 70%, indicative of polymorphism. Grafting experiments had already shown the existence of allorecognition in solitary ascidians, and investigation at the cellular level had demonstrated the occurrence of cytotoxic cells in such organisms.440 In order to shed light on allorecognition in urochordates and on the molecules involved in preventing self-fertilization, gonadal complementary DNAs of three genetically unrelated Ciona intestinalis individuals were compared by suppression subtractive hybridization. This led to the discovery of the highly polymorphic variable complement receptor–like 1 gene coding for a transmembrane protein with several short consensus repeat domains (short consensus repeat/complement control protein [CCP]), a motif shared with the variable fester receptor of Botryllus described previously.441 Genes encoding variable complement receptor– like are in the same linkage group as a set of IgSF domains with homology to nectin (CD155/poliovirus receptor [PVR], cortical thymocyte protein [CTX]/junctional-adhesion molecule [JAM] family members, etc.) and other adhesion molecules of which related members can be found also on one genetic region in vertebrate, the 19q 34 human chromosome segment with the extended LRC (see the following).442

The Meaning of Histocompatibility Reactions in the Invertebrates The association between allorecognition in Botryllus and fertilization led to the proposal that histocompatibility systems were selected during evolution to avoid inbreeding. The hypothesis made sense in the case of sessile colonial invertebrates that might have difficulty dispersing their gametes and therefore are susceptible to inbreeding depression. Indeed, the partial matching mentioned previously is a general characteristic of fusion compatibility in colonial

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invertebrates, perhaps driven by “selection operating on an error-prone genetic system for self-recognition that is perhaps constrained by derivation from a gametic function selected to reduce inbreeding.443 Furthermore, even in mammals there is a large literature suggesting a selection both at the mate-choice and pregnancy levels for preserving heterozygocity at the MHC.443a However, the possibility of a common genetic system, or linked systems, governing fusion and gametic compatibility awaits confirmation. Because animals that are neither sessile nor unable to disperse their gametes can possess alloimmune responses is inconsistent with the general hypothesis. Moreover, inbreeding avoidance can only explain the selection of histocompatibility alleles if the histocompatibility loci are genetically linked to a large fraction of its genome. This is inconsistent with the tight linkage of histocompatibility genes to a single major locus, especially in invertebrates. So inbreeding avoidance is unlikely to contribute significantly to the selection of histocompatibility alleles, although in jawed vertebrates, selection for heterozygocity at the MHC itself has obvious advantages. Another hypothesis is that alloimmunity was selected because it avoided intraspecific parasitism and/or competition for attachment sites.444 Indeed, after fusion of compatible colonies, bloodborne germline or totipotent stem cells are transferred between colonies, and can expand and differentiate in the newly arising, asexually derived individuals of the vascular partner.445 This can result in a situation where only one genotype is represented in the gametic output of the fused individuals. The FuHC polymorphism in Botryllus could function to restrict to compatible individuals the vascular fusion and the germline parasitism. The high allelic polymorphism characteristic of all invertebrate recognition systems may have evolved in response to selection for fusion with self rather than kin. Fusion with self will allow the development of a colony that benefits from fusion while eliminating the possible cost of somatic cell parasitism, and thus would be the raison d’etre of the allorecognition mechanisms.446 However, most animals are not sessile and thus are neither prone to intraspecific competition nor to compete for limited substrate attachment sites. Tunicates besides Botryllus have evolved differently (eg, in Diplosoma, large numbers of chimaeras are encountered in nature). In addition, the intraspecific competition hypothesis demands that individuals maintain expression of histocompatibility alleles, even when the expression of these alleles enables their own destruction during intraspecific competition. These considerations suggest that intraspecific competition might affect histocompatibility allele frequencies in some organisms under certain conditions. Both previous hypotheses are essentially based on observation made in colonial tunicates, but colonial living has evolved several times independently. Urochordates are most related to the vertebrates, but the data argue against any of the genes involved in their histocompatibility reactions being ancestral to MHC class I and class II. It is clear in all of these studies that a strong pressure for polymorphism is the rule, but the evidence from all of these fascinating

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reactions suggests that such “pressures” select for similar systems via convergence, at least at the level of the receptor. The selecting molecular environment can perhaps show more conservation (see the folllowing). On the other hand, as described previously, genetic regions with homology to the vertebrate MHC have been detected in the invertebrates, yet are (most likely) unrelated to the reactions detailed here (see conclusion). As described, in all of these allorecognition systems, convergent mechanisms have been encountered to reach an analogous end, and thus it is unlikely that a unique “cause” arose for selecting them in diverse organisms. However, in an attempt to find an ultimate and general explanation to the selection during evolution of highly polymorphic allorecognition systems, it has been suggested that pathogen and retroviruses are the force behind the selection of allopolymorphism.447 This does not imply intimate phylogenetic relationships between the systems observed but rather emphasizes analogous solutions when facing similar pressures. We discuss “connections” between vertebrate immunity and these histocompatibility reactions in the conclusion.

CYTOKINES AND CHEMOKINES Many cytokines/chemokines and their receptors, like most molecules of the immune system, evolve rapidly. However, consistent with the “Big Bang” theory, it is an emerging picture that the majority of cytokines and chemokines found in mouse and human are also found in the genome and EST projects of nonmammalian jawed vertebrates, best studied in chickens and certain bony fish, but now extending to the cartilaginous fish as well. This suggests that whatever the initiating pressures in the evolution of the Ig/TCR adaptive immune system, the network of cytokines and chemokines emerged (practically) full blown early in evolution. A picture starts to emerge, but the gestalt is far from clear; this is, in part, because the situation in mouse human is clouded by the plasticity of T helper cell lineages, as well as an entirely new subset of immune cells, the innate lymphoid cells (ILCs), that produce cytokines previously thought to be within the domain of adaptive lymphocytes.

The Proinflammatory Cytokines, Interleukin-1 Family Members, Interleukin-6, Interleukin-8, and Tumor Necrosis Factor ` IL-1 and related family members IL-18 and IL-33, IL-6, TNF-α , and IL-8 (CXCL8) are the prototypic cytokines associated with inflammatory responses, which are defined by induction of vasodilatation and vascular permeability, and upregulation of innate immune system–specific molecules that have direct functions or that costimulate/attract T and B cells. Classically, many of these activities can be assayed in supernatants from PAMP (eg, LPS)-stimulated phagocytes by determining whether thymocytes are induced to proliferate when one also adds suboptimal concentrations of T-cell mitogens. It was reasonable to hypothesize that such cytokines, which act both at a distance as well as in a cognate

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fashion, might be found in the invertebrates. Indeed, IL*-1–like activities have been described for echinoderm coelomocytes (either IL-1–like production by such cells, or the ability of the cells to respond to mammalian IL-1), but unfortunately no molecular data revealing the structures of the active invertebrate cytokine/cytokine receptor have been reported. In fact, no ortholog has been detected in the genome projects from protostomic invertebrates (see Fig. 4.1), and only IL-17 and TNF homologues have been detected in nonvertebrate deuterostomes and protostomes,48,448,449 and IL-8 in agnathans; thus, we may consider these as primordial cytokines related to the vertebrate versions. A molecule from earthworms capable of activating the prophenoloxidase defense pathway cross-reacted with a mAb directed to mammalian TNF-α .450 However, this molecule had no homology to TNF-α upon sequencing. IL-1 activity as measured by costimulation assays or as a consequence of PAMP stimulation has been detected in all nonmammalian vertebrates. IL-1β upregulation has been detected after treatment of macrophages with LPS, consistent with its inflammatory function in mammals. In addition, injection of gram-negative bacteria into trout induced IL-1β expression in many tissues.451 Identity with the mammalian IL-1β gene in all other species ranges from 28% to 40% (identity between mammalian IL-1α and IL-1β is about 25%). In nonmammalian species, IL-1β lacks the socalled ICE cleavage site, important for function in mouse/ human.452 IL-18 is an IL-1–related cytokine, and in contrast to IL-1 seems more focused in its function of potentiating TH1 responses. IL-18 has been detected in birds and fish, but the tissue distribution in fish seems to be expanded as compared to mammals. Additionally, IL-18 in nonmammalian vertebrates contains the ICE cleavage site, unlike its cousin IL-1β. Other IL-1–related cytokines, including IL-33 and the IL-1F series, have also been found in several jawed vertebrates, but with little study of functional activity. This will be of great interest to uncover, considering the IL-1 family members’ roles in regulating Th1, Th2, and Th17 (and others?) differentiation.453 Both chicken IL-1β and the IL-1R were identified and have been expressed as recombinant proteins.454 The IL-1R homology to mammalian orthologs is quite high (61% identity), but the highest similarity is found in the cytoplasmic domains. In addition, there are four blocks of high similarity to the cytoplasmic tail of toll/TLR proteins, and IL-1R and TLR use similar signal transduction cascades (see previous discussion). As mentioned, IL-8 (actually, the CXC chemokine CXCL8) has been identified in the jawless lamprey and seems to be expressed specifically in B cells, whereas the IL-8 receptor is found in T cells; this has been one of the mechanisms proposed to permit the cells to interact in a cognate fashion.13,291 IL-8 has also been found in various gnathostomes such as trout, flounder, and perhaps chicken; a chicken CXC chemokine called K60 clusters with IL-8 in phylogenetic trees and is upregulated in macrophages stimulated with LPS, IL-1β, and IFN. Interestingly, Marek disease virus expresses an IL-8 homologue (v-IL-8), which may be

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involved in inducing immune deviation.455 A molecule related to the IL-R was detected in the sea urchin genome, but the ligand has not been found48 ; becuase there are so many members of the IL-1 family, the search should continue. The TNF family in mammals includes the canonical TNF-α , lymphotoxin (LT) α , and LTβ, all encoded at the distal end of the MHC class III region,403 as well as a large number of other members with diverse immune functions. TNF-α is the best studied of these cytokines, and it is one of the key regulators of innate and adaptive immunity. The other two cytokines have a more limited tissue distribution and function, and are noted in their roles in lymphoid tissue development, especially in the formation of splenic white pulp and segregation of T and B cells in lymph nodes. In contrast to IL-1, TNF homologues have been detected in cnidarians, protostomes (see previous discussion), sea urchin,48 Ciona,448 and Amphioxus,456 consistent with the idea that such a multifunctional cytokine would predate the jawed vertebrate adaptive immune system. Homologues have also been cloned from several teleost species, and TNF-α expression in leukocytes is upregulated within 4 hours after treatment with LPS, IL-1β, and PMA. While there is good phylogenetic support for orthology of fish TNF-α to that of mammals, the other TNF genes seem to be teleost-specific duplicates rather than the LT genes.457 Conversely, in Xenopus, the three TNF family members described previously in mammals are closely linked in the class III region of the MHC.177 This is a bit of a surprising result as all of these family members seem to be lacking in chickens, consistent (according to the authors) with a lack of lymph nodes in these animals; however, the unusual nature of the MHC in birds (eg, the immunoproteasome genes are missing) rather is consistent with a loss of TNF genes in these animals, which is indeed surprising, especially for TNF- α.458 An excellent review detailing the evolution of all TNF and TNF receptor family members demonstrated that all of the genes are found in paralogs on four chromosomes, consistent with the 2R hypothesis. This work demonstrated that the earliest member was linked to the proto MHC, and thus it is not surprising that TNF is an ancient gene family.459 IL-6 and its related members IL-11 and IL-31 are also found in most gnathostomes examined, with few functional data.

Interleukin-2 and the fC Family of Cytokines The family of cytokines that signals through the γ C receptor includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Similar to what was described for most molecules involved in adaptive immunity, all of these cytokines (except perhaps IL-9) are present in all gnathostomes studied.460 Costimulation assays of thymocytes, as described previously for IL-1, and perpetuation of T-cell lines with stimulated T-cell supernatants are performed to detect IL-2 or “T-cell growth factor” activities. Unlike IL-1, IL-2–like factors generally stimulate cells only from the same species, and it is a “cognate” cytokine, meant for release only between closely opposed cells, or as an autocrine factor. From teleost fish to mammals, stimulated T-cell supernatants costimulate thymocyte proliferation or

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can maintain the growth of T-cell blasts, and the ortholog has been detected in bony fish and birds. The chicken IL-2 protein is only 24% identical to human IL-2 and only 70% identical to a near cousin, the turkey.454 IL-15, a relative of IL-2, has also been cloned in the chicken. A candidate IL-2R in chicken was identified by a mAb recognizing a 50-kDa molecule only on stimulated T-cells (thus an IL-2Rα homologue). This mAb blocks costimulation by IL-2–like molecules in chicken T-cell supernatants and also reduces the capacity of T-cell blasts to absorb IL-2–like activity from supernatants. IL-2 has been studied in the bony fish Fugu. In both chicken and the deduced Fugu IL-2 protein, there is a second set of cysteine residues, which are found in IL-15 and thus is a primordial feature.461 As mentioned, γ C is the signaling subunit of the IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors; absence of this chain in mammals leads to major defects in lymphocyte development (“boy in the bubble”). In fish, a γ C homologue was cloned in rainbow trout with unusually high identity (44% to 46%) to mouse/human genes.462 IL-1β, but not LPS, upregulated the trout gene in macrophage cultures and a fibroblast cell line. Since then, many other orthologs have been uncovered in fish, often duplicated in various species.460 IL-21, which is encoded adjacent to IL-2 in mammals, has been found in ectothermic vertebrates.460 Because one of its major functions is the differentiation of T-follicular cells and the GC response, it will be of interest to study it role in those animals. IL-7 is involved in lymphocyte differentiation as well as homeostasis of mature lymphocytes. Recently, mutations in Il-7R and downstream signaling molecules in the zebrafish have shown a block in T-cell development.463 These mutants will permit future study of lymphocyte production in this crucial developmental model.

TH2 Cytokines: Interleukin-4, Interleukin-5, Interleukin-9, and Interleukin-13 Reponses to extracellular pathogens, especially fi larial worms, are largely regulated by TH2 cytokines in mammals. IL-4 is the most pleiotropic cytokine in this regard, stimulating the production of neutralizing antibodies, stimulation of eosinophils and mast cells, and an antagonism of TH1 responses. IL-4, IL-5, and IL-13 (and granulocyte macrophage colony-stimulating factor) are encoded in the so-called TH2 cytokine complex in mammals, and all of the genes are coordinately upregulated after stimulation of a nearby locus-control region. This same “TH2 complex” is found in chickens but with a psuedogene for IL-5, but in Xenopus only the IL-4 gene has been identified to date. Fish have genes in which the ancestor of IL-4 and IL-13 seems to be in a preduplicated state, syntenic with genes in the TH2 complex.464 Again, duplicates of IL-4/-13 and their receptor have been found in various fish.460,465 At this stage of study, it may be that lower vertebrates do not have “full-blown” TH2 responses, consistent with the lack of canonical allergic responses in ectothermic vertebrates. On the other hand, we must be careful because cytokine genes evolve rapidly and relying on synteny is not always

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dependable, especially in the teleost fish. It will be of interest to see whether IL-4 in nonmammalian vertebrates will promote switching to isotypes that are incapable of promoting inflammatory responses. In addition, further studies of this family and its receptors in teleost and cartilaginous fish will reveal whether this facet of the adaptive immune response is indeed a relative newcomer.

Interferons IFNs, classically known for their antiviral properties, are divided into three groups: type I, type II, and type III. Perhaps surprisingly, this entire group has only been uncovered in jawed vertebrates to date. However, as described previously, viral immunity in insects is partially regulated via a JAK/STAT response.152 Types I (α and β) and III (λ) IFN are widely expressed in cells of many types and induces inhibition of viral replication in neighboring cells, as well as molecules of the innate immune system such as inducible nitric oxide synthase (iNOS) and IFN regulatory factor-1.466 In contrast, type II IFN (IFNγ or immune IFN) is synthesized by activated T cells, activates macrophages, and upregulates class I, class II, immune proteasome subunits, and TAP, and a large number of other genes. Antiviral activity is detected in supernatants from virally infected fish fibroblasts, epithelial cell lines, and leukocytes. All of the biochemical properties of mammalian type I IFN (eg, acid-stable, temperature-resistant) are present in these fish supernatants, and the putative IFN reduces viral cytopathic effects in homologous cell lines infected with virus.466 In vivo, passive transfer of serum from virally infected fish protects naïve fish from acute viral pathogenesis. There appears to be two lineages of type I IFNs in fish that are specific to this group. In chickens there are up to 10 closely related, intronless type I IFN genes.455 Sequence identity to human type I IFN ranges from 25% to 80%, with the apparent functional gene having highest similarity. Interestingly, bats have greatly expanded their type I and III IFN genes and receptors (as well as many other genes associated with viral immunity).467 These animals are highly infected with (and susceptible to) virus, and some IFN forms are constitutively expressed at low levels. While type I/III IFNs can be highly duplicated in some vertebrates, this is not true of IFN receptors, and in some cases many cytokines will used two to four receptors within a species. How can such a system work and result in different readouts? It appears that even single amino acid changes in the cytokine can induce differential conformational changes in the receptor that can modify downstream signaling.468 This paradigm will be exciting to follow in a phylogenetic context. Type II or IFNγ has been cloned in most jawed vertebrates. The chicken gene is 35% identical to human type II IFN and only 15% identical to chicken type I IFN.454,469 Recombinant chicken IFN stimulates nitric oxide production and class II expression by macrophages. The gene has been cloned in many fish, and has been studied mostly in the trout where it has been shown to upregulate CXCL10 and activate protein kinase C. Thus, at least in chickens and te-

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leost fish, type II IFN seems to have the same function as in mammals, suggesting that the TH1-type responses emerged early in vertebrate evolution.466

Heterodimeric Cytokines The IL-12 family are composed of heterodimers that share chains (p19, p40, p35, and EB13) and include IL-12, IL-23, IL-27, and IL-35. IL-12 is generally considered to be a proinflammatory cytokine produced by APCs that promotes a TH1 response after exposure to intracellular pathogens. Consistent with the ancient derivation of TH1 responses, the two subunits of IL-12 p70 (p35 and p40) have been found in chickens and several teleost species.454,470 The p40 subunit of IL-12 can also associate with p19 to form the cytokine IL23, which is involved in the perpetuation of TH17 cells. IL-27 generally inhibits all TH subsets but expands regulatory T cells and is composed of p28 and EB13; IL-35 is a relatively new regulatory T–produced cytokine composed of p35 and EB13. All of these chains have been detected in gnathostomes and several studies have been done to examine their expression at the RNA level; however, to date no reagents have been prepared to the heterodimers for functional analysis.

Transforming Growth Factor-a and Interleukin-10 TGF forms a large family with pleiotropic effects in many developmental systems. For the immune system, TGF-β isoforms are best known for their capacity to suppress adaptive immune responses (even across species barriers), although they can also stimulate lymphocytes under certain conditions, especially in mucosal surfaces. TGF-β inhibits macrophage activation in trout and growth of T-cell lines in Xenopus species. Recombinant Xenopus TGF-β, like the mammalian form, also can inhibit IL-2–like dependent growth of splenic lymphoblasts.471 Four TGF-β isoforms were isolated from chickens, as opposed to three major forms in mammals.455 The three major forms of the cytokine have been isolated in several teleost species.470 IL-10 is often considered along with TGF-β because it is mostly an immunosuppressive cytokine with multiple effects; both cytokines are expressed in subsets of “regulatory T” cells in mammals. This cytokine was originally discovered by its ability to suppress TH1 responses, but now is known to have a much expanded role in immunity. IL-10 has been found in chickens and a large number of teleosts.455,470 As usual, functional experiments have lagged behind the molecular work, but recombinant chicken IL-10 can block IFNγ production by splenocytes. IL-10 is a class II cytokine, related to IFNγ, IL-19, IL-20, IL-24, and IL-26. IL-22, a cytokine released by TH17 cells and ILCs in mammals, is upregulated in fish vaccinated with pathogens and is correlated with immune protection.472 Note that there have been very few “regulatory T” experiments reported in the comparative literature, but many older experiments that suggest that such cells exist. Over 25 years ago, experiments in Xenopus showed that graft rejection could be delayed when lymphocytes from metamorphosing animals were adoptively transferred into adult frogs.473 This

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result suggested that a “wave” of suppressor cells emerged near the time of metamorphosis that evolved to protect animals from autoimmunity when adult-specific molecules were expressed. It is time to reexamine such experiments with modern tools. In a recent experiment in zebrafish, immunization of animals with central nervous system (CNS) antigens induced an autoimmune reaction. Overproduction of foxp3 inhibited the production of IL-17 and IFNγ in these animals, ameliorating the disease.474

Interleukin-17 TH17 cells in mammals are important for inflammatory responses to extracellular bacteria via indirect stimulation of neutrophils. Despite its late appearance in the immunologic literature, IL-17 appears to be one of the most ancient cytokines, with homologues in protostomes and lower deuterostomes, and extensively amplified in sea urchins. Even the different isoforms seem to be conserved among the jawed vertebrates, which should initiate many new avenues of study.470 The family is composed of IL-17A/F, with the A and F forms being the best studied to date. IL-17A/F genes have been found in all mammals studied, as well as IL-17C/E. The most ancient IL-17 form is IL-17D, present in both oysters and agnathans.291,449 This form is expressed in lamprey T cells but not B cells, and has been proposed as a helper factor for cognate interactions.13 Finally, like many other immune genes, as many as 20 IL-17 genes have been found in sea urchins, again suggesting expanded innate immunity in this species.475

Cytokine Summary The isolation of nonmammalian cytokines and cytokine receptor genes has lagged behind molecular characterization of antigen receptors and MHC. However, with the advent of the genome and EST projects, we are rapidly acquiring a comparative view of this field, at least at the genetic and molecular level. Teleost fish and chickens have paved the way in this field, but cartilaginous fish and agnathan databases will soon be complete and provide the big picture. Already, it seems that cytokines like TNF and IL-17 seem to be most primordial, with homologues in Ciona, Amphioxus, and sea urchins, as well as deeper homologues in certain protostomes. Conversely, none of the other so-called adaptive cytokines (or even type I/III IFNs) seem to be present in the lower deuterostomes, consistent with the Big Bang theory of adaptive immunity.1,2 If it is true that the jawless fish lack these genes as well, despite their convergent adaptive immune system, one can point to the evolution of lymphoid organs and the segregation of lymphocyte subsets into discrete areas to help to explain the explosion and recruitment of these genes. We discuss this concept in more detail in the conclusion section, especially regarding the relationship of adaptive lymphocytes with ILCs. Despite our “big picture” knowledge of the emergence of cytokines, obviously functional experiments have lagged behind. Furthermore, is it true that TH2 responses are evolutionary latecomers, or are our early attempts at finding the

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genes in ectotherms because of some “missing pieces” in the databases (or our ability to find some of the genes)? Some genes may have been lost, as described previously for the chicken—again, further studies of amphibians and cartilaginous fish should help in our understanding. In mammals, the LT TNF family members are important in the development of lymph nodes and splenic white pulp, and yet the three genes are present in lymph node–less Xenopus —what are their functions in ectotherms? If we can uncover these other roles in nonmammalian vertebrates, it could be quite informative to reinvestigate such functions in mammals. Finally, chickens and fish have evolved their own paralogs of some of the well-known cytokine genes; their study should reveal selection pressures on particular species that could also be quite informative in our understanding of the gestalt of the cytokine network.

General Evolution of Chemokines and Their Receptors Chemokines and chemokine receptors are essential for many aspects of the immune system, including inflammation, the differentiation of lymphoid tissues, trafficking of hematopoietic cells during ontogeny and immune responses, and even stimulation of cells under various conditions.476 With the advent of the genome and EST projects, a seminal paper showed that, consistent with the Big Bang theory of adaptive immune system evolution, all of the major classes of chemokines and their receptors were present in the bony fish lineage, and probably will be found in cartilaginous fish as well when the genomes are completed.477 Recently, the analysis was updated to include all of the vertebrates in the databases.476 Similar to most of the immune-related genes discussed throughout the chapter, bony fish have more chemokines/receptors than any other vertebrate, including amphibians and mammals. Interestingly, no CC (so-called homeostatic) or CXC (socalled inflammatory) chemokines/receptors were detected in lower deuterostomes like Ciona and sea urchin. The chemokine receptors are member so the G protein–coupled receptor family, and of course such molecules are found in all animals. However, no members of the specialized family (G protein–coupled receptor γ) to which chemokine receptors belong were found in lower deuterostomes. The chemokine receptor genes are found on four chromosomes in mammals, four of which contain the HOX genes, one of the gene families that provided evidence for the 2R hypothesis early in the reawakening of this theory. The distribution of chemokine receptor genes on these chromosomes correlates very well with 2R, and helps to account for the large-scale gene expansion of this family in the jawed vertebrates. Thus far, very few chemokines/receptors (eg, IL-8 [CXCL8] and CXCR4) have been detected in agnathans, but as mentioned previously, agnathan T cells express IL-8R and B cells IL-8, believed to be a factor in promoting the cognate interactions between the cells.291 CXCL12, a chemokine vital for thymic differentiation, is also present in lamprey, consistent with new work on the agnathan thymus discussed previously.375 In summary, it appears that a few chemokines/receptors arose at the dawn of vertebrate adaptive immunity, and they

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were greatly expanded by both en bloc and cis duplications and became “full-blown” shortly after the 2R Big Bang.476 Currently, this is our most complete story of the evolution of a family of immune genes in deuterostomes.

ORIGINS OF ADAPTIVE IMMUNITY The immune system of vertebrates is unique because the antigen-specific receptor expressed by lymphocytes, which initiates cascades leading to activation of the adaptive immune system, is not the product of a complete germlineinherited gene. Rather, receptors are generated somatically during lymphocyte ontogeny from gene segments scattered at a particular locus. As described previously, the receptors in gnathostomes are IgSF members composed of V and C domains, with the C domains being of the rare “C1” type, which is shared by MHC class II and class I molecules. There are many specific questions: 1) Did MHC class I or class II come first?; 2) What is the origin of the MHC PBR?; 3) Was the MHC involved with innate immunity before the emergence of adaptive immunity?; 4) Did somatic rearrangement or somatic mutation come first to diversify antigen receptors?; 5) Which of the extant antigen receptors in gnathostomes, α / β TCR, γ /δ TCR, or IgH/L (if any) resembles the primordial receptor?; and 6) How did two different antigen receptor gene families emerge in evolution, after the appearance of separate lymphocyte populations? The answers to these questions are speculative, but deductions can be made based upon the wealth of molecular and emerging functional data. We base many of our arguments on the large-scale duplications that were revealed for MHC by Kasahara17 in 1996. The remarkable syntenies of paucicopy genes on the paralogous regions, and the recent fi nding that an animal that predated the duplications (Amphioxus and Ciona) has only single copy genes in the same syntenic group orthologous to the four mammalian copies, make it clear the en bloc duplications were involved.405,421 Further analysis of this region in model lower deuterostomes, as described in the histocompatibility section, will continue to be vital in our understanding of early immunity. Genetic analyses in protostome lineages have not been very informative, but we must reevaluate once we piece together data from more deuterostome species.

Major Histocompatibility Complex Origins Class I and class II molecules have been found only on the jawed vertebrates—so far, painstaking screenings of the lower deuterostome or lamprey/hagfish databases have yielded no indication of these proteins. Based upon phylogenetic analyses and thermodynamic arguments, most investigators believe that class II preceded class I in evolution.478 However, as stated previously, class I is much more plastic than class II as there are many different types of class I molecules, some that do not even bind to peptides. Because class I genes may be on two or three MHC paralogs, and they can have functions outside the immune system; this is evidence that the primordial “PBR” may not have even bound to anything. If this is true, and because class I and

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class II do bind peptides, it would suggest class I arose first. Again, genome scans of jawless fish and lower deuterostomes should be informative on this point, but to date no luck! As described previously, we should be diligent because with the discovery of T cells and a thymus-like structure in lamprey, one expects some sort of “MHC” in the agnathans291,375 ; to date, the only homologue we have uncovered to date is TAP-L, which may hold the key to antigen presentation in this group.401 From the paralog data, genes encoding the complement components C3 and Bf, TNF superfamily members, the signaling molecule Vav, B7 family members, and proteasome subunits among other genes (such as tripartite motifcontaining) should have been present in the proto-MHC, before emergence of the adaptive immune system (see Fig. 4.13). Some of these genes were found in the Amphioxus and Ciona “MHC” linkage groups, and C3 and Bf genes are linked in the sea urchin. A fifth paralogous region on human chr 12p13 contains the α2-macroglobulin gene (recall the C3/4/5 homologue), a tapasin homologue, the C3areceptor, and this “complex” is linked to the NKC. Taken together, the data suggest that the proto-MHC included vital nonhomologous genes of the innate immune system, which perhaps were linked to allow coordinate regulation of expression. After the en bloc duplications, Pontarotti et al. has suggested that “functional restraints upon the complex were relaxed” and hence the duplicated members could evolve new functions, including features indispensable to the adaptive system.405 If indeed innate immunity genes were already linked to allow upregulation at times of infection, it is no surprise that the adaptive immune system piggybacked on such a gene complex. A final point: why did the duplicate genes survive rather well over hundreds of millions of years; cis duplicates have been shown to degenerate rapidly over evolutionary time. Evidence suggests that duplicates arising from polyploidy (and by inference large en bloc duplications) survive better than cis duplicates, most likely because they cannot be inactivated by unequal crossovers; the ability of the genes to survive over very long periods, perhaps combined with strong selection pressures, would allow for subspecialization.479

Origins of Rearranging Receptors The Rearranging Machinery Most models propose that the generation of somatically rearranging receptors occurred abruptly in evolution via the generation of the RAG machinery made of two lymphocytespecific proteins, RAG1 and RAG2. RAG genes have so far been isolated in all classes of jawed vertebrates and have been quite conserved. In every case examined, RAG1 and RAG2 genes are closely linked and in opposite transcriptional orientation. Some regions of RAG1 and RAG2 are similar to bacterial recombinases or to molecules involved in DNA repair (eg, RAD16) or the regulation of gene expression (such as rpt-1r). Similarities to prokaryotic proteins and the gene structure suggest that vertebrates acquired the RAG machinery by horizontal transfer and transposition from bacteria.19,480 Indeed, RAG genetic organization has

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some transposon characteristics: the RSS are reminiscent of sequences involved in targeting excision of transposons. A class of transposons detected in several protostome and deuterostome invertebrate species that shows similarity to the catalytic domain of RAG1.481 It is believed that RSS were derived from the terminal inverted repeats of this transposon, called transib. In invertebrate genomes there are many of such “RAG1 core regions,” but only one has an open reading frame throughout the core and shows similarity to vertebrate RAG1 in other regions as well. Furthermore, a RAG2 homologue is adjacent to the urchin RAG1 gene, in a similar orientation as is found in gnathostomes.352 This finding was a big surprise and suggests that both RAG genes were in place approximaltey 100 million years before the origin of the Ig/TCR system. Their tissue distribution and expression during ontogeny are not known; nor have any candidate genes been recognized to date with RSS that might be recognized by the echinoderm homologues. It must be admitted that it is unclear how this new result fits into the puzzle of the origins of adaptive immunity. Because transib is so often found in the animal kingdom, it is certainly part of the transposon. By contrast, RAG2 is now believed to have been present in the genome, and recent data have shown it to interact with active chromatin.482,483 Thus, the new idea is that RAG2 recruits RAG1 to open chromatin, and rag1 is the active enzyme in the major rearrangement events, which is wholly consistent with the transposon hypothesis with RAG1 (ie, transib) being the genome “invader.” Another source of somatic antigen receptor diversity shared by all gnathostomes characterized to date is a unique DNA polymerase, TdT, which diversifies CDR3 during Ig and TCR gene rearrangement through the addition of nucleotides in a template-independent fashion.484 Furthermore, as detailed previously, its expression serves as an unambiguous developmental marker for the sites of lymphopoiesis. TdT has been highly conserved in both sequence (> 70% amino acid similarity, > 50 amino acid identity) and overall structure during vertebrate evolution. An amino acid alignment of all known TdT sequences reveals that some, but not all, structural motifs believed to be critical for TdT activity are particularly well conserved in all vertebrates studied. TdT protein alignments, and the crystal structure for rat β-polymerase, support the hypothesis that both evolved from a common ancestral DNA repair gene. In addition, four protein kinase C phosphorylation sites are conserved, and hence may be involved in TdT regulation. Homologues related to the ancestor of polymerase β and TdT have been found in sea urchin and other lower deuterostomes. Thus, unlike RAG, TdT has evolved by gradual evolution from a polymerase family and was recruited for immune system function.

Rearrangement or Somatic Hypermutation First? Because all antigen receptor genes use somatic rearrangement of V genes to generate diversity in CDR3 regions as well as to promote combinatorial diversity, there is no doubt that this mechanism is at the heart of adaptive immunity. Indeed, as described, most investigators believe that the introduction of the tranposable element into a V gene was the driving force in the abrupt appearance of vertebrate adaptive immunity;

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the finding of the RAG genes in sea urchins challenges this notion at some level. However, it cannot be overemphasized that SHM is also at the origins of the immune system; furthermore, all evidence to date suggests a gradual evolution of the SHM machinery (the AID/APOBEC family and associated polymerases/mismatch repair proteins) rather than the “hopeful monster” generated by the famous RAG transposon. Thus, diversity generated via SHM or gene conversion may have existed in an adaptive immune system prior to rearrangement, and V gene rearrangement was superimposed onto this already existing system.296,300 Indeed, the presence of APOBEC family members in the jawless fish (CDA1 and CDA2, as described previously) suggest that diversity generated via mutation/conversion preceded rearrangement. The RAG-induced rearrangement break and subsequent repair provided something new and novel in gnathostomes, not only diversity in sequence but heterogeneity in size300 ; this was a remarkable innovation and likely indeed heralded the sophistication of jawed vertebrate adaptive immunity. Which Antigen Receptor First? Phylogenetic analyses have suggested that among IgSF receptors γ /δ TCR-like ancestor may have predated α /β TCR and Ig H/L.485 This would suggest that direct antigen recognition, perhaps by a cell surface receptor, arose first in evolution followed by a secreted molecule and an MHC-restricted one. Hood and colleagues argue that phylogenetic analyses over such large evolutionary distances obscure true relationships among the antigen receptor genes (eg, the relationships of the molecules in the phylogenetic trees has to impose multiple loss/gain of D segments in the different antigen receptor families) and suggest a model based upon genomic organization, not so different from the Kasahara model.263 They propose an alternative phylogeny in which an ancestral chromosomal region with linked genes encoding both chains of an ancestral antigen receptor heterodimer, one having D segments the other not (eg, IgH/IgL). The α and δ TCR loci are still closely linked in all vertebrates analyzed (human chromosome 14), and a pericentric inversion is suggested to have separated the TCR β and γ loci (linked on human chromosome 7). This model predicts that D segments only emerged once, and also explains the existence of inverted V elements in both the TCR β and δ loci. This model does not predict which antigen receptor is oldest, but does provide a “simple view” of receptor evolution, consistent with the Kasahara/Ohno model. Recent data have added to this scenario. First, as mentioned previously, VH elements have been found at the TCR-δ locus in many vertebrates, including sharks, amphibians, birds, and marsupial mammals.269 In part, this is due to the close proximity of the IgH/L (lambda) and TCR alpha/delta loci in many living vertebrates (especially Xenopus268), consistent with the Hood hypothesis of ancient en bloc duplications of antigen receptor loci.268 Additionally, the idea that most TCR-δ receptors do not recognize MHCrestricted antigen suggests that they can continue to “borrow” elements from Ig loci over evolutionary time. Thus, an alternative scenario is that MHC-restricted antigen recognition arose first, perhaps derived from a NKR with a “VJ” IgSF domain that recognized SOS proteins, like the ULBPs or MIC today. The rearrangement break induced by

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RAG occurs in CDR3, which is in the center of the antigen receptor–combining site, in perfect position to interact with peptide bound to MHC, which could have been another innovation to detect foreign antigens within cells.486 Thus, such cells would be most like extant αβ T cells with an MHC-restricted antigen receptor encoded by linked genes. Once rearrangement was introduced, the combining site may have been “relaxed” so that it was no longer forced to be MHC-restricted (see the section on TCR above). The genomewide duplication then would provide two types of TCR, one MHC-restricted and the other not, like γδ TCR today. One locus, according to the Hood model, then underwent an en bloc cis duplication to give rise to the IgH/L loci. Ig, then, developed a new differentiation pathway with an alternative splicing mechanism for the transmembrane and secreted forms of BCRs. For the origin of the rearranging receptors, IgSF lineages have to be traced back through phylogeny as such receptors generated by somatic rearrangement do not exist outside the jawed vertebrates. In a quest for molecules related to elusive ancestors, without focusing on genes expressed in the immune systems of various phyla (ie, structure is more important than function in this case), the most homologous sequences and gene architectures in the various metazoan phyla must be scrutinized.

V(J) and C1 Domains C1 domains are found in the antigen receptors, MHC class I and class II, and very few other molecules (see Fig. 4.10). This IgSF domain is so far most prevalent in gnathostomes, as if C1 domains arose concurrently with the adaptive immune system and coevolved with it. What was the value of the C1 domain, and why is it found almost exclusively in adaptive immune system–related molecules? All of these molecules interact with coreceptors such as CD3 (TCR), Igα and β chains (Ig on B cells), CD4, and CD8 (with MHC on opposing cells), and it is conceivable that in sections of IgSF domain in which C1 differs from the C2 there is a specific region favoring interaction with other molecules. The G strand of Ig/TCR V domains is encoded by the J gene segment, separated from the V region-encoded A-F strands, and rearrangement is necessary in order to assemble a complete V gene. The primary structure of each Ig/TCR chain bears hallmarks of the dimeric nature of the receptor in which they participate. A diglycine bulge (GlyX-Gly), present in all V domains, is thought either to be a beneficial adaptation, or to promote dimer formation by inducing a twist in the G strand that results in V domain pairing that appropriately orients the CDR. Monitoring this feature, therefore, might reveal genes that had the ability to form dimers similar to that of modern antigen-specific receptors. In V genes that do not somatically rearrange, the G stand is an integral part of the V exon. In other remotely related IgSF genes, introns have invaded the V domain exon creating a variety of V gene families. Many examples of such events can be found in the history of the Ig superfamily, for example in the genes encoding CD8 and CTX.33,487 As described, no Ig/TCR genes have been isolated from hagfish or lampreys, although there are some tantalizing molecules potentially related to their ancestors (eg, APAR,

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see previous discussion). Were Ig and TCR “invented” in a class of vertebrates now extinct (eg, the placoderms, which are more primitive than cartilaginous fish but more advanced than agnathans)? The discovery of the three tapasin paralogs all with C1 domains suggests an origin prior to the full establishment of the vertebrate genome.33 V domains, either alone (eg, IgNAR) or in association with another V domain (eg, Ig H/L), recognize the antigenic epitope and are therefore the most important elements for recognition. For this reason, they will be the first to be traced back in metazoan evolution by asking whether V domains exist in invertebrates. Domains with the typical V fold, whether belonging to the true V-set or the I-set, have been found from sponges to insects (although not necessarily involved in immune reactions; the first ones were discovered by nonimmunologists among molecules involved in nervous system differentiation in invertebrates [eg, amalgam, lachesin, and fascicilin]). Invertebrates also use IgSF members in immunity, but so far they are not V domains, but more I- or C2-set (eg, mollusk defense molecule, hemolin, DSCAM). However, the mollusk FREPs have a V domain at their distal end, associated with a fibrinogenlike domain. As described, they are involved in antiparasitic reactions and form a multigenic family with polymorphism.135 Similarly, as mentioned previously, prochordate VCBP has a VJ domain, but it mediates innate immunity.143 Besides searching for VC1-encoding genes in nonvertebrates, surveying the human genome for such genes has been fruitful. Indeed, nonrearranging V-containing molecules, either VJ alone, VC1, or C1 alone, have been found in the human genome. Interestingly, many of them are present in the MHC class III region (human chromosome 6p21) or its paralogs (see Fig. 4.12 and 4.13). Two MHC-linked gene segments stand out: a single VJ, NKP30, and a gene containing a VJC1 core, tapasin (TAP-binding protein), involved in antigen processing. NKP30, made of a single Ig domain of the VJ type, is an NK cell–activating receptor, and it may offer a link to cell types in invertebrates. It could be a relative of an ancient receptor whose history is linked to the emergence of MHC class II and class I; unlike most NKRs, NKp30 is evolutionarily conserved, present from sharks to mammals.176 In order to resemble an ancestor, the NKP30 V domain need only be associated with a C1 domain. In fact, a C1 single-domain gene, pre–TCR-α, is also encoded in the MHC.253 Besides Ig and TCR, tapasin is one of the rare cases, if not the only other case, of a gene segment with a VJC1 structure existing on several paralogous linkage groups (6, 9q33, 19q13). In other words, while this gene is related to the rearranging receptor structure, it is undoubtedly very old, and probably predated the ancient block (genomewide) duplications. It could have acted as a donor of C1 to a V domain–containing gene in the MHC class III region (like the XMIV), which then could have been the first substrate of the rearrangement. Another set of molecules with distal VJC1 segments, the signal-regulatory proteins (VJ C1 C1), and the poliovirus receptor (VJ C1 C2) could represent another group linked to the history of the Ig and TCR (see the following). TREM1 and TREM2, receptors on monocytes/neutrophils involved in inflammatory responses, are composed of single VJ domains whose genes are MHC-linked. Myelin oligodendrocyte protein (MOG) and P0, two single V domains

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involved in the synthesis of myelin sheath, are encoded in the MHC paralogous region on chromosome 1. Chicken BG, which is related to MOG but probably has a different function, is encoded in the chicken MHC. Butyrophilin, CD83, and tapasin all have VJ domains, and butyrophilin also has a C1-type domain. More distant relatives with VJC2-based architectures are also found in MHC (receptor for advanced glycosylation end products, CTX, lectin-related genes; see Fig. 4.12), and some of these genes related to the rearranging receptor ancestors are found on several paralogs (whether the MHC paralogs or other), suggesting that the V-C1 core was generated early in vertebrate evolution subsequent to the emergence of the chordate superphylum (see Fig. 4.13). Among all these molecules, butyrophilin is perhaps not on the direct track to antigen-specific receptors. Its C domain, although proven to be C1 through its crystal structure, is more like a C2 at the primary sequence level, and belongs to the CD80/86 family, rather than the TCR. Finally, tracing the VJ NITR gene family previously described in evolution may lead us to an understanding of the original NKC/LRC/MHC, as well as identifying more candidate genes related to the ancestral gene invaded by the RAG transposon. Many invertebrate molecules not involved in immune responses are present as a distal V domain associated with one or more C2-type domains. In the vertebrates many molecules have retained this feature such as CD2 and CTX. Some members resemble “primitive” antigen receptors and several of them map, for instance in humans, to at least two tetrads of paralogous genes, the MHC on chromosomes 1, 6. 9 (12), 19 and the LRC on chromosomes 1, 3, 11, 19 (21) (see Fig. 4.13).160,442 Many form dimers and are expressed in lymphocytes, where they form a family of adhesion molecules. The crystal structure analysis of a CTX-related molecule (JAM) revealed a unique form of dimerization, suggesting that the diversity of ligand binding and domain-interactions used by different IgSF domain is extensive. Two JAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R (V, I, L) E. The receptor for advanced glycosylation end products gene has a rather “generic” receptor function as it recognizes aged cells exposing particular carbohydrate motifs. CD47, another conserved IgSF member with a CTX-like V domain, suggests an ancient function.488 Perhaps such nonrearranging VJC1 genes that regulated cytotoxicity/phagocytosis were predecessors of the antigen receptors. Some molecules with VJ/C1 cores involved in cell-cell interactions often serve as ports of entry for viruses. In a move from “property to function,” an arms race consequence could result in a virus receptor developing into an immune receptor. The best examples of such molecules are in the CTX JAM family, in which receptor interactions with viruses may trigger an apoptosis, a primitive form of antiviral immunity.33,489

have antigen receptors derived from different gene families, at first glance it seemed that the T-B split occurred twice in evolution. However, the gene expression profi les for the lamprey T and B cells are too similar to the patterns in gnathostome lymphocyte populations to be derived by convergent evolution; furthermore, the development of lamprey T cells in a thymus-like structure also suggests evolution from a common ancestor. Thus, it seems most likely that both the LRR and IgSF receptor families were present in the common ancestor,2,13 with the VLR system predominating early in vertebrate evolution (see Fig. 4.10). Such a scenario exists today with NKRs in mammals, with different gene families (C-type lectin or IgSF) predominating in different species.12 Because APOBEC family members are most likely involved in VLR assembly and perhaps in mutation, there may have been a primordial IgSF receptor that was also modified somatically by an AID-like molecule, which functioned sideby-side with VLRs. The rearrangement break caused by the RAG transposon in a primordial IgSF gene offered a great advantage, as was detailed previously, and the IgSF receptors then could have supplanted VLRs in gnathostomes, while maintaining the AID-dependent machinery to diversify it further. It is difficult to imagine how a rearrangement break in an antigen receptor gene generated by LRR insertion could be advantageous; conversely, as mentioned previously, a break in the CDR3 loop in the center of the binding site is in perfect position to interact with peptide bound to MHC. While lampreys have a thymoid for development of their “T cells,” as described previously, there was a great leap in complexity of thymus structure in the gnathostomes, consistent with a new TCR/MHC system in which positive and negative selection became a requirement for a functioning adaptive immune system. In addition, the general transcriptional and cytokine/chemokine networks for T-cell (and B-cell) development would have also been in place in the jawless vertebrates; in gnathostomes, this network became more complex in the Big Bang (eg, see the chemokine section in which the leap in sophistication can be examined with precision). In summary, a working hypothesis proposes that the divergence of lymphocytes into T and B cells occurred early in vertebrate evolution (see Fig. 4.10). LRR- and IgSF-based antigen receptors existed simultaneously as well at this time (in the same organism or organisms in the same epoch), with VLR perhaps predominating in early vertebrates. The RAG transposon insertion into an IgSF receptor (perhaps one already diversifying by mutation or conversion), along with a genomewide duplication, resulted in the Ig/TCR/MHCbased system of gnathostomes, with all of its accompanying sophistication in all aspects of adaptive immunity, including development of the thymus and spleen, complexity of cytokine and chemokine networks, and intimate interactions of cells with primary and secondary lymphoid tissues.

CONCLUSION Emergence of T and B Cells As described previously, agnathans have now been shown to have cells in the T and B lineages, similar to what is found in gnathostomes.291 Because the jawless and jawed vertebrates

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Since the last edition of Fundamental Immunology was published 5 years ago, there have been several astonishing findings, as well as other interesting discoveries and integrations, regarding the evolution of the structure and function of the

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immune system. Furthermore, there have been many genome and EST projects that have allowed us to determine whether or not particular genes or gene families are present in different taxa. Here, we briefly describe these findings and discuss their broad relevance and relationship to future studies. A new system of defense in bacteria and archaebacteria against bacteriophage, CRISPR, has been elucidated.490 Short phage DNA sequences have been acquired by prokaryotic genomes to direct sequence-specific protection against phages. These elements are transcribed into small RNAs that recruit endonucleases to cleave the phage nucleic acid. This system is widespread and its study is in its infancy. The discovery of this system fits well with studies of plants and animals over the last decade uncovering the plethora of mechanisms used to detect foreign nucleic acids (eg, TLR, NLR, RIG, APOBEC, etc.), as described previously. It should be clear to all immunologists by now that all living things must distinguish self from nonself, and have developed intricate mechanisms to achieve this end. Cytokines such as IFNγ, IL-2, and IL-4, and other adaptive cytokines (and type I/III IFNs) and their receptors are not present outside of jawed vertebrates, nor is the vast array of chemokines seen in gnathostomes. However, at the moment it appears that (almost) all of the cytokines/chemokines discovered in mammals are also present in basal jawed vertebrates: Big Bang indeed! IL-17, a cytokine garnering great attention in basic immunology, and TNF, a central cytokine in both innate and adaptive immunity, seem to be the first cytokines to have emerged in evolution based on studies of lower deuterostomes. If, as suggested from studies in mammals, IL-17 is important for responses to extracellular bacteria, nonvertebrate deuterostomes may hold the key to understanding the physiology of the entire system (ie, in the absence of the competing TH1, TH2, and regulatory T cells), and one may be able to study the dynamics of IL-17 development and function in lower deuterostomes and even protostomes. Additionally, over the past few years a new type of cell, the ILC, has come to prominence.491 These cells are predominantly found in epithelia and mucosa and are involved in homeostasis and a first line of defense. As they do not bear antigen receptors, ILCs respond via cytokine receptors or PRRs. ILCs express cytokines consistent with T-cell subsets, and it is tempting to propose that they preceded adaptive T cells in evolution, and the cytokine-production profi les were coopted by the antigen receptor–bearing cells. However, as mentioned, it appears that “adaptive” cytokines arose late in evolution, coincident with adaptive immunity. Thus, it seems more like that ILCs arose simultaneously with the adaptive cells in the immunologic Big Bang, and there has been adaptation over time. However, as IL-17 and TGF-β are old cytokines, it has been proposed that their opposing functions arose before adaptive immunity.492 TGF-β belongs to an ancient family of cytokines, and at least one of its functions in mammals is to antagonize proinflammatory responses (regulatory T differentiation, regulation of IgA synthesis, etc.). One model suggests that sophisticated innate immunity arose in mucosal tissues, regulated by the pro- and anti-inflammatory properties of IL-17 and TGF-β, respectively, by their expression in gut epithelia or

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by infi ltrating hematopoietic cells; later, this was superimposed onto adaptive lymphocytes in the vertebrate lineage. Consistent with this idea, sea urchins have an expanded family of IL-17 genes,48 suggestive of a very important function for this cytokine. So, such a system may have been the forerunner of both the ILCs and adaptive lymphocytes, perhaps found in tissues directly exposed to the environment. Additionally, we are poised for more seminal studies of mucosal immunity. Mammals have dedicated secondary lymphoid tissues in their mucosa, and thus it is often difficult to tease apart the innate from adaptive responses in these tissues, except in contrived knockout or transgenic models. By contrast, ectothermic vertebrates have no Peyer patches or mesenteric lymph nodes, and thus are excellent models to examine responses in the lamina propria, which are predominantly T-independent.493 Lastly, studies of how the microbiota interact with the immune system have fi lled journals to the gills over the past 5 years. McFall-Ngai suggested that the driving force for adaptive immunity was not so much to fight off invaders, but rather to tolerate commensals,494 perhaps not so different from Weaver’s proposal.492 This framework of interactions between leukocytes, and between leukocytes and commensals/pathogens, may be the key to understanding the genesis of adaptive immunity. A new defense system in Drosophila, important for viral defense, has been uncovered. Previously, the recognition of pathogens by TLRs in the invertebrates was believed to be indirect, interacting with self-molecules similar to a cytokine interaction. However, Drosophila Tol7 has now been shown to interact directly with viruses, similar to TLRs in vertebrates,153 but, as expected, the effector phase is different from the canonical (historical) toll pathway. There are several other tolls in the protostomes without a described function that can be studied in a new light. One of the great triumphs in the field of comparative immunology was the discovery that similar mechanisms are used to recognize extracellular pathogens and initiate immune responses. It has been approximately 15 years since the Drosophila toll/IMD systems and their relationship to vertebrate TLRs and TNF receptors has been uncovered. Over the past few years, the intracellular defense mechanism such as NOD/NLR, RIG, helicase, etc., have been elucidated in vertebrates, and we are beginning to piece together their evolutionary histories. The amazing similarities between the deuterostome and plant NOD/NLR systems will challenge us to understand their recognition events for years to come.14 The VLR system continues to amaze us.13 Most comparative immunologists never expected a unique antigen receptor system to be uncovered in jawless fish. To then realize that the divergence of lymphocytes into two lineages occurred in the ancestor of jawed and jawless fish was astonishing. Now to have uncovered a primitive type of thymus candidate in jawless fish is a total shock. All of these discoveries force us to acknowledge that we should not be surprised by future discoveries in the evolution of immunity, such as a “convergent MHC,” or an antigen presentation system not based on peptide presentation, or even a primordial type of adaptive immune system in lower deuterostomes or protostomes.

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Furthermore, from comparative studies, Boehm and colleagues have proposed a simple model for the most fundamental elements required for thymocyte differentiation, which has been superimposed onto the lamprey thymus-like structures.375,495 Further study of this minimal network of transcription factors, chemokines, and cytokines could uncover the origins of thymus differentiation in lower deuterostomes that clearly have no T/B cells. Certain groups of organisms are interesting to study for different reasons. The genome projects in cnidarians demonstrated that certain gene families were more ancient than previously believed (from studies of the classical models Drosophila and C. elegans).35 Thus, this taxonomic group has become the new rock stars of comparative immunology, not only for their immune complexity in the absence of a mesodermal germ layer, but also for elucidation of a histocompatibility reaction that has fascinated us for decades.496 Teleost fish have been shown again and again to have rapidly evolving immune systems, often expanding gene families in unprecedented ways (eg, NLR, tripartite motifcontaining), generating specific paralogs and losing others (eg, cytokines, chemokines, etc.), losing canonical syntenies found in all other vertebrates (eg, MHC class I and class II), and having unique forms of ancient molecules (eg, tetrameric IgM with no J chain). The third genomewide duplication (3R) is believed to have been a major force in this instability and rapid evolution.20 This group provides an opportunity to study the plasticity of immune systems in related organisms over a short period of evolutionary time. With the cod story, they even offer a natural class II knockout system395! Amphibians with metamorphosis offer models to study two modalities of self-tolerance, metamorphosis, and polyploidy. The larval immune system must somehow be suppressed with the appearance of adult self-determinants during metamorphosis and the refurbishing of the immune system. The amphibian Xenopus speciates by polyploidy, allowing an experimental system to examine the effects of whole-genome duplications on immune system loci (silencing, deletion, gene conversion, conservation), a key issue given the importance attributed to the genomewide duplications in the evolution of immune systems in vertebrates.328,399 Birds offer another case of rapid evolution, probably because of attack by viruses. For the MHC, birds have lost immunoproteasomes and the thymic-specific proteasome, and have modified their class I groove to be more promiscuous in peptide binding. Chickens (but not ducks) have lost the RIG-1 gene, apparently making them susceptible to influenza.497 Clearly, birds have something fundamental and unique to teach us about how pathogens (most likely viruses in this case) can force a remodeling of innate and adaptive immunity. The genomewide duplications early in the evolution of the vertebrates (the 2R hypothesis) have been confirmed in a variety of studies, and they were indeed important in the Big Bang emergence of the adaptive immune system. The 2R paradigm is useful for studying any gene family found on

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multiple chromosomes in the vertebrates; good examples of how it has aided in our understanding of immunity were the study of IgSF members involved in cell-cell interaction/ costimulation, and the evolution/emergence of chemokines and TNF family members. An emerging paradigm is the possibility that genes encoding NKC, LRC, MHC, as well as the antigen receptors and costimulatory molecules (and other immune genes) were all linked at an early point in evolution.2,498 In addition, while the agnathan adaptive system is similar to the Ig/TCR/MHC system of gnathostomes, the great sophistication that we see in the latter is not present in lampreys and hagfish. Our best guess at the moment is that the RAG transposon, in combination with the genomewide duplications, both superimposed onto an already existing adaptive framework, resulted in the Big Bang of adaptive immunity (see Fig. 4.10). With the genome projects and advances in molecular biology, we have made strides in understanding old problems in vertebrate adaptive immunity, such as 1) when the L chains emerged and what the significance of more than one isotype is; 2) which antigen receptor (if any of the extant ones) came first, and all of them came to evolve to their present state; 3) when IgD emerged and what its function is in different vertebrate phyla, especially the transmembrane form; and 4) how γ /δ T cells recognize antigen. We propose that are two arms of the γ /δ T-cell lineage, one innate and the other adaptive, similar to B cells and α / β T cells. The long-awaited molecular mechanisms of histocompatibility in nonvertebrates have been at least partially uncovered in plants, Botryllus, and Hydractinia. The common themes thus far are that the genes encoding the polymorphic receptors/ligands are closely linked and that the genes all appear to be unrelated to the vertebrate MHC. However, there are tantalizing links to immune gene clusters found throughout the animal kingdom that suggest there may be an underlying fundamental similarity in recognition, perhaps related to 1) the paucity of surface receptors available because of multiple constraints resulting in sharing of receptor/ligands, and 2) the signaling cascades. Despite the great variation in mechanisms of allorecognition across phyla, are there commonalities? Whether in cnidarians or in tunicates, the polymorphic genetic regions involved contains from one to many IgSF members resembling members of the nectin PVR/CD155 family. In Ciona, these genes are not only closely linked in a single region but also are homologs found in the LRC. Could this region be the conserved and link aspects of immunity between vertebrates and invertebrates? The frequent presence of ITIM and ITAM indeed suggest the conservation of receptors interacting among these molecules.160 It is clearly of ancient origin. With paralogs on four different chromosome regions in gnathostomes as well as an ancient connection to MHC and NKC, this region was present before 2R. It is not surprising now to detect very different molecular families involved in immune recognition events: LRR, lectins, IgSF. The comparison of allorecogniton in invertebrates has perhaps helped to focus our attention on genetic regions that may have provided the “context” for leukocyte interactions.

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III

Immunoglobulins and B Lymphocytes

5

Immunoglobulins: Structure and Function Harry W. Schroeder Jr • David Wald • Neil S. Greenspan

INTRODUCTION Immunoglobulins (Igs) are marked by a duality of structure and function.1 In common with other members of the Ig superfamily,2 they provide the immune system with a conserved set of effector molecules. These effectors can activate and fi x complement and they can bind to Fc receptors on the surfaces of granulocytes, monocytes, platelets, and other components of the immune response. Both activation of complement and binding to Fc receptors can contribute to the induction or maintenance of inflammation. They also provide the immune system with a polyclonal set of diverse ligand binding sites, which allow Igs, as a population, to recognize an almost unlimited array of self- and non–self-antigens, which may range from compounds as fundamental to life as deoxyribonucleic acid to manmade molecules that could not have played a role in the evolution of the immune system. Differential splicing allows individual Ig molecules to serve as either membrane-bound receptors for the B cell that allow antigen-specific activation or as soluble effectors, which act at a distance. In vivo, proper effector function requires more than just antigen-specific binding; it requires successful neutralization of the offending antigen while avoiding potentially pathogenic self-reactivity. The receptor and effector functions of each individual Ig can be localized to a separate region or domain of the molecule. Each variable (V) or constant (C) domain consists of approximately 110 to 130 amino acids, averaging 12,000 to 13,000 kD. A typical light (L) chain will thus mass approximately 25 kD, and a three C domain Cγ H chain with its hinge will mass approximately 55 kD.

Immunoglobulins are Heterodimers Igs are heterodimeric proteins, consisting of two H and two L chains (Figs. 5.1 and 5.2). The eponymous Ig domain serves as the basic building block for both chains. Each of

the chains contains a single amino-terminal V Ig domain and one, three, or four carboxy-terminal C Ig domains. H chains contain three or four C domains, whereas L chains contain only one. H chains with three C domains tend to include a spacer hinge region between the first (CH1) and second (CH2).3 At the primary sequence level, Igs are marked by the interspersion of regions of impressive sequence variability with regions of equally impressive sequence conservation. The V domains demonstrate the greatest molecular heterogeneity, with some regions including non-germline– encoded variability and others exhibiting extensive germline conservation across 500 million years of evolution.4 The molecular heterogeneity of the V domains permits the creation of binding sites, or paratopes, which can discriminate between antigens that may differ by as little as one atom. Thus it is the V domains that encode the receptor function and define the monovalent specificity of the antibody. The H chain CH1 domain, which is immediately adjacent to the V, associates with the single L chain C domain. Together, the CH1 and CL domains provide a stable platform for the paired set of V H and V L domains, which create the antigen binding site. The distal CH2 and CH3 domains, for those antibodies with a hinge, or the CH3 and CH4 domains, for those with an extra (CH2) domain, typically encode the effector functions of soluble antibody. Each of these Ig CH domains is encoded by a separate exon. Although the sequences of the individual CH domains are constant within the individual (and nearly constant within a species), they can vary greatly across species boundaries. The carboxy-terminal CH domain encodes a secretory tail, which permits the antibody to exit the cell. Also encoded within the germline sequence of each CH gene are two membrane/cytoplasmic tail domain exons, termed M1 and M2. Alternative splicing removes the secretory sequence typically encoded by the terminal CH3 or CH4 domain and replaces it with the peptides encoded by the M1 and M2 exons, converting a secretory antibody to a

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FIG. 5.1. Model of a Secreted Immunoglobulin (Ig)G. The germline exonic derivation of the sequence is shown at the top, and the protein structure is shown at the bottom. The location of the various cysteine residues that help hold both the individual domains and the various Ig subunits together are illustrated. Papain digests IgG molecules above the cysteine residues in the hinge that holds the two H chains together yielding two Fab molecules and an Fc, whereas pepsin digests below releasing an (Fab)′2 fragment and two individual Fcs (which are typically degraded to smaller peptide fragments). The location of some allotypic variants is illustrated. CH2 domains can be variably glycosylated, which can also affect Ig structure and effector function.

membrane-embedded receptor.5 Between species, the membrane/cytoplasmic tail region is the most highly conserved portion of the CH domains, which befits its role as a link to the intracellular signal transduction pathways that ultimately regulate B-cell function.

Paratopes and Epitopes The immunoglobulin-antigen interaction takes place between the paratope, the site on the Ig at which the antigen binds, and the epitope, which is the site on the antigen that is bound. It is important to appreciate that antibodies do not recognize antigens; they recognize epitopes borne on antigens.6 This makes it possible for Igs to discriminate between two closely related antigens, each of which can be viewed as a collection of epitopes. It also is one scenario that permits the same antibody to bind divergent antigens that share equivalent epitopes, a phenomenon referred to as cross-reactivity. It has been estimated that triggering of effector functions in solution typically requires aggregation of three or more effector domains, and thus tends to involve the binding of three or more epitopes.6 For antigens encoding repeating epitope structures, such as polysaccharides or antigen aggregates, binding of a single polymeric Ig molecule carrying multiple effector domains, such as pentameric IgM, can be sufficient to induce effector function. For antigens encoding diverse epitopes, which is more typical of monodisperse single-domain molecules in solution, triggering of inflammatory effector functions may require the binding of a diverse

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set of Ig molecules, all binding the same antigen, but at different epitopes.7

Membrane and Secretory Immunoglobulin Alternative splicing allows Igs to serve either as soluble antibodies or as membrane-bound antigen receptors. In their role as antibodies, Igs are released into the circulation from where they may traffic into the tissues and across mucosal surfaces. In their role as the B-cell antigen receptor, they are anchored to the membrane by means of their M1:M2 transmembrane domain. Soluble antibodies can also be pressed into service as heterologous cell surface antigen receptors by means of their attachment to membrane-bound Fc receptors.8 This permits the power of antibody recognition to be extended to nonlymphoid cells, such as Fc-expressing granulocytes, macrophages, and mast cells. The major difference between these two forms of cell surface receptors is that Igs as B-cell antigen receptors provide a monoclonal receptor for each B cell, whereas antibodies bound to Fc receptors endow the cell with a polyclonal set of antigen recognition molecules. This gives greater flexibility and increases the power of the effector cells to recognize antigens with multiple non-self–epitopes.

Isotypes and Idiotypes Igs can also serve as antigens for other Igs. Immunization of heterologous species with monoclonal antibodies (or a restricted set of Igs) has shown that Igs contain both common

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FIG. 5.2. Ribbon Diagram of a Complete Immunoglobulin (Ig)G1 Crystal (1 hzh in the protein data bank [PDB] from Data of Harris et al.202). The major regions of the Ig are illustrated. The heavy-chain constant regions (green) also include the hinge (yellow) between the first two domains. Cγ2 is glycosylated (also seen in yellow). The heavy- and light-chain variable regions (red and dark blue, respectively) are N terminal to the heavy- (green) and light-chain (light blue) constant regions. Complementarity determining region loops in the heavy- and light-chain variable regions (yellow and white) are illustrated as well.

and individual antigenic determinants. Epitopes recognized within the V portion of the antibodies used for immunization that identify individual determinants are termed idiotypes (Fig. 5.3), whereas epitopes specific for the constant portion are termed isotypes. Recognition of these isotypes first allowed grouping of Igs into recognized classes. Each class of Ig defines an individual set of C domains that corresponds to a single H chain constant region gene. For example, IgM utilizes μ H chain C domains and IgE utilizes ε C domains. Some V domain epitopes derive from the germline sequence of V gene exons. These shared epitopes, commonly referred to as public idiotopes or cross-reactive idiotypes (see Fig. 5.3), are, from a genetic perspective, isotypic because they can be found on many Igs of different antigen-binding specificities that derive from the same germline V region. Examples include the cross-reactive idiotypes found on

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monoclonal IgM rheumatoid factors derived from individuals with mixed cryoglobulinemia, each of which can be linked to the use of individual V gene segments.9

Classes and Allotypes Each of the various classes and subclasses of Igs has its own unique role to play in the immunologic defense of the individual. For example, IgA is the major class of Ig present in all external secretions. It is primarily responsible for protecting mucosal surfaces. IgG subclasses bind Fc receptors differently, and thus vary in effector function.10 Determinants common to subsets of individuals within a species, yet differing between other members of that species, are termed allotypes and define inherited polymorphisms that result from allelic forms of immunoglobulin C (less commonly, V) genes.11

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Glycosylation N-linked carbohydrates can be found in all constant domains as well as in some variable domains.12 The structure of the attached N-linked carbohydrate can vary greatly, depending on the degree of processing. These carbohydrates can play a major role in Ig function.13 For example, human IgG molecules contain a conserved glycosylation site at Asn 297, which is buried between the CH2 domains.14 This oligosaccharide structure is almost as large as the CH2 domain itself. O-linked sugars are also present in some Igs.12 Human IgA1, but not IgA2, possesses a 13 amino acid hinge region that contains three to five O-linked carbohydrate moieties.15 A deficiency in proper processing of these O-glycans can contribute to IgA nephropathy, which is a disease that is characterized by the presence of IgA1-containing immune complexes in the glomerular mesangium.16

A HISTORICAL PERSPECTIVE The identification of Ig as a key component of the immune response began in the 19th century. This section describes the history of the identification of Ig and introduces fundamental terminology.

Antibodies and Antigens

FIG. 5.3. Electron micrographs (top; ×350,000) and interpretive diagrams (below) of murine mAb hybridoma group A carbohydrate (HGAC) 39 (specific for the cell wall polysaccharide of Streptococcus pyogenes) in complex with anti-idiotypic mAb Fab fragments. HGAC 39 is represented in the diagrams as an open figure, and the Fab anti-Id probes are represented as solid figures. The Fab arms of the antibody targets and probes are drawn to indicate their rotational orientation as planar (oval with open center), intermediate (bone shape with or without central opening), or perpendicular (“dumbbell shaped”). Different complexes illustrate the range of Fab-Fab angles made possible by segmental flexibility. 1, anti-IdI-1 Fab; 2, anti-IdI-2 Fab; 39, HGAC 39; 3a, anti-IdI-3a Fab; K, anti-Cκ Fab; X, anti-IdX Fab. IdI designates an individual idiotope, and IdX, a crossreactive idiotope. Antibody complexes were stained with 2% uranyl formate as described by Roux et al.108 Reproduced from Proceedings of the National Academy of Sciences (from Roux et al.108 with permission).

Aristotle and his contemporaries attributed disease to an imbalance of the four vital humors: the blood, the phlegm, and the yellow and black biles.17 In 1890, Behring (later, von Behring) and Kitasato reported the existence of an activity in the blood that could neutralize diphtheria toxin.18 They showed that sera containing this humoral antitoxin activity would protect other animals exposed to the same toxin. Ehrlich, who was the first to describe how diphtheria toxin and antitoxin interact,19 made glancing reference to “Antikörper” in a 1891 paper describing discrimination between two immune bodies, or substances.20 The term antigen was first introduced by Deutsch in 1899. He later explained that antigen is a contraction of “Antisomatogen + Immunkörperbildner,” or that which induces the production of immune bodies (antibodies). Thus, the operational definition of antibody and antigen is a classic tautology.

Gamma Globulins In 1939, Tiselius and Kabat immunized rabbits with ovalbumin and fractionated the immune serum by electrophoresis into albumin, alpha-goblulin, beta-globulin, and gamma-globulin fractions.21 Absorption of the serum against ovalbumin depleted the gamma-globulin fraction, hence the terms immunoglobulin and IgG. “Sizing” columns were used to separate Igs into those that were “heavy” (IgM), “regular” (IgA, IgE, IgD, IgG), and “light” (light chain dimers). Immunoelectrophoresis subsequently permitted identification of the various Ig classes and subclasses.

Fab and Fc In 1949, Porter first used papain to digest IgG molecules into two types of fragments, termed Fab and Fc (Table 5.1).22

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TABLE Fc Fab

F(ab)’2 Fab’ Fd Fv Fb pFc’

5.1

Definitions of Key Immunoglobulin Structure Nomenclature

A constant region dimer lacking CH1 A light chain dimerized to VH-CH1 resulting from papain cleavage; this is monomeric because papain cuts above the hinge disulfide bond(s) A dimer of Fab’ resulting from pepsin cleavage below the hinge disulfides; this is bivalent and can precipitate antigen A monomer resulting from mild reduction of F(ab)’2: an Fab with part of the hinge The heavy chain portion of Fab (VH-CH1) obtained following reductive denaturation of Fab The variable part of Fab: a VH-VL dimer The constant part of Fab: a CH1-CL dimer A CH3 dimer

From Carayannopoulos and Capra206 with permission.

Papain digested IgG into two Fab fragments, each of which could bind antigen, and a single Fc fragment. Nisonoff developed the use of pepsin to split IgG into an Fc fragment and a single dimeric F(ab)2 that could cross-link antigens.23 Edelman broke disulfide bonds in IgG and was the first to show that IgG consisted of two H and two L chains.24

Two Genes, One Polypeptide The portion of the constant domain encoded by the Fc fragment was the fi rst to be sequenced and then analyzed at the structural level. It could be readily crystallized when chilled. The heterogeneity of the V domain precluded sequence and crystallographic analysis of an intact Ig chain until Bence-Jones myeloma proteins were identified as clonal, isolated Ig light chains. These intact chains could be purified and obtained in large quantities, which fi nally

FIG. 5.4. The Immunoglobulin Domain. A: A typical variable domain structure. Note the projection of the C-C’ strands and loop away from the core. B: A typical compact constant domain structure. C: The cysteines used to pin the two b-sheets together are found in the B and F strands. D: The folding pattern for variable and constant domains.2

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permitted rational analysis of antibody structure and function.25 Recognition of the unique nature of a molecule consisting of one extremely variable V domain and one highly conserved C domain led to the then-heretical Dreyer-Bennett proposal of “two genes, one polypeptide,”26 which was subsequently and spectacularly confi rmed by Tonegawa.27

THE IMMUNOGLOBULIN DOMAIN The Ig domain is the core unit that defines members of the Ig superfamily (reviewed in Williams and Barclay2 and Harpaz and Chothia28). This section describes the Ig domain in detail.

The Immunoglobulin Superfamily Each Ig domain consists of two sandwiched β-pleated sheets “pinned” together by a disulfide bridge between two conserved cysteine residues (Fig. 5.4). The structure of the β-pleated sheets in an Ig domain varies depending on the number and conformation of strands in each sheet. Two such structures, V and C, are typically found in Igs. C-type domains, which are the most compact, have seven antiparallel strands distributed as three strands in the fi rst sheet and four strands in the second. Each of these strands has been given an alphabetical designation ranging from amino terminal A to carboxy-terminal G. Side chains positioned to lie sandwiched between the two strands tend to be nonpolar in nature. This hydrophobic core helps maintain the stability of the structure to the point that V domains engineered to replace the conserved cysteines with serine residues retain their ability to bind antigen. The residues that populate the external surface of the Ig domain and the residues that form the loops that link strands can vary greatly in sequence. These solvent exposed residues offer multiple targets for docking with other molecules.

A

C

B

D

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FIG. 5.5. Sequence Conservation and Hypervariability within a Heavy Chain Variable (V) Domain. The primary sequence the V domain can be divided into four regions of sequence conservation, termed framework regions (FRs), and three regions of hypervariability, termed complementarity determining regions (CDRs). A schematic of the genomic origin of the variable domain is shown at the top of the figure. The classic separation of the sequence into FR and CDR by Kabat et al.31 is shown below the gene structure. The letter designation for individual β strands is given beneath the Chothia and Lesk nomenclature,32 which focused more on structure. The positions of each of the four invariant residues of the VH chain (FR1 Cys22, FR2 Trp36, FR3 Cys92, and FR4 Trp103) are shown as darkened circles on the Chothia and Lesk model. The ImMunoGeneTics information system® designation has attempted to rationalize sequence variability with structure and is the current nomenclature of choice.33

The V Domain V-type domains add two additional antiparallel strands to the first sheet, creating a five strand–four strand distribution. Domain stability results from the tight packing of alternately inward-pointing residue side chains enriched for the presence of hydrophobic moieties to create a hydrophobic domain core. The H and L variable domains are held together primarily through noncovalent interaction between the inner faces of the β sheets.29,30 Early comparisons of the primary sequences of the V domains of different antibodies identified four intervals of relative sequence stability, termed framework regions (FRs), which were separated by three hypervariable intervals, termed complementarity determining regions (CDRs) (Figs. 5.5 and 5.6).31 The exact location of these intervals has been adjusted over the years, fi rst by a focus on the primary sequence, 31 then by a focus on the threedimensional structure, 32 and, more recently, by a consensus integration of the two approaches by the international

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ImMunoGeneTics information system® (IMGT)33 (see Fig. 5.5). (For students of the Ig repertoire, IMGT maintains an extremely useful website, http://www.imgt.org, which contains a large database of sequences as well as a multiplicity of software tools.) The C and C’ strands that define a V domain form FR2. These strands project away from the core of the molecule (see Figs. 5.4 and 5.6) where they take on a conserved structure that is parallel and opposite to the FR2 of the companion V and adjacent to the FR4 of the complementary chain. Approximately 50% of the interdomain contacts in the hydrophobic core of the V domain are formed by contacts between the FR2 of one chain and the FR4 of the complementary chain.30 Another 30% to 45% is contributed by contacts between the CDR3 and the FR2 or CDR3 of the complementary domain. The overall interdomain contact includes between 12 and 21 residues from the L chain V domain and 16 to 22 residues from H chain V domain, most of which are contributed by the FR2, CDR3, and FR4 regions.

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highly homologous scaffold. This section describes the characteristics of the Fab domain, its component V domains, and the paratope, which is the part of the Fab that actually binds antigen.

Fab, Fv, and Fb The antigen-binding fragment (Fab) is a heterodimer that contains an L chain in its entirety and the V and CH1 portions of the H chain (see Figs. 5.1 and 5.6). The Fab in turn can be divided into a variable fragment (Fv) composed of the V H and V L domains, and a constant fragment (Fb) composed of the CL and CH1 domains (see Table 5.1). Single Fv fragments can be produced in the laboratory through genetic engineering techniques.35 They recapitulate the monovalent antigen binding characteristics of the original, parent antibody. Other than minor allotypic differences, the sequences of the constant domains do not vary for a given H chain or L chain isotype. The eponymous V domain, however, is quite variable.

Generation of Immunoglobulin Variable Domains by Recombination FIG. 5.6. The Structure of an Fab. The antigen-binding site is formed by the heavy (H) and light (L) chain B-C, C′-C″, and F-G loops. Each loop encodes a separate complementarity determining region (CDR). The location of CDRs H1, H2, H3, L1, L2, and L3 are shown. The opposing H and L chain C-C′ strands and loop help stabilize the interaction between VH and VL. This C-C′ structure is encoded by the second V framework region, FR2. The inclusion of this structure permits the variable (V) domains to interact in a head-to-head fashion. The E-F strands and loop are encoded by the FR3 region and lie directly below the antigen-binding site. The A-B strands and loop encode FR1 and lie between the CH1 and CL domains and the rest of the Vs. The beta sheet strands of the CH1 and CL domains rest crosswise to each other. The illustration is modified.203

There are approximately 40 crucial sequence sites that influence variable domain inter- and intradomain interactions.32,34 Four of these sites are relatively invariant: the two cysteines that form the disulphide bridge between the beta sheets and two tryptophan (phenylalanine in Jκ) residues, one near the beginning of the C strand and the second near the beginning of the G strand, that pack against the bridge to add stability. Beyond these and other common core residues, Ig domains can vary widely in their primary amino acid sequences. However, a common secondary and tertiary structure characteristic of the core Ig V domain tends to be preserved.

FAB STRUCTURE AND FUNCTION Introduction It is the Fab domain that allows Ig to discriminate between antigens. The Fab is individually manufactured to precise specifications by individual developing B cells. It shows an amazing array of binding capabilities while maintaining a

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Ig V domain genes are assembled in an ordered fashion by a series of recombination events.27,36–39 The elegant mechanisms used for the assembly of these genes and the Fvs they create are fully discussed in Chapter 6. However, in order to understand the relationship between antibody structure and function, a brief review is in order. In BALB/c mice, Ig V assembly begins with the joining of one of 13 diversity (DH) gene segments to one of four joining (JH) gene segments. This is followed by the joining of one of 110 functional variable (V H) gene segments.40–42 Each DH gene segment has the potential to rearrange in any one of six reading frames (RFs), three by deletion and three by inversion. Thus, these 127 gene segments can come together in 3.4 × 104 combinations. The numbers of gene segments can vary quite widely between different mouse strains, but the process of assembly and the multiplicative effect of combinatorial diversity is the same. In the final assembled V domain, the V H gene segment encodes FRs H1 to H3 and CDRs H1 and H2 in their entirety (see Figs. 5.1 and 5.5), and the JH encodes FR-H4. CDR-H3 is created de novo in developing B cells by the joining process. CDR-H3 contains the DH gene segment in its entirety, as well as portions of the V H and JH gene segments. After a functional H chain has been created, L chain assembly begins. Mice contain two L chain loci, κ and λ. In C57BL/6 mice, the κ locus includes five Jκ gene segments and 140 Vκ gene segments, of which 4 and 73 have been shown to be functional, respectively.43–45 This provides 292 combinations. There is only one Cκ .46 The BALB/c λ locus contains three Vλ, three functional Jλ, and two or three functional Cλ chains.47,48 The λ constant domains are functionally indistinguishable from each other. Due to gene organization, the λ repertoire provides at most seven combinations. Each V L encodes FRs L1 to L3, CDR L1 and L2, and two thirds of CDR-L3 (see Fig. 5.1). Each J L encodes one-third of CDR-L3 and FR4 in its entirety. Any one

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H chain can combine with any one L chain, thus 211 V, D, and J gene segments can provide approximately 1 × 107 different H:L combinations. At the V→D and D→J junctions, the potential for CDRH3 diversity is amplified by imprecision in the site of joining, allowing exonucleolytic loss as well as palindromic (P junction) gain of terminal VH, DH, or JH germline sequence. B cells that develop after birth express the enzyme terminal deoxynucleotidyl transferase (TdT) during the H chain rearrangement process.27,36 TdT catalyzes the relatively random incorporation of non-germline– encoded nucleotides between V H and DH, and between DH and JH. Each three nucleotides of N addition increase the potential diversity of CDR-H3 20-fold. Thus sequences with nine nucleotides of N addition each between the V→D and D→J junctions would enhance the potential for diversity by (20) 6, or by 6 × 107; six-fold greater than the potential diversity provided by VDJ gene segment combinations. These genomic gymnastics permit the length of CDR-H3 to vary from 5 to 20 amino acids among developing B cells in BALB/c bone marrow.49 Together, imprecision in the site of VDJ joining and N addition provides the opportunity to create nearly random CDR-H3 sequence, potentially freeing the CDR-H3 repertoire from germline sequence constraints. Although a limited amount of N addition is observed between VL and JL in human,50,51 N addition in murine L chains is distinctly uncommon. Moreover, the length of CDR-L3 appears to be under relatively strict control, greatly limiting the potential for somatic L chain junctional diversity.48,50 Thus, CDR-H3 represents the greatest focus for the initial somatic diversification of the antibody repertoire.

through mechanisms of gene conversion, and V gene segments are no exception. Sequence relationships allow grouping them into families and clans of sequences that share nucleotide homology,52 as well as structural features. Close inspection of the V H gene repertoire has shown that these family relationships reflect segmental gene conversion coupled with selection for function.4,53,54 Due to the need to maintain a common secondary and tertiary core Ig V domain structure capable of associating randomly with a complementary V chain to form a stable Fv, the core sequence of FR2, which is encoded by the V H gene segment, and the core sequence of FR4, which is encoded by the JH gene segment, are highly conserved among all Ig V domains. Conversely, the need to generate a diverse repertoire of antigen-binding sites has led to extensive diversity in the CDR-1 and CDR-2 intervals. One might presume that the FR1 and FR3 intervals, which form the external surface of the antibody, would not be under any particular constraints, but sequence comparisons suggest otherwise. Given the need to diversify the CDRs and the need to preserve FR2, it is not surprising that family identity, which might reflect ancestral relationships, can be assigned by the extent of FR1 and FR3 similarity.55 Of these, FR1 appears to be under the greatest constraints, with VH gene segments belonging to different families both within and across species barriers exhibiting extensive similarities in FR1 sequence (Fig. 5.7). Sequence similarities in FR1 and, to a lesser extent, FR3 allow grouping of human and murine VH families into three clans of related sequences, presumably reflecting an early divergence in sequence from a primordial VH gene sequence (Fig. 5.8).

Segmental Conservation and Diversity within the V Domain

Constraints on the Sequence and Structure of Variable-Encoded Complemenatarity Determining Regions

Although the large numbers of V gene segments might give the impression of a smooth incremental range of available diversity, multigene families are thought to evolve in concert

The antigen-binding site of an Ig is formed by the juxtaposition of the six hypervariable H and L chain V domain

FIG. 5.7. A comparison of two human Clan I VH sequences that belong to different VH families (modified4). Shown is a comparison of the deoxyribonucleic acid and amino acid sequences of the V5-51 and V1-2 gene segments. Each line depicts a divergence in a nucleotide or amino acid at that position. Shown at the bottom is a replacement/silent site substitution analysis by interval. Random mutation tends to exhibit an R/S ratio of 2.9. The smaller the ratio, the greater the preservation of sequence. The intervals identified by the arrows predict the family and clan of origin.

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FIG. 5.8. Evolutionary Relationships among Vertebrate VH Families. The sizes or relative placements of the evolutionary connecting lines are not to scale. VH sequences from all mammalian species analyzed to date can be placed into one of these three clans (modified4).

intervals: CDRs-H1, -H2, and -H3; and CDRs-L1, -L2, and -L3.31 The CDR sequences of V gene segments tend to be enriched for codons where mutations maximize replacement substitutions.56 This includes the RGYW motif that facilitates somatic hypermutation.57,58 While evolution appears to favor CDR1 and CDR2 sequences that facilitate codon diversity, it also appears to preserve specific loop structures. Although there is great variation in the sequence and size of these CDRs, it has been shown that five of them, CDR-H3 being the notable exception, possess one of a small set of main-chain conformations termed canonical structures.32,34,59–62 Each canonical structure is determined by the loop size and by the presence of certain residues at key positions in both the loop and framework regions. It can be calculated that the total number of possible combinations of canonical structures, or structure classes, is 300.63 However, only 10 of these combinations, or classes, are sufficient to describe the majority of human and mouse Fab sequences. Among specific classes, the lengths of CDR-H3 and -L1 appear to correlate with the type of recognized antigen. Antibodies with short loops in -H2 and -L1 appear to be preferentially specific for large antigens (proteins), whereas antibodies with long loops in -H2 and -L1 appear to be preferentially specific for small molecules (haptens).63 Given that the sequence and structure of the framework regions, which define families, influences the canonical structure of V H-encoded CDRs, it is not surprising that the structure repertoire of canonical structures is strongly associated with family and clan identity.64 This implies restrictions to the random diversification of the hypervariable loop structures (canonical structures) and their combinations within the same V H gene segment (canonical structure

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classes). It further suggests evolutionarily and structurally imposed restrictions operating to counteract the random diversification of these CDRs.

Diversity and Constraints on the Sequence and Structure of CDR-H3 The combination of VDJ assortment, variation in the site of gene segment rearrangement, and N nucleotide addition makes CDR-H3 the most variable of the six hypervariable regions. In some cases, the sequence of CDR-H3 appears designed to provide optimal flexibility.65 Correspondingly, it has been more difficult to assign canonical structures to the CDR-H3 loops similar to those observed for the V-encoded CDRs. However, insight into a gradient of possible structures has been gained. CDR-H3 can be separated into a base, which is adjacent to the frameworks, and a loop. The base tends to be stabilized by two common residues, an arginine at Kabat position 94 (IMGT 106) and an aspartic acid at Kabat 101 (IMGT 116).32,33 These form a salt bridge which, together with the adjacent residues, tends to create one of three backbone conformations, termed kinked, extrakinked, and extended.66 In some sequences with kinked or extrakinked bases, it is possible to predict whether an intact hydrogen-bond ladder may be formed within the loop of the CDR-H3 region, or whether the hydrogen-bond ladder is likely to be broken.67,68 However, for many CDR-H3 sequences, especially those that are longer, current tools provide less than optimal predictions for the structures of individual CDR-H3s. Despite the potential for totally random sequence provided by the introduction of N nucleotides, close inspection has shown that the distribution of amino acids in the

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CDR-H3 loop is enriched for tyrosine and glycine,31,69 and relatively depleted of highly polar (charged) or nonpolar (hydrophobic) amino acids, although the precise pattern depends on the species of origin.70 This pattern of amino acid utilization is established early in B-cell development, prior to the expression of Ig on the surface of the cell (Fig. 5.9)49,71–73 and reflects evolutionary conservation of JH and DH gene segment sequences. In particular, although the absolute sequence of the DH is not the same, the pattern of amino acid usage by RF is highly conserved. Of the six potential RFs, RF1 by deletion is enriched for tyrosine and glycine. RF2 and RF3 by deletion are enriched for hydrophobic amino acids, as they are by inversion. RF1 by inversion tends to encode highly polar, often positively charged, amino acids.69 Various species use different mechanisms to bias for use of RF1 by deletion, to limit use of hydrophobic RFs, and to restrict or prevent use of RFs enriched for charged amino acids. Forced rearrangement into RFs with charged amino acids yields an altered repertoire enriched for charge and depleted of tyrosine and glycine.71 The distribution of CDR-H3 lengths can also be regulated both as a function of differentiation and as a function of ontogeny.49,74 In association with long V-encoded CDRs, short CDR-H3s create an antigen-binding cavity at the center of the antigen-binding site, and CDRs of intermediate

length can create an antigen-binding groove. Each species appears to prefer a specific range of CDR-H3 lengths.75 Long CDR-H3s, which can create “knobs” at the center of the antigen-binding site, are unevenly distributed between species and reflect both divergence in germline sequence and somatic selection.

The Antigen-Binding Site is the Product of a Nested Gradient of Regulated Diversity The tension between the need to conserve essential structure and the need to emphasize diversity in an environment subject to unpredictable antigen challenge appears to create a gradient of regulated diversity in the Fv. The most highly conserved components of the Fv are FR2 and FR4, which form the hydrophobic core of the VH:VL dimer (see Figs. 5.4 and 5.5). FR1, which in the H chain presents with three conserved structures, helps form the ball and socket joint between the VH and CH1. FR3, which in the H chain defines the family and provides 7 different structures in human versus 16 different structures in mouse, frames the antigen-binding site (Fig. 5.10). The V-encoded CDRs, -H1, -H2, -L1, -L2, and most of -L3, are programmed for diversity. However, conserved residues within these CDRs, which interact with V family–associated FR3 residues, constrain diversity within a

Percent

TdT-ko

B n = 67

40 35 30 25 20 15 10 5 0

C n = 162 D n = 84 E n = 83 F

Percent

Young Adult Repertoire

n = 73

B n = 194

40 35 30 25 20 15 10 5 0

C n = 373 D n = 279 E n = 255 F

R

K

N

D

Q

E

H

P

Y

W S Amino Acid

T

G

A

M

C

F

L

V

n = 254

I

FIG. 5.9. Both in the absence or presence of N-addition, the preference for tyrosine and glycine in complementarity determining region-H3 begins early and intensifies with B-cell development. VH7183DJCμ transcripts were cloned and isolated from fractions B (pro-B cells) through F (mature B cells) from the bone marrow of 8- to 10-week-old terminal deoxynucleotidyl transferase-sufficient and terminal deoxynucleotidyl transferase-knockout BALB/c mice.73 The amino acids are arranged by relative hydrophobicity, as assessed by a normalized Kyte-Doolittle scale.204,205 Use is reported as the percent of the sequenced population. The number of unique sequences per fraction is shown.

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Binding of “Superantigens” to Nonclassic Variable Domain Antigen-Binding Sites Not all antigens bind to the paratope created by the classic antigen-binding site. Antigens that can bind to public idiotopes on V domain frameworks81 and recognize large portions of the available repertoire are termed superantigens. There are indications that B cell superantigens influence the pathogenesis of some common infections, such as those caused by Staphylococcus aureus.

ANTIGEN-ANTIBODY INTERACTIONS

FIG. 5.10. Location and Generation of Complementarity Determining Region-H3. A: A cartoon of the classic antigen-binding site (modified4). Due to its central location, most antigens bound to the antibody will interact with complementarity determining region-H3.

preferred range of canonical structures. CDR-H3, the focus of junctional diversity, lies at the center of the antigen-binding site. The conformation of its base tends to fit within three basic structures. The loop varies greatly in sequence, yet still maintains a bias for the use of tyrosine and glycine. Thus, diversity increases with proximity to the tip of the antigenbinding site but appears to be held within regulated limits. The extent and pattern of diversity in CDR-H3 can have a critical effect in the biologic function of Ig as a soluble effector molecule. Absence of N addition, with its enhancement of tyrosine and glycine usage (see Fig. 5.9) facilitates B-cell development, whereas enrichment for charged amino acids impairs it.71,76,77 However, both the absence of N addition and enrichment for charged amino acids impair immune responses and protection in vivo.71,76,78

Somatic Hypermutation and Affinity Maturation Following exposure to antigen and T-cell help, the V domain genes of germinal center lymphocytes can undergo mutation at a rate of up to 10−3 changes per base pair per cell cycle,79 a process termed somatic hypermutation. Somatic hypermutation allows affinity maturation of the antibody repertoire in response to repeated immunization or exposure to antigen. Although affinity maturation often preserves the canonical structure of the CDR loops, the distribution of diversity appears to differ between the primary and antigenselected repertoire.80 In the primary repertoire, diversity is focused at the center of the binding site in CDR-H3. With hypermutation, somatic diversity appears to spread to the V-encoded CDRs in the next ring of the binding site (see Fig. 5.10), enabling a more custom-tailored fit.

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Technological advances in biomolecular structure determination, analysis of molecular dynamics, protein expression and mutagenesis, and biophysical investigation of receptor-ligand complex formation have facilitated significant advances in the understanding of antigen-antibody interactions. Particularly valuable has been the integration of high-resolution structural data with thermodynamic and kinetic analyses on a number of antigen-monoclonal antibody complexes. In this section, some of the key insights arising from these studies will be reviewed.

Molecular Flexibility Like many other protein domains, V domains exhibit varying degrees and modes of molecular flexibility. Evidence suggests that some V modules (ie, VL-VH pairs) can adopt two or more conformations with meaningful frequencies in the unbound state. Because these different conformational states can exhibit distinguishable binding proclivities, molecular flexibility provides monoclonal antibodies with a mechanism for polyspecificity.82 Molecular flexibility can also play a role in binding a single ligand, which, in such instances, may be better understood as a process of binding rather than as a simple event.83,84 The extent of conformational adjustment by antibody or antigen required for complex formation can influence both the thermodynamics and kinetics of that process.

Role of Water A significant role for water molecules has become clear from the study of high-resolution structures and thermodynamic analyses of antigen-antibody complexes. Water molecules exhibit a broad range of association times with protein surfaces. Thus some of the more tightly protein-associated of these solvent molecules effectively behave as parts of the protein.85 Water molecules found in the antigen-antibody interface, whether constitutively bound or newly recruited, can make important contributions to both the intrinsic (ie, monovalent) affinity of the complex and to the differential affinities for different ligands (ie, specificity).86 Amino acid residues of antigen and antibody can interact, indirectly, through hydrogen bonds to one or more water molecules.86

Thermodynamics and Antigen-Antibody Interactions Specific residues in the antibody V domains or the antigen can contribute to complex formation in different ways, and some residues can contribute in multiple ways.87 In addition

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to making van der Waals contact, residues can be important due to contributions to the free energy of complex formation or to the differential free energy of complex formation for two or more different ligands. Some residues may contribute primarily to modulation of the association rate, the “relaxation” of the forming complex, or the dissociation rate.83,88 Other contact residues contribute minimally to the energetics of complex formation, and yet other noncontact residues can be significant thermodynamic contributors.89,90 Structural and thermodynamic/kinetic comparisons of antibodies with germline V domain sequences and with somatically mutated V domain sequences have provided new insights into the structural and energetic bases for affi nity maturation.89,90 Mutations in both contact and noncontact residues can have major consequences, positive or negative, for the affi nity with which an antibody binds an antigen. They can also favor tighter binding by enhancing V domain rigidity, thereby reducing the entropic penalty associated with complex formation.90 Antibodies derived from secondary or later responses that have incorporated somatic mutations have been shown to exhibit less than absolute specificity. Even these “mature” antibodies can bind multiple ligands when screened on libraries of peptides or proteins,91,92 a lesson likely to be relevant to most biomolecules. It has been routine to distinguish between interactions in which antibodies bind to protein antigens versus those in which antibodies bind to carbohydrate antigens. Recent studies of antibodies that bind to human immunodeficiency virus-1 gp120 with high affinity and that exhibit potent and broad neutralizing activity suggest that antibodies can bind to epitopes composed of both peptide and glycan elements.93,94

Hinges Some Ig isotypes contain a structural element that does not strictly correspond to the canonical structural motifs of Ig superfamily V and C domains, which is termed the hinge. Where it occurs, it is located between the C-terminus of the CH1 and the N-terminus of the CH2 domains. In isotypes such as IgG and IgA, the hinge is encoded by one (or more) separate exon(s). In isotypes with four CH domains (ie, IgM and IgE), the CH2 domain serves in place of a classical hinge. The hinge, or the CH2 domain in Igs that lack a hinge, permits an Fab arm to engage in an angular motion relative both to the other Fab arm and to its Fc stem (Figs. 5.3 and 5.11). This permits the two Fab arms to cover a range from maximal extension to an almost parallel alignment. The range of motion of the Fab arms reflects the nature of the hinge region, which in some C genes is rigid and in others, such as human IgA1, functions more as a tether for each individual Fab than as a support. This flexibility has major implications for antibody function, because it enables a bivalent antibody molecule to bind epitopes in a variety of relative spatial arrangements. Among human IgG subclasses, the most unusual hinge region is that of IgG3. Unlike other human IgG hinge regions, the IgG3 hinge is encoded by a quadruplicated hinge exon, making it the longest hinge (62 amino acids) by far (Table 5.2). The primary structure of this hinge has been divided into upper, core (or middle), and lower hinge regions with somewhat different functional associations. The upper hinge in particular has been associated with the magnitude of segmental flexibility as assessed by fluorescence emission anisotropy kinetics98

IMMUNOGLOBULIN “ELBOW JOINTS” AND “HINGES” The structure of the constant domains can affect antibodyantigen interactions by influencing the range of molecular flexibility permitted between the two Fabs. In this section, the role of the Ig hinge will be discussed.

Elbow Joints Individual Ig V and C domains tend to create rather rigid dimers. However, the antibody molecule as a whole, which consists of four or more such dimeric modules linked like beads on a string, can be viewed as a paradigm of molecular flexibility.95 Flexibility begins between the Fv and the Fb of the Fab at what is termed the elbow bend or elbow angle.96 This reflects both a ball and socket interaction between the FR1 of the H chain and the CH1 domain,97 and the identity of the L chain.96 Five residues (three in V H and two in CH1) that are highly conserved in both antibodies and T-cell receptors make the key contacts that constitute this “joint.” Elbow angles, assessed from crystal structures of homogeneous Fab fragments, range from 130 degrees to 180 degrees. λ light chains appear to permit an Fv to adopt a wider range of elbow angles than their κ chain counterparts.

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FIG. 5.11. Illustration of the Motions and Flexibility of the Immunoglobulin. Axial and segmental flexibility are determined by the hinge. The switch peptide (elbow) also contributes flexibility to the Fab. The measure of the elbow angle is defined with respect to the Fv and Fb axes of two-fold symmetry. From Carayannopoulos and Capra206 with permission.

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TABLE

Ig Type IgG1 IgG2 IgG3 IgG4 IgA1 IgD

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141

Properties of Hinges in Immunoglobulin G, A, and D

5.2

Upper Hinge Length 4 3 12 7 1

Middle Hinge Length 10 8 49 4 23

Lower Hinge Length 6 6 6 6 2

Genetic Hinge Configuration (Amino Acids/Exon)

Susceptibility to Proteolysis

15 12 17-15-15-15 12 19 34-24

High High

and with the magnitude of Fab-Fab flexibility by immunoelectron microscopy.99 The core or middle hinge appears to serve, at least in part, a spacer function. The lower hinge functions primarily to facilitate CH2-CH2 interactions. Segmental flexibility of the Ig molecule, conferred mainly by the hinge, permits or facilitates simultaneous binding through two or more Fab arms. Such monogamous bivalency or multivalency, which enhances overall binding,100,101 is a crucial factor permitting biosynthetically feasible antibody concentrations to offer adequate immunity against replicating pathogens. While it is more speculative why the Fab-Fc geometry needs to vary, it may have to do with optimizing effector function activation when antigen is bound, such as maintaining antigen binding when Fc receptors are simultaneously engaged. The attribution of flexibility control to the hinge is supported by protein engineering studies in which V domain– identical IgGs of different subclasses were analyzed.98,102 This basic conclusion is also supported by studies in which hinge regions have been selectively mutated or swapped among human or mouse IgG subclasses.103–105 One of these studies indicated that structural variation among subclasses in the CH1 domain also influenced segmental flexibility as assessed by nanosecond fluorescence polarization measurements.103 Early suggestions that IgG subclass–related differences in activating the classical pathway of complement were explained by differences in segmental flexibility 98 were not confirmed by the studies in which mutant hinge regions were created.103–105 There are several types of molecular motion attributable to the hinge region that contribute to overall segmental flexibility (see Fig. 5.11). These include flexing between Fab arms (motion toward or away from one another in the same plane), Fab arms moving in and out of the same plane, Fab arms rotating along their long axes, and Fab arms moving in or out of the same plane as the Fc region.106,107 The interFab angles observed by electron microscopy range from 0 degrees to 180 degrees.108,109 Similarly, Fab arm long-axis rotations can extend up to 180 degrees.110 Another key role of the hinge is the maintenance of the CH2-CH2 interaction (ie, effectively constraining molecular mobility within the Fc region itself). The lower hinge stabilizes CH2-CH2 contacts by providing the key cysteine residues

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Special Features

Heavy O-linked glycosylation Extensive charged amino acids; heavy O-linked glycosylation at N terminus

involved in inter-H chain disulfide bonds. Experiments in which IgG molecules were modified to eliminate the hinge region demonstrate that covalent linkage between the hinge regions just “upstream” of the two CH2 domains is critical for the preservation of IgG effector function.111

HEAVY CHAIN STRUCTURE AND FUNCTION What might be termed the fundamental strategy of humoral immunity is a two-step process that begins with the identification by antibodies (of appropriate binding specificity) of the molecules or molecular complexes that should be eliminated. Following such identification (ie, noncovalent complex formation), antibodies can then trigger other molecular systems (eg, complement) or cells (eg, phagocytes) to destroy or remove the antigenic material—guilt by association at the molecular level. Thus, antigen specificity, determined primarily by the V domains in the Fab arms, is physically and functionally linked to effector function, the activation of which is primarily attributable to the C domains of the Fc region. The effector functions associated with the humoral immune response primarily involve either complement or Fc receptor–bearing cells, such as neutrophils, macrophages, and mast cells. As might be expected, therefore, the Fc contains sites for noncovalently interacting with complement components, such as C1q, and with Fc receptors. This section focuses on the structure and function of the Fc regions of the various Ig classes and subclasses.

Structure and Function of the Fc The necessity for interacting effectively with relatively conserved molecules such as C1q and Fc receptors provides a selective basis for maintaining the primary structures of the Fc region, at least where changes would undermine such intermolecular contacts. The degeneracy possible in noncovalent molecular recognition events permits selected primary sequence variations without catastrophic alterations in function. However, there are allotypic differences in the H chain constant domains among both human and mouse Igs. In some cases, these allotypic differences are associated with variation in function, at least in vitro. For example, two recombinant V domain–identical IgG3 antibodies, of

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different allotypes, exhibited differential abilities to bind C1q or initiate antibody-dependent cell-mediated cytotoxicity.112 Nevertheless, the H chains of the human IgG subclasses are particularly well conserved, with > 90% identity of amino acid sequences. In other mammalian species that have two or more IgG subclasses, they tend to exhibit less amino acid sequence identity than the human IgG subclasses. By convention, the H chain constant domains are numbered from N-terminal to C-terminal, with CH1 residing in the Fab arm and the remaining two (IgG, IgA, IgD) or three (IgM, IgE) C domains (CH2, CH3, and, if relevant, CH4) residing in the Fc. In IgM and IgE, the CH2 domain largely plays the role of the hinge region. The C domains of different isotypes and from different species share several key structural features. In distinction from V domains, which consist of four- and five-stranded β -pleated sheets linked through an intrachain disulfide bond, C domains consist of three- and four-stranded β -pleated sheets linked through an intrachain disulfide bond (see Fig. 5.4). Across isotypes, amino acid sequence identity for CH domains is approximately 30%, while for subclasses (within an isotype) the amino acid sequence identity for CH domains is in the range of 60% to 90%. Important physical and biological properties of the human Ig isotypes are summarized in Table 5.3. As noted previously, the traditional and accepted functional anatomy of Igs attributes antigen binding (both specificity and affinity) to the V modules in the Fab arms and effector function activation to the Fc region. While this scheme is both well supported and appealing, there is considerable (perhaps not widely appreciated) evidence that in some cases structural variations in H chain domains (ie, CH domains and hinge) can influence both the affinity of the antibody for antigen and the discrimination among antigens.113–118 Although these instances of C domain influence on ligand binding (through the V domains) primarily involve multivalent antigens, there are also reports that suggest C domain influence in instances of monovalent recognition.119–121 Mechanisms for these effects in the context of binding multivalent antigens include isotype-related differences in segmental flexibility as well as the tendency for self-association. Results from a study comparing resistance to pneumococcal infection for IgG3-deficient and IgG3-producing mice are consistent with the notion that the cooperative binding permitted by murine IgG3 antibodies contributes to the effectiveness of humoral immunity.122

Fc Glycosylation All Igs contain N-linked oligosaccharides, and it is becoming increasingly clear that this glycosylation plays significant roles in Ig structure and function. Though the type and extent of glycosylation varies among isotypes, an N-linked oligosaccharide on Asn 297 in the CH2 domain is conserved on all mammalian IgGs and homologous portions of IgM, IgD, and IgE. As the average serum IgG contains 2.8 oligosaccharides, there is often glycosylation present in the V domain as well.123 The consensus sequence for the V domain N-linked oligosaccharides is not present in the germline, but it can

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be created during somatic hypermutation.124 Glycosylation in the Fc region has been shown to be important for antibody half-life and effector functions.125–127 Glycosylation of the Fc domain influences complement activation as Ig hypoglycosylation influences affinity for C1q as well as Ig binding to the FcR, possibly due to its effects on Ig structure.128 Differential sialylation of the core Fc polysaccharide has recently been shown to have dramatic effects on the proinflammatory versus anti-inflammatory activity of IgG.129,130 Furthermore, IgD N-linked glycans are necessary for IgD to bind to the IgD receptor on T cells.131 V domain glycosylation potentially affects the affinity for antigen, antibody half-life, antibody secretion, and organ targeting. Interestingly, glycosylation has been shown to be capable of both positively and negatively affecting antigen binding.132–135 The biologic significance of Ig glycosylation can be seen from studies demonstrating that IgG from patients with rheumatoid arthritis is galactosylated to a lesser extent (termed IgG G0) than IgG from normal controls. In some cases, hypogalactosylation correlates with disease activity.136 Hypogalactosylation of IgG has also been found to occur in other chronic inflammatory diseases such as Crohn’s disease and systemic lupus erythematosus.137

Immunoglobulin M IgM is an isotype of firsts. It is ontogenetically primary, being expressed first on developing B lineage cells. IgM is also the isotype that initially dominates the primary humoral immune response. It is probably, along with IgD, a phylogenetically primitive isotype in jawed vertebrates (an almost first) and may be the most phylogenetically stable isotype.138 IgM serves important immunological functions both on the surfaces of B-lymphocytes and in the fluid phase in the blood and in the mucosal secretions. On the cell surface, IgM consists of two identical μ H chains and two identical L chains (μ2L2). It is initially expressed on B lineage cells in noncovalent association with surrogate L chains, and subsequently, following successful L chain gene rearrangement, with κ or λ light chains. On the mature B-cell surface, IgM is noncovalently associated with two other polypeptide chains, Ig-α (CD79a) and Ig-β (CD79b).139–141 These integral membrane proteins serve to transduce signals when surface IgM binds to and is cross-linked by cognate antigen. In the secreted form, IgM can consist of either pentamers (μ2L2)5 or, less often, hexamers (μ2L2) 6.142 The μ2L2 monomers of the pentameric form are linked one to another by disulfide bonds in the CH4 domains. Two of these monomers are, on one side, disulfide bonded not to another μ chain but to a 15,000 Da-polypeptide, called J chain. J chain is also found in polymeric IgA. There may be multiple patterns of such disulfide bonding, such that different cysteines participate in different monomeric units.143 Application of electron microscopy to polymeric IgM molecules has suggested that IgM can adopt two different quaternary arrangements: star and staple.144,145 All of the antigen-binding sites are arrayed in radial fashion, in the same plane as one another and the Fc regions, in the star

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950(p) 19S 5 or 10 1 3 20 J chain (16 kDa) 5 0 10–12 0.25–3.1 10 3.3 5–10 0 ++++ 0 0 — Primary antibody response, some binding to pIgR, some binding to phagocytes

N-glycosylation sites O-glycosylation sites Carbohydrate average (%) Adult level range (age 16–60) in serum (mg/ml)b Approximate % total Ig in adult serum Synthetic rate (mg/kg weight/day) Biological half-life (days) Transplacental transfer Complement activation classical pathway (Clq) Complement activation alternative pathway Reactivity with protein A via Fc Allotypes Biological properties

IgM

Molecular weight of secreted form (kDa) Sedimentation coefficient Functional valency Interheavy disulphide bonds per monomer Membrane Ig cytoplasmic region Secreted Ig tailpiece Other chain

a

Properties of Immunoglobulin Isotypes

3 7 9–14 0.03–0.4 0.2 0.2 2–8 0 0 0 0 — Mature B-cell marker

175 7S 2 1 3 9 9

IgD

b

Ig, immunoglobulin; d, dimer; m, monomer; p, pentamer. Light chain molecular weight is 25 kDa. Total = 9.5-21.7 mg/ml. Compiled from Carayannopoulos and Capra,206 Lefranc and Lefranc,209 Kuby,210 and Janeway et al.211

a

5.3

Class or subclass properties

TABLE

150 6.6S 2 4 28 2 —

IgG2

2 11 28 2 —

160

IgG3

2 2 28 2 —

150

IgG4

1 1 2 1 0 0 0 0 2–3 2–3 2–3 2–3 5–12 2–6 0.5–1 0.2–1 45–53 11–15 3–6 1–4 33 33 33 33 21–24 21–24 7–8 21–24 ++ + ++ ++ +++ + ++++ 0 0 0 0 0 ++ ++ ++ +/− G1m G2m G3m — Placental transfer, secondary antibody for most responses to pathogen, binds macrophages and other phagocytic cells by FcγR

2 2 28 2 —

150

IgG1

IgA2

160(m), 300(d) 160(m), 350(d) 7S 11S 2 or 4 2 or 4 2 2 14 14 20 20 J chain (16 kDa) secretory component (70 kDa) 2 4 8 0 7–11 7–11 1.4–4.2 0.2–0.5 11–14 1–4 19–29 3.3–5.3 5–7 4–6 0 0 0 0 + 0 0 0 — A2m Secretory Ig, binds pIgR

IgA1

7 0 12–13 0.0001–0.0002 0.004 0.002 1–5 0 0 0 0 Em Allergy and parasite reactivity, binds FcεR

190 8S 2 1 28 2 —

IgE

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arrangement. In the staple form, the Fab arms bend out of the plane of the Fc regions. It has been conjectured that the staple form is utilized in binding simultaneously to two or more epitopes on multivalent antigens, such as bacterial or viral surfaces. A major pathway through which soluble IgM mediates immunity or immunopathology is the activation of the classical pathway of complement. On a per molecule basis, relative to other isotypes, IgM is highly active in activating the classical pathway and can thereby effectively opsonize bacterial pathogens. In select cases (eg, Neisseria meningitidis), binding of IgM to bacterial surfaces, followed by complement activation, can cause direct lysis of the bacteria through the insertion of the membrane attack complex into the bacterial membrane.146 IgM, like polymeric IgA, can reduce the effective number of colony- or plaque-forming units for, respectively, bacteria and viruses, through agglutination. Significant physical and biological properties of IgM and the other Ig isotypes are shown in Table 5.3.

Immunoglobulin D IgD is primarily of interest in its membrane form, as the soluble form of IgD is found in relatively modest concentrations in the blood and other body fluids. The cell surface form of IgD is found along with IgM on all mature, naïve B cells, where it appears capable of transducing activating and tolerizing signals.147 As is true for IgM, the membrane form of IgD associates noncovalently with Ig-α (CD79a) and Ig-β (CD79b). Simultaneous cell surface expression of two H chain isotypes expressing the same VH domains and the same L chains occurs via differential ribonucleic acid splicing.148 IgD exhibits greater sensitivity to proteolytic cleavage than IgM, which is consistent with a relatively short serum half-life of only 2.8 days. The relatively long hinge region is a primary target for proteolysis. Relatively modest efforts have historically been devoted to investigating of the functional roles of IgD antibodies, especially in the secreted form, in comparison to the other isotypes. However, new information suggests that IgD antibodies are produced in the upper respiratory mucosa by an unanticipated mechanism of class switch recombination and that these antibodies participate in host defense against pathogens relevant to this anatomical environment. In addition, secreted IgD antibodies can bind to basophils, through a receptor that is yet to be identified, and when these antibodies are crosslinked by cognate antigen, the basophils release potent mediators that influence immune reactivity, inflammation, and pathogen viability.149

Immunoglobulin G IgG is the predominant isotype (approximately 70% to 75% of the total Ig) in the blood and extravascular compartments. The four human IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are named in order of their relative serum concentrations, with IgG1 the most prevalent and IgG4 the least. There are differences in effector functions (eg, complement activation and Fc receptor binding) and other biological

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properties (such as serum half-life) among these subclasses. However, there are also crucial functional commonalities, such as placental passage (see Table 5.3). IgG antibodies are the hallmark of immunological memory in the humoral immune response. In addition to the isotype switch from IgM to IgG in a secondary antibody response, somatic hypermutation can lead to affinity maturation, a process by which the average affinity of antibody for the antigen eliciting the immune response can increase. IgG antibodies contribute to immunity directly and through the activation of complement or FcR-bearing cells. Important examples of immunity mediated directly through antibody binding include neutralization of toxins (eg, diphtheria toxin) and viruses (eg, poliovirus). Medically important examples of IgG-induced complement activation include immunity to encapsulated bacterial pathogens leading either to opsonization and destruction within phagocytes (eg., Streptococcus pneumoniae) or to direct complementmediated lysis (eg, Neisseria meningitidis). Activation of FcR-bearing cells by IgG antibodies has also been implicated in immunity to pathogens (eg, Cryptococcus neoformans).150 The consensus view is that human IgG1 and IgG3 isotypes are effective activators of the classical complement pathway. While some older sources state that IgG2 and IgG4 are weak or nonactivators of the classical complement pathway, more recent evidence suggests that when epitope density is high, IgG2 is effective in activating complement.151,152 One possible source for the isotype-related variation in complementactivating ability is variation in affinity for C1q (IgG3 > IgG1 > IgG2 > IgG4), the portion of the first component in the classical pathway that physically contacts the CH2 domains of antibodies. However, isotype-associated differences in complement activation have also been found to occur at steps of the cascade subsequent to the binding of C1q to antibody.112,153 For example, in one study of chimeric monoclonal antibodies engineered to express identical V domains and representing all four human IgG subclasses, the IgG3 antibody fi xed C1q better than the IgG1 antibody, but the IgG1 molecule was more effective in mediating complement-dependent cell lysis than the IgG3 molecule.153 Thus, it is probably not possible to rank the relative abilities of the IgG subclasses to activate complement in a single absolute hierarchy. The affinities of IgG subclasses for Fc receptors vary from about 5 × 105 M−1 to about 108 M−1. Recent studies in the mouse suggest that the relative contributions of IgG subclasses to various immunopathological processes depend on their relative affinities for the activating versus inhibiting isoforms of FcR.154 A remarkable attribute of IgG (for three of the four subclasses) is its serum half-life of about 23 days. This property, attributable to the Fc region and its interaction with the neonatal Fc receptor (FcγRn), has been exploited for therapeutics through the genetic fusion of solubilized receptors, (eg, cytotoxic T-lymphocyte antigen 4) to IgG Fc regions.155 A recently derived insight into the function of IgG4 antibodies relates to their apparently unique ability to spontaneously dissociate into half-molecules and to form antibodies composed of different half-molecules with distinct antigen

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specificities (ie, bispecific antibodies that cannot cross-link antigen and thus have anti-inflammatory activity).156 This phenomenon may be of particular relevance to specific immunotherapy in the setting of allergy.157

Immunoglobulin A While IgG is the clearly predominant isotype in the blood, IgA is the dominant Ig isotype in the mucosal secretions as well as in breast milk and colostrum.158 In the blood, 10% to 15% of the Ig is IgA (vs. 65% to 75% IgG). Moreover, IgA has a shorter half-life than IgG in serum. The predominant form of IgA in human serum or plasma is monomeric (ie, α2L2), but there are small quantities of dimers [(α2L2)2] and fewer still trimers and tetramers. Secretory IgA consists of dimers and lesser amounts of trimers and tetramers associated with one joining (J) chain (distinct from the J region in the heavy chain V domain) and one SC chain (see following discussion). The latter is the extracellular portion of the polymeric Ig receptor (pIgR), which is expressed by mucosal epithelial cells and transfers polymeric IgA or IgM from basolateral to apical surfaces, thereby providing most of the Ig content of the mucosal secretions. The J chain is disulfide bonded to the tail pieces, short C-terminal extensions of the CH3 domains, of the two IgA monomers of a dimer, while SC forms a disulfide bond to a cysteine in one CH2 domain of one monomer. The two α H chain C region genes correspond to two IgA subclasses, IgA1 and IgA2. IgA1 is the predominant (> 80%) IgA subclass in the serum. While IgA2 is the major form in

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some human mucosal secretions, such as those in the large intestine and the female genital tract, there is variation in the relative proportion of IgA1 and IgA2 in different secretions. The shorter hinge region of IgA2 confers increased resistance to bacterial proteases that might be encountered in the mucosal environment. The extended hinge region of IgA1 is believed to permit molecules of this isotype to accommodate variable epitope spacings on multivalent antigens. While the long hinge region of IgA1 molecules might be expected to confer relatively high susceptibility to proteolysis, relative protection against the activity of bacterial proteases is provided by heavy O-linked glycosylation in the hinge (Fig. 5.12). Nevertheless, the IgA1 hinge is uniquely susceptible to IgA proteases produced by certain pathogenic bacteria.159 Secretory IgA (S-IgA) has been shown to participate in immunity against a range of viral, bacterial, and parasitic pathogens at mucosal surfaces. The relative absence of functional complement and phagocytes in mucosal secretions is consistent with a different mix of mechanisms for mediating immune effects associated with S-IgA versus, for example, serum IgG. Mechanisms associated with IgA are less dependent on inflammation-producing molecules or cells, such as inhibition of microbial adherence through V module-mediated specific binding to microbial adhesins, agglutination of microbes, blocking of microbial receptors for cell surface carbohydrates with IgA-associated glycans, and mucus trapping (in which binding of S-IgA to bacteria makes them more adherent to host-generated mucus). There is also evidence that polymeric IgA can neutralize viruses

FIG. 5.12. A comparison of an x-ray and neutron-solution–scattering theoretical model (human immunoglobulin (Ig)A1) and x-ray crystal (murine IgG1 and IgG2a) structures. Light chains (yellow), heavy chains (red and dark blue), and glycosylation (light blue) are illustrated. The extended length of IgA1 over that of IgG can be seen along with extensive glycosylation that characterizes this isotype. From Boehm et al.207 with permission.

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in some circumstances by interfering with steps postattachment, such as internalization. In some cases, posttranslational modifications of viral surface molecules, related to proteolytic events associated with epithelial cell transit, lead to IgA antibodies expressing protection-related antigen specificities that have no parallel in the IgG pool.160,161 Some of the contributions of IgA to immunity are mediated through binding to FcαRI (CD89) on human neutrophils, monocytes/macrophages, and eosinophils. For example, cells bearing FcαRI on their plasma membranes can phagocytose IgA-antigen complexes. FcαRI binds to the IgA Fc region between the Cα2 and the Cα3 domains.162 Amino acid residues in the IgA Fc region critically involved in the interaction with CD89 are indicated in Figure 5.13. The ability of IgA to activate complement is controversial. At present, the preponderance of evidence suggests that IgA does not activate the classical complement pathway and only weakly, and under some pathophysiological circumstances, activates the alternative complement pathway. However, there is evidence suggesting that, in vitro at least, polymeric, but not monomeric, IgA can activate the complement pathway dependent on mannose-binding lectin.163 Additional properties of the polymeric forms of IgM and IgA are considered in the following section.

Immunoglobulin E IgE is best known for its association with hypersensitivity reactions and allergy, but this isotype is also of interest in the context of immunity to parasites. In the blood, IgE is

present at the lowest concentration of any of the Ig isotypes (with roughly five orders of magnitude less IgE than IgG) and has the shortest half-life. The unimpressive quantitative representation of IgE in the blood is related to the high affinity of IgE antibodies for Fc εRI, often referred to as the high-affinity Fc receptor for IgE. Fc εRI is expressed on mast cells, basophils, Langerhans cells, and eosinophils. Due to the high affinity of Fc εRI for its IgE ligand, mast cells and basophils are covered with relatively long-lived Fc εRI-IgE complexes. The interaction between IgE and Fc εRI has an affinity of ∼1010 L/M. It primarily involves contacts between Fc εRI and amino acids in the CH3 domains, with some contributions from amino acids in the CH2 domains. Although each IgE potentially has two sites for interacting with the FcεRI, the stoichiometry has been shown to be 1:1. Furthermore, it has been suggested that IgE binds to Fc εRI in a kinked conformation.164 Upon ligation with bivalent or multivalent antigens specifically recognized by the bound IgE molecules, the FcεRI molecules transduce signals that activate the mast cells or basophils to secrete potent mediators of inflammation, such as histamine. These mediators are responsible for the symptoms associated with asthma, allergic rhinitis, and anaphylaxis. There is a second receptor for IgE. Fc εRII (CD23), a type II membrane protein, is expressed on monocytes/ macrophages, B-lymphocytes, natural killer cells, follicular dendritic cells, Langerhans cells, eosinophils, activated epithelial cells, and platelets. It binds monomeric IgE with an affinity of ∼107 L/M, roughly three orders of magnitude lower than the affinity of Fc εRI for IgE. Functional consequences of FcεRII-IgE interaction on macrophages include secretion of mediators of immediate hypersensitivity as well as cytokines and chemokines. There is also evidence suggesting that the Fc εRII-IgE interaction can contribute to antigen capture and presentation to both B and T cells.

Fc Receptor Immunoglobulin Interactions

FIG. 5.13. Illustration of the Residues Essential for the Binding of immunoglobulin (Ig)A to Fc`R (CD89). Residues represent mutations made in IgA CH regions as mapped on an Fcy fragment. Of the residues mutated, L465 and L266 were found to be important for binding to CD89. From Carayannopoulos et al.162 with permission.

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There are four receptors that bind the Fc regions of IgG molecules in humans and five such receptors in mice. Three of the human receptors are expressed primarily on hematopoietic cells directly involved in immune responses: FcγRI, FcγRII, and FcγRIII. Among the FcγR involved in antibody effector functions, there is important variation in affinity for IgG molecules. FcγRI binds IgG with relatively high affinity, permitting the binding of monomeric IgG. In contrast, FcγRII and FcγRIII bind to IgG with relatively low affinity. Consequently, these latter two receptors do not bind significant quantities of monomeric IgG but preferentially interact with IgG that has been effectively aggregated through interaction with bivalent or multivalent antigens (ie, immune complexes). Key amino acid residues involved in the binding FcγRs and Fc εR1 with their corresponding Ig can be seen in Figure 5.14. Allotypic variations in FcγRIIA can also influence affinity for IgG ligands and subsequent effector function.165 There are different isoforms of FcγRII and FcγRIII. Of particular functional relevance, FcγRIIA is activating,

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A

B FIG. 5.14. Certain residues are conserved between Fcγ Rs and FcεRl as well as between immunoglobulin (Ig)G and IgE that facilitate binding. Two sites participate: site 1 in (A) and site 2 in (B). Heavy lines indicate the highest number of contacts and dashed lines indicate the least. Of considerable note are residues W87 and W110 in site 2 of the receptors and P426 in the Ig that form a core “praline sandwich” in the interaction between Ig and receptor. From Garman et al.208 with permission.

whereas FcγRIIB is inhibiting. In mouse models, deficiency of FcγRIIB can be associated with an autoimmune syndrome similar to human lupus.166 Studies in mice have also suggested that the effectiveness of antibodies of the various murine IgG subclasses in mediating FcγR-dependent effector functions are correlated with the ratio of affi nities of antibodies of those subclasses for activating versus inhibiting FcγR.154 FcγRIIIA is expressed on natural killer cells, whereas FcγRIIIB is expressed on neutrophils. The former receptor is attached to the membrane by a standard transmembrane polypeptide, whereas the latter is attached via a glycophospholipid tail. The fourth human receptor, the neonatal FcγR, or FcγRn (sometimes referred to as the Brambell receptor), transports

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IgG across the placenta. It also plays a crucial role in protecting IgG from proteolytic degradation, thereby prolonging serum half-life. While FcγRI, FcγRII, and FcγRIII are members of the immunoglobulin superfamily, FcγRn is structurally similar to major histocompatibility complex class I molecules, including noncovalent association with β2-microglobulin. The interaction between FcγRn and IgG involves amino acid residues in the CH2-CH3 interface167 and is pH-sensitive. This latter property is consistent with the ability of FcγRn to bind IgG in acidic vesicular compartments and then release it into the neutral-pH environment of the blood. A crystallographic structure of the rat FcγRn in a 1:1 complex with a heterodimeric Fc (containing only one FcγRn-binding site) reveals that there are conformational changes in the Fc on binding to FcγRn.168 The investigators also identified three titratable

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salt bridges that confer pH-dependent binding of the IgG Fc to the FcγRn. There are other FcR that interact with the non-IgG isotypes: IgM, IgD, IgA, and IgE. The FcR for IgA and IgE are covered in the respective sections devoted to the corresponding isotypes. In mice, an additional Fcα /μR binds both IgA and IgM, whereas in human these receptors are distinct.169,170 Functional attributes of these receptors and the FcδR are continuing to be studied. There are functional as well as structural parallels among FcR for IgG and non-IgG isotypes. Some of the features of Fc-FcR interaction that are conserved across isotypes are illustrated in Figure 5.14.

Transmembrane and Cytoplasmic Domains Igs are expressed in both membrane and secreted forms. In contrast to the secreted form, the membrane Ig contains a transmembrane and a cytoplasmic domain. The transmembrane domain is a typical single-pass polypeptide segment consisting of 26 hydrophobic residues, extending from the C-terminus end of the C-terminal CH domain and which forms an alphahelix followed by a variable number of basic amino acids. The cytoplasmic portion of the Ig H chain ranges in length from 3 amino acids for IgG to 30 amino acids for IgE. Both membrane expression of Ig and the integrity of the cytoplasmic domain are important in antibody function. As the cytoplasmic domain is rather short, membrane-bound IgM is not thought to “signal” directly, but through the associated Igα and Igβ molecules.171 However, disruption of either membrane expression of IgG1 in mice or the cytoplasmic tail results in the failure to generate an effective IgG1 response and IgG1 memory.172 Mice lacking IgE membrane expression exhibit significant impairment in IgE responses and have extremely low levels of secreted IgE.173 For those Igs with longer membrane:cytoplasmic tails, membrane Ig affects signaling beyond its association with Igα and Igβ. Specific residues in the transmembrane domain have been identified that are crucial for signal transduction while having no effect on the association with Igα and Igβ.174

HIGHER ORDER STRUCTURE Many of the biological functions of IgA and IgM are dependent on their ability to form multimeric structures. This section will discuss the role of multimeric Ig in immune function.

Dimers, Pentamers, and Hexamers The majority of multimeric IgA exists as dimers and, less commonly, trimers and tetramers, while IgM forms pentamers and occasionally hexamers. The polymeric structures of these antibodies enhances their functional affi nity (avidity) for antigen, is essential for their active transport (both IgA and IgM) across epithelial cells to mucosal secretions, and in the case of IgM, enhances the activation of the classical pathway of complement. Once multimerized, IgA or IgM in a complex with J chain can bind to pIgR and cross

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mucosal epithelial cells.175,176 Though IgM can undergo transcytosis to the mucosal secretions, its principal action is in the serum. The ability of IgA and IgM to multimerize is due to a tailpiece, an additional C-terminal segment of 18 amino acids in the secreted forms of the μ and α heavy chains. Tailpieces of both IgM and IgA contain a penultimate cysteine (residue 575 in IgM and 495 in IgA) that forms two different disulfide bonds important for multimer formation. In an Ig monomeric unit containing two identical H chains, one cysteine residue forms intermonomeric subunit bonds, whereas the remaining cysteine residue on the other heavy chain bonds to a cysteine on the J chain.177–180 Another cysteine residue, Cys414, also forms intermonomeric subunit disulfide bonds in IgM, and this bond is important in hexamer formation.181 Besides disulfide bonds, the highly conserved glycan linked to Asn563 in IgM (and the homologous region in IgA) is also important for multimerization.182 Domain-swapping experiments demonstrate that the tailpiece regulates multimerization in the context of the specific H chain. While addition of the α tailpiece to IgM has little effect on IgM polymerization, the introduction of the μ tailpiece to IgA leads to higher-order IgA polymers.183 Based on this finding, it has been proposed that IgM polymerization is more efficient than IgA polymerization.

The J Chain The J chain, an evolutionarily conserved 137 amino acid polypeptide produced by B-lymphocytes, functions to regulate multimer formation and to promote linkage of multimeric Ig to pIgR on epithelial cells. The J chain consists of a single domain in a beta barrel conformation and does not show sequence similarity to Ig domains.184 It contains eight cysteine residues that participate in disulfide bonds with two tailpiece cysteines, as described previously, as well as function to stabilize its own structure through intramolecular bonds.183,185 The J chain influences the polymerization of the multimers, as in the absence of J chain, IgA forms fewer dimers and IgM forms fewer pentamers.186 The J chain exists in all polymeric forms of IgA and is important in IgA polymerization and secretion across the mucosa. J chain is not required for IgM polymers but is required for external secretion. While IgM pentamers contain J chain, hexamers almost always lack it. The makeup of IgM is biologically significant because IgM hexamers have about 20-fold greater complement-activating activity than IgM pentamers. The presence of increased levels of hexameric IgM has been postulated to play a role in the pathogenesis of Waldenström macroglobulinemia and cold agglutinin disease.187

Immunoglobulin Transport Transport of dimeric IgA and pentameric IgM to the mucosal secretions occurs after binding to pIgR that is present on the basolateral surface of the lining epithelial cells. The J chain is essential for the secretion of IgA and IgM and,

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as described previously, influences the polymeric structure of the Ig.175 pIgR is a transmembrane receptor synthesized by mucosal epithelial cells that contains seven domains including five extracellular V-like domains, a transmembrane domain, and a cytoplasmic domain.188 Once bound to pIgR, polymeric Ig is endocytosed and transported to the apical surface of the cell. pIgR is then proteolytically cleaved between the fifth and sixth domains to release a complex (termed secretory Ig) containing the H, L, and J chains, and the SC, which represents the cleaved extracellular portion of pIgR.189,190 As pIgR undergoes constitutive transcytosis in the absence of polymeric Ig, free SC is also released into the mucosal secretions. SC has several biological functions, including protecting the Ig from degradation by proteases and binding bacterial antigens such as the Clostridium difficile toxin A.191,192 SC also functions to localize sIgA to the mucus layer to help protect against invasion by pathogens.193 Results from pIgR-null mice demonstrate that alternate pathways exist to transport polymeric Ig to the mucosal secretions as some secretory Ig still crosses the epithelial cells in the absence of pIgR.194 Results from pIgR-null mice also demonstrate the importance of high levels of secretory antibodies as these mice are more susceptible to mucosal infections with pathogens such as Salmonella typhimurium and Streptococcus pneumoniae.195,196 The epithelial transcytosis of polymeric Ig has several biological implications. First by delivering the Ig to the mucosal surface, it enables antibodies to bind to pathogenic agents and prevent them from penetrating the mucosa, a process termed immune exclusion. Second, transcytosing antibody can neutralize viruses intracellularly.197,198 Finally, polymeric Ig can bind to antigens in the mucosal lamina

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propria and excrete them to the mucosal lumen (where they can be removed from the body) by the same pIgR-mediated transcytosis process.199,200 Some pathogens can exploit the pIgR-mediated transcytosis process in reverse to penetrate the mucosa. For example, the pneumococcal adhesin, CbpA, can bind pIgR at the epithelial apical surface, leading to bacterial penetration of the mucosa.201

CONCLUSION Igs are extremely versatile molecules that can carry out many biological activities at the same time. The need to be able to recognize unique antigen structures prior to any previous exposure coupled with the need to maintain host cell receptor or complement recognition properties presents a truly unique challenge for the system. As has been described, the system incorporates diversity within specific constraints. The precise biological niches may differ, but the overall design for these molecules is the same. The flexibility and biologic properties of Igs have made them a major focus of molecular engineering. Igs are being used as therapeutic agents, as well as for biotechnology applications. These opportunities have led to a resurgence of interest in the structure–function aspects of antibodies as we approach “designer antibodies.” Both the variable and constant portions of these molecules are current substrates for engineering purposes, offering the potential for altering both receptor and effector function. The study of antibodies began with the need to understand how sera could neutralize toxins. It is likely that antibodies will continue to be a major focus for those who seek to take fundamental principles of protein chemistry to the bedside.

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6

Immunoglobulins: Molecular Genetics Edward E. Max • Sebastian Fugmann

INTRODUCTION To respond to a foreign molecule (antigen) on an invading pathogen, the “humoral” immune system generates antibodies, or immunoglobulins (Igs), that can bind specifically to the offending antigen. Each antibody molecule is composed of two identical light (L) chains and two identical heavy (H) chains, all linked by disulfide bonds to form a symmetric Y-shaped tetramer. The ability of the immune system to generate an antigenspecific antibody depends on the fact that, before exposure to antigen, millions of naïve resting B cells circulate in the individual, each cell displaying on its membrane several thousand identical copies of a single unique species of antibody; these serve as B-cell receptors (BCRs) for that lymphocyte. Only a tiny fraction of the B cells express a BCR capable of binding to any particular antigen. When these B cells bind their antigen, they become activated to proliferate and mature into antibodysecreting plasma cells, which manufacture large amounts of antibody specific for the activating antigen. To be able to generate antibodies against a universe of diverse pathogens, this “clonal selection” mechanism for specific antibody secretion requires an enormous diversity of Ig species expressed on naïve B cells prior to antigen exposure. Indeed, in the 1960s the number of different antibody sequences in the repertoire of typical mouse was estimated in the millions. To encode this many sequences seemed to require an unreasonably high percentage of the mammalian genome (now estimated to contain only about 30,000 genes). Understanding the genetic source of Ig diversity—Ig gene assembly—was the first major challenge and achievement of the molecular biologic investigations of antibody genes, and this will be discussed first in this chapter. A week or so after antigen administration, the B-cell response changes in two ways that generally improve the protective functions of antibodies. B cells initially express antibodies of the IgM isotype, but cells that migrate into germinal centers receive T-cell-derived stimuli that can induce them to switch to production of IgG, IgA, or IgE without changing their antigen specificity; this switch results from a deoxyribonucleic acid (DNA) recombination event known as class switch recombination (CSR). In addition, over the course of an immune response, the affinity of antibody for antigen gradually improves as a result of somatic hypermutation (SHM) of antibody genes, coupled to selection for B cells expressing high-affinity antibodies. CSR and SHM are discussed later in this chapter. In this chapter, well-established facts about Ig genes are summarized concisely, while areas currently under investigation are considered in more detail, with particular attention to topics expected to interest immunologists.

OVERVIEW OF IMMUNOGLOBULIN GENE ASSEMBLY In the 1960s, investigators determined the amino acid sequences of Igs secreted by several mouse myelomas (clonal tumors of B-lymphocytes that secrete a single pure species of Ig). The N-terminal domains of the L and H chains— each roughly 100 amino acids—were highly diverse between different myeloma proteins and were designated variable (V) regions. In contrast, sequences of the remaining domains of the proteins were essentially identical for every myeloma Ig of a given class (and so they were designated constant [C] region domains). The advent of recombinant DNA technology allowed comparisons of V region genes expressed in different myelomas with the corresponding sequences in nonlymphoid DNA (commonly referred to as “germline” DNA). It was found that each myeloma V gene is composed of several segments that are separated in germline DNA; these germline segments must undergo one or more DNA recombination events to assemble a complete V region.1 For example, each complete Vκ gene from a myeloma or B-lymphocyte encodes roughly 108 amino acids and is assembled by linking one of about 40 germline Vκ segments (encoding amino acids 1 through 95) to one (of five) “joining” or Jκ segments encoding residues 96 to 108. Similarly, a complete Vλ gene is assembled from one germline Vλ segment and one Jλ segment. H chains are assembled from three segments; a diversity (D) segment is interposed between V H and JH. In developing B cells, the germline gene segments are assembled into functional V exons by a process named V(D)J recombination (Fig. 6.1). V(D)J recombination is a “cut and paste” process in which the DNA between two recombining V, D, or J gene segments is excised from the chromosome, and the two remaining DNA segments are joined together to reseal the DNA break. The two principal proteins executing the “cut” phase of this process are encoded by the recombination activating genes (RAG)1 and 2. These proteins recognize unique sequences, known as recombination signal sequences (RSSs), that flank and mark each eligible V, D, and J gene segment (RSSs are described further in the following). After the RAG proteins cut the DNA, the subsequent “joining” of the gene segments relies largely on ubiquitous DNA repair factors.

How Recombination Contributes to Diversity V(D)J recombination contributes in several distinct ways to the diversity of antigen-binding specificities. First, there is

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A

B FIG. 6.1. Variable Assembly Recombination. A: In the κ locus, a single recombination event joins a germline Vκ region with one of the Jκ segments. B: In the immunoglobulin H locus, an initial recombination joins a diversity segment to a JH segment. A second recombination completes the variable assembly by joining a VH to DJH.

combinatorial diversity, as each Ig locus contains multiple V, D (in case of the IgH locus), and J segments that can be combined in many ways. The total number of theoretically possible combinations of V H, DH, JH, V L, and JL, is the multiplication product of the numbers of possible H chains— about 40 (V H) × 27 (DH) × 6 (JH) or 6480 combinations in humans—times the number of possible L chain combinations (about 290), or almost 2 million. This repertoire is vastly larger than could be achieved by devoting the same total lengths of DNA sequence to preassembled variable region exons. Second, there is junctional diversity generated by flexibility in the position of joining between gene segments. This was initially recognized by comparisons of nucleotide sequences of various myeloma Vκ genes to their germline Vκ and Jκ precursors. As shown in Figure 6.2A, these comparisons revealed that the crossover point between sequence derived from a germline Vκ region and a Jκ region could vary in different myelomas, increasing the diversity of amino acids around codons 95 and 96. H chain VDJ exons exhibit this flexibility at both V-D and D-J junctions, yielding striking variation in the lengths D region– derived segments, from zero to about 14 amino acids. And additional junctional diversity is produced by the addition of nucleotides not present in any germline elements: “N” and “P” nucleotides, discussed below. Importantly, the three-dimensional structures of Igs established by X-ray

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crystallography reveal that the V L-JL junction and the V HDH-JH junction each encode one of the three “complementarity determining region” loops of L or H chain that can contact antigen; thus, this junctional diversity is directly functionally relevant for diversifying antigen binding. The imprecision of V(D)J recombination increases Ig diversity, but at a cost. Because the precise boundaries between V, D, and J result from independent stochastic events, only about one-third of all recombination events maintain the correct reading frame through the J segments. Gene rearrangements leading to functional Ig genes are often referred to as “productive,” while out-of-frame rearrangements are labeled “nonproductive.”

Function of Recombination Signal Sequences Analysis of DNA sequences flanking the germline V, D, and J gene segments revealed highly similar sequence motifs that have subsequently been shown to define targets for V(D)J recombination: the RSSs, which serve as the recognition sequences for the V(D)J recombinase proteins RAG1 and RAG2, as mentioned previously. Notably, RSSs lie adjacent to L- and H-chain Ig gene segments and to T-cell–receptor (TCR) gene elements throughout phylogeny. RSSs consist of a conserved seven base pairs (bps) long “heptamer” (consensus: CACAGTG) and a nine bp long “nonamer” sequences

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A

B

FIG. 6.2. Vk-Jk Recombination at Single Base Resolution. A: The sequence of the recombined MOPC41 κ gene around the VJ junction is shown (center) with the sequences of the two germline precursors (Vκ41 and Jκ1) shown above and below. The germline origins of the recombined gene are indicated by the vertical lines and the shading of the V-derived sequence. B: The consequences of joining the same germline sequences (from part A) at four different positions are shown. Of the four alternative recombination products illustrated, the top one is that actually found in MOPC41. The second example has a single nucleotide difference but no change in encoded amino acid sequence. The third and fourth alternatives yield Arg or Pro at position 96; both of these amino acids have been found at this position in sequenced mouse κ chains.

(consensus: ACAAAAACC) that are separated by less wellconserved spacers of either approximately 12 or 23 bp in length (Fig. 6.3). Based on the spacer lengths, the two classes of RSSs are referred to as 12-RSSs and 23-RSSs, respectively. (Note that some laboratories use the term recombination signal instead of RSS in their publications.) Recombination occurs almost exclusively between coding sequences associated with RSSs of different spacer lengths, a requirement referred to as the “12/23-rule” (i.e., the recombination between two 12-RSSs [or two 23-RSSs] is “forbidden” and does not occur in vivo). Within each gene locus, all gene segments of one class (e.g., all Vs in the Igκ locus) carry RSSs with the same spacer length. Thus the 12/23 rule drives appropriate recombination events leading to functional VJ and VDJ products, and prevents futile recombination events, such as between two V or two J gene segments. While the

heptamer and nonamer are the major determinants of RSS function necessary for V(D)J recombination, increasing evidence suggests that spacer sequences can modulate recombination efficiencies of compatible gene segments (e.g., they affect the non-random usage of human Vκ elements2).

THE THREE IMMUNOGLOBULIN GENE LOCI To understand the contribution of the germline V, D, J element repertoire to Ig diversity, several laboratories undertook cloning and sequence analysis of individual V region genes from the IgH, Ig κ, and Igλ loci of human and mouse. More recently, the complete sequences of all human and mouse Ig loci have been determined as part of the genome sequencing projects for these two species (available online at www.ncbi.nlm.nih.gov, though annotation that describes

FIG. 6.3. Conserved Elements Flank Germline Variable (V), Diversity (D), and Joining (J) Region Genes. Conserved heptamer and nonamer recombination signal sequences (RSSs) lie adjacent to V, D, and J coding sequences and are important for targeting V(D)J recombination. The heptamer and nonamer elements are separated by spacer regions of about 12 basepairs (bp) (thin lines) or 23 bp (thick lines). Depending on the locus, V regions may be flanked by 12 bp or 23 bp RSS; and similarly for J regions. But one of each type of element must be present for recombination to occur, a requirement that prevents futile recombination events (e.g., J to J).

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TABLE

6.1

Locus

Species

IgH

Mouse Human Mouse Human Mouse Human

Igκ Igλ

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Overview of the Number of Variable, Diversity, and Joining Segments in Each of the Three Immunoglobulin Loci in Humans and Mice V

D

Functional

Pseudogenes

Functional

110 40 95 46 3 36

85 83 45 87

10 24

56

J Pseudogenes

3

Functional

4 6 4 5 3 4–5

Pseudogenes

3 1 1 2–3

D, diversity; Ig, immunoglobulin; J, joining; V, variability. Pseudogenes are recognized based on sequence defects that would preclude function (premature stop codons, defective recombination signal sequences, defective splice sites). The numbers in this table are approximate, owing to variation between mouse strains and between individual humans.

function and refers to earlier literature is incomplete). It is important to point out that Ig gene loci are not identical between individuals (humans) or between individual strains of inbred mice. Several Internet resources are devoted to providing convenient updated access to Ig germline gene sequences. The international ImMunoGeneTics database (http://imgt.org) includes a database for Ig and TCR genes from a variety of species, and includes maps, sequences, lists of chromosomal translocations, and multiple helpful links. IgBLAST (www.ncbi.nlm.nih.gov/igblast/) is a service of the National Center for Biotechnology Information and allows a submitted sequence to be searched against known annotated germline V, D, and J sequences.

The Murine Immunoglobulin H Germline Variable, Diversity, and Joining Gene Segments VH Segments The murine V H region extends over about 2.5 megabases on chromosome 12 and includes roughly 100 functional

segments (depending on mouse strain) plus additional V H pseudogene segments (Table 6.1). All V H elements are in the same transcriptional orientation as the D, J H, and CH regions.3 The V H segments are classified into 16 distinct families based on sequence similarity; V H elements within a family show more than 80% nucleotide sequence identity. Elements of individual V H families are largely clustered together, though some interdigitation occurs (Fig. 6.4). The V H families can be grouped into three “clans” based on sequence conservation primarily of their framework regions (framework region 1, codons 6 to 24, and framework region 3, codons 67 to 85, respectively), which form the more conserved structural backbone of the Ig variable region. Importantly, these clans are conserved between man, mouse, and frog, suggesting that their emergence in the repertoire preceded the amphibian-reptile divergence.4

Diversity and JH About 50 kb downstream of the most 3′ V H element resides the murine D cluster spanning about 80 kb, depending on

FIG. 6.4. Maps of the Murine and Human VH Loci. The 15 known murine VH gene families are shown in their approximate map positions. Each rectangle represents a cluster of VH genes of the indicated family; the clan identification of the VH families is indicated by the color of the rectangle: black for clan I, gray for clan II, and white for clan III. Although some interdigitation is shown by overlapping families (e.g., the Q52 and 7183 families), the murine VH families are largely clustered. In contrast, all human VH genes (vertical lines) of a prototypic haplotype are shown in the right panel; extensive interdigitation of families is apparent.

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the mouse strain (see Table 6.1). Each D segment is flanked by 12 RSSs on both sides, so that the 12/23 rule ensures that all assembled V genes carry a D element between their V and J segments (which are both flanked by 23 RSSs that prevent direct V to J rearrangements). The murine D elements are classified into four families: DSP2, DFL16, DST4, and DQ52. Although D regions could theoretically contribute to Ig diversity by being read in all three frames, the mouse has evolved mechanisms that strongly favor one of them.5 Four functional germline JH sequences reside about 0.7 kb downstream of the most 3′ D region, DQ52.

The Human Immunoglobulin H Germline Variable, Diversity, and Joining Gene Segments VH Segments The human V H locus spans 1.1 Mb at the telomeric end of chromosome 14 (14q32.33) (see Table 6.1). The human germline V H segments—numbering roughly 40 to 45—fall into seven families that, in contrast to the family clusters characteristic of the murine locus, are extensively interdigitated (see Fig. 6.4). Some human V H sequences are polymorphic owing to V H insertions or deletions in different allelic chromosomes. Twenty-four additional germline V H sequences have been mapped to chromosome 15 and 16 and represent nonfunctional “orphans” that were apparently duplicated from the IgH locus on chromosome 14. 6

Diversity and JH Regions

Twenty-six human D elements are located in an ∼40 kb region about 20 kb downstream of VH6, the most 3′ of the VH genes.7 This D cluster is comprised of four tandem duplications of a 9.5 kb segment containing a representative of each of six D families. The twenty-seventh D element—DHQ52— is the only one showing sequence similarity to a mouse segment (DQ52) and shares a homologous location just 5′ to JH1. In contrast to mice, humans use all reading frames of D elements.7 One reading frame encodes primarily hydrophilic residues, one encodes hydrophobic residues, and one includes frequent stop codons. Some D elements contain stop codons that can be removed by nuclease trimming during VDJ assembly. As in mice, the human JH cluster is immediately downstream of DHQ52.

Heavy Chain Constant Regions Murine and human genomic clones containing C region H-chain (CH) genes include separate exons encoding the ∼100 to 110 amino acid Ig domains. These domains were independently identified by internal homologies of amino acid sequences and by three-dimensional structural analysis (X-ray crystallography). The exons are separated from each other by introns of roughly 0.1 to 0.3 kb. Thus, for example, the mouse γ 2b protein has three major domains (CH1, CH2, and CH3) with a small hinge domain between CH1 and CH2. The gene structure may be summarized as follows: CH1 - intron - hinge - intron - CH2 - intron - CH3 (292) (314) (64) (106) (328) (119) (322)

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where the numbers in parentheses represent the number of nucleotides in each segment. As an interesting contrast, the hinge region of the α gene is encoded contiguously with the CH2 domain with no intervening intron, while the unusually long human γ 3 hinge is encoded by three or four hinge exons.

Genomic Organization of the CH Region Each B-lymphocyte initially produces IgM by expressing an assembled variable region linked to C μ, but may use CSR (discussed later in this chapter) to replace Cμ with one of the several CH regions lying downstream, thereby allowing expression of IgG, IgA, or IgE (Fig. 6.5A). Eight murine CH genes span about 200 kb of DNA on chromosome 12; these genes were linked by contiguous clones in 1982. 8 Several γ pseudogenes lie within the clustered γ functional genes 9 (Fig. 6.5B). The coding sequences of all CH genes are oriented in the same direction. The human CH genes were similarly cloned, and then eventually completely linked by the Human Genome Project. The human IgH locus contains a large duplication, with two copies of a γ–γ–ε– α unit separated by a γ pseudogene (see Fig. 6.5B). One of the duplicated ε sequences is also a pseudogene, and a third closely homologous εrelated sequence—a “processed” pseudogene—is present on chromosome 9. The IgH locus has also been examined in several other species besides mouse and human, and several notable differences have been observed. Rabbits, for example, have 13 Cα sequences and only a single Cγ gene10 ; this unusual expansion of genes contributing to mucosal immunity may be related to the peculiar habit of coprophagy in these animals. In contrast to the multiplicity of rabbit Cα genes, pigs have only one Cα gene and eight Cγ genes. Camels are unusual in having H chains that function in the absence of L chains.11

Membrane versus Secreted Immunoglobulin Igs are found either as secreted molecules in the serum or as membrane-bound receptors. The membrane-bound μ chains contain a C-terminal hydrophobic transmembrane domain consisting of 26 uncharged hydrophobic amino acids encoded by additional membrane exons, and these residues anchor the protein in the cell membrane lipid bilayer. The membrane (μm) and secreted (μs) forms are derived from the same gene by alternative splicing (Fig. 6.6). The same general gene structure has been found for other CH genes, suggesting that differential splicing accounts for the two forms of all Ig isotypes. Early B cells make roughly similar quantities of both μm and μs, whereas maturation to the plasma cell stage is associated with strong predominance of μs production, facilitating high-level secretion of circulating Ig. The balance between the two ribonucleic acid (RNA) splice forms of μ has been interpreted as a competition between splicing of the CH4 and M1 exons versus the cleavage/polyadenylation at the upstream μs poly(A) site. These processes are mutually exclusive because CH4-M1 splice removes the μs poly(A) site, while cleavage at the μs poly(A) site removes the membrane exons. Cis-regulatory elements (and corresponding transacting RNA binding proteins) control the balance between these

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A

B FIG. 6.5. Deletional Isotype Switch Recombination. A: The expression of “downstream” heavy chain genes is accomplished by a recombination event that replaces the Cμ gene with the appropriate heavy chain constant gene (Cε is shown as an example), deleting the deoxyribonucleic acid between the recombination breakpoints. B: The murine and human heavy chain constant region genes are diagrammed with the approximate intergene distance indicated below (in kb); various literature values for these distances differ somewhat, possibly due to allelic polymorphisms. The human locus shows a large duplication of γ γ εα sequences.

processes. They include a GU-rich element downstream of the μs poly(A) site,12 the polyadenylation factor cleavage stimulator factor 64,13 and the U1A protein.14 These factors likely function downstream of B-lymphocyte–induced maturation protein-1 (BLIMP-1) whose expression in Ig-secreting plasma cells was also found to be critical for μs poly(A) site utilization.15 Cis-acting sequences affecting the ratio of alternative splice forms have been described for other isotypes besides Cμ, particularly Cα .16 Membrane Ig serves as the antigen-recognition component of the BCR that is critical for initiating the signal

for lymphocyte activation following contact with antigen. Transduction is mediated by an associated protein dimer composed of the BCR components Igα and Igβ (CD79a and CD79b) whose cytoplasmic domains contain immunoreceptor tyrosine-based activation motifs similar to those found in the CD3 chains mediating TCR signaling. Additional signaling is mediated by conserved tyrosines in the cytoplasmic tails of the IgG and IgE H chains, which serve as a phosphorylation-dependent docking sites for the signaling adapter Grb2.17 Binding of Grb2 enhances BCR signaling and subsequent B-cell proliferation.

FIG. 6.6. Two Ribonucleic Acids (RNAs) Generated from the m Gene by Alternative Processing. The top line illustrates the exons of the μ gene (black rectangles) in an expressed, rearranged μ gene. A primary transcript including all the exons present in the deoxyribonucleic acid can be processed as shown to yield either μs RNA (containing a C-terminal “secreted” sequence) or μm RNA (containing the two membrane exons).

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Kappa Light Chain Genes Murine Germline Vk Locus The murine Vκ locus spans about 3.2 mb on chromosome 618 and contains 20 Vκ families, some of which are shared by human and mouse (see Table 6.1). Vκ sequences within a single family are largely clustered together. Some Vκ elements lie in the opposite orientation to that of the Jκ and Cκ elements, and these Vκ segments undergo VJ recombination by an inversion rather than deletion (Fig. 6.7). A few Vκ sequences have been localized to chromosome 16 and 19 and are considered orphan genes. Human Germline Vk Locus The human Vκ locus (see Table 6.1) lies on the short arm of chromosome 2 (2p11-2) spanning ∼2 mb of DNA.19 The locus includes a large inverted duplication, so that most Vκ sequences exist in pairs with one copy lying in the cluster proximal to Jκ (and in the same orientation) and a second copy (inverted) in the distal cluster. The average sequence similarity between duplicates is 98.9%, suggesting the duplication occurred less than 5 million years ago. This is consistent with the absence of such duplication in chimpanzees, which diverged from the human lineage approximately 6 million years ago. Interestingly, about 5% of human alleles also lack the distal duplication. Outside the Igκ locus, at least 25 orphan Vκ segments have been identified in clusters on chromosome 1, 2, and 22. The orphan cluster located in the long arm of chromosome 2 was probably separated from the major locus—on the short arm of this chromosome—by a pericentric inversion (which must have occurred rather recently in evolution as it is absent from chimpanzee and gorilla).

A

B

FIG. 6.7. The Same “Micro” Mechanism of Recombination can Join Vk and Jk by Deletion or Inversion, Depending on the Relative Orientation of the Two Precursors in Germline DNA. A: When V coding sequence (shaded rectangle) and J coding sequence (white rectangle) are oriented in the same 5′ → 3′ direction in germline DNA (as indicated by the internal arrowheads), the recombination yields a VJ coding joint plus a DNA circle containing the signal joint (apposed triangles). B: If V is oriented in the opposite direction in germline DNA, an identical recombination reaction at the “micro” level (inside shaded circle) leaves the signal joint linked to the recombined VJ coding joint.

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Jk and Ck Elements In comparison to the H chain genes, the organization of the C region segments in the κ locus is relatively simple (see Table 6.1). A single Cκ gene with a single exon and with no reported alternative splice products is found in both mouse and human. While all five Jκ elements are functional in humans, the third J element in mice has not been observed in functional κ L chains. Apart from the typical Vκ-Jκ rearrangements, an additional recombination event occurs uniquely in the κ locus. The event is mediated by V(D)J recombination utilizing a 23-RSS element—designated Recombining Sequence in the mouse 20 and Kappa Deleting Element in the human21—that is positioned in an intergenic region downstream of Cκ ; the recombination results in the deletion of the Cκ exon. Hence, Cκ fragments are undetectable on Southern blots of DNA from λ-expressing human lymphoid cells, 22 as in most B cells the Cκ genes are apparently deleted from both chromosomes before Igλ gene rearrangement begins.

Lambda Light Chain Genes Murine l Locus In laboratory mouse strains, only about 5% of the B-lymphocytes utilize Igλ L chain, and the diversity of these L chains is meager due to the very small number of V region genes (Fig. 6.8). Complete sequence analysis23 of the murine locus revealed two V-J-C clusters (Vλ2-Vλx-Jλ2Cλ2-Jλ4Cλ4 and Vλ1-Jλ3Cλ3-Jλ1Cλ1) separated by about 110 kb. Each Jλ is linked to its own Cλ region gene, but Jλ4 is nonfunctional. Recombination occurs largely within each cluster, although Vλ2Cλ1 products are occasionally observed. The ancestry of the Vλx element is uncertain, as it is rather dissimilar to the other Vλ segments; indeed, it resembles Vκ as much as Vλ. In contrast to the Igκ locus, the Vλ segments are flanked by 23RSS and the Jλ gene segments by 12-RSS (see Fig 6.3). Human l Locus The human Vλ region was characterized by intensive cloning, sequencing, and mapping of Vλ elements and ultimately by the complete sequence analysis of 1 Mb covering the entire locus 24 (see Table 6.1). Within the Vλ cluster lies the human VpreB gene (discussed below), as well as several genes and pseudogenes unrelated to the Igλ system. λ L chains are much more abundant in man than in mouse (about 40% of human L chains are λ versus about 5% in mouse). Four forms of human λ chains have been classified serologically, with differences residing in a small number of amino acids in the C region. The serologic classification of Kern+ corresponds to a glycine at position 152 versus a serine in Kern−. The Oz + designation corresponds to a lysine at position 190 versus an arginine in the Oz− variant. Similarly, Mcg + λ chains (versus Mcg−) contain asparagine 112 (versus alanine), threonine 114 (versus serine) and lysine 163 (versus threonine). Four functional human Jλ-Cλ segments and three pseudogenes are clustered within an approximately 33 kb region of DNA (see Fig. 6.8) and the four major expressed human λ isotypes correspond to the functional JCλ1, JCλ2, JCλ3, and

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FIG. 6.8. Germline l Genes. The maps in this figure are schematic (i.e., not to scale). A: The murine λ gene system includes four JC complexes and three V genes, as shown. B: The human λ locus includes multiple V genes, of which only three are shown. The human VpreB “surrogate” light chain gene is located within the Vλ cluster. The Cλ locus includes a segment of seven JC complexes plus three additional unlinked sequences. The hatched JC complexes diagrammed above the seven linked λ sequences represent polymorphic variants with additional duplications of the JC unit. The 14.1 sequence—the human λ5 “surrogate” light chain homolog—lies downstream of the JC cluster. Exon 1 of the 14.1 gene is homologous to an exon upstream of Jλ1 (as indicated by the unlabeled white rectangle).

JCλ7, with the latter encoding an isotype provisionally designated Mcp.25 JCλ6 may be functional in some individuals, and the common allele—which has a 4 bp insertion leading to a deletion of the C-terminal third of the Cλ region— can nevertheless undergo Vλ-Jλ recombination, encoding a truncated protein that can associate with H chains. A variety of polymorphic variants of the human λ locus have been detected, apparently the result of gene duplication, as shown in Figure 6.8.26 Lastly, three Cλ-related sequences have been discovered near the major Jλ-Cλ cluster. One of these, designated λ14.1, represents the human homolog of the murine “surrogate” L chain λ5 (see following discussion).

l-Related “Surrogate” Light Chains Ig H chains cannot reach the cell surface without pairing with Ig L chains. However, Ig μ H chains can be detected on the surface of pre–B cells whose Ig κ and Igλ loci are still in their germline configuration and thus do not produce L chains. In these cells, a “surrogate L chain” (SLC) composed of two smaller proteins, VpreB and λ5, facilitates the surface expression of the μ H chain protein. The first component (λ5) was identified as the product of a gene expressed exclusively in pre–B cells that showed high sequence similarity to the J and C regions of the λ locus, 27 As four murine Cλ genes were already known, it was designated λ5. The second component of the SLC was identified as a gene residing about 4.7 kb upstream of λ5 in the mouse genome. Based on its similarities to both Vλ and Vκ (and its expression in pre–B cells), it was called VpreB1. A second, nearly identical sequence in the mouse genome is named VpreB2 and

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appears to be functional, 28 and a less similar VpreB3 has also been described. Neither λ5 nor VpreB genes show evidence of gene rearrangement in B or pre–B cells, and homologs have been found in every mammalian species examined. The two SLC proteins form a L chain–like heterodimer that is able to fulfill some functions of a true L chain, including association with μ H chains to permit surface μ expression prior to the availability of κ or λ L chains. Thus, when a μ H chain gene was transfected into an Ig-negative myeloma line, no surface μ expression was observed unless λ5 and VpreB genes were also transfected.29 Surface μ chains are covalently linked to the λ5 protein, while the VpreB1 protein is noncovalently associated. The expression of μ-SLC on the surface of pre–B cells triggers the onset of Vκ-Jκ rearrangement, as discussed below. In humans, three λ5-like sequences are located downstream of the human Cλ cluster on chromosome 22 (see Fig. 6.8), but only one—designated 14.1—appears to be functional. The human VpreB homolog lies within the Vλ cluster30 in contrast to murine VpreB, which lies close upstream of λ5.

V(D)J RECOMBINATION The mechanism by which germline variable region segments (V L and JL, or V H, D, and JH) are assembled in the DNA to form a complete active V region has been pursued ever since Ig gene recombination was first discovered. In this section we will address 1) the molecular mechanism of the reaction, 2) the topology of the recombination events, 3) the components of the recombinase machinery, and 4) the regulation of that machinery during B-cell development.

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Molecular Mechanism of V(D)J Recombination Recombination Model Overview A model for the detailed mechanism of the V(D)J recombination event must account for the observed features of the recombination products and of their germline precursors. In the germline precursors, the RSSs with their heptamer, nonamer, and appropriate 12 or 23 bp spacers are necessary and sufficient to create efficient recombination targets; model substrates in which RSSs fl ank DNA sequences completely unrelated to Ig genes are competent to undergo recombination. The model shown in Figure 6.9 will serve as a framework for discussion of the recombination mechanism. The recombination is thought to begin with binding of the RAG1-RAG2 complex to the RSSs that flank the two gene segments to be recombined. Simultaneous DNA cleavage occurs precisely between the RSSs and the gene segments. The two ends of the RSSs (frequently named “signal ends”) are joined directly, forming “signal joints.” In contrast, the ends of the gene segments (also referred to as “coding ends”) are processed prior to joining and are ultimately ligated together, giving rise to “coding joints” and completing the recombination event. Recombination Products: Coding Joints and Signal Joints In the recombination products, signal joints are typically direct ligation products of the signal ends: the RSSs are joined directly at the heptamers (“back-to-back”), and nucleotide additions or deletions at these junctions are quite rare. The properties of the coding joints, however, are more complex, as the joining reaction at these DNA ends is “imprecise.” The following features are frequently present: 1. Deletions: variable number of bases are deleted from the ends of the coding regions (in comparison to the “complete” sequence in the germline precursor) 2. Nongermline (“N”) nucleotides: random nucleotides (with a bias toward G and C) are added by a templateindependent DNA polymerase (discussed below). The sequence of these N nucleotides has no relationship to the germline V, D, or J sequences. 3. Palindromic (“P”) nucleotides: the ends of the coding gene segments are sealed by a DNA hairpin structure (see Fig. 6.9, and discussed in the following). “Opening” of these hairpins frequently occurs by nicking at some distance away from the hairpin tip leading to single-stranded overhangs. Filling in of such overhangs by DNA polymerases generates DNA palindromes that mirror the nucleotides at the end of the V, D, or J segment.31 P nucleotides are generally only one or two bps, but they can be longer, especially in mice with the severe combined immunodeficiency defect (SCID) disorder in which the opening of the hairpins occurs in an aberrant manner. Recombination Intermediates: Blunt Signal Ends and Hairpin Coding Ends To study broken DNA ends as intermediates in V(D)J recombination, several laboratories employed ligation-mediated–

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FIG. 6.9. Model for V Assembly Recombinations. All V assembly recombination reactions (in immunoglobulin and T-cell receptor genes) may proceed by a common mechanism, illustrated here by D-J recombination. The recombination signal sequences (RSSs) are included in triangles, which is the conventionally used RSS graphic. Hairpin loops are created on coding ends dependent on the action of the two Recombination Activating Genes: RAG1 and RAG2. After the opening of the hairpin loops, the pictured diversity coding sequence shows the effects of “nibbling” by exonuclease, but the joining coding sequence is spared and shows P nucleotide generation; N region addition is pictured in this example as occurring only on the diversity region end. In reality, exonuclease digestion and N nucleotide addition can occur on either (or both) ends. The steps in the proposed mechanism are discussed in the text.

polymerase chain reaction (LM-PCR) to detect signal ends. This technique involves ligating blunt double-stranded oligonucleotide linkers to blunt genomic DNA breaks, and then amplifying the ligation junctions between a primer in the ligated oligonucleotide and a primer based on known sequence from the ligated genomic DNA; amplification products can

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then be cloned and sequenced. LM-PCR analyses of both TCR and Ig genes undergoing V(D)J recombination showed the signal ends to be blunt double strand breaks (dsbs), usually exactly at the heptamer border.32 Similar LM-PCR experiments failed to detect the coding ends unless they were pretreated with mung bean nuclease, a single-strandspecific endonuclease that recognizes the distortion of DNA at a hairpin structure. Sequences of these LM-PCR products from coding ends suggested that the hairpins are precisely at the end of the coding elements, usually without loss or gain of a single nucleotide.33 By Southern blot analyses, coding ends were found to have two properties suggestive of a hairpin-like structure: 1) resistance to exonuclease treatment, and 2) doubling of the apparent length of restriction fragments under denaturing electrophoresis conditions.34 Hairpin ends represent V(D)J recombination intermediates that, in wild-type cells, are opened at the hairpin tip (or a few nucleotides away from it) by the Artemis nuclease (discussed below). P nucleotides result from opening the loop at an asymmetric position (see Fig. 6.9); this model would explain why P nucleotides are never observed at coding ends that have been “nibbled” after opening of the hairpin. P nucleotide segments in the rare coding joints observed in SCID mice are unusually long and likely result from resolution of hairpins by nicking enzymes that, unlike Artemis, do not focus on the area near the tip of hairpin loops but instead nick in variable positions in the double-stranded hairpin “stem.”34

Topology of V(D)J Recombination Deletion versus Inversion If a V segment and a J segment are both oriented in the same direction, they can recombine by excising the DNA between the coding sequences and ligating the two coding ends. Ligation of the two signal ends produces a DNA circle that generally lacks replication origins and therefore fails to replicate as cells divide after V(D)J recombination. Such excision circles are therefore generally absent in mature B-lymphocytes that have already undergone several rounds of proliferation after completing the Ig gene assembly. By isolating circular DNA from cells actively undergoing Vκ-Jκ rearrangement, it is possible to isolate and characterize the circular molecules bearing signal joints.35 As mentioned previously, some germline Vκ genes are oriented in the opposite direction from the Jκ-Cκ region. In these cases, VJ recombination occurs by an inversion of the DNA between the recombining V and J segments, leaving both the VκJκ coding joint and the signal joint (formed by ligating the RSSs) retained in the chromosome (see Fig. 6.7). This demonstrates that the enzymatic machinery “sees” only the DNA in the immediate vicinity of the recombination site and is insensitive to the topology of the DNA strands far from this site.

Nonstandard Joints In addition to the canonical coding and signal joints, several “nonstandard” recombination joints have been documented, that, though not contributing to physiologic Ig gene assembly, represent tell-tale signs of a recombination event.36

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In the first phase of V(D)J recombination, the DNA is cut at both gene segment–RSS boundaries that participate in the reaction, thereby generating four DNA ends. In principle, there are three possible topologies in which these DNA ends can be rejoined: 1. “Signal and coding joints”: the standard reaction product in which the two coding ends get joined generating the assembled VJ gene and the 12-RSS/23-RSS signal joint. 2. “Open and shut joints”: the RSSs get ligated back to the gene segments from which they were released. These joints are topologically identical to the starting DNAs, but can be distinguished from them if nucleotides have been added or deleted at the junctions. 3. “Hybrid joints”: joints in which the RSSs have traded places so that the 23-RSS that was flanking the Vκ segment is now linked to the Jκ segment, and vice versa. “Hybrid” and “open and shut” joints have been observed in transfected plasmids bearing artificial recombination substrates36 as well as in endogenous Ig loci in vivo.37

Secondary V(D)J Recombination As discussed previously, imprecise joining of gene segments causes about two-thirds of all recombination products to be out-of-frame. Thus, a B-lymphocyte could end up with nonproductively rearranged Igκ genes on both alleles. However, germline Vκ segments lying upstream of an initial Vκ-Jκ recombination junction can recombine with Jκ segments lying downstream of the junction, producing a “secondary” recombination event, as shown in Figure 6.10A.

A

B FIG. 6.10. Secondary Recombinations. A: In the κ light chain system, a primary recombination can be followed by recombination between an upstream V and a downstream J. B: Analogous secondary recombinations can occur in the heavy chain system between upstream D and downstream J segments. After V(D)J recombination eliminates all 12-RSS signal elements from the chromosome, secondary recombination can still occur between VH (23-RSS) and an internal heptamer within the VH coding sequence of the VDJ unit.

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Such secondary recombination also occurs in cells that have assembled a productive Vκ-Jκ joint if the encoded antigen binding domain recognizes an autoantigen. This type of secondary recombination, known as “receptor editing,” is considered in more detail later in this chapter. In the IgH locus, secondary D-J rearrangements sometimes occur, but only until V H-DJH recombination removes all unused upstream DH segments (Fig. 6.10B). VDJ rearrangement eliminates all the 12-RSSs from the IgH locus that could pair with the 23-RSSs flanking the upstream V H elements. Sometimes, these V H segments do, however, recombine with an established VDJ unit, displacing most of the originally assembled V H element,38 a process sometimes called V H replacement. Such events are mediated by cryptic RSSs (mainly a heptamer sequence) that is present near the 3′ end of about 70% of all V H genes (see Fig. 6.10B). Such internal cryptic RSSs are not generally found in L chain genes. As discussed previously for the L chain, secondary recombination represents a rescue mechanism for cells with nonproductive rearrangements on both H chain chromosomes, and for cells whose encoded antibody recognizes an autoantigen.

The V(D)J Machinery Since the discovery of V(D)J recombination as the process that assembles the germline antigen receptor gene segments into functional genes, one major question was the identity of the enzymatic machinery catalyzing this complex set of reactions. Genetic and biochemical work by a large number of laboratories led to identification of a total of 13 different proteins that have been shown to be directly involved in V(D)J recombination: RAG1, RAG2, HMG1, Ku70, Ku80, DNA-PKcs, Artemis, pol μ, pol λ, TdT, XRCC4, Cernunnos/XRCC4-like factor (XLF), and DNA ligase IV. The only lymphoid-specific factors are RAG1, RAG2, and TdT; all others are ubiquitously expressed in all cell types, and this feature allows investigators to study aspects of V(D)J recombination by ectopically expressing the RAG proteins in nonlymphoid cells. A recent biochemical tour de force study showed that coding joint formation could be recapitulated in vitro using artificial recombination substrates and highly purified preparations of all 13 proteins.39 The respective coding joints showed all of the features typically observed in vivo (nucleotide deletion, N nucleotide, and P nucleotide addition), suggesting that most, if not all, of the factors involved in the coding end processing steps of V(D)J recombination have been identified. In contrast, signal joint formation was not observed. This step seems to require the removal of the RAG proteins after the cleavage reaction and is likely to require additional factors as yet unidentified.

Recombination Activating Gene Proteins: Mediators of Early Steps in V(D)J Recombination A major advance in the investigation of V(D)J recombination was the identification of two genes whose products are critical for this process in the B and T cell lineages. In the pioneering experiments, Schatz and Baltimore40 stably transfected fibroblasts with a construct containing a selectable marker whose expression was dependent on V(D)J recombination;

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as expected, no measurable recombination occurred in this nonlymphoid cell. However, when either human or murine genomic DNA was transfected into these fibroblasts, a small fraction of recipient cells stably expressed recombinase activity, activating the selectable marker. This suggested that a single transfected genomic DNA fragment was able confer recombinase activity in a fibroblast. (Presumably the fibroblast contained endogenous copies of the same genes, but their expression was repressed by mechanisms that could not repress the transfected genes.) This active fragment was cloned and turned out to contain two closely linked genes, designated RAG1 and RAG2, respectively. Both RAG1 and RAG2 are essential for recombination; therefore, these genes would not have been discovered by this transfection technique if they had not been closely linked in the genome. The genes are notable for having no introns splitting up their open reading frame in most species, and for their opposite transcriptional orientation in all species examined. A crucial role for the RAG genes in V(D)J recombination was supported by the conservation of these genes in all jawed vertebrate species analyzed thus far, from shark through man. RAG1 and RAG2 are expressed together in developing B and T cells, specifically at the stages at which V(D)J recombinase activity is required for the assembly of Ig and TCR genes. Moreover, mouse strains in which either gene has been eliminated by homologous recombination (gene “knockouts”) have no mature B or T cells, as the result of their inability to initiate V(D)J recombination.41,42 Similarly, a subset of human patients with SCID syndrome characterized by the complete absence of T- or B-lymphocytes have been found to have null mutations in RAG genes.43 Patients with hypomorphic alleles often have a complex set of features (oligoclonal T cells, hepatosplenomegaly, eosinophilia, decreased serum Ig but elevated IgE) known as the Omenn syndrome, which can also be caused by defects in other genes involved in V(D)J recombination. Interestingly, the same RAG mutation in different patients can cause either Omenn syndrome or SCID, depending on unknown factors.44 RAG1 shows intrinsic binding affinity for the RSS nonamer sequence via its nonamer binding domain even in the absence of RAG2. Exhaustive mutational analysis has revealed that RAG1 contains the catalytic center of the RAG complex, composed of three amino acids critical for all enzymatic activity: D600, D708, and E962.45,46 RAG2, on the other hand, serves as a regulatory cofactor; it has no intrinsic binding affinity for RSSs, but once bound to RAG1 improves the strength and specificity of RAG1 RSS contacts.47,48 It is also enhances RAG activity on chromosomal substrates and it restricts V(D)J recombination to the G0/G1 stage of the cell cycle (both features are discussed below). Attempts to determine the molecular role of the RAG proteins in cell-free recombination assays were initially hampered by poor solubility of the proteins, but functional analyses of truncated RAG genes (using RAG expression vectors cotransfected into fibroblasts along with recombination substrate plasmids) revealed that surprisingly large segments of both proteins could be deleted without eliminating recombinase activity, and some of the remaining core regions were

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soluble and could be handled relatively easily in experiments. This work allowed the demonstration that in a cell-free in vitro system, core regions of the two RAG proteins together are capable of carrying out cleavage of substrate DNAs as well as hairpin formation on the coding end.49 The RAG-mediated cleavage occurs in two steps: first a nick is introduced on the top strand between a gene segment and the adjacent heptamer (see Fig. 6.9), then the 3′-hydroxyl group participates as the nucleophile in a direct transesterification reaction to attack the phosphodiester bond adjacent to the heptamer on the bottom strand (see Fig. 6.9), yielding a DNA hairpin structure on the coding end and a new 3′-hydroxyl group on the 3′ end of the bottom heptamer strand.50 After DNA cleavage, the RAG proteins remain in a complex with the DNA ends and facilitate aspects of the joining phase. Mutant forms of RAG1 or RAG2 have been reported that are competent for cleavage but show impairment in coding or signal joint formation.51 While nicking can occur asynchronously at the 12-RSS and 23-RSS, hairpin formation is “coupled” and occurs synchronously at both RSSs. In vitro, coupled cleavage requires only the RAG proteins, HMG1/2 (discussed below) and Mg 2+ as the divalent metal ion in the reaction buffer. In vivo, DNA dsb formation at an individual RSS is dangerous as it could give rise to translocations, and it is thought that Mg 2+ promotes an optimal molecular “architecture” for controlled V(D)J recombination. In vivo experiments indeed suggest that RAG proteins may bind to and introduce a nick at a single 12-RSS, but do not complete DNA cleavage until a matching 23-RSS is captured into the RAG-RSS complex.52 In addition to the “classical” activities of RAG proteins on DNA segments containing RSSs, these proteins can also catalyze DNA strand cleavage on “nonstandard” substrates. 1. Transposition. In vitro, purified recombinant core RAG proteins can catalyze the excision and insertion of a DNA fragment with signal ends into foreign DNA, acting as a transposase.53,54 This property provides additional support for the early speculation that the V(D)J recombination system may have originated by insertion of transposon-like DNA fragment encoding RAG genes (and bearing RSSs at its ends) into a primordial antigen receptor gene, thereby generating a pair of separated V and J gene segments. This model of the origin of V(D)J recombination is consistent with the many mechanistic similarities at the molecular level between Ig gene rearrangements and transposition,55 and the recent identification of the Transib transposase family that shows striking sequence similarity to RAG1 and is widespread in insect, echinoderm, helminth, coelenterate, and fungal genomes.56 The recent finding of an apparent homolog of the entire RAG1 and RAG2 gene locus in a sea urchin genome suggests that the two RAG genes may have entered the genome of a common ancestor of all deuterostomes far earlier than the Ig-/TCR-based adaptive immune system developed.57 It remains unclear whether the primordial RAG transposon encoded solely RAG1 (which would then have integrated

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next to the primordial RAG2 gene) or both RAG1 and RAG2. The transposase activity of RAGs, however, seems to be almost completely suppressed in vivo, and the C-terminus of RAG2 may have evolved to control this potentially deleterious activity.51,58–60 2. VH replacement. As mentioned previously, recombination events can occur between a VH 23-RSS and cryptic RSS within rearranged VH coding sequences. An in vitro model suggests that in VH replacement, the RAG proteins nick both DNA strands without forming a hairpin coding end.61 Whether this is indeed a completely different activity is unclear. 3. Translocations at non-RSS sequences. The RAG complex also generates two nicks to cleave within the major breakpoint region of the Bcl2 gene. This 150-bp segment is the target of a common RAG-catalyzed translocation between the IgH locus and the Bcl2 gene occurring in most follicular lymphomas. In this segment, there are no RSSs, and the RAG proteins recognize an unusual sequence-dependent DNA conformation different from the normal B-form double helix.62 Although the “core” RAG proteins have been useful for elucidating the molecular mechanism of the cleavage step of V(D)J recombination in biochemical studies, it is clear that the “noncore” portions of each protein confer important functions, as expected from their sequence conservation across species. Broadly speaking, the “noncore” regions ensure regulated and efficient recombination on the physiological substrates (i.e., imperfect RSSs deviating from the perfect consensus heptamer and nonamer) in the context of chromatin. The functions of the “noncore” regions have largely been inferred by comparing V(D)J recombination products from cells expressing core RAG proteins versus full-length versions, and more recently by in vitro studies using full-length RAG proteins that are now available for such analyses. The C-terminal region of RAG2 has multiple functions and is important for achieving normal numbers of B- and T-lymphocytes in vivo, 63 for the formation of precise signal joints during IgH recombination, 64 and for protecting against RAG-mediated DNA transposition.51,65 These functions are thought to be conferred at least in part, by a plant homeo domain (PHD) zinc finger fold that is formed by amino acids 414 to 487 in murine RAG2. This PHD domain binds specifically to the tails of histone H3 that are trimethylated at lysine 4 (H3K4Me3), 66–68 a histone modification that is associated with “open” chromatin and that is uniquely present on “accessible” RSSs in Ig loci (discussed below). In vitro studies suggest that the binding of the RAG2 PHD domain to histone tails causes a conformational change that increases the catalytic activity of the RAG complex. 69 Furthermore, the RAG2 C terminus regulates RAG2 protein levels—and hence V(D)J recombinase activity—across the cell cycle to prevent dsbs during DNA synthesis or mitosis, when such breaks could lead to chromosomal deletions.32 RAG1 protein and messenger RNA (mRNA) transcript levels of both RAG genes vary little across the cell cycle, but phosphorylation of RAG2 at Thr490 by the cyclin-dependent

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kinase cdk2 mediates its destruction via ubiquitination and proteasomal degradation during S phase.70 Mice expressing RAG2 with a T490A mutation (which cannot be phosphorylated) showed RAG2 protein and dsbs throughout the cell cycle, demonstrating the importance of the RAG2 degradation signal in cell-cycle control of V(D)J recombination.71,72 The N-terminal noncore region of RAG1 is required in vivo for optimal RAG1 activity and for the formation of precise signal joints in D-J recombination.64 This region of RAG1 contains a RING finger domain that seems to be required for ubiquitination of several proteins, including histone H3.73 Apart from the obvious importance of the RAG proteins in understanding the initial steps of V(D)J recombination, knowledge of these proteins and their genes has allowed two major technical advances that have opened the way to many additional experiments. First, various nonlymphoid cell lines with known defects in various DNA repair genes have been transfected with the RAG genes to identify genes involved V(D)J recombination (these factors are described below). Second, availability of the RAG1 and RAG2 knockout mice has been instrumental in a large number of immunology studies. These mice completely lack functional B cells or T cells, and are not “leaky” like SCID mice, which develop some functional B and T cells, especially as the animals age. Thus the RAG-deficient mice can be used to study the importance of the “innate” immune system (i.e., responses that occur in the absence of antigen-specific lymphocytes) in particular immune responses. They can also be used as recipients for various lymphocyte populations to explore the roles of different cell types. They can also be used as recipients for various lymphocyte populations to explore the roles of different cell types. They can be transfected with transgenes encoding specific Ig genes to study the roles of specific antibodies in B cell development and in immune responses. Finally, they can be used in “RAG complementation” experiments designed to assess the phenotype—in lymphocytes—of various other gene knockouts.74 In RAG complementation, embryonic stem cells in which the gene of interest has been knocked out by homologous recombination are injected into homozygous RAG2 knockout (RAG2−/−) blastocysts. This procedure yields chimeric mice in which all B and T cells derive from the embryonic stem cells deleted for the gene of interest, as these are the only source of intact RAG genes to support lymphocyte development. Such animals can be made more easily than a knockout mouse line, and can be used to study the effect of gene deletion in lymphocytes independent of effects the deletion may have in other cells. In particular, for cases where the gene knockout causes embryonic lethality due to effects on nonlymphoid cells, RAG complementation allows the selective knockout in lymphocytes to be studied in the background normal gene expression in nonlymphoid cells.

High Mobility Group Proteins The search for RAG cofactors that stimulate cleavage activity in biochemical assays led to the identification of HMG1.75 HMG1 (and the closely related HMG2) are abundant and

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ubiquitous proteins that bind DNA in a non–sequence-specific manner and to cause a local bend in DNA. The two RAG proteins can form a stable signal complex with a 12RSS, but efficient complex formation with a 23-RSS requires the addition of either HMG1 or HMG2.76 HMG1/2 apparently stabilizes the bending of the 23-RSS that is induced by the RAG proteins themselves.77

Nonhomologous End Joining Components The RAG proteins are the essential lymphocyte-specific factors in the DNA cleavage phase of V(D)J recombination, but DNA repair factors that are part of a DNA repair pathway known as nonhomologous end joining (NHEJ) are essential for the joining phase. NHEJ is the major pathway for repair of dsbs (such as those induced by ionizing radiation or reactive oxygen species) during the G0-G1 phases of the cell cycle. (In the S and G2 phases, the additional chromatid genome copy enables breaks to be repaired by homologous recombination.) The six classical core components of NHEJ are Ku70, Ku80, DNA-PKcs, XRCC4, DNA Ligase IV, Artemis, and Cernnunos/XLF, but additional proteins play a role in some models of NHEJ. The DNA-PK Complex . The first gene for an NHEJ component to be recognized as participating in V(D)J recombination was the SCID gene. This gene was originally identified as being mutated in the scid mouse strain that is immunodeficient due to a marked impairment in V(D)J recombination of both Ig and TCR genes. Lymphocytes from scid mice are able to perform the RAG-mediated cleavage reaction, and can also form signal joints, but are markedly defective in coding joint formation. Subsequently, it was found that the scid mutation also impairs NHEJ, causing radiosensitivity. The gene mutated in the scid mouse strain encodes DNAPKcs, a large protein (460 kD) with a kinase domain near its C terminus that is related to phosophoinositide-3-kinase (PI3K). This kinase is DNA-dependent and represents the catalytic subunit (hence “cs”) of a heterotrimer known as the DNA-PK complex. The other components are Ku70 and Ku80 (also referred to as Ku86), which were originally identified as the autoantigens recognized by a patient antiserum (Ku was the coded name of the patient, and the numbers refer to the approximate size of the proteins, 70 kD and 80 to 86 kD, respectively). Together, these two very abundant proteins form a heterodimer that binds to the ends of double-stranded DNA independent of the nucleotide sequence of the DNA. The DNA-Ku complex can then recruit DNAPKcs and activate autophosphorylation of this protein.78 In vitro activation of DNA-PKcs was found to be efficient when DNA ends either were at high concentration or, if at low concentration, were on DNA fragments long enough to circularize readily. In contrast, when the DNA-PKcs was located on the ends of DNA fragments too short to circularize (and too dilute for efficient intermolecular interactions with other DNA ends), the DNA-PKcs activation was much reduced. These observations suggest that kinase activation can occur only after two DNA ends are brought together by DNA-PKcs in “synapsis.”79,80 Further phosphorylation of

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DNA-PKcs inactivates the protein and may prepare it for removal once DNA ends have been sealed. Ku genes are highly conserved through evolution, and homologs are even found encoded in the genome of some bacteria, consistent with a function in general NHEJ not restricted to V(D)J recombination. While mice with a targeted deletion of DNA-PKcs resemble the original scid mutation (i.e., defective coding but functional signal joint formation81,82), Ku70 and Ku80 mutant cell lines are defective in both signal and coding joint formation, and Ku70and Ku80-deficient mice exhibit a complete block in B- and T-cell development due to their inability to undergo V(D)J recombination. 83–85 DNA Ligase IV and XRCC4 . An important role of activated Ku-DNA-PKcs complex is to recruit the additional components of NHEJ. One such component is DNA ligase IV, which is recruited to the Ku complex and activated by the protein XRCC4. 86,87 The evidence suggests that DNA ligase IV is the essential ligase that joins DNA ends in V(D)J recombination and NHEJ. Human patients with ligase IV deficiency (characterized by hypomorphic alleles) have a severe phenotype including chromosomal instability, developmental and growth retardation, radiosensitivity, and immunodeficiency with a T–B–NK+ phenotype. 88 The rare DH-JH junctions detected show extensive nucleotide deletion consistent with delayed ligation and prolonged exonuclease digestion.89 In mice, disruption of either the XRCC4 or the DNA ligase IV gene causes embryonic lethality associated with neuronal apoptosis. Crossing these mice with p53 mutants does not improve V(D)J recombination, but rescues the mice from embryonic lethality, suggesting that neuronal cells may be unusually susceptible to p53-triggered apoptosis induced by normal low-level DNA damage during brain development; a similar mechanism may explain the severe human phenotype.90 DNA ligase IV is the only NHEJ component absolutely required to join compatible sticky DNA ends in vitro, though XRCC4 can stimulate this activity significantly. 87 Cernunnos/XRCC4-like Factor. The next NHEJ component was independently discovered by two laboratories. One group used yeast two-hybrid screening to search for proteins interacting with XRCC4.91 The other group searched for the gene causing a syndrome of T+ B lymphocytopenia, increased radiosensitivity, and microcephaly in a Turkish family; these investigators used functional cDNA rescue of a patient’s cell line from a radiomimetic drug to identify the gene.92 The protein identified by both groups is a 299 amino acid nuclear protein, which was named Cernunnos or XLF. The protein has a predicted secondary structure similar to that of XRCC4, to which it binds in cells93 as expected from its isolation via two-hybrid screen. When Cernunnos/XLF-deficient fibroblasts were transfected with RAG genes and a recombination substrate, imprecise signal joining was observed, similar to the defect in patients with hypomorphic DNA ligase IV mutations. These experiments all suggest a role for Cernunnos/ XLF linked to the function of XRCC4 and ligase IV. Artemis. The coding ends generated by RAG cleavage cannot be directly ligated because of their hairpin structure,

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and therefore V(D)J recombination requires a single-strand endonuclease activity to cleave the hairpins. This activity is conferred by the protein named Artemis, which was discovered through positional cloning of the genetic defect in a group of human SCID patients with defects in V(D)J recombination and increased radiation sensitivity.94 Patients with homozygous null mutations of Artemis survive (no embryonic lethality) and show sensitivity to γ irradiation as well as defects in coding joints, while signal joint formation is normal. Hypomorphic Artemis mutations can cause features of the Omenn syndrome similar to those observed with hypomorphic RAG gene mutations.95 Purified recombinant Artemis protein has an intrinsic exonuclease activity in vitro; however, when complexed with DNA-PKcs in the presence of DNA ends, it gains a single-strand endonuclease activity and, in an ATP-dependent step, becomes phosphorylated at multiple sites in the C-terminal region of the protein.96,97 The Artemis endonuclease can cleave synthetic and RAG-generated hairpin ends as well as other singlestranded DNA near a transition to double-strand DNA.98 DNA Polymerase X Family Members. If a hairpin opening leaves blunt ends or complementary sticky ends (like the ends generated by many restriction enzymes), in vitro joining experiments suggest that these ends can be joined by ligase IV without any additional processing.99 However, as Artemis probably opens most hairpins noncomplementary DNA overhangs, further processing of DNA ends generally occurs before ligation completes the recombination. This processing may include further nuclease digestion (by Artemis or exonucleases) and apparently also involves variable DNA extension by three DNA polymerases—polymerase λ , polymerase μ, and terminal deoxynucleotidyl transferase (TdT)—all of which are members of the polymerase X family. Interestingly, all three proteins contain a Brca1-C-terminus domain, which is thought to confer binding to Ku.100 Terminal Deoxynucleotidyl Transferase and N Regions. TdT, the primary source of untemplated “N region” additions in VDJ junctions, is an enzyme uniquely expressed in the thymus and bone marrow; in the B lineage, it is expressed almost exclusively in pro-B cells. It catalyzes the nontemplated addition of nucleotides to the 3′ end of DNA strands. Though no template determines the nucleotides added, the enzyme adds dG residues preferentially, consistent with N region sequences observed in VDJ joints. Both TdT expression and N nucleotide addition are characteristically absent from fetal lymphocytes.101 N region addition is common in H chain genes (recombined in pro-B cells) but rare in murine L chain genes (recombined in pre-B cells), though perhaps somewhat less rare in human.102 This is consistent with the observation that in mice the expression of a μ H chain may downregulate TdT expression,103 contributing to the reduced level during the stage of L chain recombination. Lymphocytes with engineered defects in their TdT genes produced rearranged Ig V regions with almost no N additions. Conversely, when TdT expression was engineered in cells undergoing κ or λ L chain rearrangement, the level of

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N nucleotide addition to these coding joints was dramatically increased. Furthermore, mice engineered to undergo premature Vκ-Jκ joining in pro-B cells show an increased frequency of N region nucleotides in their recombined Vκ genes.104 These results suggest that the low frequency of N region sequences in normal κ or λ recombinations is caused by the reduced levels of TdT at this stage of B-cell development (see following discussion). The absence of N region addition in TdT mutant mice, as well as in normal fetal lymphocytes, is associated with an increase in the frequency of recombination junctions with microhomologies. These are short stretches of nucleotides that are present close to the end of both germline gene segments involved in the recombination event. These junctions suggest a joining intermediate in which the complementary single-stranded regions from the two coding ends hybridize to each other, much as “sticky ends” generated by restriction endonucleases can facilitate ligation of DNA fragments. This alternative joining pathway may restrict the diversity of neonatal antibodies; the resulting antibodies are possibly enriched in specificities for commonly encountered pathogens, or have broadened specificity, as has been reported for TCRs lacking N regions.105 Decreased N region nucleotides and a high incidence of homology-mediated recombination have also been found in the rare coding joints formed in Ku80−/− mice, consistent with a role for Ku in recruiting TdT or supporting its action.106 Polymerase m and Polymerase l. Polymerase μ and polymerase λ are ubiquitously expressed polymerases. Both readily fi ll in single-strand gaps in DNA and apparently participate in V(D)J recombination by fi lling in single-strand 3′ overhangs generated by asymmetric hairpin opening. Without this fi lling in, such overhangs might be resected by nucleases. Indeed, when in vitro NHEJ reconstitution experiments are performed using purified proteins and DNA fragments with overhanging ends, the omission of polymerase μ or polymerase λ increases the deletional trimming at junctions.100 Similar excessive deletions at VDJ junctions are observed in mice lacking polymerase μ or polymerase λ. Remarkably, however, polymerase μ knockout mice show abnormalities only in their L chains,107 whereas the deletions in polymerase λ knockouts are restricted to their H chains.108 This selectivity may be explained by corresponding changes in the relative mRNA levels for these two polymerases at different stages of B-cell development.

Other Participants in V(D)J Recombination DNA Damage Response Factors. In eukaryotic cells, DNA breaks initiate signals that halt cell division, induce DNA repair, and in some cases trigger apoptosis. Several proteins apart from NHEJ components can be detected at DNA breaks induced by V(D)J recombination or irradiation, including γ-H2AX, a phosphorylated form of the histone H2AX; ATM, the product of the gene mutated in the disease ataxia telangiectasia; Nbs1 (or nibrin), the product of the gene mutated in Nijmegen breakage syndrome; and 53BP1, p53 binding protein 1. The importance of these proteins in V(D)J recombination is not clear because defects in all three

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are compatible with near normal V(D)J recombination. Possibly, they participate in backup mechanisms to prevent aberrant V(D)J recombination and thus translocations. Pax5/B-Cell–Specific Activator Protein. Pax5 (also known as B-cell–specific activator protein; BSAP) is a transcription factor required for normal B-cell development. Pax5deficient mice are able to complete DJH recombination, but V H to DJH recombination is impaired except for certain V H genes located proximal to the D regions. Interestingly, 94% of human and mouse V H coding genes were found to have potential Pax5 binding sites. Surprisingly, Pax5 was found to coimmunoprecipitate with RAG proteins, to potentiate in vitro cleavage of a V H gene RSS, and to enhance V H to DJH recombination in RAG-transfected fibroblasts; the latter enhancement required intact Pax5 binding sites in the V H sequence.109

REGULATION OF V(D)J RECOMBINATION IN B-CELL DEVELOPMENT The expression of only one antigen binding specificity by each B-lymphocyte is a crucial requirement of the clonal selection model of the humoral immune response. Thus, the recombination events that occur between Ig gene segments are carefully regulated so that most B cells express only one L chain isotype, either Ig κ or Ig λ (isotype exclusion), and use only one of the two alleles of H and L chain genes (allelic exclusion). These constraints ensure that each B cell expresses a single H2L2 combination. Current evidence suggests that V(D)J recombination is controlled largely at two levels: regulation of the RAG protein activity and regulation of accessibility of the germline V, D, and J elements to the recombinase machinery. Both of these are controlled by the stage of B-cell development; conversely, the expression of Ig provides a signal critical for regulating maturation of B cells. A brief scheme of B-cell development is presented in the following as background. B- and T-lymphocytes differentiate from pluripotent hematopoietic stem cells in the fetal liver and bone marrow (Fig. 6.11). The primordial lymphoid progenitor has the potential to differentiate into B- or T-lymphocytes or natural killer cells. Among the earliest markers that indicate B-lineage specificity are the non-Ig components of the preBCR: Igα , Igβ, and λ5. CD19, which functions as a coreceptor in signal transduction, first appears in large proliferating “pro-B” cells, which also express several other distinguishing surface markers including c-kit, B220, TdT, and CD43. RAG gene expression in pro-B cells initiates D to J rearrangements on both alleles. Subsequently, recombination with germline V H elements occurs; if the recombination is “productive” (i.e., yielding an “in-frame” VDJ junction), a μ H chain protein can be produced. This protein appears on the B-cell surface along with SLC in a pre-BCR (also named μ-SLC) complex that also includes Igα and Igβ. As the resulting large pre-B cells proliferate, RAG gene expression declines. After several rounds of division, the cells become smaller, stop dividing, turn up RAG gene expression once more, undergo L chain recombination, and express surface

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FIG. 6.11. Immunoglobulin (Ig) Gene Recombination in B-Cell Development. A simplified scheme of B-cell development is presented as a background for discussion of Ig gene recombination. The stages occurring in the bone marrow versus in the periphery (e.g., lymph nodes, spleen) are shown, along with the status of IgH and IgL genes at each stage. A graphic depicting the Ig-related proteins displayed on the surface at each stage is presented; at the bottom, the stage-dependent expression of recombination activating genes and terminal deoxynucleotidyl transferase—both important in V(D)J recombination—is schematically depicted.

IgM. These “immature B cells” again turn down RAG expression. In these IgM + IgD− immature B cells, contact with autoantigens may upregulate RAG expression again to facilitate receptor editing (discussed in more detail below). When immature B cells eventually also express surface IgD, they become “mature B cells” and migrate into the periphery, ready to be triggered by antigen exposure.

Allelic Exclusion and Regulated V(D)J Recombination The previous description of B-cell development serves as a background to understand an explanation of allelic exclusion that was first proposed by Alt and colleagues110 and has been supported by subsequent experiments. According to this model the functional rearrangement of an L (or H) chain gene in a particular B cell would inhibit further L (or H) chain gene rearrangement in the same cell. If the inhibition occurred promptly after the first functional rearrangement, then two functional Igs could never be produced

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in the same cell. An initial nonproductive rearrangement would have no inhibitory effect, so recombination could continue until a functional product resulted or until the cell used up all its germline precursors. In pro-B cells, the first Ig gene rearrangements join D to JH segments (commonly on both chromosomes), and this is followed by V H to DJH recombination. If the first V H to DJH recombination in a pro-B cell produces a functional VDJ gene, a functional μ H chain will be expressed on the cell surface paired with the SLCs. The expression of this preBCR complex has been shown to have two consequences. First, it blocks further H chain recombination by decreasing RAG gene expression111 and by reducing target accessibility, as reflected in decreased V H gene transcription.112 The latter is important for rendering the IgH locus inaccessible during subsequent rearrangement of the Ig κ and Igλ loci. If the initial V H to DJH rearrangement is nonfunctional (e.g., out of frame), subsequent V H to DJH recombination occurs on the other allele. If the VDJ recombination product on the

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second chromosome is also nonproductive, then the cell has reached a dead end and is eliminated by apoptosis.113 The second consequence of pre-BCR expression is the initiation of Ig L chain recombination. This effect was originally deduced from the rarity of κ-expressing cells without H chain gene rearrangement, suggesting that H chain expression is required for κ recombination. As additional evidence, a functional μ gene introduced into early B-lineage cells can cause RAG gene expression and turn on transcription of unrearranged Vκ genes. These are designated “sterile” transcripts because they cannot encode a κ protein, but they are required for Vκ-Jκ recombination. When this recombination ensues, the possibilities for functional and nonproductive Vκ-Jκ rearrangements resemble those discussed previously for the H chain. Expression of a functional κ chain that can associate with μ to form a surface-expressed IgM molecule results in the downregulation of RAG gene expression and suppression of further κ rearrangements. By this mechanism, functional rearranged VκJ-Cκ transgenes can suppress rearrangement of endogenous κ genes.114 Most B cells show isotypic exclusion (i.e., they express either κ or λ but not both). Furthermore, κ rearrangement seems to occur before λ. Thus in normal and malignant human B-lymphoid cells, κ-expressing cells generally have their λ genes in germline configuration, while in λ-expressing cells, κ genes are either rearranged (rarely) or deleted (most commonly) by recombination signal recombination events discussed previously in this chapter.22 The mechanisms that dictate the order of L chain recombination remain unknown. Plausible models include either the selective suppression of λ recombination until all options on the Ig κ locus are exhausted or differences in the timing of the developmental programs controlling κ and λ accessibility.

Regulation of RAG Expression A complete explanation of RAG gene expression would explain its lymphoid specificity, the two waves of RAG expression (during IgH and IgL rearrangements) and the autoantigen-induced upregulation associated with receptor editing. Although our current knowledge is still incomplete, several cis-regulatory elements that regulate RAG expression have been characterized. Surprisingly, the elements and mechanism for regulating expression during B- and T-cell development are distinct. RAG1 and RAG2 are transcribed toward each other in opposite directions, driven by promoters near the respective transcription start sites. Three B-cell–specific enhancers—designated Erag, D3, and Ep—have been reported, lying about 23 kb, 8 kb, and 1.6 kb, respectively, upstream of RAG2.115–117 The B-cell–specific function of these regulatory regions is likely explained by the intersecting specificities of transcription factors that interact with them, including Pax5, E2A, FoxP1, FoxO1, NFATc1, and Ikaros. NFκ B, which binds at several locations in the RAG enhancers, and FoxO1 (binding to Erag) were found to be important mediators of the upregulation of RAG expression in cells undergoing receptor editing.118,119 Regulation of RAG2 protein across the cell cycle has been discussed previously in this chapter.

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Parameters Affecting Recombinational Accessibility and Transcription V(D)J recombination is triggered by RAG expression in the development of both B and T cells, yet Ig gene recombination is largely confined to B cells (exception: early T cells typically show D-JH recombination); TCR gene recombination is exclusive to T cells. A widely accepted explanation for this locus specificity is provided by the “accessibility” model.120 This model proposes that only those gene segments programmed for recombination at a given stage of B- and T-cell development are “accessible” to the RAG recombinase. One clue suggesting this model was that susceptibility to recombination and transcription of germline gene elements seem to be tightly correlated.120 For example, many germline V H genes are transcribed at the pre-B cell stage, just at the time when these genes are targets for recombination; these transcripts— designated “sterile” like the Vκ transcripts mentioned previously—are not seen in more mature B cells in which H chain recombination has been terminated. In support of the accessibility model, recombinant RAG proteins incubated with nuclei purified from pro-B cells (which generate sterile transcripts in the IgH locus) were found to cleave DNA at Ig JH RSSs, but not at TCRδ RSSs; conversely, in pro-T nuclei the TCRδ RSS was cleaved, but not an Ig gene RSS.121 One molecular correlate of accessibility is the epigenetic state of DNA in the nuclear chromatin. The minimal repeat unit of chromatin is the nucleosome, which consists of eight core histones (two copies each of H2A, H2B, H3, and H4) with 146 bp DNA wrapped around it. In vitro, RAG proteins are unable to bind to and cut DNA wrapped around nucleosomes,122,123 and hence nucleosomes have to be shifted or removed (a process called chromatin remodeling) to allow access. An alternative but not mutually exclusive approach to gain access is posttranslational modification of the histone tails, which regulates the tightness of DNA-nucleosome contacts. The following section provides an overview of how accessibility of the Ig gene loci for RAG activity is regulated by several distinct but interconnected epigenetic mechanisms. We discuss a few important examples for each mechanism and refer to comprehensive review articles for an in-depth discussion.

Subnuclear Localization In general, inactive genes tend to be located in the periphery of nuclei, while active genes are recruited to a more central nuclear location.124 It is unclear whether the location per se dictates the chromatin state of a locus or whether the movement is a consequence of a locus being “opened.” Fluorescence in situ hybridization (FISH) with large (∼100 kb) probes specific for Ig loci is routinely used to reveal the position of Ig gene loci and control genes in the nucleus. The IgH and Igκ loci are located at the nuclear periphery in hematopoietic progenitors and pro-T cells, but move to central areas of the nucleus in pro-B cells.125 As only the IgH locus gets rearranged at this stage, the correlation of position with accessibility is not perfect. Transcription Transcription typically occurs in “open” chromatin, and an emerging theme suggests that while some locus “opening” has

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to precede transcription, transcription per se also positively reinforces this chromatin state. As mentioned previously, transcription of individual elements within Ig gene loci correlates well with their availability for V(D)J recombination at that stage. In early pro-B cells when D to JH recombination takes places a “μo” transcript starts upstream of DQ52 and proceeds all the way through the JH elements126,127 ; and only after DJH rearrangement do VH sterile transcripts appear.120 It is currently unclear whether transcription per se is directly linked to recombination or whether the correlation is largely mediated by similar requirements for gaining access to chromatinized DNA.

Histone Modifications Posttranslational modifications of histone tails are important epigenetic marks of the chromatin state (also referred to as the “histone code”). Distinct marks correlate well with actively transcribed and inactive (or repressed) gene loci. As these patterns hold true for Ig loci as well, active marks (e.g., histone acetylation) and inactive marks (e.g., the methylation of lysine 9 on histone H3, H3K9Me2) correlate well with both transcription and recombination accessibility.128 Importantly, one particular mark for open chromatin, H3K4Me3, is directly linked to the RAG recombinase. As discussed previously, histone tails with this modification are recognized by the PHD domain of RAG2; strikingly, the distribution of RAG2 in Ig loci matches exactly the pattern of H3K4Me3 (which mark accessible gene segments).129 Beyond facilitating the recruitment of the recombinase, this histone modification also increases cleavage activity in vitro.69 Methylation Most cytosine residues within CpG dinucleotides are methylated in mammalian DNA, but genes that are actively expressed in a particular cell are generally relatively hypomethylated in that cell type, implying that DNA methylation inhibits transcription. DNA methylation also seems to inhibit V(D)J recombination. The developmental maturation from pro-B to pre-B cells is associated with progression from a κ locus that is largely methylated, nontranscribed, and nonrearranging to one that is hypomethylated, transcribed, and rearranging.130,131 Furthermore, methylation of artificial recombination substrates blocked V(D)J recombination when transfected into a recombination competent B-cell line132 ; V(D)J recombination of a transgenic construct occurred only when it was unmethylated.133 Methylation and histone acetylation are interrelated; for example, the methylCpG-binding protein MeCP2 recruits histone deacetylases, which reduce acetylation of histones. Gene Localization in the Nucleus While accessibility allows the RAG proteins to selectively bind to appropriate sets of RSSs at each developmental stage, V(D)J recombination also requires that a pair of compatible gene segments (and their RSSs) are in close physical proximity. This becomes a particularly daunting requirement for gene segments > 1 mb apart in linear DNA sequence. Such distant segments are apparently brought close together in the nucleus, a process of “locus compaction” that loops out large

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regions of intervening DNA. Evidence for this model derives from FISH experiments showing greater compaction of the IgH locus in pro-B cells poised to undergo V(D)J recombination than in their earlier hematopoietic progenitors.125 B cells deficient in Pax5 are impaired in recombination of the most distal V H regions and show less movement of these regions towards the JH-C locus than normal cells.134 Data from a recently developed high-resolution FISH method provide a detailed model of the three-dimensional structure of the IgH locus, revealing rosette-like structures with central hubs from which several loops extend.135

cis Mediators of Accessibility and Looping All previously described properties are dependent on cisregulatory elements within the Ig loci, including classic promoters and enhancers. Individual promoters are present upstream of all V elements in all Ig loci, while the downstream D and J elements share a smaller number of promoters. Enhancers are present in each Ig locus, and they are thought to confer the transcriptional activation of each locus at the appropriate stage of B-cell development. Murine κ and IgH loci have intronic enhancers in the intron between J and C (iEκ and Eμ, respectively), and all three loci have enhancers downstream of C coding regions. (For example, downstream of the murine IgH locus is a complex of four enhancers, collectively known as the 3′regulatory region.) Promoters and enhancers were originally defined based on their role in regulating transcription, but these and other recently reported elements appear to play additional roles in Ig gene recombination. Transcriptional activation and the correlated locus “opening” is mediated by the recruitment of transcription factors that in turn recruit histone modifying and chromatin-remodeling enzymes. In addition, promoters and enhancers regulate transcription through the formation of DNA loops, some of which are critical determinants of the three-dimensional structure of Ig loci, thereby affecting V(D)J recombination as well as CSR (as discussed below). An enhancer may activate transcription of several genes within a given gene locus, but its effects may be deleterious if it can activate other nearby genes requiring different patterns of expression. To prevent enhancer function beyond appropriate domains, boundary elements known as insulators establish borders between gene loci that are differentially regulated. The protein CCCTC binding factor (CTCF) is commonly found at insulators and also functions by creating DNA loops. One such insulator apparently lies downstream of a complex of enhancers at the 3′ end of the IgH locus, the 3′-regulatory region, where it may protect genes further downstream from being regulated by the Ig enhancer elements.136 A recently discovered regulatory element with insulator properties is the intergenic control region-1 (IGCR1), which participates in CTCF–dependent looping between the Eμ and 3′ regulatory region enhancers.137–139 Based on results of deletion of this region, the IGCR1 element suppresses recombination of VH to D segments not already joined to JH, prevents VH to DJH recombination in T cells, and mediates the BCR-induced signal that terminates sterile VH transcription and recombinational accessibility of VH segments after a productive VDJ recombination leads to expression of a μ protein.

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mice provided evidence of receptor editing leading to virtually 100% λ L chain expression.143

Late RAG Expression: Receptor Editing and Receptor Revision Although the RAG genes are generally downregulated by a signal mediated by the appearance of membrane IgM at the end of the pre–B-cell stage, RAG gene expression and V(D)J recombination can recur later during “receptor editing” of autoreactive B cells in the bone marrow. One estimate suggests that about 25% of Igs are products of receptor editing.140 After production of an initial Ig κ . protein, receptor editing by L chain rearrangement can occur three ways: an initial VκJκ junction could be deleted by recombination between an upstream V and downstream J on the same chromosome (“secondary recombination,” as discussed previously), VκJκ recombination could occur on the other (allelic) copy of the κ locus, or V λ-Jλ recombination could be activated. Most replacement of productively rearranged L or H chain genes likely serves to extinguish an antibody that was autoreactive, thus complementing two other mechanisms to silence autoantibodies: anergization and cell deletion by apoptosis. Early studies with transgenic autoantibodies suggested that anergy or deletion were the main fates of self-reactive B cells, but these conclusions may have depended on the nonphysiologic inability of the cells to silence the transgenic autoantibody by receptor editing. More recent studies involving autoantibody “knockin” genes—i.e., productive V[D]J recombined genes swapped into the physiologic positions within the IgH or Ig κ gene loci using homologous recombination —have shown that receptor editing is the major mechanism for B cell tolerance.141,142 This conclusion was also supported by a study of mice expressing a transgenic antibody against the murine Cκ constant region, a model of a self-superantigen; these

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IMMUNOGLOBULIN GENE ALTERATIONS IN GERMINAL CENTERS Several days after exposure to an antigen, B cells accumulate in local lymph nodes, gut-associated lymphoid tissue, and spleen, and begin additional maturation steps in germinal centers (GCs). During the GC response, antigen-driven B cells undergo cycles of proliferation and their Ig genes undergo two unique alterations. 1) Lymphocytes switch from making IgM to making a new H chain isotype by the process of CSR. This process introduces dsbs upstream of Cμ in a specific repetitive noncoding DNA segment—the “switch region”—and in a similar switch region upstream of the new target Cx region; the DNA between the breaks is then deleted, and the ends of the remaining chromosomal DNA are rejoined so that the assembled VDJ region now lies upstream of the new Cx gene (Fig. 6.12). 2) In the other GC-associated gene alteration, the affinity of the antibody for its antigen increases by a process that introduces random mutations in VH and VL—somatic hypermutation (SHM)—and then selects for B cells producing higher-affinity antibodies. For many years, CSR and SHM were considered to be unrelated processes, but several lines of evidence provided hints that they might share some mechanistic features. First, while dsbs are expected intermediates for the DNA recombination underlying class switching, DNA breaks in V regions were also detected accompanying SHM. Second (and conversely), in addition to the mutations occurring in V regions associated with SHM, mutations were also observed surrounding the recombination junctions of CSR. Third, RNA transcription was found to be required for both processes.

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FIG. 6.12. Switch Regions and Composite Switch Junctions. The recombination breakpoints in isotype switch recombination fall within repetitive “switch” (S) regions. Stimuli that activate switch recombination (IL-4 and CD40 activation in the example shown) generally promote transcription across the target S region, initiating just upstream at the “I” exon. Recombination between Sμ and Sε produces two composite switch junctions: an Sμ-Sε junction retained in chromosomal DNA, and a reciprocal Sε-Sμ junction found in fractions of circular DNA. Polymerase chain reaction amplification across either composite junction can be used to study switch recombination.

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Activation-Induced Deaminase A fourth and dramatic link between CSR and SHM was the discovery that both processes require the protein known as activation-induced deaminase (AID). The gene encoding AID (known as Aicda, for activation induced c ytosine deaminase) was discovered144 by a subtractive strategy designed to screen for transcripts that were expressed in a murine B-cell line when induced to undergo CSR, but that were not expressed in uninduced cells. AID is expressed almost exclusively in GC B cells and in B cells activated in vitro, though exceptions to this generalization will be discussed later. Mice engineered with a targeted defect in the Aicda gene are completely deficient in CSR and SHM. The same defects are seen in patients with a homozygous defect in the human Aicda gene, a condition known as the hyper-IgM syndrome-2.145 These patients have elevated serum levels of IgM because their B cells cannot undergo efficient CSR. AID is not only necessary for CSR and SHM, but apparently sufficient (in a mammalian cell at least), as overexpression of AID in fibroblasts can confer transcription-dependent CSR of an artificial switch construct146 and transcriptiondependent SHM of a transfected model mutation target DNA.147 These experiments suggest that AID is the only B-cell–specific protein required for SHM and CSR. AID is also required for somatic Ig gene conversion in those species (e.g., rabbit and chicken) that use that process to somatically diversify Ig genes. As translated from the cDNA, AID is a ∼24 kD 198 amino acid protein that forms homodimers. AID shows 34% amino acid identity with the RNA editing enzyme APOBEC1, which catalyzes the deamination of a cytosine residue to uracil in a specific position in the mRNA encoding apolipoprotein B. The human Apobec1 and Aicda genes are genetically linked, both lying at chromosome 12p13. Three other APOBEC1related genes on other chromosomes have also been identified, but are not thought to participate in SHM or CSR. Like APOBEC1, recombinant AID protein has a cytidine deaminase activity in vitro; it was initially proposed that, by analogy with APOBEC1, AID functions by deaminating cytidines in specific RNAs to produce novel edited transcripts encoding one or more proteins required for CSR and SHM. However, no evidence of AID-dependent edited RNAs has been reported, nor does AID deaminate RNA cytidines in vitro. Instead, current evidence indicates that AID acts on DNA, deaminating cytidines to uracil—in V regions for SHM, and in switch regions for CSR. The resulting uracils would be then recognized as both Watson-Crick mismatches and abnormal DNA bases by the cell’s genetic surveillance machinery, triggering error-prone repair of V regions to produce SHM, or DNA cleavage to mediate CSR. This DNA-deamination model for AID is consistent with various properties reported for this protein in vitro or in cells. 1. In transfected Escherichia coli cells, AID mutates cytidine to uracil in DNA, a result that would be unexpected by the RNA-deamination model, as bacteria presumably lack the specific mammalian RNA targets predicted by that model.148,149

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2. Defective SHM and CSR are observed with inhibition or genetic inactivation of uracil-N-glycosylase (UNG), an enzyme that removes uracil residues from DNA,150,151 consistent with the idea that CSR and SHM involve a step in which AID-produced uracil in DNA is removed by UNG. Moreover, B cells from Ung−/− mice accumulate uracils in V and in switch regions in an AID-dependent manner.152 3. AID isolated from B-lymphocytes can deaminate cytidine to uracil in single-strand DNA in vitro153–155 or in doublestrand DNA that is being transcribed in vitro, presumably because transcription causes localized regions of singlestrandedness.155–157 4. AID is found to be associated with IgH genes in vivo in B cells undergoing CSR,158–160 as assessed by Chromatin ImmunoPrecipitation (ChIP) using an anti-AID antibody. 5. A DNA sequence motif WRC—where W is A or T (weak Watson-Crick pairs) and R (purine) is A or G—that has been recognized as a hotspot target of SHM is also a preferred target for AID deamination in vitro,154,157 and the hotspot preference can be altered by engineering amino acid changes in the segment of the AID protein thought to recognize the hotspot target in DNA.161–163 In light of these and other observations, the DNA deamination model for AID action is now almost universally accepted. The AID protein is highly conserved from fish to human, with all species having approximately 200 amino acids and sharing sequence similarities throughout the protein. In all species, AID contains a motif common to the active site of all cytidine deaminases: H[A/V]E − X(24–36) − PCXXC. This motif is also found in other members of the AID-APOBEC gene family, including APOBEC2, APOBEC3 (with several distinct paralogs in human), and APOBEC4.164 AID is encoded in the five exons of the Aicda gene. Structure-function relationships of AID have been probed by examining cross-species sequence comparisons and the effects of natural and engineered mutations in the protein. Remarkably, mutations in the N-terminus of AID impaired SHM but not CSR, whereas mutations or deletion in the C-terminus selectively impaired CSR,165–167 suggesting the possibility that the N- and C-terminal regions of the protein contact specific cofactors required (respectively) for SHM and CSR function. Other functional features of the protein include a dimerization domain, several phosphorylatable residues, segments affecting nuclear localization, and target sequences for association with other proteins. Although AID was initially recognized for its participation in SHM and CSR in GC cells, more recent reports have reported several other roles for this protein, both beneficial and deleterious. 1. Epigenetic methylation. Selective DNA methylation of cytidines is an epigenetic mechanism that participates in the regulation of transcription, and the methylation pattern of DNA is normally replicated when DNA is replicated prior to cell division. In primordial germ cells of

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early embryogenesis, a global erasure of methylation occurs as part of the reprogramming to pluripotency, and AID apparently participates in this process.168,169 Germline mutation. AID can deaminate methylcytosine to thymine, which unlike uracil is a natural DNA base. This reaction in germ cells may contribute to the most frequent germline point mutation observed in mammals: the transition from CpG to TpG.170 Genomic protection. AID and other APOBEC family members protect cells against retroviral infections and against the spread of endogenous retroviruses.171–173 Tolerance, apoptosis. AID appears to be important for the establishment of B-cell tolerance in humans and mice, in that the low levels of AID expression observed in immature and transitional B cells appear to be necessary for suppressing the appearance of autoantibodies.174–176 This effect may be related to the apparent requirement of AID for normal levels of apoptosis, which contribute to the elimination of B cells expressing autoantibodies. Oncogenic mutations and translocations. Soon after AID was discovered, it was observed that overexpression of the protein caused tumors in transgenic mice,177 and even normal expression of AID in B cells contributes to tumorigenic translocations and oncogenic mutations.178 A notable example of AID-stimulated translocation is the recurrent c-myc/Ig translocation seen in Burkitt lymphoma and murine myelomas. Other cancers where AID plays a role through mutation include intestinal and lung cancer.

Based on studies of AID + / − heterozygous mice expressing roughly 50% of normal AID, the level of AID activity seems to be the limiting factor for both SHM and CSR, but is also limiting for oncogenic translocations and mutations.179,180 Therefore, AID activity must be regulated robustly to balance between its multiple beneficial and deleterious actions. The regulation of AID activity is extremely complex, involving multiple levels of control, and is still incompletely understood.

Transcriptional and Posttranscriptional Regulation of AID Expression Early studies showed that AID expression in B cells is upregulated by IL-4 and CD40 engagement. In more recent investigations, four DNA regions that regulate Aicda gene expression have been identified. They include an upstream enhancer responsive to T-cell signals, a promoter, a regulatory region just downstream of exon 1, and an additional enhancer downstream of the Aicda gene.181–186 The microRNA miR-155 has been shown to suppress AID expression.187,188 MicroRNAs are short (21 to 23 nt) RNAs that hybridize to complementary sites in numerous target RNAs, triggering their degradation or inhibiting their translation. A target for miR-155 lies in the 3′ untranslated region of the AID transcript. AID expression was increased in miR155–deficient mice and in mice whose Aicda gene was mutated to disrupt the 3′UTR target sequence. In both cases, consequences of the increase in AID expression included

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increased off-target mutations and increased c-myc-IgH translocations. Perhaps to protect against such effects, miR155 is itself upregulated by the same signals that induce Aicda transcription in B cells.

Posttranslational Regulation The level of AID activity in the nucleus is affected by its distribution between cytoplasm and nucleus, by the protein half-life, and by phosphorylation at various positions in the protein. AID was found to shuttle between nucleus and cytoplasm.189 Most of the protein is cytoplasmic and only a small fraction is in the nucleus, where it can act on DNA to cause CSR and SHM as well as “off-target” deleterious effects. Movement between cytoplasm and nucleus is controlled by at least three independent mechanisms: active nuclear import mediated by an N-terminal nuclear localization signal,190 active nuclear export mediated by a C-terminal nuclear export signal,189,191 and a cytoplasmic retention mechanism apparent when active import and export mechanisms are both blocked. Two proteins that may facilitate AID import independently of the nuclear localization signal are GANP192 and CTNNBL1.193 Nuclear-cytoplasmic distribution affects AID stability, because in the nucleus AID is targeted for ubiquitination and proteasomal degradation, shortening its halflife.194 Conversely, in the cytoplasm, the chaperone protein Hsp90 protects AID from proteasomal degradation.195 In addition to ubiquitination, AID is also subject to posttranslational phosphorylation at several distinct residues in the protein, including Ser3, Thr27, Ser38, Thr140, and Tyr184. Of these, the functional relevance of the Ser38 site is best understood. Phosphorylation of AID Ser38 is essential to recruit replication protein A (RPA), a trimeric singlestrand DNA binding protein159,196 that may be required to stabilize a single-strand DNA target for AID. Preparations of AID purified from transfected E. coli or from nonlymphoid eukaryotic cells lack Ser38 phosphorylation; they can deaminate single-strand DNA in vitro, but are inactive in an assay for deamination of double-strand DNA transcribed in vitro by T7 polymerase. Activity in the latter assay requires AID that is phosphorylated at Ser38 and associated with RPA. In the AID protein, Ser38 lies within a conserved phosphorylation site [RRXX(T/S)] for the cyclic-AMP dependent protein kinase A (PKA), and multiple experiments have confirmed that PKA phosphorylates AID at Ser38 in vitro and physiologically in vivo.196,197 The physiologic importance of Ser38 phosphorylation is strongly supported by observations of the effect of mutating this serine to alanine. The AIDS38Α protein has essentially normal activity in deaminating single-stranded DNA in vitro, but severely reduced activity in deaminating transcribed double-strand DNA in vitro. Less is known about the other sites of AID phosphorylation. At Thr140, alanine replacement causes more modest inhibition of AID activity than Ser38Ala, preferentially affecting SHM in vivo, but without significant effect on in vitro catalytic activity.198 In contrast to Ser38 and Thr140, phosphorylation of Ser3 apparently suppresses in vivo AID activity; alanine replacement at this position increases SHM,

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CSR, and oncogenic translocation, also without affecting in vitro catalytic activity.199 Clearly, multiple mechanisms regulate the level of activity of AID in B-cell nuclei, but these mechanisms cannot explain how—within nuclei—AID acts on Ig V genes and switch regions at a much higher frequency than on the rest of the genome. Targeting mechanisms differ somewhat between SHM and CSR, and will be discussed in the context of those processes.

Somatic Hypermutation The hypothesis that antibody genes inherited in the germline might be subject to somatic mutation in lymphocytes during the life of an individual was suggested as an explanation for the diversity of antibodies several years before recombinant DNA technology became available to clarify the role of V(D)J recombination. Persuasive evidence for somatic mutation was reported in the 1970s: analyses of Vλ1 amino acid sequences of murine myeloma antibodies showed many instances of a particular prototype sequence, plus several variants containing unique single amino acid substitutions that could be explained by single nucleotide changes. The prototype was interpreted as reflecting the germline sequence, with the variants arising by somatic mutation.200 Subsequent investigations at the DNA level revealed myeloma V region sequences that deviated from their germline counterparts, verifying the principle of somatic mutation. Somatic mutations are much rarer in IgM than in antibodies with “switched” isotypes (IgG, IgA, and IgE) made by B cells that have been exposed to AID in GCs, but they do occur; antibodies with “switched” isotypes without mutations are also found. These observations suggest that though both SHM and CSR normally occur in GC B cells, the two processes are unlinked.

Role of Hypermutation in Immune Responses To understand the role of SHM in the antibody response, several groups have studied the extent of Ig gene mutation at different times after the immunization of mice. Studies of the responses to p-azophenylarsonate (Ars), phosphorylcholine, influenza hemagglutinin, oxazalone, and several other antigens have all indicated that the initial response after primary immunization is established by antibodies showing no somatic mutation. About 1 week after immunization, mutated sequences begin to be observed, increasing during the next week or so. Booster immunizations yield sequences showing additional mutations. Many hybridomas made late in the immune response produce mutated antibodies with a higher antigen affinity than the unmutated (sometimes loosely called “germline”) antibodies made early after immunization. The shift to higher affinity is a phenomenon long recognized at the level of (polyclonal) antisera and has been termed “affinity maturation.” This phenomenon can now be explained as the result of an “evolutionary” mechanism selecting antibodies of progressively higher affinity from the pool of randomly mutated V sequences. According to this model, at the time of initial antigen exposure an animal has a set of naïve B-lymphocytes

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expressing IgM with germline (unmutated) versions of Ig variable regions resulting from gene rearrangements that occurred prior to immunization. Because of the diversity of available V H, D, JH, V L, and JL sequences as well as the impressive recombinational potential described previously, some B cells will express Ig molecules capable of binding the antigen with modest affinity. Antigen binding stimulates these B cells to proliferate and to move to lymphoid follicles, where they form GCs. In the GC environment, AID is expressed and SHM machinery is activated, generating random mutations in the Ig genes of stimulated GC B cells. Many of these mutations reduce the affinity of the encoded antibody for antigen, 201 and some may induce autoantibody specificities (i.e., the ability to bind to self-molecules202). As clearance of the antigen lowers the antigen concentrations, only the cells displaying high affinity for antigen will be stimulated effectively; cells displaying lower-affinity antibodies or antibodies with affinity for self-antigens are subjected to programmed cell death (“apoptosis”).203,204 The preferential proliferation of the high-affinity cells and their maturation to secreting plasma cells causes an increase in the average affinity of the antibodies in the serum. Some high-affinity cells become memory cells, persisting long after the initial antigen exposure, ready to respond to a subsequent antigen exposure with rapid production of high-affinity antibody. In this model, the driving force for affinity maturation— analogous to natural selection in the evolution of species—is selection for high-antibody affinity in the face of falling antigen concentration. The importance of this selective force is suggested by the observation that affinity maturation can be inhibited by repeated injection of antigen (which removes the selective pressure for high affinity) 205 or by overexpression of the antiapoptotic protein Bcl-XL (which allows survival of B cells expressing low affinity antibody).206

Cellular Context of Somatic Mutation Each GC appears to be populated by a small number of antigen-specific founder B cells207 and an unusual Thy-1 negative T-cell population, also antigen-specific, 208 The GC environment promotes contact between the B cell and follicular dendritic cells— which store, process, and present antigen—and T-lymphocytes, which activate somatic mutation in part via CD40-CD40Ligand interaction.209 In a widely accepted model of GC function, SHM occurs in a subpopulation of B cells known as centroblasts. These cells proliferate in the “dark zone” of the GC and bear characteristic surface markers including IgD, CD38, and the receptor for peanut agglutinin. Proliferating GC centroblasts give rise to nondividing centrocytes in the “light zone” of the GC; there centrocytes are programmed for apoptosis unless they are rescued by follicular dendritic cell–presented antigen and T-cell activation via CD40 engagement. Selection for high-affinity antibodies occurs because cells expressing high-affinity antibodies are most efficiently rescued from apoptosis. Surviving centrocytes may return to the dark zone to undergo several successive cycles of mutation and proliferation followed by selection. This model is supported by direct observation (by two-photon microscopy) of B cells moving between light zone and dark zone.210 Migration of

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B cells to follicles or GC zones is thought to be controlled by a complex system of chemotactic receptor /ligand pairs including CXCR5/CXCL13, CXCR4/SDF1, CCR7/CCL9 (or CCL21), S1P1/sphingosine-1-phosphate, and EBI2/its lipid ligand.211 Although certain features of this classical model have been challenged, the broad outlines have received strong support from studies in which cells in the dark zone or light zone of individual GCs can be marked by a photoactivatable green fluorescent protein (GFP) and then followed for several hours using two-photon imaging as the cells migrate through a mounted lymph node.212 The notion that GC B cells compete to avoid apoptosis on the basis of antigen affinity of their BCR has been has been supported by a study that directly compared caspase activation (an apoptosis marker) in B cells expressing transgenic antibodies with higher versus lower affinity for the same antigen; the lower-affinity cells were found to undergo a significantly higher rate of apoptosis.213 The mechanism by which high-affinity antigen binding selects for survival is not fully understood. One possibility is that a higher-affinity BCR directly delivers a stronger activation signal to the B cell, inhibiting apoptosis. However, a second possibility is that a higher-affinity BCR could capture antigen more efficiently, enabling stronger antigen presentation to GC T cells, which could then deliver stronger survival signals to the B cell via secreted or surface protein interactions. Consistent with the second model, experiments using two-photon microscopy have documented that that B cells from mice immunized with a fluorescent antigen can capture that antigen from follicular dendritic cells in the GC.214 To test whether B-cell antigen capture might confer survival independently of the BCR, investigators engineered B cells in ovalbumin-primed mice to deliver ovalbumin via a surface lectin instead of via the BCR; they observed that the internalized ovalbumin antigen was capable of conferring a B-cell survival advantage in the GC in the absence of BCR engagement.212 However, effective BCR signaling can enhance antigen processing and presentation,215 so internalization/presentation and BCR signaling may work together to promote GC B-cell survival and mediate selection for high affinity. Most experiments on SHM have focused on GC cells, as does the discussion in this chapter. However, T-cell– independent SHM may also occur in a population of less mature cells, which may populate the splenic marginal zone and which may increase the repertoire of circulating lymphocytes prior to antigen exposure, especially in young individuals, 216,217 or perhaps function in tolerance induction, as discussed previously. Also, mice lacking histologically detectable GCs as a result of lymphotoxin-α deficiency are capable of SHM and affinity maturation.218 T-cell–independent antigens can induce a low level of SHM in B cells.219

Molecular Mechanism of Hypermutation AID deaminates cytidine to uracil, an analog of thymine. Thus, if replicated before repair, an original C:G base pair would, in the daughter cell receiving the uracil-bearing DNA strand, mutate to a T:A. But it was initially not clear how cytidine deamination could affect A:T base pairs, which are targeted in 50% to 60% of mutations observed in SHM. By

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analyzing abnormalities in SHM that are observed in various mutant B cells, Di Noia and Neuberger150 formulated a model for SHM that has explained this question and has gained wide acceptance (Fig. 6.13). The model proposes that after AID-catalyzed deamination creates a uracil residue in the target DNA, the possible outcomes depend on how the resulting mismatch is resolved. 1) The U:G mismatch may be replicated as described above, resulting in what are known as phase 1A mutations. 2) The uracil base may, before replication, be excised by UNG, creating an abasic site. Normally, the creation of such abasic sites is the first step of the base excision repair pathway, in which subsequent steps remove the sugar-phosphate backbone, leaving a single nucleotide gap that is then restored to a C:G bp by DNA polymerase β and DNA ligase. If the DNA replicates before the abasic site is repaired, the strand with the abasic site may directly engage translesional polymerases (which are error-prone, as discussed below) to insert an unpaired nucleotide (i.e., any nucleotide) opposite the abasic lesion, leading to phase 1B mutations (see Fig. 6.13). 3) The original U:G mismatch— or possibly the abasic site created by UNG action—may be recognized by the mismatch repair (MMR) system of the cell. MMR triggers excision of the DNA strand for several nucleotides surrounding the mismatch, and this strand is then resynthesized by polymerase β, in the case of faithful repair, or by error-prone polymerases, in the case of SHM. Error-prone repair inserts mispaired bases, which may become fi xed on one strand by replication, creating mutations (designated phase 2) from both A:T and G:C bps at some distance from the position of the original U:G mismatch. If a mispaired base is recognized by MMR before replication, a new cycle of MMR may be initiated, extending mutation even farther from the initial deamination event. Role of Uracil-N-Glycosylase in Somatic Hypermutation. The UNG gene encodes two proteins that differ in their N termini as a result of alternative promoters that generate different initial coding exons. UNG1 is expressed in mitochondria, whereas UNG2 is nuclear. Hydrolytic deamination of cytosines to uracil occurs at a significant rate in all eukaryotic and prokaryotic cells, and misincorporation of dUTP during replication further contributes to the load of uracil in DNA. UNG2 plays a major role in mitigating this load by initiating faithful base excision repair. In SHM, the faithful repair of uracil is somehow subverted to introduce mutations. Ung − / − mice and human patients with a rare form of HyperIgM immunodeficiency due to UNG mutations have similar immunologic phenotypes. First, as discussed in the following, Ung − / − individuals are profoundly defective in CSR, as expected if this process requires AID-catalyzed deamination of cytidine followed by UNG-catalyzed removal of uracil. Although the frequency of mutation is roughly normal in UNG-deficient individuals, mutations at C are almost exclusively transitions of C to T (phase IA). As suggested by Figure 6.13, Ung − / − individuals would not create the abasic sites that lead to C → G and C → A mutations by replication, though some mutations of these kinds could be produced by MMR. Indeed, the frequencies of mutations

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FIG. 6.13. Mechanistic Model of Reactions Triggered by Activation-Induced Deaminase (AID) in Somatic Hypermutation and Class Switch Recombination (CSR). The graphic at the top depicts a small region in a VH gene showing a targeted C:G basepair and a nearby T:A pair. After AID-catalyzed deamination of a C residue, the DNA may be subjected to various modifications as shown, leading to mutations indicated by nucleotides in gray rectangles. The model is described in the text. The Phase II mutations occur when mismatched bases incorporated by an errorprone polymerase are replicated before they can be corrected by MMR. The same mechanism explains two switchregion–associated events occurring during CSR: DNA strand breaks (see center panel at bottom) and mutations..

from A, T, and G in Ung − / − individuals are normal, apparently resulting from MMR activated by the U:G mismatch in many cells. Mismatch Repair in Somatic Hypermutation. MMR is a highly conserved mechanism that detects abnormalities in DNA, including mispaired nucleotides and abnormal bases, and repairs them. The eukaryotic MMR system has two main components. MutS binds tightly and specifically to DNA defects and recruits MutL. The Mut complex then activates a latent nuclease activity to remove a segment of the DNA strand including the mismatched base; this gapped strand is then resynthesized by a DNA polymerase. Mammals have several MutS homologs, including three reported in somatic cells—MSH2, MSH3, and MSH6—which exist in the cell as heterodimers. MSH2-MSH6 (also known as MutSα) is the most abundant form and is specialized for recognizing single base gaps or mismatches, while MSH2-MSH3 (MutSβ) recognizes larger gaps and insertion/deletion loops. The mammalian MutL proteins are heterodimers consisting of MLH1 paired with PMS1, PMS2, or MLH3. Apart from their

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effects on SHM and CSR, mutations in MMR genes, especially MSH2 and MLH1, underly hereditary nonpolyposis colorectal cancer. Recent efforts in several laboratories have led to in vitro reconstitution of mammalian MMR with purified components, enabling powerful analysis of this complex mechanism.220 In addition to the MutS and MutL proteins, the system requires the following components: Exo1 to excise the gap; replication protein A, which binds to the single strand DNA in the gapped region (and is known to bind to phosphorylated AID, as discussed previously); proliferating cell nuclear antigen (PCNA), which promotes processivity by encircling DNA in a sliding ring clamp and by recruiting other components; DNA polymerase δ ; DNA ligase I; and several other proteins. Knockouts of MSH2, MSH6, and Exo1 have been studied by several laboratories and show consistent decrease in B-cell SHM from A:T bps (with a corresponding increase in the percentage of mutations from G:C bps).221,222 These results, in the context of the model of Figure 6.13, suggest that MMR triggered by the MSH2-MSH6 heterodimer primarily introduces mutation at A:T bps. The predilection for

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mutating A:T bps matches the activity of polymerase η, as discussed in the next section, implying that this polymerase may be the one most frequently engaged by the MMR machinery in repairing AID-generated lesions. Indeed, MSH2MSH6 is capable of binding to a U:G mismatch, MSH2 can bind to polymerase η in cell extracts, and MSH2-MSH6 can stimulate the activity of polymerase η in vitro.223 Error-Prone Polymerases. Although avoidance of error is a high priority for most DNA replication, error-prone DNA synthesis served an important function long before SHM evolved to mutate Ig genes; indeed, error-prone polymerases have been most thoroughly studied in E. coli and yeast. These polymerases are useful in all cells for replication of DNA containing focal lesions that would block replication by stringent high-fidelity polymerases. However, the tolerance of these error-prone polymerases to abnormalities in DNA structure is accompanied by tolerance of non–Watson-Crick basepairing in the replication of normal DNA and by absence of DNA proofreading activity. More than a dozen of these polymerases have evolved in eukaryotes, each specializing in different aspects of “translesion DNA synthesis” (TLS) and showing differing spectra of infidelity in replication of normal DNA. Clear evidence for participation of several TLS polymerases in SHM has come from comparing the known in vitro activities of a particular polymerase with the SHM abnormalities seen in mice or humans with mutations in the corresponding polymerase gene. Polymerase η characteristically inserts mismatched bases opposite T nucleotides, and individuals lacking this polymerase show the predicted abnormality: decreased mutations at A:T base pairs. This was first demonstrated in patients with xeroderma pigmentosum variant disease, whose defects in polymerase η subject them to sunlight-induced skin cancers in addition to their abnormal SHM; a similar defect in mutations from A:T bps was subsequently shown for polymerase η knockout mice.224,225 Polymerase η binds to MSH2-MSH6, as mentioned previously, and is upregulated in cells undergoing SHM. In addition to polymerase η, the TLS polymerase REV1 has been shown to participate in SHM. REV1 is known to preferentially insert cytosine residues opposite uracil or abasic sites, and Rev1 knockout B cells were found to be impaired in C to G mutations, especially in the nontranscribed (coding) strand.226 Polymerase ζ, 227,228 polymerase κ,229 and polymerase θ230–232 knockouts have also been reported to show altered SHM patterns, but these effects are smaller or not seen in all circumstances. Error-prone repair apparently operates in competition with faithful repair carried out by DNA polymerase β. The human B-cell line BL2 is known to undergo SHM, but in subclones of this line with higher SHM proficiency, polymerase β levels were very low; overexpression of polymerase β in a proficient subclone suppressed SHM activity.233 Compared to wild-type B cells, polymerase β – deficient B cells (developing in a wild-type recipient) showed increased switch region mutation after induction of CSR in vitro.234 A fundamental question about the action of error-prone polymerases is how they are specifically engaged for SHM of Ig genes, given that most spontaneous cytosine deamination

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in nonlymphoid cells is accurately repaired. One apparent clue is that some TLS polymerases are upregulated in B cells undergoing SHM. In addition, when the sliding clamp protein PCNA is monoubiquitinated at lysine residue K164, it is known to bind and activate TLS polymerases including polymerase η. When this ubiquitination is prevented, either by knockout of the specific E3 ubiquitin ligase of PCNA or by a K164R mutation at the ubiquitination site in PCNA, SHM is abnormal. PCNA Κ164Ρ mice were found to have a dramatically decreased ability to mutate A:T sites during SHM, similar to the phenotype of the polymerase η knockout, as though polymerase η requires monoubiquinated PCNA in order to participate in SHM.235 However, these clues do not fully explain why Ig genes in GC B cells are less faithfully repaired than other genes in the same cells. This question is part of the larger issue of mutational targeting, as discussed below.

Targeting and Distribution of Mutations The mutation rate of Ig genes in GC B cells undergoing SHM may reach as high as 10−3 mutations/bp/generation, or about 10 6 times higher than the normal genomic mutation rate; 236 this elevated rate could be lethal to B cells if mutations were not carefully targeted specifically to Ig genes. Several other genes highly expressed in GC B cells were also found to be mutated at lower levels (e.g., Bcl6 and Cd95237), though several other genes expressed in GC B cells at comparable levels are not mutated. Apparently, some of the features that target SHM to Ig genes may be shared by other genes. Recently, a genome-wide analysis has made it clear that AID binds much more widely to the genome than was previously appreciated. When chromatin from mouse B cells incubated with lipopolysaccharide (LPS) and IL-4 was analyzed by ChIP with an anti-AID antibody, 5910 genes were found by deep sequencing of the immunoprecipitated DNA (ChIP-seq),160 including many previously described AID targets. Although this is a large number, it is still a small minority of the genome. As judged by the number of sequence tags recovered, AID binding to the IgH locus was substantially higher than binding to any other region. A sample of genes identified as AID-binding by ChIP-seq showed significant mutation frequency, whereas control genes did not. The AID-binding genes were associated with high levels of mRNA transcription (by RNA-seq), by a chromatin mark commonly associated with transcription—histone H3 trimethylated on Lys4 (H3K4me3)—and by density of Pol II binding. However, only the IgH locus, with its uniquely high levels of AID occupancy and mutations, bound to RPA; this binding was AID-dependent and was diminished in the S38A and T140A mutants of AID described previously. The difference between AID-targeting and SHM frequency has been reinforced by analysis of SHM in panels of non-Ig genes isolated from murine Peyer patch B cells.238 All the non-Ig genes were mutated at levels much lower than the 2.2×10 −2 mutations per bp detected near JH4, but substantial numbers of mutations were found at Bcl6, Pim1, CD79B, and several other previously reported targets of SHM. The mutations were almost completely AID-dependent, as in cells from Aicda −/ − mice, all genes showed very low mu-

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tation frequencies that were barely above the sequencing error rate. However, different genes varied widely in the way their mutation rates were affected by the double knockout Ung −/ − Msh2−/ −, in which only phase 1A mutations should be possible. At one extreme, c-myc, which is known to mutate at a very low rate in GC B cells, showed a mutation rate in the Ung −/ − Msh2−/ − cells almost 17-fold higher than in the wild-type, as though almost all AID-induced deamination in the wild-type B cells had been faithfully repaired by UNG- and Msh2-dependent mechanisms. In contrast, Bcl6 mutations were only 1.3-fold higher in the double knockout, suggesting that faithful repair of this gene was dramatically less active. Thus SHM susceptibility depends not only on differential AID targeting across the genome but also on differential ratios of error-prone versus faithful repair. The distinction between these two variables (deamination and repair) has only recently been recognized, so it was not taken into account in many earlier studies described in the following. RNA Transcription. A relationship between SHM and RNA transcription is suggested by the observation that unrearranged V H and Vκ genes are generally neither transcribed nor mutated, but become susceptible to both processes when V(D)J recombination moves them close to their (respective) intronic enhancers, Eμ and iEκ. In contrast, the λ locus lacks an enhancer between J and C; unrearranged Vλ regions are transcribed in B cells 239 and can be mutated.240 The Ig coding sequences are apparently not specifically required to target SHM, as transgenic V coding sequence can be replaced by a human β -globin gene or prokaryotic neo or gpt gene without affecting the hypermutation rate downstream of the promoter. Mutations around a given V region are distributed in a domain that begins roughly 100 to 200 bps downstream of the promoter, extends for ∼1.5 to 2 kb downstream, and then tapers off long before the RNA transcriptional termination. This distribution led to the hypothesis that after transcriptional initiation, a “mutator factor” attaches to the transcriptional machinery, attacks DNA as the transcription complex moves downstream, and eventually falls off, so that further transcription proceeds without mutations.241 Consistent with this model, a VκJκ-Cκ transgene bearing a second Vκ promoter engineered upstream of the Cκ region was found to incur mutations over a second domain extending into the Cκ region, in addition to the usual V region mutations.242 Conversely, the insertion of an irrelevant 2 kb DNA fragment between a Vκ promoter and the leader (signal peptide) exon prevented mutation within the Vκ transgene, which now apparently lay downstream of the mutational domain.243 (Mutations also occur in domains surrounding repetitive switch regions upstream of CH genes. The frequency of these mutations is similar to that in V regions, but the domains are larger, correlating to some extent with the length of the switch region, as discussed in the following.) The key mutator factor is apparently AID, which can deaminate double-strand DNA

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in vitro only when the DNA is transcribed. One hypothesis suggests that DNA near the advancing RNA polymerase complex becomes negatively supercoiled or partially single-stranded, which might facilitate access of the DNA to AID. 244 Another explanation, involving R loops, is described below. Evidence for patches of single-stranded DNA in transcribed V regions undergoing SHM has been obtained using a bisulfite technique.245 In vivo dependence of SHM frequency on transcriptional activity has been confi rmed by several fi ndings. In one study, SHM was studied in knockin mice with a prerecombined V HDJ H region driven by either of two V region promoters; SHM rates in these knockin strains were highly correlated with transcription driven by the different promoters.246 In another study, a tetracycline-inducible GFP reporter gene engineered with a stop codon was stably transfected into a B-cell line. The rate of reversion of the stop codon by SHM (allowing GFP expression) was found to be directly related to the transcription rate, as regulated by a tetracycline analog. 247 Other studies examining SHM in models where transcription was altered by mutating enhancers have supported the relationship between SHM and enhancer-induced transcription. Some non-Ig enhancers could support SHM in a stably transfected cell line, 248 but others could not, leading to the suggestion that specific elements in enhancers might confer susceptibility to SHM. One candidate element is the sequence CAGGTG, which is a target for E-box transcription factors and which is found in several Ig enhancers. When a murine κ enhancer containing CAGGTG was linked to a GFP gene inactivated by a premature stop codon, stable transfectants of chicken B-cell line DT40 were found to produce mutants that expressed GFP, but a single mutation in the CAGGTG motif prevented SHM without changing transcription. 249 Inactivation of the E-box transcription factor E2A in DT40 cells was found to reduce SHM of endogenous Ig genes without significant effects on Ig gene (or AID) transcription.250 However, some conflicting conclusions have been drawn about enhancer-dependence of SHM depending on whether experiments investigated transgenes versus constructs created in the endogenous context using homologous recombination (i.e., knockouts or knockins). For example, the downstream IgH enhancers HS3b and HS4 were found important for SHM in transgenes251 but dispensable in the context of endogenous genes.252 In one case, transcriptional enhancer activity has been clearly separated from an associated SHM stimulating activity. Just downstream of the chicken Ig λ 3′ enhancer lies a DNA segment whose deletion in the DT40 B-cell line severely impaired SHM without dramatically affecting transcription; this segment could confer SHM when inserted into a non-Ig locus.253,254 To summarize, it appears that transcription is necessary but not sufficient for targeting hypermutation, but the additional elements required for SHM have not been defi ned as of this writing (though E2A function may be among them).

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Chromatin Marks. A parameter related to transcription of Ig genes (i.e., their context in chromatin) has also been studied in relation to SHM targeting. Culture of a B-cell line under conditions that upregulate SHM (in the presence of T cells and anti-IgM) led to increased acetylation of histone H3 and H4 at V region but not C region DNA, as assessed by ChIP analysis 255 paralleling the distribution of mutations. The increased histone acetylation was not a consequence of AID action as it occurred when AID expression (and SHM) was inhibited by antisense treatment; moreover, when cells were treated with the deacetylase inhibitor trichostatin A, the C region was both acetylated and subjected to SHM. Other histone modifications that may be correlated with SHM are phosphorylation or monoubiquitination of histone H2B.256,257 Hotspot Focusing. Mutations occurring within V region genes expressed in vivo in B cells may be highly selected for antigen-binding function of the expressed antibody. To analyze the spectrum of mutations produced by SHM unbiased by selection, investigators have studied nonproductively rearranged VDJ alleles or “passenger” transgenes engineered with stop codons to prevent expression as a protein. In these genes, mutational “hot spots” as well as “cold spots” have been recognized, apparently due to local sequence features that may promote or suppress somatic mutation. The consensus sequence WRC (i.e., [A/T][G/A][C]) is the most consistent hotspot for mutation, presumably reflecting the predilection of AID for in vitro deamination of this sequence. It is possible that evolution has concentrated mutational hot spot frequencies in CDR regions to enhance the potential for diversity generation in the parts of the protein critical for antigen contact.258 The previous discussion has identified several factors influencing the targeting of SHM, a process in which a unique triggering event—AID-dependent deamination of DNA—is followed by a cascade of other events that depend on mechanisms common to most cells. All of these steps, including the AID-dependent trigger, can be affected by biologic parameters common to all cells, such as transcription, enhancers, epigenetic state, DNA repair mechanisms, etc. Many of these same mechanisms affect CSR as well as SHM. Recently, three additional biologic parameters have been found to influence AID-triggered events in CSR: RNA polymerase stalling, the RNA splicing in spliceosomes, and RNA degradation by exosomes. It is possible that these three processes impinge on both SHM and CSR, but because they were discovered in the context of CSR, they are discussed in the following section.

Heavy Chain Switch Switch Regions and Switch Junctions As briefly mentioned previously in this chapter, isotype switching involves removal of C μ from downstream of the rearranged H chain VDJ gene and its replacement by a new downstream CH region. This occurs by a deletional recombination—CSR—in which the recombinational breakpoints generally occur within G-rich repetitive DNA

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sequences known as switch (or S) regions lying 5′ of each CH region (except C δ). While most switch breakpoints fall in the S regions, some are in nearby nonrepetitive DNA. The S region of the mouse μ gene, S μ , is located about 1 to 2 kb 5′ to the C μ coding sequence and is composed of numerous tandem repeats of sequences of the form (GAGCT) n(GGGGT), where n is usually 2 to 5 but can range as high as 17. All of the S regions of downstream isotypes include pentamers similar to GAGCT and GGGGT embedded in larger repeat units rather than precisely tandemly repeated as in S μ . In support of the critical role of S regions for CSR, knockout of Sγ1 by homologous recombination essentially abolished expression of IgG1 from that allele, 259 and mice with a deletion of S μ were also dramatically impaired in CSR. 260,261 A switch recombination between, for example, μ and ε genes produces a composite Sμ-Sε sequence (see Fig. 6.12). From a comparison between the sequence of an Sμ-Sε composite switch region and the sequences of the germline Sμ and Sε, one can localize the exact recombination sites between Sμ and Sε that occurred in each allele. Such comparisons have indicated that there is no specific site, either in Sμ or in any other S region, where the recombination always occurs, although clusters of recombination sites have been reported at two specific regions within the tandem repeats of murine Sγ 3 region.262 Thus, unlike the enzymatic machinery of V-J recombination, the switch machinery can break and join sequences in a broad target region, and as the recombination targets are in intronic DNA, there are no reading frame complications. Often both IgH alleles in a single cell undergo switching to the same downstream isotype. Some alleles undergo sequential switching events; for example, a common pathway to IgE expression is an initial μ → γ1 switch, followed by a CSR between the composite Sμ-Sγ1 switch region and the S ε region.263 Sγ1- Sε switching may even occur independently of Sμ.264 Although most CSR occurs as a deletion within a single IgH allele, switching between two allelic chromosomes was detected at a frequency of roughly 7% to 10% in mouse and rabbit.265,266 DNA fragments excised by switch recombination have been cloned from fractions of circular DNA isolated from cells actively undergoing isotype switch recombination. Thus, at least some of the excised DNA segments ligate their ends to form “switch circles”; these contain composite switch junctions that are in theory reciprocal to the composite switch junction retained on chromosomal DNA (see Fig. 6.12). Because switch circles are not linked to centromeres and do not apparently contain origins of replication, they are not efficiently replicated. Therefore, they are not found in cells that have divided many times after switching (e.g., in myelomas or hybridomas). Many composite switch junction sequences show mutations near the recombination breakpoint when compared to the corresponding germline switch sequences. Indeed, many features of CSR are shared by SHM (as indicated previously in this chapter), including the requirements for transcription, AID, UNG2, and MMR components for normal CSR and SHM. However, CSR is more complex in that it involves simultaneous targeting to two DNA regions (i.e., switch

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regions of Cμ and the target Cx), it requires dsbs, and it consequently requires mechanisms to repair the breaks. AID can theoretically trigger single-strand breaks on both DNA strands by the mechanisms discussed for SHM, including cleavage by APE-1. If these breaks are close enough together, they can effectively form a dsb with staggered ends, and if dsbs occur in two switch regions, the DNA repair mechanisms common to all cells may rejoin the broken ends to complete a CSR event. Indeed, B cells in which Sμ and Sγ1 were deleted and replaced by recognition sites for the yeast homing endonuclease I-SceI were capable of mediating some μ → γ1 CSR in the absence of AID if I-SceI was expressed, 267 highlighting the participation of ubiquitous AID-independent repair mechanisms for CSR. These mechanisms depend on the NHEJ components described previously in this chapter plus backup participation by alternative end-joining mechanisms discussed below.

Regulation of Isotype Switching: Proliferation and AID Expression Isotype switching occurs physiologically in animals about 1 week after immunization with T-dependent antigens, at about the same time that somatic mutation of Ig genes begins. Both processes normally occur in GCs of lymphoid organs, a location that facilitates interactions between B cells, T cells, and follicular dendritic cells presenting antigen. As demonstrated by in vitro switching experiments, T cells promote switching by secretion of cytokines (especially IL-4 and transforming growth factor β) as well as by cell-to-cell contact. A major component of the cell contact signal is mediated by an interaction between the B-cell surface marker CD40 and its ligand—designated CD40L, CD154, or gp39—which is expressed on activated T cells (primarily CD4 +). CD40 is a member of the TNF–receptor family, while CD40L belongs to the TNF–ligand family. The dependence of switching on the CD40-CD40L interaction is highlighted by the genetic disease known as the X-linked hyper-IgM syndrome-1, which was found to be caused by a defect in the human gene encoding the CD40L/gp39.268 Like AID-deficient patients with hyper-IgM syndrome-2 described previously, patients with X-linked hyper-IgM syndrome-1 have elevated concentrations of IgM in their serum and almost no Igs of other isotypes. In addition, their antibodies fail to show affinity maturation or evidence of B-cell memory responses. Similar defects are seen in humans with mutations in their CD40 gene, an autosomal recessive disease designated hyper-IgM 3.269 CSR impairment may also be caused by abnormal function of CD40 signaling pathway components including IKKγ (also known as NEMO), NFκ B proteins, and C-Jun N-terminal kinase. Mouse strains with engineered defects in CD40 are defective in SHM and T-dependent CSR, but respond with normal isotype switching to T-independent antigens. The T-independent switching pathway may be especially important for gut-associated switching to IgA.270 One role of the CD40 engagement is to induce B-cell proliferation. Indeed, other proliferative stimuli (e.g., LPS or IgM or IgD crosslinking) can support cytokine-induced isotype switching in vitro in the absence of T cells and CD40

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activation. The relationship of CSR to cell division is supported by evidence that switching is linked to the cell cycle 271 and to the number of cell divisions after stimulation, 272 a phenomenon which may reflect cell division-related regulation of AID expression.273 However, apart from activating proliferation, CD40 has additional effects that may facilitate switching, including upregulation of IL-4 responsiveness and IL-4 receptor number, 274 upregulation of sterile “switch transcripts” (discussed below), and upregulation of AID expression. Activated B cells also express CD40L, which can not only trigger CD40 signaling but also transduce a “reverse” signal affecting B-cell function.275 Independent of CD40, B-cell activation can independently be stimulated by TLR ligands and cytokines such as APRIL, another member of the TNF family.276

Regulation of Class Switch Recombination to Specific Isotypes: Promoters, Enhancers, and Chromatin Different isotypes are known to predominate in different immune responses depending on the antigen, route of antigen administration, and several other parameters. These different parameters act in part by influencing the cytokine milieu of the B cells. IL-4, for example, promotes the expression of IgE and IgG1, whereas TNF-β promotes switching to IgA. These lymphokines have been proposed to act by making the target isotype “accessible” to switch recombinase machinery that may be largely non–isotype-specific. The accessibility is associated with expression of a “sterile” or “germline” RNA transcript that initiates upstream of a target S region (see Fig. 6.12) and extends through the target C region. The germline transcript is spliced so that a noncoding upstream exon known as an I (“intron”) region is joined to the first coding exon of the C region. (This contrasts with the “productive” transcript containing VDJ spliced to the C region.) The same experimental conditions—particularly the same cytokines—that favor the accumulation of germline transcripts from a particular isotype generally also stimulate switch recombination to the same isotype. In many cases, the signals transduced by the cytokine receptor have been elucidated. For example, IL-4 stimulates germline transcription by activating the transcription factor STAT6, which binds to one of several IL-4 response motifs in the promoter region upstream of Iε and Iγ1. CD40 engagement also acts in part through NFκ B-mediated binding to I region promoters.277 Gene targeting experiments have shown that mouse strains lacking the I region (and its promoter) of a particular isotype do not switch to that isotype, reinforcing the idea that sterile transcription is necessary for CSR.278 The low extent of sequence conservation of the I exons and the lack of consistent open reading frames suggest that these transcripts do not encode a functional protein. Indeed, the exact sequence of the I region may be irrelevant as an I region can be replaced by an unrelated sequence and still support CSR.279 However, the transcribed exon upstream of the S region apparently needs a splice donor site allowing splicing to the downstream C region, as a targeted construct lacking such a splice donor site was reportedly unable to support CSR even though transcription through the S region occurred.280

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Apart from I region promoters, germline transcription and isotype switching are also regulated by IgH enhancers. A combined deletion of the murine 3′-regulatory region enhancers HS3b and HS4 was found to cause a significant impairment in switching to most isotypes, although switching to IgG1 was unaffected (and IgA only moderately decreased).281 The diminished switching was associated with diminished germline transcription of the same isotypes, suggesting that an important function of the enhancers in CSR is to increase germline transcription. The relative independence of γ1 from regulation by enhancers may be related to the putative locus control region associated with that gene.282 Enhancers are believed to function via physical interactions between enhancer-bound proteins and promoterbound proteins, creating a DNA loop that brings enhancers into close proximity to promoters. Such looping may have special significance in promoting DNA recombination between segments of DNA lying great linear distances apart in the chromosome, as has been discussed in the context of V(D)J recombination. Chromosome conformation capture experiments have shown that Eμ and 3′-regulatory region enhancers are in close proximity in mature resting B cells, and that when B cells are stimulated to switch to γ1 by LPS + IL4 (or to γ 3 by LPS alone), the corresponding I region promoter moves close to the two enhancers.283,284 This looping would bring Sμ, which lies just downstream of Eμ, close to the Iγ1 (or Iγ 3) promoter, presumably facilitating recombination between Sμ and the Sγ region. Another parameter correlated with regional transcriptional regulation is the chromatin context of the genes, including specific modifications of histone proteins, as discussed previously in this chapter. For example, acetylation of histone H3 (H3Ac) is associated with DNA regions of increased “accessibility” to transcription (as well as to experimental digestion by restriction enzymes), while transcriptional promoters tend to be marked with trimethylation of H3 at lysine4 (H3K4me3). In B cells stimulated to switch to γ1 by LPS + IL4 (or to γ 3 by LPS alone), the corresponding I region and switch regions show increased H3Ac and H3K4me3 marks.285,286 The importance of the H3K4me3 mark for transcription and CSR is highlighted by evidence that preventing this mark—by B cell–specific knockout of PTIP, a component of the machinery that catalyzes this modification—leads to impaired germline γ transcription and defective CSR to γ isotypes.287 PTIP knockout B cells show decreased DNA looping between the 3′ enhancer region and Iγ region promoters, suggesting that PTIP contributes to this looping.288 Knockdown of other factors that are necessary to maintain H3K4me3 also reduce CSR efficiency, including the components of the complex known as FACT ( facilitates chromatin t ranscription).289 Surprisingly, a histone modification generally associated with gene silencing—trimethylation of H3 lysine 9 (H3K9me3)—has also been reported to mark switch regions targeted for CSR in both mouse and human B cells.290,291 Recently, a screen for nuclear proteins associating with AID in vitro identified KAP1 (KRAB domain–associated protein 1),292 a transcriptional repressor that associates with

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heterochromatin protein 1, as binding to H3K9me-modified chromatin. By coimmunoprecipitation, KAP1 was confirmed to bind to AID in vivo, and B-cell–specific conditional knockout of KAP1 was found to diminish AID binding to Sμ and to impair CSR efficiency by about 50%.

Switch Region Targeting of AID in Class Switch Recombination: R Loops, Paused Polymerase II, and AGCT A model in which AID loads onto an RNA polymerase complex and acts on DNA as the transcription complex travels downstream was discussed previously in the context of AID function in SHM, and a similar model apparently applies in the context of CSR. The domain of AID susceptibility in switch regions can be deduced from the distribution of C:G → T:A mutations in B cells that are defective for both UNG and MMR, because (as shown in Fig 6.13) in these cells the only AID-dependent mutations would be U:G mismatches resolved by replication to T:A. A study of the distribution of such mutations in clones from Ung−/−, Msh2−/− B cells found that a domain of mutations began about 150 bp 3′ of the Iμ transcription start site and extended 4 to 5 kb downstream, with diminishing mutation frequency near Cμ.293 The location of AID binding in B-cell DNA can also be directly determined by ChIP-seq analysis, as discussed previously. AID was found to bind to many loci that are transcribed in B cells; indeed, the patterns of AID and RNA polymerase II binding detected by ChIP are very similar.160 RPA, however, was efficiently bound only at IgH switch regions, and this binding was inhibited by the Ser38Ala mutation that blocks the critical Ser38 phosphorylation discussed previously. One likely consequence of germline transcription in facilitating CSR involves the formation of a stable RNA:DNA complex known as an R-loop. In this structure, RNA transcribed from the template strand of a DNA molecule binds tightly to that strand with Watson-Crick basepairing, displacing the other DNA strand, which forms a single-stranded loop. In support of the R-loop model, cell-free transcription across G-rich switch regions was found to generate a stable association of the transcript RNA with the template DNA 294 ; significantly, no substantial association occurred when the switch region was transcribed in reverse orientation, leading to a C-rich transcript, or when the transcribed template was a DNA fragment other than a switch region. The displaced DNA strand in S region R loops was susceptible to deamination by AID.155 Evidence that such R-loops form in vivo over S regions has been obtained from bisulfite analysis of singlestrand DNA regions in B cells.295,296 The tightly bound RNA-DNA complex on the template strand of an R-loop may explain both the high level of polymerase II accumulation and the AID-induced mutations in the vicinity of switch region DNA.297 Progression of the polymerase II complex transcribing through the switch region might be impeded by the RNA bound to the template strand, leading to an accumulation of “stalled” polymerase II molecules; the “stalling” of AID molecules associated with the transcription complex might prolong exposure of the DNA to deamination by AID. Some support for this scheme was reported from an in vitro model in which AIDtriggered mutations in a transcribed DNA substrate were

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increased when transcription was slowed by reducing the nucleotide triphosphate concentration.298 The importance of stalled polymerase II for CSR was reinforced by the results of a screen for factors whose knockdown by specific shRNAs would inhibit CSR 299 : one protein recovered in this screen was the murine homolog of suppressor of Ty 5 (Spt5), a transcription elongation factor known to be associated with stalled polymerase II. Spt5 knockdown decreased both CSR and switch region hypermutation, as well as AID occupancy of the Sμ region, without affecting cellular levels of AID protein or germline transcripts. By coimmunoprecipitation experiments, AID and Spt5 were found to associate. And genes with high Spt5 occupancy by ChIP analysis were found to be most susceptible to mutations induced by AID overexpression. Polymerase II stalling has been extensively studied in yeast and drosophila, and is found in many genes that require more rapid changes in expression than can be achieved by modulating transcriptional initiation.300 Apparently, the complex of proteins mediating polymerase II stalling has been adapted in B cells for the special function of regulating CSR of IgH genes. The R-loop model explains how the “upper” nontranscribed DNA strand would become single-stranded and accessible to AID, but it raises the question of how AID might gain access to the “lower” template DNA strand, held in a tight RNA-DNA hybrid. This strand is known to be accessible to AID because it undergoes C → T mutations in Ung − /− Msh2− /− double knockout B cells.293 A likely answer to this question has come from an analysis of proteins bound to AID when mixed with a B cell extract plus in vitro–transcribed switch region DNA.301 The complex of AID and transcribed DNA was found to bind to components of the multisubunit RNA exosome. The exosome is an evolutionarily conserved structure containing nine core proteins that can associate with RNA nucleases, leading to degradation of RNA from template DNA; the exosome could thus potentially expose the template DNA strand to AID. Indeed, shRNA knockdown of one exosome component in the murine B cell line CH12F3 inhibited CSR. Moreover, exosome components were found to associate with AID in vivo by immunoprecipitation experiments and were detected (by ChIP) bound to switch region DNA in cells activated for CSR. Finally, in a deamination assay of a model switch region DNA transcribed in vitro by T7 polymerase, the addition of AID + RPA + PKA led to deamination of only the nontemplate strand, but the further addition of exosome components led to deamination on the template strand as well. These experiments highlight the exosome as a likely candidate for explaining AID action on the template strand of R-loops, though an unhybridized “lower strand” may also be produced as a result of transcription-dependent supercoiling or antisense transcription. However, the propensity for R-loop formation is not the only property of switch regions that facilitates CSR. When the Sγ1 region was inverted, it retained about 25% of the wildtype CSR activity.302 As this inverted, and now C-rich, DNA segment could not form an R-loop, this result suggests that while the R-loop contributes to CSR, other features of the S region that are preserved in the inverted sequence also play a

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role. Replacement of a natural murine Sμ sequence with the AT-rich frog Sμ sequence, which cannot form an R-loop, supported somewhat reduced but still substantial frequencies of CSR, and it functioned equally well in either orientation. Significantly, in either orientation, the recombination junctions were clustered in a portion of the S region that is rich in repeats of the sequence AGCT, which is a special case of the WRC consensus sequence for AID targeting, being present on both strands as a self-complementary palindrome. Indeed, the AGCT-rich region was a good substrate for in vitro deamination by AID when transcribed in association with RPA. The AGCT motif is enriched in all mammalian S regions and may be particularly effective as a target for CSR because its presence in clusters on both strands promotes closely spaced nicks on opposite strands, or even a dsb with a single base overhang if the cytosines in both strands of the same AGCT motif are targeted.303 The density of AGCT in S regions correlates with the location of switch junctions better than the density of WRC or the boundaries of G-richness or R-loops.296 These results all suggest that clusters of AGCT may represent a target for CSR that evolved in amphibians, with R-loop formation evolving later in mammals to further enhance AID accessibility to S region DNA.

DNA Breaks as Intermediates in Class Switch Recombination The recombination event that underlies isotype switching includes DNA breaks and rejoining events that must involve both strands of DNA. Although the RAG-induced DNA breaks that initiate V(D)J recombination occur at the corresponding position on the two strands (yielding a blunt end and a hairpin), the nature of the ends in the initial CSR cleavage is not so clear. An early compilation of switch junctions304 found only infrequent instances of microhomology at the junction (i.e., short sequence segments that are identical in the unrearranged S sequences near the recombination breakpoint). As these microhomology examples would be consistent with invasion of one DNA strand from Sμ targeting a short homologous region in a downstream S region (or vice versa), the rarity of such junctions has been interpreted as an indication that CSR only rarely occurs by strand invasion and instead usually proceeds by ligation of blunt DNA ends. However, dsbs with staggered ends would generally be the result of the widely accepted mechanism shown in Figure 6.14 (bottom left): a DNA break on one strand might result from AID-catalyzed cytosine deamination, removal of the resulting uracil by UNG, and singlestrand cleavage 5′ to the abasic site by an endonuclease, probably apurinic-apyridinic endonuclease 1 (APE1). The initial staggered ends could be converted to blunt ends through exonuclease trimming of a 5′ or 3′ single strand overhang, through fi lling in of a shorter 3′ end by a DNA polymerase, or through a combination of both processes. Filling in by error-prone polymerases could explain the mutations commonly observed around the switch junction, as mentioned previously. Evidence supporting initially staggered DNA breaks in CSR was reported from occasional switch junctions observed in a model CSR substrate designed with two oppositely

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AID RNA pol

AID, bound to RNA polymerase, deaminates Cs in DNA S region repeats

Transcription through S region leads to R loop formation and extensive deamination of upper DNA strand U U U

A RN RNA is removed from “lower” template strand by exosome, allowing AID to target this strand U U U U

e U U

U

U U:G mismatch recognized by MMR; Exo I clears DNA patch on strand with U; apposed gaps form dsb dsb

U

U

U

UNG removes uracils; abasic sites cleaved by APE1; closely spaced nicks lead to dsb

dsb

FIG 6.14. Mechanistic Model of Class Switch Recombination. This figure incorporates features of the most widely accepted models, but some aspects are uncertain at present. Two strands of deoxyribonucleic acid (DNA) near the 5′ boundary of a switch region are shown in black, with the S region repeats indicated by dots. DNA with normal Watson-Crick basepairing is indicated by the gray shading between the DNA strands. Ribonucleic acid (RNA) polymerase moves to the right, transcribing the sequence into an RNA strand; multiple other proteins accompany the RNA polymerase, but only activation-induced deaminase (AID) (black circle) is shown. AID can deaminate C residues to U in single-stranded R-looped DNA displaced by the RNA-DNA complex. The exosome complex (e in the drawing) degrades RNA in the RNA-DNA hybrid, leaving an unpaired lower strand where AID can deaminate C residues. DNA cleavage triggered by processing of uracils can lead to double-strand breaks in pathways dependent or independent of mismatch repair components as shown.

oriented S regions such that CSR would occur by inversion, preserving both recombination junctions on the same chromosome. Several duplications at the ends of inverted DNA after CSR suggested that complementary overhangs at dsbs had been fi lled in before joining.305 Additional evidence for staggered breaks has come from several investigators using ligation-mediated–polymerase chain reaction (LM-PCR) to detect double-strand DNA ends at switch regions in cells undergoing CSR.306–308 LM-PCR protocols involving ligation of a blunt linker directly to blunt ends from genomic DNA were successful in amplifying blunt ends from DNA of B cells activated for CSR, but when the DNA was pretreated with T4 polymerase, which would convert staggered end cuts to blunt, the yield of amplified LM-PCR products was significantly increased, suggesting that most of the ends in the isolated genomic DNA (before T4 polymerase treatment) were staggered.

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When the CSR-associated dsbs in Sμ were examined by LMPCR in B cells from normal and AID − /− individuals, the dsbs were significantly fewer in AID − /− B cells, though not completely absent. Furthermore, microscopic foci of the modified histone γH2AX (which rapidly accumulates at dsbs) were found at IgH genes (localized by FISH) in B cells undergoing CSR; these foci were strikingly diminished in AID − /− B cells, consistent with AID-dependence of dsbs in CSR.309 These foci, as well as switch region dsbs, occur predominantly in the G1 phase of the cell cycle.310 Like the off-target deamination by AID discussed previously in the context of SHM, off-target DNA breaks are another potential consequence of AID. B cells from mice overexpressing AID showed a high incidence of chromosomal translocations and DNA breaks compared with Aicda− /− mice; in the context of homozygous p53 deficiency (which allows cell growth despite DNA damage), many of the mice developed B-cell lymphomas.311 In a study designed to systematically examine the locations of dsbs in mouse B cells stimulated to undergo CSR, ChIP was used to identify DNA associated with Nbs1, a protein that rapidly binds to dsbs in vivo, as discussed below.312 This analysis detected hundreds of reproducible AIDdependent DNA break locations, some of which were syntenic with DNA rearrangements found in human B-cell lymphomas. Many of the AID-dependent dsbs occurred in nontranscribed regions, unlike the CSR-related dsbs in IgH switch regions. Evidence indicates that AID-dependent off-target dsbs are normally repaired largely by homologous recombination: B cells defective for the homologous recombination component XRCC2 were found to harbor many more γ-H2AX foci than were found in Xrcc2+/+ cells or in Aicda− /− Xrcc2− /− cells.313 B cells apparently protect against these breaks by upregulating Xrcc2 transcription when activated for CSR.

Ubiquitously Expressed Components of Class Switch Recombination Machinery UNG and Mismatch Repair. The model of Figure 6.13 suggests that after AID-catalyzed deamination of a cytidine residue, the resulting uracil is removed by UNG, creating an abasic site that is converted by APE1 to a single-strand DNA break (nick), which can become double-stranded if there is a nearby nick on the opposite strand. This model predicts that both CSR and the creation of dsbs would be severely impaired in the absence of UNG. Indeed, B cells from Ung− / − mice showed almost complete inability to switch in vitro to IgG1 or IgG3 secretion, and significant impairment in IgA secretion.151 dsbs in Ung−/ − B cells, as detected by LM-PCR, were also significantly reduced, but not abolished.308 Human patients with homozygous UNG deficiency due to mutations in both UNG alleles showed a hyper-IgM phenotype, with a more profound defect than in Ung−/ − mice: the patients showed essentially no IgG, IgE, or IgA secretion by stimulated B cells, and no dsbs (by LM-PCR) in Sμ.314 A role for APE1 or APE2 in CSR is supported by the observation that in mice with engineered deficiencies these genes, B cells induced for CSR show decreased switching and decreased induction of dsbs.315 The complementary pathways of UNG and MMR action in SHM were discussed previously (see Fig. 6.13); current evidence suggests a similar participation of MMR in CSR. Although

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CSR was dramatically impaired in Ung− / − B cells (e.g., in vitro switching to IgG1 was reduced to about 6% of wild-type), the double knockout Ung− / − Msh2− / − caused significant further impairment (to 1.5% of wild-type IgG1 switching).316 Evidence suggests that the C-terminal 10 amino acids of AID that are required for CSR but not SHM may function by stabilizing the interaction of UNG2 and the MSH2-MSH6 dimer to DNA.317 If a single-strand break created on one strand by UNG and APE1 is too far away from the the closest single-strand break on the opposite strand to create a dsb, then Exo1 engaged by MMR can chew from a nick on one strand toward a nick on the opposite strand, creating a dsb.310,318 Consistent with this idea, mice engineered with a knockout of the MMR component Exo1 show a significantly decreased efficiency of CSR, to roughly 15% to 30% of normal.319 Thus, many of the same AID-triggered mechanisms that induce mutations in V H regions operate in switch regions to induce dsbs. Once created, these dsbs are resolved by mechanisms distinct from the mechanisms resolving mismatches in SHM; however, as discussed in the following, some of the components that resolve dsbs generated in V(D)J recombination play a similar role in CSR. End Joining Proteins in Class Switch Recombination. If AID triggers DNA cleavage at switch regions, ubiquitous DNA repair and ligation enzymes could participate in the subsequent DNA repair steps of CSR, as in V(D)J recombination. This possibility has been tested by engineered gene knockouts. However, as NHEJ knockouts would impair V(D)J recombination, thus blocking B-cell development prior to the stage of CSR, investigators have studied NHEJ in CSR using mouse strains with knockins of productive recombined H and L chain genes, which can undergo CSR. IgH/IgL knockin mice with intact Ku genes were able to switch to downstream isotypes, but the corresponding Ku-70– or Ku-80–deficient mice were reported to be dramatically impaired in CSR, although decreased cell proliferation could have contributed to this effect as more recent studies using different conditions report 30% to 50% residual CSR activity.320 The other “core” factors of NHEJ—XLF/Cernunnos, Ligase4, and XRCC4—catalyze the DNA ligation in NHEJ and have also been investigated for participation in CSR.320–324 B cells deficient in any one of these genes show variably decreased CSR efficiency, with increased junctional microhomologies compared with normal B cells. This observation suggests that one or more fairly robust backup pathways— commonly called alternative NHEJ—can repair dsbs using microhomology-based ligation when “classic” NHEJ is inoperative. This conclusion is consistent with studies of alternative NHEJ repair of dsbs unrelated to CSR.78 The proteins CtIP,325 PARP1,326,327 and XRCC1328 apparently contribute to alternative NHEJ during CSR, as experimentally reduced expression of each protein decreases microhomology at CSR junctions. Apart from these core NHEJ components, Artemis and DNA-PKcs are required for joining ends that require processing, like the hairpin coding ends produced in V(D)J recombination discussed previously in this chapter. Artemisdeficient murine B cells have no obvious impairment in CSR efficiency329 but show several-fold increases (compared to

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wild-type) in the number of chromosome aberrations in the IgH locus detected by FISH assays330 ; these aberrations are AID-dependent and only observed after activation of B cells for CSR. In humans, Artemis deficiency results in a SCID syndrome with severe defects in B- and T-cell development, but rare Sμ-Sα recombination junctions amplified from patient B cells were found to show a high index of microhomologies,331 suggesting that Artemis is required for normal classic-NHEJ resolution of dsbs in human CSR. Studies on the role of DNA-PKcs in CSR have yielded somewhat conflicting conclusions on whether this protein is required for maximally efficient CSR, with the results of individual studies perhaps depending on the knocked-in Ig genes used, the specific mutations of DNA-PKcs, or the genetic background of mice studied.332,333 However, DNAPKcs − / − B cells showed evidence of AID-dependent chromosome aberrations in the IgH locus, similar to those seen in Artemis−/− cells but more numerous.330 Thus it appears that both Artemis and DNA-PKcs are required for efficient repair of at least a subset of the dsbs associated with CSR. DNA Damage Response Proteins in Class Switch Recombination. NHEJ is one component of a larger mechanism for detecting and repairing dsbs, collectively known as the DNA damage response (DDR). Because DNA dsbs are potentially damaging to the cell, and occur in all cells through accidents of DNA metabolism, toxic chemicals, and radiation, DDR appeared early in evolution, and many components are conserved from yeast to mammals. Defects in DDR can cause developmental abnormalities, cancer predisposition, and sensitivity to radiation, as well as immunodeficiency resulting from impaired V(D)J recombination or CSR. DDR factors participate in a baroque cascade of interactions to cluster at dsbs and initiate repair. The complexities of the DDR are beyond the scope of this chapter, but are described in several recent reviews.78,334 Several DDR components have been documented to participate in CSR and are listed below; mutations in some of these cause specific genetic syndromes in humans, often associated with immunodeficiency. 1. Nbs1, product of the gene that is defective in the human disease Nijmegen breakage syndrome.309,335,336 Nbs1 functions as part of a complex that also includes Mre11 and Rad50 (MRN complex). Mre11 has a nuclease activity, and may contribute to DNA cleavage in CSR; mice and humans with defective Mre11 also show impaired CSR.337,338 2. ATM, product of the gene mutated in the human disease ataxia telengectasia and a member of the phosphatidylinositol-3′-kinase family.339,340 3. γ-H2AX—the phosphorylated form of histone H2AX— which rapidly accumulates in foci at dsbs, and helps to assemble other proteins at dsbs to prevent the breaks from progressing to chromosome translocations.341 4. The E3 ubiquitin ligase RNF8 (Ring Finger 8), which is known to monoubiquitinate histones at dsbs.342 5. The E3 ubiquitin ligase RNF168, found to be mutated in the RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features, learning difficulties) syndrome.343

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6. 53BP1 (p53 binding protein 1) originally discovered as a protein binding to the tumor suppressor p53, but subsequently found to function in checkpoint control and to localize rapidly to DNA breaks in vivo. 53BP1−/− murine B cells show dramatic impairment in CSR (to 55 to 25% of wild-type), but increases in the frequency of AID-triggered deletions within Sμ,344 suggesting that formation of dsbs and repair of closely-spaced breaks does not require 53BP1, but joining of more distant dsbs depends on this protein. Although the we currently lack a detailed model explaining the functions of all the DDR factors in dsb repair and specifically in CSR, the emerging evidence suggests that these proteins have mutual interactions and distinct but related roles, so that the elimination of any one protein reduces CSR efficiency but permits residual CSR to occur by pathways that remain intact. Other Proteins that may Target AID for Class Switch Recombination. As discussed in the context of SHM, accurate targeting of AID is important since off-target activities of this protein can be deleterious, and off-target dsbs triggered by AID could be particularly dangerous. Several searches for proteins that might contribute to targeting of AID to switch regions have uncovered candidates for this function. In one study,345 biotinylated AID was used as bait to fish for in vitro binding proteins, which were identified by mass spectrometry. One protein identified by this screen was PTBP2 (polypyrimidine tract-binding protein 2) which is considered to be a regulator of RNA splicing. Knockdown of PTBP2 by shRNA in CH12 B cells caused a substantial decrease in binding of AID to Sμ (determined by ChIP) and significant impairment of CSR. The possible role of a splicing regulator in CSR could be related to the puzzling observation that germline transcripts must be spliced in order for efficient CSR, 280 as mentioned previously. A screen for proteins that could bind selectively in vitro to AGCT sequences—which are found clustered in many switch regions—identified another protein that might target AID in CSR: the 14-3-3 proteins, a family of seven members widely expressed and known to bind to many signaling proteins.346 ChIP assays on B cells stimulated for CSR showed that 14-3-3 proteins bind to switch regions in an isotypespecific way (depending on stimulus conditions) in both normal and Aicda−/− cells, and reduction of 14-3-3 activity by either a peptide inhibitor or by genetic engineering decreased both CSR efficiency and AID binding to switch region DNA. Finally, a bimolecular fluorescence complementation assay revealed that in B-cell nuclei AID and 143-3 molecules form a complex that is dependent on the AID C-terminal amino acids required for CSR. These experiments suggest that 14-3-3 proteins are additional candidates for AID targeting molecules. PKA, the cAMP-regulated protein kinase responsible for phosphorylation of AID Ser38 discussed previously, has also been suggested as a candidate that targets AID-dependent DNA cleavage to switch regions.347 PKA was found by ChIP to bind to switch regions of stimulated B cells from normal and Aicda−/−mice, whereas the binding of RPA depended

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on Ser38-phosphorylated AID. Cellular concentrations of cAMP were found to rise rapidly after stimulation to CSR, and a genetic inactivation of PKA activity that prevented its activation by cAMP reduced both RPA binding and CSR. These data suggest a model in which CSR stimuli recruit both PKA and nonphosphorylated AID to switch regions, and AID gains the ability to recruit RPA and trigger dsbs efficiently only when cAMP-activated PKA phosphorylates Ser38. Additional components of CSR may be discovered by analysis of patients with a hyper-IgM phenotype unexplained by defects in known components.314

CONCLUSION Recombinant DNA technology has revolutionized the study of the antibody response. Initial investigations used powerful cloning and sequencing methods to define the structure of the Ig genes as they exist in the germline and in actively secreting B-lymphocytes. Subsequent experiments have begun to shed light on the mechanisms of the processes unique to these genes: V(D)J recombination, CSR, and SHM. The knowledge of Ig genes gained so far has answered some of the most puzzling mysteries about antibody diversity, as discussed previously, and has also led to many practical ramifications involving these genes that are beyond the scope of this chapter. As one example, cloned Ig genes have allowed the production of recombinant monoclonal antibodies and the bioengineering of Ig-fusion proteins that exploit the exquisite specificity of antibody V region binding (e.g., antibody-toxin fusions) or the ability of Ig C region domains to extend serum half-life or engage Fc receptors. Other engineered derivatives utilizing Ig genes include single-chain antibodies, bispecific antibodies, and “intrabodies” designed not to be secreted from a cell but rather to bind to intracellular targets.348–350 Ig V gene fragments cloned into bacteriophage so as to express single-chain V regions on the phage surface (phage display libraries) can be used to obtain specific monoclonal antibodies without immunization or use of mammalian cells, and in vitro mutation and selection protocols can mimic affi nity maturation to yield high-affi nity antibodies.351 Even Ig gene regulatory regions have been exploited to achieve B-cell–specific expression of oncogenes352 and of intracellular toxins that could be used to target B lymphomas.353 Apart from these biotechnology applications, Ig gene probes have led to the identification of numerous proto-oncogenes that become activated by translocation into Ig gene loci.354 For instance, Bcl2 was initially discovered as the target of Ig H chain translocation in follicular lymphoma, and provided an entry into an entire family of apoptosis-related genes. A fi nal example of medical benefit from Ig gene technology has been the use of patient-specific Ig gene rearrangements of leukemias or lymphomas to monitor disease status by PCR.355,356 Further practical applications of Ig genes can be anticipated in the future, as well as a deeper scientific understanding of their molecular biology and their contribution to the immune system.

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7

Antigen–Antibody Interactions and Monoclonal Antibodies Jay A. Berzofsky • Ira J. Berkower

INTRODUCTION The basic principles of antigen–antibody interaction are those of any bimolecular reaction. Moreover, the binding of antigen by antibody can, in general, be described by the same theories and studied by the same experimental approaches as the binding of a hormone by its receptor, of a substrate by enzyme, or of oxygen by hemoglobin. There are several major differences, however, between antigen– antibody interactions and these other situations. First, unlike most enzymes and many hormone-binding systems, antibodies do not irreversibly alter the antigen they bind. Thus, the reactions are, at least in principle, always reversible. Second, antibodies can be raised, by design of the investigator, with specificity for almost any substance known. In each case, one can fi nd antibodies with affi nities as high as and specificities as great as those of enzymes for their substrates and receptors for their hormones. The interaction of antibody with antigen can thus be taken as a prototype for interactions of macromolecules with ligands in general. In addition, these same features of reversibility and availability of a wide variety of specificities have made antibodies invaluable reagents for identifying, quantitating, and purifying a growing number of substances of biologic and medical importance. One other feature of antibodies that in the past proved to be a difficulty in studying and using them, compared to, say, enzymes, is their enormous heterogeneity. Even “purified” antibodies from an immune antiserum, all specific for the same substance and sharing the same overall immunoglobulin (Ig) structure, will be a heterogeneous mixture of molecules of different subclass, different affinity, and different fine specificity and ability to discriminate among crossreacting antigens. The advent of hybridoma monoclonal antibodies1–3 has made available a source of homogeneous antibodies to almost anything to which antisera can be raised. Nevertheless, heterogeneous antisera are still in widespread use and even have advantages for certain purposes, such as precipitation reactions. Therefore, it is critical to keep in mind throughout this chapter, and indeed much of the volume, that the principles derived for the interaction of one antibody with one antigen must be modified and extended to cover the case of heterogeneous components in the reaction. In this chapter, we examine the theoretical principles necessary for analyzing, in a quantitative manner, the interaction of antibody with antigen and the experimental techniques that have been developed to study these interactions

as well as to make use of antibodies as quantitative reagents. Furthermore, we discuss the derivation, use, and properties of monoclonal antibodies.

THERMODYNAMICS AND KINETICS The Thermodynamics of Affinity The basic thermodynamic principles of antigen–antibody interactions, as we indicated previously, are the same as those for any reversible bimolecular binding reaction. We review these as they apply to this particular immunologic reaction.

Chemical Equilibrium in Solution For this purpose, let S = antibody binding sites, L = ligand (antigen) sites, and SL = the complex of the two. Then for the reaction S + L D SL

(1)

the mass action law states [SL] K A = ______ [S][L]

(2)

where K A = association constant (or affinity) and square brackets = molar concentration of the reactants enclosed. The import of this equation is that for any given set of conditions such as temperature, pH, and salt concentration, the ratio of the concentration of the complex to the product of the concentrations of the reactants at equilibrium is always constant. Thus, changing the concentration of either antibody or ligand will invariably change the concentration of the complex, provided neither reactant is limiting, that is, neither has already been saturated, and provided sufficient time is allowed to reach a new state of equilibrium. Moreover, because the concentrations of antibody and ligand appear in this equation in a completely symmetrical fashion, doubling either the antibody concentration or the antigen concentration results in a doubling of the concentration of the antigen–antibody complex, provided the other reactant is in sufficient excess. This proviso, an echo of the one just discussed, is inherent in the fact that [S] and [L] refer to the concentrations of free S and free L, respectively, in solution, not the total concentration, which would include that of the complex. Thus, if L is not in great excess, doubling [S] results in a decrease in [L] as some of it is consumed in the complex, so the net result is less than a doubling of [SL]. Similarly, halving the volume results in a doubling of the total concentration of both antibody and

183

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FIG. 7.1. Schematic Plot of Bound Ligand Concentration as a Function of Free Ligand Concentration at a Constant Total Concentration of Antibody-Combining Sites, [S]t. The curve asymptotically approaches a plateau at which [bound ligand] = [S]t.

ligand. If the fraction of both reactants tied up in the complex is negligibly small (as might be the case for low-affinity binding), the concentration of the complex quadruples. However, in most practical cases, the concentration of complex is a significant fraction of the total concentration of antigen or antibody or both, so the net result is an increase in the concentration of complex but by a factor of 20 years for HAE, new formulations now FDA approved Made in Tg rabbits, in phase 3 trials for HAE (Rhucin) Models only, no significant human trials

Inhibitor of C1r/s, MASPs, kallikrein, and other plasma proteases As above Small molecules, specific inhibitors of fD protease activity

Soluble forms of natural complement regulators

sCR1 (TP10; TP20) APT070 (Microcept) CAB-2 (MLN-2222) Recombinant MBL Recombinant fH

Phase 2 trials CPB, early trials in stroke, MI, others Preclinical in renal transplantation, development stages in others Phase 1 trials in CPB Preclinical as substitution therapy Preclinical, substitution therapy in AMD

TT30

Preclinical for AP-mediated diseases

CP/AP C3 convertase regulator SCR 1–3 of CR1 on membrane-targeting tail, convertase regulator DAF:MCP hybrid, convertase regulator Replacement of deficient protein Increasing levels of “protective” fH; convertase regulator CR2-fH hybrid, targeted convertase regulator

Available for PNH, aHUS; other indications in development Short-acting variant of above; phase 2 for MI, CPB Preclinical, inflammation Preclinical, arthritis, stroke Preclinical, AMD Preclinical Preclinical

Humanized mAb, C5 cleavage blocker; inhibits C5a production, MAC As above Humanized mAb, binds/blocks C5a Humanized mAb, blocks C5aR Humanized mAb, blocks fD enzyme Humanized mAb, blocks fB binding Humanized mAb, blocks properdin

Compstatin (POT-4) OmCI

Phase 1, AMD Preclinical, AMD

ARC1905 PMX-53 JPE-1375

Preclinical, AMD Phase 2, arthritis, psoriasis Preclinical, inflammation

Cyclic peptide, binds C3, blocks cleavage Tick-derived C5-binder, inhibits C5a production, MAC RNA aptamer; binds/blocks C5 Peptide C5a antagonist Peptide C5a antagonist

Antibodies

Anti-C5 (Eculizumab) Anti-C5 (Pexelizumab) Anti-C5a (TNX-558) Anti-C5aR (Neutrazumab) Anti-fD (TNX-334) Anti-fB (TA106) Antiproperdin Complement protein blockers

aHUS, atypical hemolytic uremic syndrome; AMD, age-related macular degeneration; AP, alternative pathway; CP, classical pathway; CPB, cardiopulmonary bypass; CR1, complement receptor 1; CR2, complement receptor 2; DAF, decay accelerating factor; fB, factor B; fD, factor D; FDA, U.S. Food and Drug Administration; fH, factor H; HAE, hereditary angioedema; MAC, membrane attack complex; MASP, mannan-binding lectin–associated serine protease; MBL, mannan-binding lectin; MCP, membrane cofactor protein; MI, myocardial infarction; PNH, paroxysmal nocturnal hemoglobinuria; RNA, ribonucleic acid; SCR, short consensus repeat.

in a myocardial ischemia model, sCR1 delivered at the time of reperfusion markedly reduced the size of the resultant infarct, effectively protecting the myocardium from further injury by complement. Soluble CR1 was tested in many other animal models of disease, spanning the spectrum from ischemia through autoimmune to degenerative, and proved effective in most conditions.116,121 Despite its capacity to inhibit complement activation in vivo in man, clinical trials of sCR1 (developed latterly by Avant Therapeutics, Needham, MA, and known as TP10) have been disappointing. Development for the original application, adult respiratory distress syndrome, was abandoned after a single, small trial. Some success was obtained in use post-lung transplant where sCR1 treatment significantly shortened time on respirator postoperatively,122 while in a large-scale trial in highrisk patients on cardiopulmonary bypass, sCR1 reduced the incidence of post-bypass myocardial infarction and death in men but not women—an unexpected finding confirmed

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in a second trial.123 Although still in development for this and a few other applications, the history of sCR1 remains one of unfulfi lled promise. Modified forms of sCR1, including addition of carbohydrate groups (sLex) to target sites of inflammation (TP20), and the engineering of small (three SCRs), membrane-localizing forms (APT070), may prove of more clinical benefit in the future. The success of sCR1, at least in animal models, led to the engineering of soluble forms of other membrane C3 convertase regulators, DAF, MCP, and hybrids of DAF and MCP (CAB-2), all of which have proved effective in at least some animal models, but none have progressed beyond the earliest stages of clinical testing. Similarly, attempts to develop soluble forms of CD59 as therapeutics have been unsuccessful, likely because of the small size of the molecule and its propensity to bind plasma proteins. Very recently, a fusion protein in which the C3d-binding SCRs of CR2 are fused to the complement APinhibiting amino terminal SCRs of fH has been described.124

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This agent, which binds C3d at sites of complement activation and there inhibits complement activation, has excellent AP-inhibitory activity in vitro and in vivo in monkeys, and is being fast-tracked for treatment of AP-mediated diseases.

Plasma Regulators as Anticomplement Drugs The plasma regulators of complement activation are also potential therapeutics; indeed, the exception referred to previously is the enormous success achieved in treating HAE, caused by deficiency of C1inh, with plasma-derived C1inh. Frank and coworkers showed 30 years ago that C1inh purified from plasma could terminate acute attacks in patients with HAE.125 Commercial preparations of pasteurized, purified C1inh were widely available within a few years and have been used since in Europe and some other locations to great effect, saving the lives of many sufferers. Unfortunately, use of C1inh in the United States fell foul of suspicions that it was a vector for transmission of hepatitis and other viruses, removing from use the best means of treating attacks. Events in the last few years have changed the scene. New formulations of plasma-derived C1inh, subjected to ultrafi ltration to further reduce risk of virus contamination, and recombinant forms of C1inh are now approved by the U.S. Food and Drug Administration (FDA) and in use both for treatment of acute attacks and prophylaxis in HAE.126 Both plasma-derived and recombinant fH are also in development as potential therapeutics, driven in large part by the strong genetic link, noted previously, between the fH-Y402H variants and AMD. Treatment of AMD patients with the “protective” Y402 variant, either local (intraocular) or systemic, is suggested but remains untested clinically.

Monoclonal Anticomplement Antibodies as Drugs A number of blocking monoclonal antibodies, capable of switching off complement activation in vivo, have been tested with good effect in animal models of complementmediated disease. Monoclonal antibodies targeting either the AP (anti-fB, anti-fD) or TP (anti-C5, anti-C6) have proved particularly effective in models. Several of these reagents are being developed as human therapeutics; the first of these to reach the market is a humanized monoclonal anti-C5 antibody, eculizumab, marketed under the trade name Soliris™.127 This monoclonal antibody binds human C5 with high affinity and prevents its cleavage by the convertase enzyme, thus blocking C5a generation and MAC assembly. Eculizumab was first approved for use in the rare hemolytic disorder PNH in which erythrocytes and other blood cells are rendered susceptible to complement damage because they lack the GPI-anchored regulators DAF and CD59. Erythrocytes hemolyse spontaneously, releasing hemoglobin into the plasma and, via renal fi ltration, the urine, leading to the characteristic dark urine. Patients become anemic as erythrocyte loss exceeds rate of production and are often dependent on large, frequent blood transfusions. Thrombotic episodes and strokes can prove fatal. Administration of eculizumab dramatically reverses these symptoms; erythrocyte hemolysis ceases, patients become

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transfusion independent, and risk of stroke markedly decreases. Maintenance with twice-weekly dosing maintains this spectacular remission for many months or years.128 In 2011, eculizumab was approved by the FDA for treatment of aHUS, a major advance for a disease that was previously untreatable except by dialysis.129 Epidemic HUS, caused by infection with Shiga toxin–producing strains of Escherichia coli, closely resembles aHUS in terms of pathology but is an acute disease ending either in recovery or renal failure. Emergency use of eculizumab in a very large outbreak of HUS in Germany proved remarkably effective, rescuing many critically ill patients.130

Small Molecule Complement Inhibitors as Drugs A limiting factor in the use of recombinant proteins and monoclonal antibodies as anticomplement agents is cost; eculizumab treatment of PNH currently costs in excess of $500,000 per patient per year, making it the most expensive FDA-approved drug! The quest for an inexpensive small molecule inhibitor of complement, preferably orally active, has been a long one, and there are still no ideal agents available. Most promising among the current candidates is a C3-/C3b-inhibiting peptide, compstatin, which tightly binds its ligand thereby blocking complement activation via all pathways.131 Compstatin has been shown to be an effective complement inhibitor in vivo in primate models (it does not work in rodents). Compstatin is currently in phase I trials for treatment of AMD where local (intraocular), slowrelease formulations may prove particularly useful. A number of other synthetic and naturally occurring molecules have been shown to be effective inhibitors of complement activation in vitro and in models, but none have yet entered clinical trials. The G-protein–coupled heptaspan receptors for C3a and C5a are excellent drug targets, and a number of small molecule blockers of the C3a and/or C5a receptors have been reported.132 In animal models, these act to reduce the inflammation associated with complement activation. A cyclic hexapeptide, F[OpdChaWR], blocks the C5a receptor and inhibits inflammation in a broad range of animal models and is in development for human use with sepsis likely to be the first target.133

NOVEL ROLES OF COMPLEMENT In addition to its roles as innate immune effector, complement is increasingly being implicated as an important player in a number of other physiological and pathological processes. The list of novel roles is long and growing, and here I will briefly describe just a few as an illustration of this burgeoning field of interest.

Complement as a Regulator of B-Cell Responses The fi rst indication that complement influenced adaptive immunity came from the work of Pepys who, in the 1970s, showed that C3 depletion in mice treated with cobra venom factor or other complement activators caused a marked reduction in antibody response to a range of antigens.134

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iC3b

C3b C3d Ag

CR2/ CD21

CR1/CD35

CD19 mIg

CD 79

CD 79

Although a number of mechanisms were suggested at the time, it was more than a decade before Fearon and coworkers showed that C3 fragment–coated (opsonized) antigen was a much more efficient activator of B cells than unopsonized antigen because it simultaneously engaged the BCR (surface IgM) and CR2, assembled in a signaling complex on the B-cell surface.135 The signaling complex comprised surface IgM tightly linked to the phosphatase enzyme CD45, adjacent to CR2, and linked to the Ig superfamily member CD19 and the tetraspanin molecule tetraspanin CD81, which connects the complex to the intracellular signaling moiety PI-3 kinase (Fig. 36.16). Although antigen-binding BCR can alone cause B-cell activation, large amounts of antigen are needed; if the antigen is coated with iC3b/C3d, it binds both CR2 and the BCR, lowering the B-cell antigen response threshold by up to 1000-fold! CR1 on the B cell also plays an important role by fi rst capturing C3b-coated antigen, then acting as cofactor for the fI-mediated cleavage of C3b into iC3b and C3d, the ligands for CR2. Recognition of the importance of CR2 in regulating B-cell responses led to a flurry of interest in using C3d, coupled to pathogen-derived antigens, as super-vaccines; promising results in animal models have yet to translate to humans.

Complement and T-Cell Activation Most published work suggests that T cells do not express classical complement receptors in significant amounts. Expression of CR2 is controversial, with some reporting expression, particularly on activated T cells, and suggesting that CR2 might play a role in driving T-cell activation. CD8 T cells certainly express receptors for C3a, and several studies have suggested that local activation of C3

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CD81

FIG. 36.16. The B-cell Signaling Complex. Antigen coated with C3 fragments (opsonized) binds the B-cell receptor (mIg) to trigger B-cell activation. C3d fragments on the antigen, produced by factor I degradation in the presence of complement receptor 1 as cofactor, bind complement receptor 2, present on the B cell in a signaling complex with CD19 and CD81. Co-ligation of the B-cell receptor and complement receptor 2 signaling complexes markedly lowers the threshold for B-cell response to antigen.

and production of C3a, and C5a, might be an important part of T-cell help and a trigger to T-cell activation.136 The complement regulator MCP (CD46) is expressed on T cells and also plays roles as a costimulator in T-cell activation; coligation of CD3 and CD46 in the presence of interleukin-10 drives T cells to differentiate into T regulatory cells that in turn dampen activation of bystander cells.137 The natural ligand for MCP in this context is C3b, further implicating C3 products in T-cell control. Indeed, a growing body of evidence indicates that MCP and its ligand C3b are key controllers of T-cell fate and modulate T-cell cytokine production in health and disease.

Interactions with Toll-like Receptors The toll-like receptor (TLR) family of pattern recognition molecules are an ancient immune defense system that act by sensing and signaling cell responses to infection. TLRs recognize a broad range of bacterial and viral products, present either outside or inside the cell, and set in train cytokine production and other appropriate cell responses to infection. Several of the pathogen TLR agonists, including lipopolysaccharide (from bacterial cell walls) and zymosan (from yeast cell walls) are efficient activators of complement; hence, these agonists trigger a double-hit through the TLR and through complement activation. Complement activation products, specifically C5a and, less strongly, C3a, enhance the cytokine response triggered by engagement of the TLRs, although the precise mechanism remains a subject of debate.138 Some studies strongly implicate the C5L2 receptor in these events and, indeed, TLR activation of cells markedly increases their subsequent response to C5a in a C5L2-dependent manner.139 Cross-talk between TLRs and complement via C5a has been implicated in T-cell

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C3/C3b cleavage: Captured fH (C4bp) Bacterial proteases

C5b Bb

C6 C7

C3b C3d

C3b Inhibition of MAC: Capsule Captured S protein

Evasion and Hijacking of Complement by Pathogens Complement exists to rapidly identify and destroy invading bacteria, a vital pillar of innate immune defense. Driven by this selective pressure, bacteria and some other pathogens have evolved numerous ways of evading complement activation and killing (Fig. 36.17).140 First among these are the physical barriers; for example, gram-positive bacteria possess cell walls that protect from MAC killing. Many pathogens have evolved the capacity to mimic or steal the human complement regulators to subvert complement attack. Recruitment of host plasma complement regulators C4bp and/or fH is a particularly common occurrence and many pathogens, notably Borellia , Neisseria, and Streptococcus, have evolved specific membrane proteins, virulence factors that act to capture these regulators, thereby protecting the pathogen surface.141 Some pathogens acquire membrane regulators such as CD59 and DAF from host cells, either by physical interaction with the cells, or for some viruses, during their release from infected cells. Several viruses have evolved soluble complement regulators that are structural and functional analogues of the human regulators; for example, vaccinia virus and smallpox virus both encode complement control proteins made up of SCRs that have both decay accelerating and cofactor activities,142 while Borellia burgdorferi expresses a CD59like MAC inhibitor.143 Other pathogens have evolved enzymes that efficiently degrade complement proteins or activation products as a means of avoiding complement

Bacterium

C8 C7

Capsule

C6 b

activation, and other complement molecules, including the gC1qR, CR3, and MCP, have been shown to independently interact with TLRs. The growing understanding of the interactions between these two innate immune effector systems will likely inform better approaches to therapeutic intervention in the future.

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C5

FIG. 36.17. Bacterial Complement Evasion Strategies. Many bacterial species have evolved elaborate defense strategies to protect against complement opsonization and killing; some of the more common are illustrated here. The capsule, where present, provides a physical barrier to bacterial membrane damage but may itself be a target for opsonization. C3 fragments and C3 convertases are targeted by secreted proteases and by C3 convertase regulators; they are either pirated from the host or expressed in the bacterial genome. Membrane attack complex formation is inhibited not only by capsule but also by terminal pathway regulators, either captured (such as S protein) or expressed by bacteria (CD59like molecules).

COMPLEMENT

Inhibition of MAC: Bacterial “CD59” Captured CD59

damage; still others possess binding proteins that mop up complement proteins onto the pathogen surface. Among pathogens, Staphylococcus aureus stands out as an escape artist extraordinaire, expressing factors that inhibit complement activation, digest complement proteins, block complement receptors, and mimic complement regulators at multiple stages.144 Some viruses go one step further: not content with avoiding complement killing, they turn complement to their advantage. Several viruses use complement regulators and receptors as entry routes into the cell; the measles virus binds MCP, the Epstein-Barr virus binds CR2, and the Coxsackie virus binds DAF. MCP is used as a receptor by many different pathogens, leading to its colorful description as a “pathogen magnet.”145 Human immunodeficiency virus takes yet further liberties, inviting complement activation in order to acquire surface C3 fragments that are then used as a passport for cell entry by binding CR3 on host cells.146

Complement and Lipid Metabolism In the late 1980s and early 1990s, two separate lines of research converged to implicate complement as a major regulator of lipid metabolism in adipose tissue. Speigelman’s group showed that an adipose tissue–derived protein they termed adipsin, implicated in regulation of fat mass, was identical to the complement AP enzyme fD.147 They also showed that adipocytes synthesized not only fD but also the AP components fB and C3, everything needed to generate AP activity locally. Meanwhile, Cianflone was investigating a remarkable plasma protein termed acylation-stimulating protein, which was a powerful stimulus for triacylglycerol synthesis in adipose tissue; molecular characterization showed that acylation-stimulating protein was identical to the complement activation product

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Adipocyte

↑Triglyceride synthesis

C3, fD, fB

C5L2

Local AP cycling

C3a CPN C3adesArg

↑uptake of glucose, fatty acids, cholesterol

FIG. 36.18. Complement and Adipocyte Activation. Adipocytes synthesize and secrete the alternative pathway (AP) proteins factor D, C3, and factor B into the local environment. Local AP activation, initiated through tick-over or other triggers, generates C3a, which is rapidly processed to C3adesArg by carboxypeptidase N. C3adesArg binds C5L2 on adipocytes, initiating a signaling cascade that results in increased uptake of glucose, fatty acids, and cholesterol; increased triglyceride synthesis; and increased lipid accumulation in adipose tissue.

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C3adesArg, generated when the C3a anaphylatoxin is “inactivated” by carboxypeptidase-N.148 It is now clear that local production of AP proteins and AP cycling in adipose tissue is a major regulator of lipid turnover through production of C3adesArg, which binds specific receptors (likely C5L2) on adipocytes to mediate its effects (Fig. 36.18).149 The link between AP activation and adipose tissue activities might explain the association, between NeF-driven AP dysregulation and the loss of adipose mass in the disease partial lipodystrophy. The broader implications of the effects of complement products on lipid handling, adipose tissue mass, and atherosclerosis are now coming under the spotlight.150

CONCLUSION Complement, an evolutionarily ancient system discovered more than 120 years ago, continues to surprise with increasing realization of its importance in diverse diseases, growing capacity to regulate its activation in vivo, and expanding list of roles outside of innate immunity. The decades to come will likely bring further revelations. I hope that this brief chapter provides some of you with the background and enthusiasm to contribute to the quest.

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37

Cell-Mediated Cytotoxicity Judy Lieberman

INTRODUCTION The most effective way the immune system can control the threats of intracellular infection and cellular transformation is by destroying infected and cancerous cells.1–7 When killer lymphocytes recognize harmful cells, they can target them for elimination by triggering programmed cell death. The main killer cells are natural killer (NK) cells of the innate immune sytem and cluster of differentiation (CD)8 + T lymphocytes of adaptive immunity, although some CD4 + T lymphocytes, particularly TH1 and regulatory T (Treg) cells, also express and deploy the specialized cell death machinery. All killer lymphocytes contain specialized secretory lysosomes, called cytotoxic granules, that are fi lled with deathinducing enzymes, called granzymes (“granule enzyme”). When the killer cell is activated, the cytotoxic granules move to the immune synapse formed with the target and fuse their membranes with the killer cell membrane, dumping their contents into the immune synapse in a process termed granule exocytosis. Perforin, a pore-forming protein in the granules, delivers the death-inducing granyzmes into the cytoplasm of the target cell to initiate its death. In this encounter, the killer cell remains unharmed.8 It is a serial killer that can detach from one target to seek and destroy others.9 Killer cells can also activate programmed cell death by using cell surface receptors to ligate cellular death receptors, such as Fas, on target cells. Granule-mediated cell death is key to control viral and intracellular bacterial infection and cancer because perforin-deficient mice and humans homozygous for perforin mutations or deficient in molecules needed for granule exocytosis are highly vulnerable to infection with intracellular pathogens and prone to develop spontaneous lymphomas.10 The death receptor pathway regulates lymphocyte homeostasis. Patients genetically deficient in the death receptor Fas or its ligand FasL develop autoimmunity.11 Target cells destroyed by cytotoxic granules or death receptor ligation die a highly regulated death (programmed cell death or apoptosis) rather than by necrosis. Programmed cell death minimizes inflammation and damage to nearby tissue as target cells undergoing programmed cell death are rapidly recognized and cleared by immune phagocytes, especially macrophages.12 The topic of this chapter was reviewed in more depth in a recent issue of Immunological Reviews.13 In this chapter we first describe the killer cells: Which immune cells are able to kill and how they develop this capacity and are regulated. Because of its destructive potential, cytotoxicity needs to be carefully regulated. We next

focus on the death machinery used for granule- and death receptor–mediated cytotoxicity and how it is mobilized and used to destroy the target cell. We also discuss what is known about how killer cells are protected against their own weapons of destruction. Some granzymes are expressed without perforin in nonkiller cells. We also discuss the increasing evidence for noncytotoxic proinflammatory roles of killer molecules.

THE KILLER CELLS The major killer cells are NK cells in innate immunity and CD8 T cells in adaptive immunity. Naïve T cells that have not previously seen antigen do not express either granule effector molecules or death receptors and are incapable of cell-mediated cytotoxicity.14 Within about 5 days of activation, naïve CD8 T cells differentiate into effector cytotoxic T lymphocytes (CTLs) that express both types of cytotoxic molecules. At the same time, these cells downregulate adhesive and chemokine receptor molecules that retain them in lymph nodes and acquire receptors that allow them to traffic to tissue sites of infection and tumor invasion. Activation to cytotoxic effector cells is tightly regulated. It requires not only antigen-receptor activation but also costimulation, and is greatly enhanced when antigen-presenting cells are stimulated by danger and pathogen-associated pattern motif receptors or when naïve T cells are stimulated by exogenous inflammatory and antiviral cytokines, including type I interferons (IFNs), interleukin (IL)-1, and IFNγ. Upon activation, effector CD8 T cells also begin to express the Fc γ receptor CD16, also present on NK cells, which enables them to recognize and lyse target cells that have been coated with IgG antibodies in a process called antibody-dependent cell-mediated cytotoxicity.15 In situations of persistent and extensive antigen, however, such as occur in tumors and chronic viral infection, many of the CD8 T cells that have the surface protein expression of CD8 effector cells no longer express perforin and are not cytotoxic.16–18 Effector CD8 T cells that lack cytotoxicity have been termed “exhausted.” Most effector cells in an immediate immune response die within a few weeks, but some survive and develop into memory cells. Memory cells downregulate expression of cytotoxic effector proteins, but the kinetics of downregulation varies with the molecule and with the particularities of the immunostimulatory environment.14,19 In particular, activation of CD8 T cells without CD4 T-cell help leads to an unimpaired primary cytotoxic response but greatly impairs the development of antigen-specific memory cells.20

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The immunosuppressive drug rapamycin directs antigenstimulated CD8 T cells to differentiate preferentially into memory cells rather than to effector CTLs.21 Memory CD8 T cells rapidly reacquire cytotoxic capability within hours of restimulation. The molecular basis for this rapid response is not well understood, although recent studies suggest that in memory CD8 T cells, the chromatin of cytolytic effector gene promoters and of eomesodermin, the master transcription factor that regulates CD8 effector genes, bears epigenetic marks that poise them for transcription compared to naïve T cells.22–26 These cells may also store messenger ribonucleic acids (mRNAs) for perforin and granzyme that can be rapidly translated upon activation. Some types of activated CD4 T cells, especially TH1, NKT, and Treg cells, also express granzymes and perforin and have cytotoxic activity. Murine Tregs express granzyme B but probably not granzyme A.27,28 Although immunosuppression by Tregs is mediated by soluble factors, there is also a poorly understoood component that requires cell-to-cell contact. Direct lysis of cognate T cells and potentially other immune cells by granule-mediated and death receptor pathways by Tregs is likely an important mechanism for suppressing immune activation.29–31 Because it takes a week to 10 days for naïve CD8 T cells to proliferate and differentiate into a large population of antigen-specific CTLs, the immediate response to intracellular infection in individuals that have not been vaccinated or previously exposed is mediated by NK cells. Although freshly minted NK cells were previously thought to immediately express granzymes and perforin, it now seems clear that—at least in mice—resting NK cells have minimal cytotoxic activity.32 They constitutively express mRNAs for granzymes A and B and perforin but only have granzyme A protein. Because they lack substantial perforin protein, cytotoxicity is limited. However, perforin and granzyme proteins and cytotoxicity are upregulated rapidly when NK cell–activating receptors are stimulated. Less differentiated NK cells that highly express the neural cell adhesion molecule or CD56 are poorly cytotoxic, whereas more differentiated CD56dim NK cells are potent killer cells.33 In the circulation, CD56dim NK cells have about a log more perforin than CD56bright NK cells. NK-activating receptors recognize cell surface changes in tumors, stressed cells, and infected cells, such as downregulation of major histocompatibility complex/human leukocyte antigen molecules or cell surface expression of nonclassical major histocompatibility complex molecules, such as MICA and MICB, which are induced by stress. A longstanding dogma of innate immunity is that innate immune responses are not altered by antigen exposure. However, it is now clear that NK cytotoxicity to infection and other stimuli can be greatly increased by previous antigen exposure.34 NK cell memory of prior exposure probably results from the expansion of NK cells bearing activating receptors specific for different important pathogens. These receptors, many of which are poorly conserved during mammalian evolution, may have coevolved with important pathogens. The link between individual NK receptors and pathogen recognition remains to be defined.

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CYTOTOXIC GENE EXPRESSION There are 5 human granzymes and 10 mouse granzymes expressed from three gene clusters that arose by gene duplication. In humans, the genes encoding granzymes A and K, tryptases that cleave after basic amino acids, are clustered on chromosome 5; the genes for granzyme B, which cleaves after aspartic acid residues such as the caspases; and granzyme H (or C in mice), which cleaves after hydrophobic residues, are clustered with myeloid cell proteases like mast cell chymase on chromosome 14; and the gene for granzyme M, which is highly expressed in NK cells and cleaves after Met or Leu, is found on chromosome 19 (Fig. 37.1). The mouse granzyme B cluster is uniquely expanded by multiple gene duplications to encode, in addition, granzymes D, E, F, G, L, and N. Nothing is known about these mouse-specific enzymes, but they may have evolved to defend against specific common mouse pathogens.1 Granzyme A and B are the most abundant granzymes and the most studied. Killer cells, including NK cells, cytotoxic CD4 and CD8 T cells, and even some Treg cells, express highly individualized and tightly regulated patterns of granzymes that depend on both cell type and mode of activation.35,36

Expression in Noncytolytic Cells Perforin is only expressed by cytotoxic cells. Although granzymes were previously also thought to have similarly restricted expression, noncytotoxic cells can express granzymes without perforin.37 Granzyme transcripts can be amplified from prothymocytes in fetal liver and double negative thymocytes.38 Although granzyme A transcripts are detected in thymocytes with the potential to develop into CD8 + cells, granzyme A activity is detected only in the most mature CD4–CD8 + thymocytes. These results suggest posttranscriptional regulation of granzyme translation (see the following for additional examples). Granzyme B, but not granzyme A, is expressed in Treg cells and plays an important perforin-dependent role in Treg function in mice. Benign and transformed B cells can be induced to express granzyme B by IL-21 alone or when combined with anti–B-cell receptor antibody.39 Granzyme B is also expressed without perforin in many different types of myeloid cells. Within the immune system, granzyme B is expressed in human plasmacytoid dendritic cells (pDCs).40 There are comparable levels of granzyme B transcripts in resting and activated pDCs but significantly higher amounts of granzyme B protein in activated cells, suggesting posttranscriptional regulation of expression. Granzyme B is also expressed in both normal and neoplastic human mast cells in vitro and in vivo.41 It localizes to mast cell granules and is secreted when they are activated. In mice, skin-associated mast cells and bone marrow–derived in vitro differentiated mast cells express granzyme B but lung mast cells do not.42 Neither granzyme A nor perforin are detected in mouse mast cells. The granzyme B gene is encoded within a few hundred kilobases of mast cell proteases. Thus, the granzyme B/mast cell chymase and tryptase genomic region is likely open and active in mast cells. In human basophils, IL-3 induces granzyme B, but not granzyme A or perforin,

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FIG. 37.1. The Human Granzymes are Encoded in Three Clusters. (Figure reprinted with permission from Chowdhury D, Lieberman J. Death by a thousand cuts: granzyme pathways of programmed cell death. Annu Rev Immunol. 2008;26: 389–420.)

expression.43 Expression of granzyme B in mast cells and basophils suggests a role of granzyme B in mediating allergic disease. In fact, granzyme B has been found in bronchoalveolar lavage fluid after allergen exposure. Several studies have suggested that granzyme B and perforin are expressed in human neutrophils, but this is controversial.44–47 Granzyme B is also expressed in the absence of perforin in the human reproductive system in developing spermatocytes and in placental trophoblasts48 and by granulosa cells of the human ovary in response to follicle stimulating hormone.49 In addition, granzyme B has been detected in a subset of primary human breast carcinomas and in chondrocytes of articular cartilage.50 The granzyme M transcript is expressed at low levels in the photoreceptor cells of the retina in the mouse.51 An alternatively spliced form (aGM) is exclusively expressed in these cells at much higher levels. Like granzyme M, granzyme K has an alternatively spliced form exclusively expressed in the brain.52 The physiologic significance of the alternative transcripts of granzymes M and K is unclear.

Extracellular Signals Regulating Granzyme Expression The kinetics and expression of the individual granzymes and perforin vary in different clonal populations in vitro and in vivo and depend on how they are activated.53–55 Most circulating CD8 + T lymphocytes that express any granzyme express both granzyme A and granzyme B, but some cells are positive for only one granzyme. Single-cell expression profi les of granzymes, perforin, and IFNγ have been investigated in in vitro or in vivo activated CD8 + T cells

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using reverse transcription-polymerase chain reaction in mice35 and intracellular staining and flow cytometry in humans.56 Individual T cells show diverse expression of these genes. Although some pairs of genes (perforin and IFNγ) are coexpressed more frequently than others, no specific combination of genes is consistently coexpressed. During in vitro activation of mouse naïve lymphocytes with antibodies to CD3, CD8, and CD11a and IL-2, the expression of granzyme A and granzyme C is delayed compared with cytolytic activity and expression of perforin and granzyme B.35 When mouse CTLs are activated in vivo by influenza virus infection, most antigen-specific CD8 T cells found in the lung 1 week after infection express both granzymes A and B, and about a third of them also express perforin. Moreover, there is no in vivo difference in the kinetics of induction of granzyme A, granzyme B, or perforin. Granzyme C is not induced by influenza infection in vivo. The diversity of expression of individual granzyme and perforin genes suggests that each gene is regulated independently, although it is likely that these genes will share some common transcription factor recognition sites and epigenetic changes. Differences in T-cell receptor (TcR) avidity, costimulatory and inhibitory receptor engagement, danger and innate immune receptor activation, cytokine milieu, type and state of activation of the antigen-presenting cell, and presence of helper or regulatory CD4 T cells will likely influence the induction of the granzyme and perforin genes. Moreover, the cell’s prior history of activation will affect cytolytic gene expression during subsequent encounters with antigen. Surprisingly, little is known about this subject.

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The perforin and granzyme genes are induced during T-cell activation. However, the only signal shown consistently to upregulate granzyme A and B and perforin is IL2.57 IL-2 regulates perforin and granzyme expression directly and independently of its effect on CD8 + T-cell survival and proliferation.58 Mice genetically deficient in IL-2 retain the ability to elicit a CTL response against many viruses, tumors, and allografts,59,60 although there are deficiencies in cytotoxicity under certain conditions.61 The other γc-dependent cytokines (IL-4, IL-7, IL-9, IL-15, and IL-21) likely substitute for IL-2 in its absence. IL-15 is particularly important because it also shares the γ-chain with the IL-2 receptor. IL-15 induces the expression of perforin, granzymes A and B, IFNγ, and Fas ligand in primary mouse lymphocytes.62 IL-21 works synergistically with IL-15 to upregulate granzyme A and B expression in mouse CD8 T cells.63 In vivo in mice, IL-21 exhibits potent antitumor function by enhancing NK and CD8 T-cell cytotoxicity.64 Similarly in human peripheral blood CD8 T cells, IL-15 and IL-21 both activate granzyme B and perforin expression, but IL-21 does so without inducing CD8 T-cell proliferation.65 Members of the IL-6/IL-12/IL-27 family also can upregulate granzyme and perforin expression.66,67

Transcriptional Regulation of Perforin and Granzymes Two key transcription factors, T-bet (TBX21) and eomesodermin (EOMES), which belong to the T-box family, are the key master regulators of cytotoxic gene expression and survival of committed CD8 memory cells.68–71 After naïve CD8 T-cell activation, T-bet is induced before eomesodermin.72 Notch signaling and the Runx3 transcription factor upregulate eomesodermin, but also directly upregulate expression of perforin and granzyme B genes.72,73 Mice deficient in both T-bet and eomesodermin genes are unable to control tumors and intracellular infection.74–76 They develop a wasting syndrome caused by anomalous differentiation to IL-17– secreting cells, suggesting that these two genes not only positively regulate cytotoxic gene expression and other genes required for CTL survival and function but also suppress differentiation to alternate lineages. Chromosome transfer experiments have shown that expression of the perforin gene (PRF1/prf1) is regulated by cis-regulatory regions extending about 150 kb around the gene.72,77,78 These include a core promoter located 120 bp upstream of the transcription start site and two enhancer regions and a locus control region (LCR) that are altered during T-cell differentiation and activation. The LCR is open for transcription specifically in cytotoxic cells. The region around the presumed LCR is more accessible to deoribonuclease (DNase I) digestion (and therefore its chromatin is open) in murine CD8 CTLs than in CD4 TH1 cells, likely explaining their approximately 20-fold increase in prf1 mRNA. Increased IL-2 does not enhance the accessibility of the LCR. The enhancers are both activated by IL-2R signaling mediated by signal transducer and activator of transcription (STAT)5 binding to two sites in each enhancer. Other STAT family members activated by alternate cytokines can also activate them. Activation of the more

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proximal enhancer also depends on IL-2–activated NF-κ B binding. Both enhancers also contain binding sites for AP-1 and ETS transcription factors, whereas the distal enhancer has an E-box and NFAT binding site, and the proximal enhancer contains eomesodermin, Ikaros, and CREB binding sites. Recruitment of ribonucleic acid (RNA) pol II to the transcription start site and activation of transcription increase with IL-2 stimulation. The key factors involved in activating transcription at the prf1 promoter are Runx3 and eomesodermin. T-bet does not appear to play a direct role in activating prf1 transcription but likely acts indirectly by increasing IL2-Rβ expression and enhancing IL-2 signaling. The current model suggests that Runx3 is needed to open the extended prf1 locus during T-cell differentiation, whereas eomesodermin plays a more direct role in activating transcription near the promoter. Other transcription factors also likely participate in transactivating the perforin promoter, including an ETS transcription factor, probably myeloid elf-1–like factor (MEF). Much less is known about the details of gene regulation of the granzymes. Enhancers or other long-range regulatory region of granzyme genes remain to be defined. Granzyme B is the only granzyme whose expression has been studied. A distal DNase hypersensitivity site 3.9 kb upstream of the granzyme B transcription start site is accessible only in activated, but not resting, CD8 T cells.79 Inclusion of this region in a GFP reporter in transgenic mice enhances CTL-specific expression, suggesting that this region may have enhancer activity. Induction of the expression of granzyme transcripts requires at least two independent stimuli: activation of the TcR and costimulation by cytokines of the γc family. The signals from several distinct signal transduction pathways are integrated in the nucleus in the form of transcription factors that bind to granzyme gene regulatory elements and activate transcription. Early studies identified a 243-bp fragment upstream of the mouse granzyme B transcription start site that potentially regulates granzyme B transcription.80 This region contains binding sites for two ubiquitous transcription factors: activating transcription factor/cyclic AMPresponsive element binding protein and activator protein-1, and two lymphoid specific factors, Ikaros and core-binding factor (PEBP2).81 Several of these transcription factor binding sites are evolutionarily conserved between the human and mouse granzyme B promoters.82,83 Analysis of reporter assays using promoters that had been systematically mutated at these sites in primary cells and cell lines revealed subtle differences in the importance of some transcription factors in primary cells versus cell lines. For example, activator protein-1, cyclic AMP-responsive element binding protein, and core-binding factor were not as important for transcription in primary cells as they appeared to be in cell lines.82,84 These studies suggested that combinations of transcription factors (particularly, activator protein-1 and corebinding factor) activate granzyme B expression in primary cells. The most compelling difference between the mouse and human granzyme B gene promoter is the importance of the Ikaros site only in human granzyme B expression.82,84 Studies in Stat1-deficient mice indicate that STAT1 mediates

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CHAPTER 37

granzyme B induction by IFNα or IL-27.67,85 IL-27–induced augmentation of granzyme B expression also depends on T-bet.67 Eomesodermin also drives granzyme B expression.71 Direct binding of T-bet and eomesodermin to the granzyme B promoter has not been examined.

Posttranscriptional Regulation Several examples of cells expressing perforin and/or granzyme transcripts, but not protein, were described previously, including resting NK cells, thymocytes, and unactivated pDCs and mast cells. Murine memory CTLs also express abundant granzyme B mRNA but no protein.86 All these results point toward a general mechanism of “prearming” cytotoxic lymphocytes with effector mRNAs, allowing these cells to rapidly respond to external stimuli. This type of gene regulation is well known to regulate cytokine expression, presumably for the same purpose. Two recent studies provide evidence for negative regulation of granzyme B and perforin expression by microRNAs miR-27* and miR-223 in NK cells.87,88 It will be interesting to see if expression or processing of these microRNAs declines rapidly after NK cell activation.

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GRANULE-MEDIATED CELL DEATH Killer Cell Granules Killer cells contain cytotoxic granules that are acidic, electron-dense, specialized secretory lysosomes89 (Fig. 37.2). These granules are mobilized like secretory vesicles in other secretory cells, such as neurotransmitter-containing vesicles near the synapses of neurons and melanin-containing vesicles of melanocytes. Cytotoxic granules contain the granzymes, trypsin-like serine proteases, whose major job is to initiate programmed cell death in cells marked for immune elimination. Cytotoxic granule proteins also regulate the survival of activated lymphocytes and may also cause inflammation by acting on extracellular substrates. The granzymes are trypsin-like serine proteases that use a classic histidine, serine, aspartic acid catalytic triad to cleave their substrates. Human granzymes A, B, C, and M; rat granzyme B; and human progranzyme K have all now been crystallized with high resolution.90–95 The active granzymes are produced by cleavage of a dipeptide from the N-terminus of the proenzyme. Activation is accompanied by a radical conformational change. Progranzyme K has a more rigid structure lacking an open active site than the active granzymes.

FIG. 37.2. Key Components of Cytotoxic Granules. The cytolytic effector molecules, perforin, granzymes, and granulysin, are bound to the serglycin proteoglycan. Cytotoxic granules also contain molecules found in all lysosomes, such as Lamp1 (CD107a) and cathepsins, as well as membrane-associated proteins specific to secretory lysosomes, such as vesicle-associated soluble N-ethylmaleimide-sensitive factor accessory complex component (VAMP)7 or VAMP8, Munc13-4, and Rab27a, which are essential for granule exocytosis. Cathepsins B and C play a special role in cytotoxic granules: Cathepsin C processes the progranzymes to the active enzyme, and membrane-associated cathepsin B helps protect the killer cell from membrane damage in the immune synapse by perforin. Other cathepsins may substitute for these cathepsins when they are absent or mutated.

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Detailed information about the conformation surrounding the active sites of granzyme A and B has provided the structural basis for understanding how subtle differences in the active site conformation lead to substantial differences in substrate specificity. As a consequence, mouse granzyme B is preferentially able to cleave mouse procaspase-3, whereas human granzyme B is better able to cleave the human orthologue. Granzyme A differs from the other granzymes in forming a covalent homodimer; the other granzymes are monomeric. Dimerization creates an extended site for substrate binding that is believed to confer a high degree of specificity to granzyme A for its substrates.90,96 In particular, because of the extended exosite for substrate binding, granzyme A substrates do not share a common short peptide sequence around the cleavage site. The cytotoxic granules also contain perforin, a poreforming molecule that delivers the granzymes into the target cell. Another pore-forming molecule, granulysin, which is homologous to the saposins, is cationic and selectively active at disrupting negatively charged bacterial and possibly fungal and parasite cell membranes. Granulysin is expressed in humans and nonhuman primates, and orthologues are found in some other species (pigs, cows, and horses), but not in mice. The positively charged cytotoxic effector molecules are bound in the granule to an acidic proteoglycan, called serglycin, after its many Ser-Gly repeats.97,98 In addition to these specialized molecules, the cytotoxic granules also contain lysosomal enzymes, the cathepsins, and internal lysosomal membrane proteins, such as CD107 (Lamp1). The outside of the granule membrane binds soluble N-ethylmaleimidesensitive factor accessory protein receptor (SNARE) proteins, synaptotagmins and Rab GTPases, which regulate vesicular trafficking and cytotoxic granule release. Some of these molecules, including Rab27a and Munc13-4, which

are important for granule exocytosis, are only incorporated into cytotoxic granules as they mature by fusion of cytotoxic granules with specialized exocytic vesicles, formed in secretory cells by fusion of late endosomes and recycling endosomes. Some of the granule-associated molecules associate with lysosomes in all cells, whereas some have a specialized function in killer cells.

Steps in Granule Exocytosis When CTLs and NK cells form an immune synapse with a target cell, engagement of activating receptors, including the TcR, NK cell–activating receptors, and Fc receptors, stimulates the killer cell to destroy the target cell7 (Figs. 37.3 and 37.4). Activation for cytolysis is enhanced by binding of CD8 or CD4, costimulatory receptors, and adhesion molecules such as LFA-1, which cluster in well-defined concentric rings within the immune synapse. Killer cell activation causes a calcium flux that induces lytic granules to cluster around the microtubule organizing center and then align along the immunologic synapse.99–103 Granules move to the immune synapse via both the microtubule network and actin cytoskeleton. The latter interaction is via myosin IIA in NK cells.104 The actin meshwork thins around the site of the synapse to make room for granules to move through it.105–107 Cytotoxic granules then dock to the killer cell plasma membrane in the central region of the immune synapse (c-SMAC). In T cells, granule docking and fusion may localize to a distinct (secretory) region of the central cluster (c-SMAC) of the immune synapse that is separate from the signaling domain containing the T-cell receptor and associated kinases.108 Recent studies did not observe a separation of signaling and secretory domains in the c-SMAC of NK cells. Cytotoxic granule docking is orchestrated by binding

FIG. 37.3. Steps in Granule-Mediated Cytotoxicity. After the killer cell recognizes a target cell (1), an immune synapse is formed at the interface and the microtubule organizing center (MTOC) moves to the synapse, reorganizing the microtubule network (2). Cytotoxic granules move along microtubules to dock at the killer cell membrane in the central supramolecular activation complex (c-SMAC) of the immune synapse. Granule membranes fuse with the killer cell plasma membrane, releasing their contents (magenta) into the immune synapse (3). Perforin delivers the granzymes into the cytosol of target cells (4) where they initiate apoptotic death (5). The granzymes concentrate in the nucleus of target cells. The killer cell then detaches from the dying cell and is free to seek out additional targets.

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FIG. 37.4. Model of Granule Exocytosis. In response to antigen recognition, the mature cytotoxic granule moves along microtubules, probably with assistance from the actin-myosin cytoskeleton (not shown) to dock at the cell membrane at the immune synapse. A cytotoxic granule vesicle-associated soluble N-ethylmaleimide-sensitive factor accessory (SNARE) complex component (VAMP) protein binds to Munc18-2, which is associated with plasma membrane syntaxin 11. Cytotoxic granule proteins Rab27a and Munc13-4, in association with a synaptotagmin SLP1 or SLP2, help anchor the granule to the membrane. A SNARE complex forms between plasma membrane SNAP23 and syntaxin 11 and granule membrane VAMP to initiate fusion of the granule membrane to the plasma membrane. Following membrane fusion, the cytotoxic granule contents are released into the immune synapse. After fusion, granule membrane-associated cathepsin B (not shown) is displayed on the killer cell membrane and protects it from perforin membrane damage. Figure adapted from de Saint Basile et al.7

of Rab27a on the cytosolic side of the mature granule membrane with synaptotagmin-like proteins, SLP1 or SLP2, which are anchored in the cell membrane. Docked granules are then primed for fusion by the interaction of Munc13-4 on their surface with syntaxin 11 on the killer cell membrane. This triggers the formation of a SNARE complex, the molecular machine for granule membrane fusion, between a cytotoxic granule vesicle-associated SNARE complex component (VAMP) protein with syntaxin 11 and SNAP23 on the cell membrane. Of the seven human VAMP proteins, studies in cytotoxic T cells have suggested that VAMP8 is required, whereas in NK cells both VAMP4 and VAMP7 are needed for different steps leading to granule exocytosis.109,110 Granule membrane fusion also requires participation of Munc18-2 to trigger the conformational activation of the SNARE complex. Although the general mechanism of granule exocytosis described previously is used by all killer cells, some of the details of granule trafficking and fusion at the synapse may differ between killer T cells and NK cells (although apparent differences may disappear when the same high-resolution techniques are applied to both types of killer cells). Cytotoxicity and granule fusion may occur even in the absence of a stable synapse.111

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Genetic Diseases Caused by Defects in Perforin or Granule Exocytosis Inherited deficiencies in perforin or the genes encoding syntaxin 11, Munc13-4, and Munc18-2 that orchestrate cytotoxic granule trafficking and release are linked to defective cytotoxicity and profound immunodeficiency.112–118 Patients with mutations in these genes develop the familial hemophagocytic lymphohistocytosis (FHL) syndrome. These patients are handicapped in controling viral infections and develop a severe immune activation syndrome that is often fatal in childhood unless treated with bone marrow transplantation. Some patients with milder perforin mutations that do not completely eliminate cytotoxic function are not diagnosed until adulthood. These adult patients with FHL not only have impaired antiviral immunity but are also more prone to develop lymphoma (such as perforin-deficient mice). The most prominent and sometimes fatal clinical manifestation of FHL is an inflammatory syndrome caused by uncontrolled activation and expansion of CD8 T cells, often in response to poorly controlled herpes virus infections, which leads to systemic activation of macrophages, which infiltrate tissues and overproduce proinflammatory cytokines. Macrophage activation is driven by excessive production of IFNγ by activated CD8 T cells.10,119 Sequencing

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of perforin mutations in patients with FHL has identified nonsense, frameshift, and missense mutations that disrupt perforin synthesis, folding, or activity.5,120 The importance of some of these have been validated by testing cytolytic function of rat basophilic leukemia cells engineered to express mutant perforin and granzyme B. Defects in genes encoding the AP-3 adaptor—needed to shuttle cargo from the Golgi to secretory lysosomes—the lysosomal trafficking regulator LYST, or Rab27 lead to human syndromes (Hermansky-Pudlak syndrome type 2, Chediak-Higashi syndrome, and Griscelli syndrome type 2, respectively) and corresponding mouse models (pearl, beige, and ashen mice, respectively) in which cytotoxicity as well as other processes involving secretory lysosomes are defective. In fact, mice and humans with defects in these genes have defects in pigmentation due to defective melanosome transport.

Lessons from Knockout Mice Mice genetically deficient for granzymes A, B (and the granzyme B cluster), and M and perforin provide important tools for probing the importance of these effector molecules in immune defense.1 Perforin-deficient mice112 closely recapitulate the symptoms of humans with genetic perforin deficiency. They are severely immunodeficient and compromised in their ability to defend against viruses and tumors and develop the inflammatory syndrome of FHL when infected with mouse cytomegalovirus.121 Mice deficient in any 1 of the 10 granzymes, or even of the granzyme B cluster, only have subtle differences compared to wild-type animals. These experiments highlight the functional redundancy of the granzymes. While only one molecule (perforin) effectively delivers the granzymes into target cells, each of the granzymes can trigger cell death. However, target cells may be selectively resistant to one or another of the granzymes (ie, by bcl-2 overexpression or by expression of viral serpins). Requirements for a single granzyme have been shown in some cases by specific immune challenges. For example, granzyme A–deficient mice are more susceptible to the poxvirus ectromelia122 and granzyme B–deficient mice have less GvHD.123 In constructing genetically deficient mice, genetic alterations of one gene can affect the expression of nearby granzyme genes. In the original granzyme B knockout mice, the PGK-neo cassette remaining in the granzyme B locus impedes the expression of other granzyme cluster genes (granzymes C, D, and F). The granzyme B gene has also been deleted keeping the expression of granzymes C, D, and F intact.124 Cytotoxic T cells from the granzyme B–specific deletion mouse are significantly more effective at inducing apoptosis than those from the granzyme B–cluster knockout animal, underlining the importance of the other granzyme B cluster granzymes, especially when granzyme B is absent. Because granzyme A and B are the most abundant granzymes in T cells, granzyme A/B doubly deficient mice are more immunodeficient than the single knockouts.125–127 CTLs from granzyme A/B–deficient mice, although somewhat impaired in cytotoxicity relative to wild-type cells, nonetheless largely retain the ability to kill target cells.128–130 However, the timing of key molecular events during apoptosis, such as externalization of phosphatidylserine (annexin V staining),

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is delayed during cell death induced by granzyme A/B–deficient CTLs versus wild-type CTLs.131 Cytotoxic T cells lacking granzyme A and B induce a modified form of cell death that seems morphologically distinct from either perforin-mediated necrosis or wild-type CTL–mediated apoptosis.131 Granzyme A/B–deficient animals do not develop spontaneous tumors and clear many viruses normally. The likely explanation of these results is that the other “orphan” granzymes (particularly H/C, K, and M),132–134 substitute for granzyme A and B. Although granzyme M is highly expressed in innate immune killer cells, including NK cells, NKT cells, and γδ T cells, granzyme M–deficient mice have normal NK and T cell numbers and NK activity against tumors.135 Defense against the mouse poxvirus ectromelia and implanted NKsensitive tumors is unimpaired in granzyme M–deficient mice compared to wild-type mice. Deficient mice are somewhat impaired in responding to mouse cytomegalovirus infection as they have higher viral levels, but they are eventually able to clear the infection. Thus, granzyme M does not appear to be essential for NK cell–mediated cytotoxicity.

Perforin Delivery of Cytotoxic Molecules into Target Cells When the granule membrane fuses with the killer cell membrane, the granule contents are released into the synapse. Granzymes and perforin probably dissociate from serglycin in the immune synapse before they enter target cells.136 Granzymes bind to the target cell membrane by electrostatic interactions (granzymes are very positively charged and the cell surface is negatively charged)137–139 and also by specific receptors, such as the cation-independent mannose-6-phosphate receptor.140 However, specific receptors are not required for binding, internalization, or cytotoxicity.137,139,141,142 The lack of a receptor enables all types of cells to be eliminated and limits escape from immune surveillance. The granzymes are delivered into the target cell (but not the killer cell) by perforin where they initiate at least three distinct pathways of programmed cell death. Although perforin is essential for granule-mediated cytotoxicity to deliver the granzymes into cells, the granzymes are redundant, as each granzyme can independently activate cell death. Although genetic deficiency of one or a few granzymes does not lead to severe immunodeficiency, mice lacking one or another granzyme display subtle differences in their ability to control specific viral infections. Why are there so many granzymes? The immune system needs to contend with a wide variety of tumors and infections, some of which have elaborated strategies to evade apoptosis and immune destruction. Some of the granzymes may have evolved to disarm specific intracellular pathogens. The interplay between granzyme B and H and adenovirus illustrates how multiple granzymes may have evolved to eliminate important pathogens.143–145 Although both enzymes can cleave and inactivate at least two adenoviral proteins, the virus has also developed a way of inactivating granzyme B. Granzyme H potentiates the effect of granzyme B by destroying an adenoviral granzyme B inhibitor. Perforin delivers granzymes and other effector molecules into the target cell cytosol.146,147 At high concentrations,

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perforin multimerizes in a cholesterol- and calcium-dependent manner in the plasma membrane of cells to form 5- to 30-nm pores.148–152 Recent cross-linking and biophysical studies suggest that perforin may form at least two types of pores in membranes: small pores composed of about seven monomers that are not stable and much larger stable pores.153,154 Cryoelectron microscopy reconstructions suggest that the large pores are composed of approximately 19 to 24 subunits and have a lumen large enough for granzyme monomers or granzyme A dimers to readily pass through. The precursor of human perforin is a 555 amino acid protein synthesized with a 21 amino acid leader sequence. The N-terminal region of the mature 67-kDa protein (residues 44-410 of the human protein) is homologous to domains in complement proteins C6, C7, C8a, C8b, and C9 that form the complement membrane attack complex (MAC). The crystal structure of monomeric mouse perforin was recently solved.152 The complement homology domain, termed the MAC/perforin domain (MACPF), is similar in structure to that of bacterial pore-forming cholesteroldependent cytolysins, although they insert into membranes in opposite orientations. The MACPF domain of perforin is followed by an epidermal growth factor (EGF)-like domain; a C2 domain, a domain present in synaptotagmins and other calcium-dependent proteins, which becomes able to bind to lipid membranes after a conformational change in response to calcium; and a short 12 amino acid C-terminal peptide. The docking of the calcium-bound C2 domain is the first step in pore formation. Docking likely triggers both multimerization and a major conformational change in which two clusters of α-helices in the MACPF domain jackknife into the membrane. It is unclear whether multimerization to form a pore occurs before or after this conformational change. Perforin is glycosylated at two sites: one in the MACPF domain and one in the C-terminal peptide. Glycosylation of at least one site is needed for targeting perforin to cytotoxic granules, probably via binding of the glycan to the mannose-6-phosphate receptor.155 En route to or in the granule, the glycosylated C-terminal peptide is removed from human (but not mouse) perforin by an undefined cysteine protease to produce the mature active protein.156 The original model for how perforin delivers granzymes into cells was that granzymes entered cells through perforin pores in the target cell plasma membrane. This model predicts that granzymes directly pass and disperse into the target cell cytosol. However, granzymes do not directly enter the cytosol but instead are first endocytosed into clathrincoated vesicles and transported to endosomes.154,157,158 Thus, the original model is not correct. Current data suggest that perforin indeed forms target cell plasma membrane pores, but these pores are small and transient (Fig. 37.5). However, calcium flows into the target cell through these pores and remains elevated for a few minutes. Because intracellular calcium is low in cells with an intact cell membrane, the cell senses a calcium influx as a sign of disruption of the plasma membrane. The elevated calcium triggers a cellular membrane damage response (also known as cellular wound healing) in which intracellular vesicles move to the plasma membrane and fuse their membranes to patch holes, and any damaged membrane is rapidly removed and

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internalized into endosomes.158–161 At the same time, membrane-bound granzymes, granulysin, and perforin are endocytosed. Elevated cytosolic calcium activates endosomal fusion, and granzyme- and perforin-containing endosomes fuse to form giant endosomes approximately 10 times larger than normal endosomes that have been termed gigantosomes. In the endosomal membrane, perforin forms larger and more stable pores through which granzymes begin to leak out into the cytosol. About 15 minutes after cell death has been triggered, the gigantosomes rupture, releasing any remaining cargo to the cytosol where they begin to activate programmed cell death. When the membrane repair response is inhibited, because the cell remains leaky, target cells die by necrosis instead of by slower, regulated, and energy-dependent programmed cell death. Although perforin is the major molecule responsible for granzyme delivery, under some circumstances other molecules might serve that function. For example, bacterial and viral endosomolysins can substitute for perforin in vitro (and are widely used as laboratory reagents for intracellular delivery162) and potentially might play a similar role in vivo. The heat shock protein (Hsp)70, which chaperones some peptides across cell membranes, can also carry granzyme B (and presumably other granyzmes) into cells.163 Hsp70 is on the surface of some stressed cells or tumor cells and might help to remove these cells from the body.

Programmed Cell Death Pathways Activated by Granzymes Once in the cytosol, the granzymes independently activate several parallel pathways of programmed cell death6 (Table 37.1). Granzyme B cleaves and activates the caspases and also directly cleaves many important caspase substrates. Granzyme B can activate cell death that mimics caspase activation, even when the caspases are inhibited or in cells in which the caspase mitochondrial pathway is deficient. Granzyme A activates a distinct programmed cell death pathway that does not involve the caspases or disrupt the mitochondrial outer membrane. The substrates of the two major granzymes are largely nonoverlapping. The exceptions, lamin B and PARP-1, may indicate common features needed for cells undergoing all forms of programmed cell death, such as disruption of the nuclear membrane, inhibition of deoxyribonucleic acid (DNA) repair, or maintaining cellular adenosine triphosphate levels. What is known about cell death executed by the other (so-called orphan) granzymes is briefly described in the following. The orphan granzymes may be more highly expressed under conditions of prolonged immune activation.164 The orphan granzymes are functionally important as mice genetically deficient in the whole granzyme B cluster are less efficient at clearing allogeneic tumors than mice deficient in just granzyme B.124 Although some key granzyme proteolytic substrates are in the cytosol (ie, Bid, caspase-3, and ICAD for granzyme B), other important targets are in other membrane-bound cellular compartments, especially the nucleus and mitochondrion. In the cytosol, granzymes B, H, and possibly K also directly cleave the proapoptotic BH3-only Bcl-2

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FIG 37.5. Current Model of Perforin Delivery of Granzymes into the Target Cell. A: Perforin treatment of HeLa cells causes dramatic membrane perturbation and blebbing. B: Killer cell degranulation causes a transient calcium influx in target cells that persists for a few minutes. In this experiment from Keefe et al.,157 PHA-activated human cytotoxic T lymphocytes were incubated with Fura-2loaded, anticluster of differentiation three-coated U937 cells, and images were obtained every 30 seconds. The Fura-2 indicator dye is blue when calcium is low and green when it is elevated. C: Perforin and granzyme B are endocytosed into giant EEA-1–staining endosomes (image courtesy of Jerome Thiery). D: When HeLa cells are treated with perforin and granzyme B, within 5 minutes, granzyme B (green) concentrates in gigantosomes and is released beginning after about 12 minutes. The released granzyme concentrates in the target cell nucleus. E: Model for perforin delivery. After cytotoxic granule exocytosis into the immunologic synapse (1), perforin multimerizes in the target cell membrane to form small transient pores through which calcium enters (2), triggering a plasma membrane repair response (3) in which lysosomes fuse with the damaged plasma membrane and perforin and granzymes are rapidly internalized by endocytosis. Perforin and granzyme-containing endosomes then fuse in response to the transient calcium flux (4) to form gigantosomes. Within gigantosomes, perforin continues to multimerize to form larger pores, preventing acidification and causing some granzyme release (5) before inducing endosomal rupture and complete granzyme release into the target cell cytoplasm (6 ). (D,E: Reprinted from Thiery J, Keefe D, Boulant S, et al. Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells. Nat Immunol. 2011;12:770–777, with permission.)

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TABLE

37.1

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Features of the Distinct Cell Death Pathways Induced by the Granzymes

Granzyme

A

B

C/H

K

M

Expression Cytolytic CD8 T cells Cytolytic CD4 T cells CD4 Tregs NK cells Myeloid cells

++ + − + −

++ + + +/− +

+

+

+/−

Common features Rapid loss of membrane integrity Annexin V staining Chromatin condensation DNA damage Mitochondrial depolarization

+ + + + +

+ + + + +

+ + + + +

+ + + + +

? ? ? ? ?

Caspase activation

−

+

−

−

?

Type of DNA damage Oligonucleosomal DNA fragmentation Single-stranded DNA nicks TdT labeling Klenow labeling

− + + +

+ − + +

− + + +

− + + +

? − ? −?

Type of mitochondrial damage Inhibition by Bcl-2 overexpression Cytochrome c release Mitochondrial swelling

− − +

+ + +

? +? ++

? ? +

? ? +

Autophagy

−

−

−

−

+?

++

CD, cluster of differentiation; DNA, deoxyribonucleic acid; NK, natural killer; TdT, terminal deoxynucleotidyl transferase; Treg, regulatory T. Table modified from Chowdhury and Lieberman.6

family member Bid to initiate the classical mitochondrial apoptotic pathway that leads to mitochondrial outer membrane permeabilization (MOMP) and release of cytochrome c and other proapoptotic proteins from the intermembrane space.165–171 Granzymes A and B (and possibly other granzymes) enter mitochondria through an unknown mechanism to cleave important substrates including electron transport proteins.172–174 Granzyme C (in mice) and granzyme H (in humans) activate caspase-independent cell death with a pronounced mitochondrial phenotype. All of these events cause mitochondrial depolarization and production of superoxide anions and other reactive oxygen species, which is a key first step in killer lymphocyte–mediated death, as superoxide scavengers block granzyme-mediated cell death.173 Granzyme A and B rapidly translocate to and concentrate in the nucleus,175,176 where proteolytic cleavage of key substrates is important to induce programmed cell death by both granzyme A (SET, Ape1, lamins, histones, Ku70, PARP-1) and B (lamin B, PARP-1, NuMa, DNA-PKcs). Nuclear translocation of the granzymes may be mediated by importin-α .177

Granzyme A Granzyme A induces caspase-independent cell death, which is morphologically indistinguishable from apoptosis178–180

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(Fig. 37.6). Granzyme A is the most ancient of the granzymes; tryptases homologous to granzyme A are found in cytotoxic cells in bony fish.181 Granzyme A was the first granzyme described and is the most widely expressed. Cells treated with granzyme A and perforin die rapidly: Within minutes, they undergo membrane blebbing and have evidence of mitochondrial dysfunction (increased reactive oxygen species, loss of mitochondrial transmembrane potential [ΔΨm], disruption of mitochondrial morphology).172,173 Within half an hour, externalization of phosphatidyl serine (measured by annexin V staining) occurs; and DNA damage, chromatin condensation, and nuclear fragmentation become apparent within 1 to 2 hours. DNA is damaged by single-stranded cuts into megabase fragments that are much larger than the oligonucleosomal fragments generated during caspase or granzyme B–activated cell death.182 Because the DNA fragments are too large to be released from the nucleus, assays that measure DNA release into culture supernatants are typically negative. Mitochondria are damaged without MOMP or release of proapoptotic mediators, such as cytochrome c, from the mitochondrial intermembrane space.173 In mitochondria, granzyme A cleaves Ndufs3 in electron transport chain complex I to interfere with mitochondrial redox function, adenosine triphosphate generation and maintenance of ΔΨm and to generate superoxide anion.129,172,173 The superoxide generated

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FIG 37.6. The Granzyme A Pathway of Cell Death. Reactive oxygen species generated by granzyme A (represented by scissors) cleavage of Ndufs3 in electron transport complex I in mitochondria drives the endoplasmic reticulum–associated SET complex into the nucleus. Granzyme A enters the nucleus by an unknown pathway. In the nucleus, Granzyme A cleaves three components of the SET complex (SET, HMGB2, and Ape1) to activate two nucleases in the complex to make single-stranded deoxyribonucleic acid (DNA) lesions; NM23-H1 makes a nick, which is extended by the exonuclease Trex1. Granzyme A also degrades the lamins and the linker histone H1 and removes the tails from the core histones, opening up chromatin and making it more accessible to these nucleases. DNA repair proteins Ku70 and PARP-1 are also targets.

by damaged mitochondria drives an endoplasmic reticulum (ER)-associated oxidative stress response complex, called the SET complex, into the nucleus where it plays a critical role in granzyme A–induced nuclear damage.173,182 The SET complex contains three nucleases (the base excision repair endonuclease Ape1, an endonuclease NM23-H1, and a 5′-3′ exonuclease Trex1), the chromatin modifying proteins SET and pp32, which are also inhibitors of the tumor suppressor protein phosphatase 2A, and a DNA binding protein that recognizes distorted DNA, HMGB2.183–187 One of the normal functions of the complex is to repair abasic sites in DNA generated by oxidative damage. Recent studies also implicate the cytosolic SET complex as binding to the human immunodeficiency virus preintegration complex and facilitating human immunodeficiency virus infection.188 The SET complex exonuclease Trex1 digests cytosolic DNA produced by endogenous

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retroviruses and infectious viruses to inhibit the innate immune response to cytosolic DNA.189–192 Mutations in Trex1 that inactivate its nuclease activity or cause its mislocalization are linked to human inflammatory and autoimmune diseases, including Aicardi-Goutiere syndrome and systemic lupus erythematosis.193–197 Granzyme A, which traffics to the nucleus by an unknown mechanism, converts this DNA repair complex into an engine for DNA destruction by cleaving SET, an inhibitor of the endonuclease NM23-H1.185 This allows NM23-H1 to nick DNA; the exonuclease Trex1 then extends the break.184 At the same time, granzyme A cleaves and inactivates HMGB2 and Ape1 to interfere with base excision repair.186,187 In addition to disabling base excision repair, granzyme A also interferes with DNA repair more generally by interfering with the recognition of damaged DNA by cleaving and inactivating Ku70198 and PARP-1.199 Within the

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nucleus, granzyme A also opens up chromatin by cleaving the linker histone H1 and removing the tails from the core histones, making DNA more accessible to any nuclease, and disrupts the nuclear envelope by cleaving lamins.200,201

Granzyme B Granzyme B is unique among serine proteases because it cleaves after aspartic acid residues like the caspases202,203 (Fig. 37.7). It induces target cell apoptosis by activating the caspases, particularly the key executioner caspase, caspase-3.204,205 Human granzyme B, but not the mouse enzyme, also activates cell death by directly cleaving the key caspase substrates, Bid and ICAD, to activate the same mitochondrial and DNA damage pathways, respectively, as the caspases.92,166–168,206–208 As a consequence, caspase inhibitors

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have little effect on human granzyme B–mediated cell death and DNA fragmentation while the same inhibitors significantly block the action of the mouse enzyme. Thus, human CTLs and NK cells may be more effective than mouse killer cells at eradicating virus-infected cells or tumors that have developed methods for evading the caspases. Both human and mouse enzymes cleave many of the same substrates as the caspases (including PARP-1, lamin B, NuMa, DNAPKcs, tubulin) and have substrate specificity close to that of caspases-6, -8, and -9.209 However, human granzyme B cleaves optimally after the tetrapeptide IEPD, whereas mouse granzyme B has somewhat different peptide specificity, preferring to cleave after IEFD.92,206 Moreover, other regions including the P’ region (C-terminal to the cleavage site) and more distal regions contribute to substrate specificity. Because of subtle

FIG 37.7. The Granzyme B Pathway of Cell Death. Human granzyme B (represented by scissors) cleaves Bid to produce truncated Bid (tBid), which initiates the classical mitochondrial apoptotic pathway characterized by reactive oxygen species generation, loss of the mitochondrial transmembrane potential, and disruption of the mitochondrial outer membrane to release proapoptotic mediators in the intermembrane space, including cytochrome c (blue balls, cyt c), SMAC/Diablo, and HtrA2/Omi. Cytochrome c binds to apoptotic protease activating factor 1 (Apaf-1) to form the apoptosome that activates caspase-9. Caspase-9 activates caspase-3 and the apoptotic cascade that includes cleavage of ICAD, the inhibitor of the caspase-activated DNase (CAD), allowing CAD to enter the nucleus and make oligosomal DNA double strand breaks. Human granzyme B on its own can directly activate caspase-3 and some key downstream caspase targets, including ICAD, lamin B, tubulin, and the DNA repair proteins DNA-PKcs and PARP-1. Mouse granzyme B lacks direct proteolytic activity on some important substrates (see text).

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differences in sequence, the human and mouse granzyme B can differ in important ways with respect to their substrates and the efficiency with which they are cleaved. The granzyme B (and caspase) mitochondrial pathway leads to reactive oxygen species (ROS) generation, dissipation of ΔΨm, and MOMP with release of cytochrome c and other proapoptotic molecules from the mitochondrial intermembrane space. Human granzyme B activates this pathway directly by cleaving Bid, whereas mouse granzyme B activates it indirectly. However, granzyme B targets mitochondria in other ways, including by cleaving antiapoptotic Mcl-1 and Hax-1, a protein that helps maintain the mitochondrial transmembrane potential.174,210 Loss of ΔΨm, but not cytochrome c release, occurs in the presence of pancaspase-inhibitors (even using mouse granzyme B) and in mice genetically deficient for Bid, Bax, and Bak (the latter two Bcl-2 family members are required for Bid-induced mitochondrial damage).132,169,211,212 Granzyme B can also activate ROS by activating extramitochondrial nicotinamide adenine dinucleotide phosphate-oxidase.213 DNA damage by granzyme B is mediated primarily by the activation of the caspase-activated DNase (CAD) following proteolytic cleavage of its inhibitor ICAD either directly by human granzyme B or indirectly by executioner caspases, such as caspase-3. In humans, there is a common polymorphism of granzyme B in which three amino acids (Q48, P88, Y245) are mutated to R48A88 H24.214 This polymorphism does not seem to affect cytotoxicity and does not have any known clinical significance.215

Granzymes C and H Mouse granzyme C and human granzyme H, homologous granzymes encoded downstream from granzyme B, are predicted to cleave after aromatic residues.133,216 Granzyme H arose during primate evolution, independently of granzyme C, in an intergenic recombination event between granzyme B and a mast cell chymase.217 Both induce caspase-independent death with hallmarks of programmed cell death: ROS generation, dissipation of ΔΨm, chromatin condensation, and nuclear fragmentation.133,216 DNA destruction by granzyme C (and probably granzyme H as well) is via single-stranded nicks and does not involve CAD. Rapid mitochondrial swelling and disruption of mitochondrial ultrastructure are particularly striking in cells treated with granzyme C. The mitochondrial pathways activated by granzyme C and H may be different; granzyme C triggers cytochrome c release, a sign of MOMP, whereas granzyme H does not.133,216 Granzyme H cleaves two adenoviral proteins: a DNA binding protein (also a granzyme B substrate) and the adenovirus 100K assembly protein, a previously described inhibitor of granzyme B.143,145 Cleavage of DNA binding protein interferes with viral DNA replication, whereas cleavage of 100K restores granzyme B function in adenovirus-infected cells. Granzyme H also cleaves the cellular La protein, an RNA binding protein that participates in the posttranscriptional processing of mRNAs transcribed by RNA polymerase III and some transfer RNA (tRNA) and viral RNAs.218 Cleavage mislocalizes La from the nucleus and decreases translation of hepatitis C virus proteins. Therefore,

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granzyme H may play a special role in immune defense against certain viruses. Because granzyme H is expressed in NK cells, it may help eliminate these viruses early in infection before adaptive immunity has had a chance to develop.

Granzyme K Granzyme K is another tryptase in mice, rats, and humans that is encoded downstream near granzyme A on human 5q11 to 5q12 (or the syntenic region of mouse chromosome 13). It is much less expressed than granzyme A, and unlike granzyme A, is a monomer, not a dimer. Mice genetically deficient in granzyme A express granzyme K, which may explain the lack of a significant phenotype of granzyme A− / − mice, except when challenged with some viruses.219,220 Purified rat and recombinant human granzyme K have been available for some time,221,222 but little was known about its cell death activation until recently. Like granzyme A, purified rat granzyme K efficiently induces caspase-independent cell death, characterized by mitochondrial dysfunction without MOMP (ROS and loss of ΔΨm, but without cytochrome c release).132 However, unlike granzyme A, rat granzyme K–induced cell death was originally reported to be inhibited in cells overexpressing Bcl-2.132 This finding was surprising, as Bcl-2 inhibits MOMP, which leads to cytochrome c release, which was not detected in granzyme K–treated cells. In fact, a more recent study found that cell death by recombinant human granzyme K did not activate caspase-3 and was unaffected by caspase inhibitors or Bcl-x L overexpression.171 Granzyme K mimics granzyme A DNA damage171: It causes caspaseindependent nuclear fragmentation and nuclear condensation and single-stranded DNA breaks by targeting the SET complex. Like granzyme A, granzyme K causes SET complex nuclear translocation and hydrolyzes and inactivates SET, Ape1, and HMGB2 in the SET complex.171 Presumably, cleavage of SET, the inhibitor of NM23-H1, triggers DNA damage by the granzyme A–activated DNases, NM23-H1, and Trex1 in the SET complex.184,185 The same group recently reported that granzyme K causes mitochondrial damage that includes not only ROS generation and dissipation of ΔΨm but also Bid cleavage (to a fragment that appears to be the same size as is generated by granzyme B) and MOMP with release of cytochrome c and endonuclease G (endoG).171 This needs to be verified because rat granzyme K does not cause cytochrome c release,132 and this same group showed that caspases are not activated by granzyme K, and overexpression of Bcl-xL does not interfere with human granzyme K–induced cell death,171 as would be expected if MOMP is triggered. Although granzyme K appears to duplicate the nuclear damage pathway of granzyme A, further studies are needed to determine whether the mitochondrial granzyme K pathway resembles that activated by granzyme A (no MOMP) or granzyme B (Bid cleavage, MOMP), or is a hybrid of both. A proteomics analysis that compared granzyme A and K suggested that although the two enzymes share many substrates, some may be unique to granzyme K.223 In fact, recent studies suggest that granzyme K cleaves and inactivates p53, which should interfere with cellular repair pathways, and interferes with the ER unfolded protein response by cleaving multiple components of the ER degradation complex.224,225

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Granzyme M Granzyme M is the most distinctive of the granzymes. It likely arose from a gene duplication of a neutrophil protease, as it is encoded near a cluster of other neutrophil proteases in human chromosome 19p13.3 (or a synteic region of mouse chromosome 10) and is slightly more homologous to one of them (complement factor D) than to the other granzymes.226 Unlike the other granzymes, granzyme M cuts after Met or Leu.227,228 None of the serine protease inhibitors that block the other granzymes, including the pan-granzyme inhibitor 3,4-dichloroisocoumarin, effectively inhibit granzyme M.229 Moreover, granzyme M appears to function primarily in innate immunity, as it is expressed mostly in NK cells and γδ T cells and only in the subset of CD56 + T cells.230,231 Until recently, it was not clear whether granzyme M induces cell death.134 Granzyme M− / − mice have unimpaired NK- and T-cell development and NK cell–mediated cytotoxicity but are less able to defend against mouse cytomegalovirus infection.135 The literature does not agree about the type of cell death activated by granzyme M. Kelly et al.,134 using recombinant human granzyme M expressed from baculovirus in insect cells, found that granzyme M induced rapid, caspaseindependent cell death that looked like autophagic death and did not find evidence for DNA fragmentation, mitochondrial depolarization, phosphatidyl serine externalization, or caspase activation. On the other hand, using human granzyme M expressed in yeast, the Fan laboratory argued that granzyme M activated caspase-dependent cell death, in part by cleaving and inactivating both the apoptosis inhibitor survivin and ICAD, with phosphatidyl serine externalization, caspase activation, CAD activation with oligonucleosomal DNA laddering, PARP cleavage, and mitochondrial disruption with MOMP (mitochondrial swelling, dissipation of ΔΨm, ROS generation, cytochrome c release).232–234 This group also suggested that another granzyme M substrate may be TRAP75, a Hsp that inhibits granzyme M–induced ROS generation.233 However, one aspect of this study that may not be completely consistent with what is known about granzyme M is that the Fan paper232,233 claims that granzyme M cleaves ICAD after a Ser residue, whereas peptides containing Ser at the P1 site are not substrates of granzyme M expressed in yeast. Therefore, further work will be needed to determine whether granzyme M activates granzyme B– like caspase-dependent cell death or a novel pathway distinct from that activated by the other granzymes. The mouse and human isoforms may also have diffeent substrates.235 Examining cell death induced by native purified granzyme M may be necessary to determine what type of cell death is induced by these enzymes. One intriguing other activity of granzyme M might be to cleave and inactivate the granzyme B serpin inhibitor SerpinB9 (PI-9), which it has been shown to do in vitro.228 If this proves to be a physiologically relevant substrate in cells, then one function of granzyme M might be to potentiate the activity of granzyme B. Mice genetically deficient in granzyme M are more susceptible to cytomegalovirus infection, and granzyme M cleaves a cytomegalovirus structural protein and inhibits its replication.236 Thus, an important function of granzyme M may be to help protect us from this important human pathogen.

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Granulysin Human cytotoxic granules of cytotoxic T cells and NK cells also contain another effector molecule: the membrane perturbing saposin-like molecule granulysin.237 The granulysin gene (GNLY ) was first identified as a late activation gene expressed 3 to 5 days after T-cell activation, which coincides with the expression of the other cytotoxic effector molecule genes in naïve T cells.238 Granulysin is synthesized as a 15-kDa protein that is cleaved at both ends to produce a 9-kDa peptide. Both forms can form membrane pores in membranes. The larger form is secreted by NK cells and cytotoxic T cells, whereas the 9-kDa form is stored and released from cytotoxic granules during NK-cell or cytotoxic T-cell attack. Granulysin preferentially disrupts bacterial membranes and has been postulated to play a role in immune elimination of bacteria, fungi, and parasites.239–242 It may also have some antitumor activity, but this requires very high granulysin concentrations in vitro that may not be physiologically relevant. There is also some evidence that secreted granulysin can act as a chemoattractant for dendritic cells and other immune cells and can induce them to express proinflammatory cytokines.243,244 Purified granulysin is only active as a cytotoxic agent against bacteria and other pathogens when experiments are performed at high micromolar concentrations of granulysin under hypotonic or acidic conditions that are not found extracellularly. Thus, granulysin’s membrane perturbing activity likely only operates within cells, perhaps to target intracellular bacteria and other pathogens located in acidic intracellular vesicles, such as phagolysosomes. Perforin is needed to deliver granulysins into target cells.239,245,246 Understanding the importance of granulysin in antibacterial defense and immunopathology (it is overexpressed at sites of immune activation and in blistering skin diseases) will be facilitated by the recent generation of transgenic mice that express granulysin.247

How is the Killer Cell Protected from its Cytotoxic Molecules? The killer cell is not injured by its own granules. It delivers the “kiss of death” and escapes the encounter with the cell targeted for elimination unharmed and then can find and destroy other targets. How the killer cell determines that it has killed its target and is ready to detach is unknown. Several mechanisms ensure that the killer molecules are inactive during protein synthesis, processing, and storage within the granule. Within the killer cell, the cytotoxic molecules are synthesized as proenzymes that are only processed to their active form within the granule. The granzymes and perforin are expressed with a signal sequence that directs them to the ER. The high concentration of calreticulin in the ER likely serves as a sink for free calcium, which prevents perforin activation.248–251 Cleavage of the signal peptide of the granzymes produces an inactive proenzyme that contains an N-terminal dipeptide that needs to be removed to produce an active protease. During synthesis, perforin is also rapidly transported from the ER to the Golgi. This is facilitated by a conserved C-terminal tryptophan residue by an unknown mechanism.155 Mutation of the terminal tryptophan leads to enhanced death of the killer cell. In the Golgi, mannose-6-phosphate–containing glycans are added

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to progranzymes and perforin, which serve as sorting signals for transport to lysosomes. Within the cytotoxic granule, the N-terminal dipeptide on all progranzymes is removed by cathepsin C (dipeptidyl peptidase I) to generate the active enzyme.252 However, mice and humans genetically deficient in cathepsin C have only partially reduced granzyme activity and cytolytic function and modestly reduced immune defense against viral infection.253,254 This suggests that alternate ways can activate progranzymes. In fact, IL-2 treatment stimulates cathepsin C–independent dipeptide cleavage in NK cells from patients with Papillon-Lefevre syndrome, who have loss of function of cathepsin C.255 Cathepsin H and probably other cathepsins can process progranzyme B.256 Granzymes, which are highly basic, are bound, as are perforin and presumably granulysin, to the acidic serglycin proteoglycan within the granule, which helps keep them inactive. Serglycin is responsible for the electron dense core and may also enhance effector protein storage in the granules.257 Granzyme proteolytic activity and perforin pore formation is also negligible at the acidic pH (pH 5.1 to 5.4) of the granule. Although granzyme and perforin trafficking within cytotoxic cells minimizes leakage of active death effector molecules out of granules, any stray molecules in the cytoplasm could cause cell death.258 During granule exocytosis, some granzymes might inadvertently reenter effector cells. Because CTLs typically kill several targets in succession without harming themselves, an important question is how CTLs protect themselves from their own cytotoxic molecules. An important protective mechanism against killer cell suicide is serpin expression in the killer cell cytoplasm.259 Serpins are members of a superfamily of protease inhibitors with more than 1,500 family members.260,261 Serpins inactivate their target proteases either by covalently and irreversibly binding to the active site of the enzyme or by forming noncovalent complexes that are so strong they resist the denaturing conditions of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).261,262 Serpins that inactivate granzyme B (SerpinB9, also known as PI-9, in human cells263 or its ortholog Spi6 in mouse cytotoxic cells264) are expressed in killer cells. Mice genetically deficient in Spi6 have reduced numbers of memory CD8 T cells, suggesting that CTL survival is compromised by their own granzyme B.265 In fact, cytotoxic T cells from these mice show granule membrane instability and have increased cytosolic granzyme B and apoptosis.266 However, no killer cell serpins are known that inactivate the other granzymes. A recent report suggests that SerpinB4 is a potent granzyme M inhibitor that may render some tumors resistant to granzyme M.267 However, NK cells or activated killer lymphocytes are not known to express this serpin. When perforin and granzymes are released into the immune synapse, why is killing unidirectional? How is the killer cell membrane protected from perforin damage? During granule exocytosis, the cytotoxic granule membrane fuses with the killer cell plasma membrane, exposing internal granule membrane–associated proteins. These include cathepsin B, which inactivates by proteolysis perforin redirected toward the killer cell.8 However, killer cells genetically deficient in cathepsin B survive unscathed when they kill targets.268 This suggests

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that other membrane-bound granule cathepsins (or perhaps other CTL surface proteases or other perforin inhibitors) might also inactivate perforin redirected at the killer cell.

Cellular Resistance to Granule-Mediated Death The human granzyme B SerpinB9 is not only expressed by lymphocytes259,269 but also by dendritic cells,270 cells at immune privileged sites (testis and placenta),48,271,272 endothelial and mesothelial cells,273 and mast cells.274 Similar results hold for Spi6 in mice.264,265,275 Modulators of inflammation such as LPS, IFNγ and IL-1 β276,277 induce SerpinB9 expression. SerpinB9 expression is enhanced by estrogen and hypoxia because of estrogen responsive elements and hypoxia-inducible factor 2 binding sites, respectively, in its promoter.278,279 In particular, SerpinB9 is induced by hypoxia in neuroblastomas.280 This expression pattern suggests that SerpinB9 not only protects killer cells and myeloid cells that express granzyme B from autodestruction but also may protect antigen-presenting cells, bystander cells at sites of inflammation, and cells in immune privileged sanctuaries from granzyme B–mediated killing. It may also be a mechanism for tumor evasion of immune surveillance. No intracellular inhibitors of granzyme A have yet been identified. However, some trypsin inhibitors also inhibit granzyme A. Granzyme A is bound and irreversibly inhibited in the circulation by two trypsin inhibitors, α-2 macroglobulin and antithrombin III.281 Extracellular granzyme A complexed to proteoglycans is resistant to these two protease inhibitors.282 A recent study identified another granzyme A inhibitor, pancreatic secretory trypsin inhibitor, from pancreatic secretions.283 Pancreatic secretory trypsin inhibitor is found in the blood, particularly in patients with severe inflammation and tissue destruction.284,285 Unlike the other two granzyme A inhibitors, pancreatic secretory trypsin inhibitor inhibits granzyme A complexed to proteoglycans.283 It is unclear whether any of these granzyme A inhibitors are expressed in cytotoxic lymphocytes.

Viral Granzyme Inhibitors A number of viruses produce inhibitors of apoptosis or Bcl-2– like proteins that inhibit caspase-mediated apoptosis, which consequently also inhibit granzyme B–mediated cell death. The pox virus–encoded cytokine response modifier A gene (CrmA) inhibits granzyme B.286 CrmA directly binds and inhibits granzyme B both in vitro and in vivo. Overexpression of CrmA in target cells inhibits CTL-mediated cell death. CrmA also strongly binds and inhibits caspases-1 and -8 and weakly inhibits other caspases such as caspase-3.287 Parainfluenza virus type 3 specifically inhibits granzyme B by degrading granzyme B mRNA in infected T cells.288 Importantly, granzyme A transcripts are not affected by this virus. The mechanism of virus-mediated granzyme B mRNA decay is not known. Human granzyme B is inhibited by the adenoviral assembly protein (Ad5-100K) by a unique “unserpin”-like mechanism.143 Ad5-100K rapidly complexes with granzyme B and gets cleaved very slowly at specific sites. Granzyme B that enters the infected target cell during killer cell attack is

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saturated by the abundant Ad5-100K protein. Importantly, the slow kinetics of the cleavage reaction ensures that there is always a molar excess of Ad5-100K protein relative to granzyme B. Ad5-100K, which is also needed for virus assembly,143 does not inhibit caspases or other apoptotic pathways.144 In fact, the inhibitory activity of Ad5-100K is specific for human granzyme B and not its mouse or rat ortholog.

EXTRACELLULAR ROLES OF GRANZYMES Although most research has focused on the cell death–inducing properties of granzymes, there is increasing evidence of extracellular functions of granzymes in promoting inflammation and coagulation and degrading extracellular matrix. Low amounts of granzymes A, B, and K are detected in the serum of healthy donors.289 During inflammation and infection, elevated levels of granzymes are found in serum and other bodily fluids, including the serum of patients undergoing acute cytomegalovirus infection and chronic human immunodeficiency virus infection, the joints of patients with rheumatoid arthritis, and the bronchoalveolar lavage fluid of allergen-challenged patients with asthma and patients with chronic obstructive pulmonary disease.43,282,290–294 Elevated granzyme levels have also been found in the serum of patients with endotoxemia and bacteremia, reflecting the fact that granzymes (but not perforin) are expressed and secreted by activated myeloid cells and a few other cell types, not just by lymphocytes.41,44,45,295–298 In fact, in patients with sepsis, not only is serum granzyme K elevated, but its natural inhibitor (inter-α protein) is depleted, so the free active form of the enzyme is circulating and might cause damage.299 Granzyme B has also been detected in macrophages of atheromatous lesions and rheumatoid joints.296 Proteolysis by extracellular granzymes will be inhibited by serum and extracellular protease inhibitors, such as the trypsin inhibitors, antithrombin III and α-2 macroglobulin.282 Some conditions that induce extracellular granzymes may also increase the release of intracellular serpins.300 Extracellular granzymes might arise from direct secretion —bypassing granule exocytosis—or by leakage from the immune synapse or by release from necrotic cells. Most directly secreted granzymes are secreted as proenzymes, which are inactive.301 However, some of the proenzymes might be activated extracellularly by serum proteases. It is not known whether the immune synapse forms a perfectly tight gasket that completely prevents granzymes from leaking into the extracellular space during degranulation. Asymmetric synapses termed kinapses, which are less stable and less tight, and are formed by cytolytic CD4 T cells and probably under circumstances where the integrated activating signal from the target cell is weaker, may be leakier than the canonical stable synapse.302 Although extracellular granzymes are not likely to get into the cytoplasm of cells to induce cell death without a high local concentration of perforin, they can proteolyze cell surface receptors or extracellular proteins. Recent studies suggest extracellular granzymes A and K activate macrophages to produce and secrete inflammatory cytokines, although the mechanism for this is not known.303–305 These experiments performed with recombinant and purified granzymes need to be confirmed using cytotoxic cells because macrophages

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are exquisitely sensitive to immune activation by endotoxin, and it is not possible to verify the absence of endotoxin in preparations of these trypsin-like enzymes because the endotoxin assay measures trypsin activity.180 Some of the reported extracellular functions/substrates of the granzymes are summarized subsequently, but it is likely that these proteases, despite their high degree of substrate specificity, could have multiple, as yet unappreciated, destructive effects, particularly if present at high concentrations at inflamed sites in the absence of natural inhibitors. The physiologic significance of these extracellular activities is still unclear. One provocative study found a dramatic increase in granzyme B–sufficient versus granzyme B–deficient mice in the rate of rupture of aortic aneurysms in atherogenic mice (deficient in apolipoprotein E [Apo E]) that were also perforin deficient.306 This result suggests that extracellular granzyme B contributes to the pathogenesis of atherosclerosis. The known extracellular activities of granzymes suggest a proinflammatory effect. Granzyme A can activate the proinflammatory cytokine IL-1β directly,307 and granzyme B can convert pro–IL-18 to its active form.308 Granzyme K can activate proinflammatory cytokine production from lung fibroblasts, probably by cleaving their surface protease–activated receptor-1.309 Granzymes also degrade extracellular matrix. Granzyme A may be able to degrade heparin sulfate proteoglycans, collagen type IV, and fibronectin.310–312 Granzyme B can remodel the extracellular matrix by cleaving vitronectin, fibronectin, and laminin.313 Proteolysis of the extracellular matrix might facilitate lymphocyte migration to sites of infection or inflammation or cause tissue destruction at sites of inflammation.313,314 Granzyme A may also inhibit clotting by cleaving the thrombin receptor and von Willibrand factor315–317 or by activating prourokinase to activate plasminogen.318 In the central nervous system, granzyme B cleaves a glutamate receptor (GluR3), potentially contributing to immunoneurotoxicity, excitation, and autoimmunity in the brain.319,320 Granzyme B on its own causes death of neurons in a pertussis toxin–sensitive manner, suggesting possible cleavage or involvement of G protein–coupled receptors.321 Other potential granzyme B receptor targets are Notch1 and fibroblast growth factor receptor 1 (FGFR1), which might inhibit growth signals to developing or malignant cells.322

DEATH RECEPTOR PATHWAYS NK cells and cytotoxic T cells can also trigger apoptosis by ligating and activating cell surface tumor necrosis factor (TNF) receptor family members that contain a cytoplasmic approximately 80 amino acid long death domain on target cells323,324 (Fig. 37.8). Death-by-death receptor ligation can be distinguished from granule-mediated cell death because it is calcium independent and is not inhibited by calcium chelation. The death receptors on target cells form trimers when they are activated. In humans, six members of the larger TNF receptor family contain death domains: Fas (CD95, activated by Fas ligand [FasL, CD95L]), TNFR1 (activated by TNF), DR3 (activated by TNF ligand–related molecule 1 [TL1 or TNFS15]), DR4 and DR5 (activated by TNF-related apoptosisinducing ligand [TRAIL]), and DR6 (unknown ligand). There

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FIG. 37.8. Death Receptor Pathways of Apoptosis. Ligation of a death receptor trimer on target cells recruits the deathinduced signaling complex, which activates caspase-8, releasing it to the cytoplasm where it can cleave Bid to activate mitochondrial apoptotic pathways and cleave and activate the effector caspases-3, -6, and -7. In type I cells, apoptosis does not require mitochondrial amplification, whereas type II cells die only if mitochondrial mediators of apoptosis are released. The caspases activated downstream of caspase-8 are represented by numbered dimers. Cytochrome c required for caspase-9 activation in the apoptosome is represented by a blue ball. Caspase-8 activated by death receptor signaling and granzyme B–mediated death are very similar, although the granzyme B–mediated death is much more rapid. Fas-associated death domain can also recruit an alternate signaling complex that leads to cell activation rather than apoptosis (not shown).

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are mouse orthologs for all of these, except DR5. The death domains recuit one of two adapter molecules, Fas-associated death domain (FADD) or TNF receptor–associated death domain (TRADD), which serve as a platform for recruiting signaling complexes. Depending on the cellular context, signaling by the death receptors can either trigger caspasemediated apoptosis or proliferative and proinflammatory responses. In general, the receptors that predominantly recruit FADD (Fas, DR4, and DR5) are more likely to trigger apoptosis, whereas signaling from the TRADD-associated receptors (TNR1, DR3, and possibly DR6) is more likely to activate cell survival and proliferation pathways via activation of the NF-κB transcription factor and the JNK and p38 MAP kinase pathways. When cell death is triggered in cells in which the caspase pathway is inhibited, targeted cells undergo an alternate programmed cell death pathway termed necroptosis, mediated by a kinase (receptor-interacting protein 1 [RIP1]) that is recruited by TRADD.325,326 After death receptor activation and recruitment of FADD, FADD recruits the apical caspases-8 and/or -10 (the latter has no mouse ortholog) to form the death-induced signaling complex (DISC) at the cell membrane. Within the DISC, these caspases are autoproteolyzed and activated. The activated caspases are then released to the cytoplasm where they can cleave the Bcl-2 family member Bid to activate mitochondrial damage and also cleave and activate the executioner caspases (3, 6, and 7). The mitochondrial pathway amplifies caspase activation by activating caspase-9. Some cells (called type I [eg, thymocytes]) undergo apoptosis without requiring activation of the mitochondrial pathway, whereas others (type II cells [eg, B lymphocytes]) are resistant to cell death if the mitochondrial pathway is blocked. Humans and mice that are genetically deficient in either FAS or its ligand are able to defend against intracellular pathogens but develop an autoimmune syndrome called autoimmune lymphoproliferative syndrome.11,327 Fas-mediated death is required to eliminate chronically activated T cells and contributes to elimination of self-reactive immune cells. Mice with genetic deficiencies in these genes develop similar symptoms. Although caspase-8 is considered the main initiator caspase that associates with and is activated by death receptor signaling, humans bearing caspase-8 mutations have defects in T-cell activation and immunodeficiency rather than autoimmunity, which highlights the importance of the nonapoptotic signaling that results from death receptor engagement.328 Of note, humans with caspase-10 mutations develop autoimmune lymphoproliferative syndrome, suggesting that under certain circumstances, caspase-10 substitutes for caspase-8 in initiating death receptor–mediated apoptosis.329 Nonapoptotic death receptor signaling, mediated by activation of NF-κB, JNK, and MAP kinase pathways, not only promotes cell proliferation but also has a proinflammatory effect, which involves activating chemokine and cytokine production by macrophages and dendritic cells. The relative strength

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of proapoptotic and nonapoptotic signaling is determined in part by cellular expression of c-FLIP, an inhibitor of caspase-8, which is recruited to the DISC and promotes recruitment of RIP1 and TNF receptor-associated factor 2 (TRAF2) to activate nonapoptotic pathways. The c-FLIP mRNA can be spliced into alternate isoforms: c-FLIPL (long) and c-FLIPS (short). DISC recruitment of c-FLIPS, which is homologous to caspase-8 but is enzymatically inactive, inhibits apoptosis, whereas the concentration of c-FLIPL determines whether it is proapoptotic (at low concentrations) or antiapoptotic (at high concentrations). Some tumor cells as well as some activated T cells and NK cells overexpress c-FLIPL, which renders them insensitive to death receptor–mediating apoptosis and promotes their survival. Mouse studies suggest that DR5 (the ortholog of human DR4 and DR5) and its TRAIL may play an important role in innate immune tumor surveillance.330 TRAIL may also participate in eliminating activated CD8 CTLs that were primed in the absence of effective CD4 help.20 Immature NK cells express TRAIL, and DR5-deficient mice are prone to develop tumors and metastases in several endogenous mouse tumor models. As a consequence, soluble TRAIL and agonistic antibodies to DR4 or DR5 are currently being developed for potential tumor immunotherapy.331,332

CONCLUSION Killer lymphocytes in the innate and adaptive immune responses protect us from infection and cellular transformation by releasing cytotoxic granules and help control immune cell proliferation and autoimmunity by both cytotoxic granule release and death receptor–activated cell death. Killer cells trigger multiple programs of cell death, which ensures that the immune system can control pathogens that have devised strategies to resist individual cell death pathways. Lymphocyte-targeted cells are recognized by scavenger cells such as macrophages that rapidly engulf them and remove them to limit inflammation that occurs when cells die by necrosis. Research in the next few years should provide a better understanding of how cytotoxic gene expression is regulated, how killer cells are protected from their own molecules of destruction, the alternate cell death pathways activated by the multiple granzymes, and how the granzymes overcome the strategies by which viruses and tumors try to evade elimination. Further research will clarify the mechanisms and physiologic importance of inflammatory noncytotoxic effects of killer cell enzymes.

ACKNOWLEDGMENTS I thank members of the Lieberman laboratory past and present for many useful discussions. This work was supported by National Institutes of Health grant AI45587. Parts of this chapter were modified from a recent review article on the granzymes.6

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Immunity to Infectious Agents

38

The Immune Response to Parasites Thomas A. Wynn • David L. Sacks • Alan Sher • Eleanor M. Riley

PARASITES AND THE IMMUNE SYSTEM Distinct Features and Global Health Importance of Parasitic Pathogens The term “parasite” is formally used as a designation for eukaryotic protozoan and metazoan pathogens residing within or upon their hosts. The origin of this usage is not clear but almost certainly relates to the common historical period and tropical disease context in which many of these agents were identified. Indeed, parasites are the most phylogenetically diverse category of pathogens and at the lower end of their evolutionary tree are often difficult to distinguish from fungi and other protista in both their morphology and genomic organization. Although the taxonomic basis of their classification into a single group is under question, parasites as infectious agents do share many biologic characteristics. They frequently (although not always) display complex life cycles consisting of morphologically and antigenically distinct stages and produce long-lived or chronic infections to ensure transmission between their hosts. The induction of severe morbidity or mortality is an atypical outcome. However, in the tropical and subtropical regions where transmission is high, the low frequency of disease translates into a major global health and economic problem because of the sheer numbers of people exposed and because of the confounding issues of malnutrition and coinfection. As illustrated by outbreaks in the past decades of disease caused by the protozoa Giardia, Cryptosporidia, Cyclospora, and Toxoplasma, parasites also represent a continuing threat to populations in wealthier countries. Indeed, all of the major food- and water-borne protozoa have been classified as Category B bioterrorism pathogens because of their potential to cause acute epidemic illness. The human immunodeficiency virus (HIV)/acquired immune deficiency syndrome epidemic has also increased the impact of parasitic disease in both developed and developing regions because immunocompromised hosts become highly susceptible

to some normally tolerated parasites such as Cryptosporidia, Toxoplasma gondii, and Leishmania. The danger also exists for many of these organisms to spread into new geographical regions, as environmental degradation and climate change become ever-increasing threats. Finally, parasitic disease remains an important problem in livestock, causing annual economic losses in the billions of dollars and, in the case of trypanosomiasis, limiting the agricultural development of huge areas of potential grazing lands on the African continent. The immune system plays a central role in determining the outcome of parasitic infection establishing a critical balance meant to ensure both host and pathogen survival. As with other infectious agents, disease emerges when the scales tip toward either a deficient or excessive immune response. Manipulation of that response by means of vaccination or immunotherapy remains a key approach for global intervention in parasitic disease. A list of the most important parasitic infections of humans, along with estimates of their prevalence, annual mortality, and current control methods, is presented in Table 38.1. The data testify to the continued enormity of the problem reflected in the numbers of people annually infected and dying of diseases such as malaria, schistosomiasis, and trypanosomiasis as well as the high level of morbidity in those surviving. A striking situation reflected in the data is the complete absence of effective vaccines for protecting human populations. In the case of malaria, the need for a global immunization strategy has become particularly acute as drug resistance spreads worldwide. Clearly, the development of vaccines to prevent parasitic diseases remains one of the major unachieved goals of modern immunology and one of its greatest and most difficult challenges. The scientific challenge lies with the extraordinary complexity of parasites as immunologic targets and their remarkable adaptability to immunologic pressure. The field of immunoparasitology is focused on developing a basic understanding of this important host–pathogen interface for

910

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TABLE

38.1

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Global Impact of Parasitic Disease and Current Control Measuresa

Malaria Schistosomiasis Soil-transmitted helminthsb Leishmaniasis African trypanosomiasis Chagas disease Lymphatic filariasis Onchocerciasis a

THE IMMUNE RESPONSE TO PARASITES

Estimated Prevalence (Millions)

Annual Deaths (Thousands)

216 207 1200 12 0.3–0.5 10 120 18

1200 20–280 12–135 50–80 10–48 10 0 0

Control Methods Currently Used Vector control, chemotherapy Chemotherapy, hygiene Chemotherapy, hygiene Vector control, chemotherapy Vector control Vector control Vector control, chemotherapy Vector control, chemotherapy

Data compiled from World Health Organization Fact Sheets (www.who.int/mediacentre/factsheets/en/) and other miscellaneous sources. Hookworm, ascariasis, and trichuriasis. The data are combined values for all three infections.

the ultimate purpose of intervention. At the same time, the work in this area—particularly in recent years—has provided immunology with a series of major insights concerning effector and regulatory responses as they occur in vivo. Indeed, because of their years of close encounter with and adaptation to the vertebrate immune system, parasites can be thought of as the “ultimate immunologists,” and there is much to be learned from them about the fundamental nature of immune responses.

Some Hallmarks of the Immune Response to Parasites The interaction of parasites with the immune system has several distinguishing features that are of special interest to fundamental immunologists. Most parasitic pathogens are able to survive the initial host response and produce longlasting or chronic infections designed to promote transmission. In the case of many protozoa (eg, Toxoplasma, Leishmania), chronicity is characterized by a state of latency in which replication of the parasite is minimal and infection cryptic. The development of chronicity depends not only on the ability of the parasite to escape protective immune responses (immune evasion) but also on the generation of finely tuned mechanisms of immunoregulation that serve both to prevent parasite elimination and suppress host immunopathology. As discussed in detail later in this chapter, the study of these immunomodulatory pathways in both human and experimental parasitic infections has yielded important insights concerning the mechanisms by which regulatory cells and cytokines control immune effector functions in vivo. An additional prominent feature of the immune response to parasites is Th1/Th2 polarization. For reasons that are not entirely clear, parasitic infections often induce cluster of differentiation (CD)4 + T-cell responses that are highly polarized in terms of their Th1/Th2 lymphokine profi les. This phenomenon is particularly striking in the case of helminths, which in contrast to nearly all other pathogens, routinely trigger strong Th2 responses leading to high immunoglobulin (Ig)E levels, eosinophilia, and mastocytosis. At the opposite pole, many intracellular protozoa induce CD4 + T-cell responses with Th1-dominated lymphokine secretion patterns. This striking difference presents a beautiful example of immunologic class selection. Interestingly,

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in murine Leishmania major infection, CD4 + cells polarize to either Th1 or Th2 depending on the strain of parasite or strain of mouse infected, and the association of these responses with healing or exacerbation provided the first demonstration of a functional role for this dichotomy.1,2 Parasite models have also been used to reveal new effector functions, such as the ability of eosinophils to kill pathogens and, as discussed subsequently, are now being used extensively to study microbial innate recognition and immune response initiation. This ability to uncover and investigate basic immune and immunopathogenic mechanisms while studying the host response to a group of phylogenetically unique pathogens of global importance is perhaps the most engaging and rewarding aspect of research in immunoparasitology.

INNATE RECOGNITION AND HOST DEFENSE Innate recognition plays an important role in determining the outcome of the host–parasite encounter by both providing an initial barrier to infection and by influencing the magnitude and class of the subsequent adaptive immune response. At the same time, from the parasite’s point of view, innate immune defenses must be subverted for infection to be established; it is clear that many parasitic pathogens have evolved specific mechanisms for evading them, and these evasion mechanisms can also provide an explanation for virulence differences amongst parasite strains. Moreover, in some cases, parasites appear to actually hijack the process of innate recognition to deviate adaptive immunity to facilitate their own persistence.

Humoral Mechanisms Innate resistance against parasitic infection is mediated in part by preexisting, soluble factors that recognize and destroy invading developmental stages or target them for killing by effector cells. The alternative pathway of complement activation provides a first line of defense against extracellular parasites and because of this, the infective stages of parasitic protozoa and helminths have developed a various strategies to subvert complement-mediated attack. In some instances, blood and tissue parasites have evolved redundant mechanisms to ensure their survival

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during serum exposure. For example, infective metacyclic and bloodstream trypomastigotes of Trypanosoma cruzi express multiple stage-specific surface glycoproteins, such as gp160 and the 87-93kDa trypomastigote decay accelerating factor, which are actively released by the parasite and are functional homologues of human decay accelerating factor that interferes with assembly of C3 convertases by binding to C3b.3 Another trypomastigote glycoprotein, gp58/ gp68, inhibits alternative pathway C3 convertase assembly by binding to factor B. T. cruzi trypomastigotes have also been found to continuously shed acceptor molecules with covalently bound C3 fragments, thought to be due to an endogenous phospholipase that cleaves glycosylphosphatidylinositol (GPI)-anchored membrane proteins. In addition, trypomastigotes export calreticulin to the parasite surface where, by binding C1, the protein can both inhibit activation of the classical pathway and promote parasite invasion.4 T. cruzi amastigotes, on the other hand, have been shown to resist complement lysis by preventing membrane insertion of the membrane attack complex (MAC), C5b-9. An analogous mechanism of resistance has been observed for the infective metacyclic promastigote stage of Leishmania, which expresses an elongated form of the major surface and released glycolipid on Leishmania promastigotes, lipophosphoglycan (LPG), such that it behaves as an effective barrier to membrane insertion and pore formation by MAC.5 Metacyclics also increase expression of the surface metalloproteinase gp63,6 which can cleave C3b to the inactive iC3b form, thus preventing deposition of MAC.7 Both C3b and iC3b effectively opsonize the complement resistant forms for uptake by macrophages, its host cell of choice. Tissue-invasive strains of Entamoeba histolytica also activate the alternative complement pathway but are resistant to lysis due to the action of a Gal/GalNAc lectin, which mediates adherence of trophozoites to host cells and binds to C8 and C9 terminal components.8 Interestingly, the lectin shares sequence similarities with CD59, a membrane inhibitor of MAC in human blood cells. The damage caused to worms as a consequence of alternative pathway activation is due primarily to the bound C3 activation products that act as ligands for cellular adherence and killing by eosinophils, neutrophils, and macrophages. In addition to synthesizing their own complement regulatory proteins to subdue the activation cascade, helminths also acquire endogenous regulatory molecules from the host. For example, schistosomes can inhibit complement activation through surface-expressed parasite proteins that bind C2, C3, C8, and C9 but also do so by acquiring decay accelerating factor from the host and incorporating it into their teguments.9 Similarly, the infective L3 stage larvae of Onchocerca volvulus, the causative agent of river blindness, were shown to bind the main human fluid phase regulator factor H, thereby promoting C3b inactivation.10 Other parasitic nematodes such as Toxocara canis, Brugia malayi, and Trichinella spiralis appear to block complement attack by secreting proteases that attack the complement pathway11 or regulatory proteins that inhibit its function.12 A well-characterized set of soluble mediators providing a barrier to parasitic infection are the primate-specific

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trypanosome lysis factors (TLF1 and TLF2) present in serum that contribute to the innate resistance of humans to Trypanosoma brucei infection.13 The key active components of these serum complexes are haptoglobin-related protein and apoliprotein L (ApoL)-1 that together are cytotoxic to T. b. brucei and act synergistically to provide enhanced trypanosome killing when assembled into the same high-density lipoprotein (HDL) particle. Haptoglobin-related protein and ApoL-1 have different proposed activities; ApoL-1 is able to form ion pores in lysosomal membranes, whereas haptoglobin-related protein is able to accelerate lysosomal membrane peroxidation. Whereas the TLFs are capable of killing T. brucei, the species that infect humans, T. b. gambiense and T. b. rhodesiense, are both refractory to TLF-mediated cytolysis. This property has been correlated with the expression of a serum resistance– associated gene that is homologous to the variant surface glycoprotein. Importantly, transfection of serum resistance–associated gene from T. b. rhodesiense into T. brucei confers resistance to lysis by human serum, arguing that its expression may have been a critical step in the adaptation of the former parasite for infection of primates.14 Interestingly, mice cotransfected with TLF components display enhanced resistance to T. brucei infection, suggesting that such a genetic modification strategy might be useful in protecting livestock against this parasite, which still is a major impediment to cattle farming in many parts of Africa.15

Cellular Mechanisms Phagocytosis by macrophages represents an innate first line of defense against protozoan pathogens. Macrophages possess primary defense mechanisms, including activation of oxidative metabolism, which are induced by the attachment and engulfment of microbial agents, and the early survival of intracellular parasites will depend on their ability to avoid or withstand oxidative stress conditions. The major source of reactive oxygen species is the multimeric enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Early studies suggested that Leishmania parasites avoid triggering the oxidative burst by actively inhibiting macrophage (PKC) activation,16 which is required for phosphorylation of several sites on the cytosolic oxidase subunit, p47phox. The inhibition of the respiratory burst has also been linked to leishmanial LPG, which is rapidly transferred to the inner leaflet of the phagosomal membrane and prevents translocation of the NADPH oxidase cytosolic components.17,18 As some LPG-deficient Leishmania strains still manage to survive in macrophages, it is clear that redundant mechanisms exist for the parasite to avoid macrophage triggering. These include opsonic ingestion through receptors that are uncoupled from the activation of NADPH oxidase. A number of “silent” entry receptors have been described that are variably used by different species and developmental stages of Leishmania, including complement and mannose receptors,19 and receptors for apoptotic cells.20,21 In contrast to Leishmania, Toxoplasma enters all nucleated cells, including macrophages, by an active invasion mechanism that excludes most host cell proteins, including membrane components of the NADPH oxidase, from the

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parasitophorous vacuole.22 Malaria parasites are also sensitive to oxidative stress, and knockout mice lacking NADPH oxidase suffer more rapid increases in malaria parasite densities than wild-type mice.23 In this case, reactive oxygen species are generated primarily as a result of the degradation of host hemoglobin within parasitized red blood cells (RBCs).24 The detoxification of reactive oxygen species is achieved with a range of low–molecular weight antioxidants, including the tripeptide glutathione, and a number of host- and parasite-encoded enzymes.25 The maturation of phagosomes into digestive organelles represents the heart of the defensive machinery of macrophages, and intracellular parasites have evolved diverse strategies to avoid, escape from, or withstand the acidified, hydrolytic environment of phagolysosomes. For Toxoplasma, the integral membrane proteins that are excluded from the nascent vacuole include those involved in acidification and fusion with the endosomal network.26 If instead the parasite is forced to enter the cell by a phagocytic pathway, as a consequence of, for example, antibody opsonization, it is targeted through the normal phagolysosomal system and is killed.27 For T. cruzi, which trigger a wound repair pathway involving lysosome exocytosis to enter into cells,28 the early vacuole is acidified and potentially fusogenic. Intracellular survival of T. cruzi is dependent on its ability to escape from the vacuole, a process facilitated by its expression of a putative pore-forming protein that is immunologically cross-reactive with human C9, and that can disrupt the phagosome membrane allowing egress of the parasite into the cytoplasm.29 Leishmania promastigotes, again via transfer of their surface LPG which increases the periphagosomal accumulation of F-actin and disrupts phagosome microdomains, transiently inhibit normal phagosome maturation.30 The delay in phagosome maturation may be necessary to allow sufficient time for metacyclic promastigotes transmitted by the sand fly to differentiate into more acidophilic, hydrolase-resistant amastigotes. The various strategies employed by parasitic protozoa to evade the innate defenses of host macrophages are depicted in Figure 38.1. Neutrophils have been an understudied component of the innate cellular response to protozoan pathogens, despite the fact that they are rapidly and massively recruited to the site of parasite delivery by the bites of arthropod vectors. They have been clearly revealed by intravital two-photon microscopy to be the first cells to take up Leishmania in the skin during the first hours of infection following inoculation by needle or vector sand flies.31,32 Their localized recruitment is triggered by the vascular damage caused by the needle injection or the sand fly bite in addition to signals derived from sand fly saliva and the parasite.33,34 The survival of Leishmania following their phagocytosis by neutrophils, similarly to macrophages, is dependent on their ability to inhibit fusion of tertiary granules with the parasite-containing phagosome, which was again shown to be linked to the expression of the promastigote surface LPG.35,36 Interestingly, macrophages have been observed to phagocytose infected neutrophils in vitro,35,37 and the exploitation of the apoptotic cell clearance function of macrophages is the basis for the “Trojan horse” infection model whereby infected, apoptotic

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neutrophils are proposed to silently deliver Leishmania to host macrophages to initiate productive infections in these cells. At later stages of infection, neutrophils may help to defend against Leishmania, in part, by releasing fibrous deoxyribonucleic acid (DNA)-based extracellular traps that ensnare and kill the parasite.38 DNA-based extracellular traps are also elicited by T. gondii tachyzoites, with evidence that they exert direct microbicidal effects and interfere with host cell invasion and parasite spread.39 Although neutrophils are not required to control malaria infections (and may, indeed, contribute to tissue damage), their oxidative burst is severely compromised following exposure to the heme detoxifying enzyme heme oxygenase, leaving malaria patients at increased risk for systemic, gram-negative, bacterial infections.40 Unlike protozoa, helminths are too big to be engulfed by phagocytes and can only be killed by these cells when the latter have been activated by products of the adaptive immune response. Instead, eosinophils, which frequently accumulate in tissues soon after worm invasion, may mediate innate cellular defense against helminth larvae by means of discharge of the major basic protein and cationic proteins present in the granules of these cells.41,42 In contrast to intracellular killing by phagocytes and extracellular killing by eosinophils, some innate cellular defenses do not eliminate parasites directly but instead trigger other effector cells to do so. Perhaps the best studied example of this form of innate immunity is the natural killer (NK) cell pathway of cytokine production. NK cells become activated as a consequence of various parasitic infections; Leishmania promastigotes; Plasmodium falciparum –infected RBCs; components of T. gondii, T. cruzi, E. histolytica, and Cryptosporidium parvum ; and excretory–secretory proteins of the hookworm Necator americanus43 all activate human peripheral blood NK cells to produce interferon (IFN) γ. Despite occasional reports of direct binding of parasite ligands to NK-cell receptors, the emerging consensus is that NK-cell activation is secondary to pattern recognition receptor (PRR)-mediated activation of myeloid dendritic cells (DCs) and monocyte/macrophages and requires both contact-dependent and cytokine-mediated (interleukin [IL]-12, IL-18) signals.44 Moreover, in the absence of Th2 responses, NK cells may become an important source of the protective type-2 cytokine, IL-13, during murine gastrointestinal nematode infections.45 These findings suggest that NK cells may provide a T-lymphocyte independent pathway for cytokinemediated defense and as such serve to prevent parasites from overwhelming the host prior to the development of adaptive responses. Nevertheless, there is increasing evidence that NK responses are markedly enhanced by T-cell–derived IL-2, revealing a novel pathway by which adaptive immune responses may augment innate responses.46,47 Trafficking of NK cells to parasite-infected tissues is critically dependent upon chemokines binding to CCR5.48 Both IL-10 and transforming growth factor (TGF)-β have been shown to serve as negative regulators of NK cell IFNγ production by means of their suppression of monokine and B7 expression by antigen-presenting cells (APCs) or, in the case of TGF-β by directly affecting NK-cell function.49 Such suppression

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T. gondii tachyzoite Rhoptry contents

moving junction parasitophorous vacuole membrane

T. cruzi trypomastigote GIPLs Lysosomes

PMN’s p50 p65

SHP-1

NF-kB

cruzain

STAT1 JAK2

PS

TLR

CR3

IFNγ

GP63 LPG Coat

MR

Leishmania promastigote FIG. 38.1. Evasion of Innate Immune Mechanisms in Infected Macrophages by Parasitic Protozoa. Macrophages possess potent antimicrobial functions that are initiated by uptake of pathogens. Receptor-mediated phagocytosis of Leishmania promastigotes is accomplished by CR3 or mannose receptors that are unlinked from the signaling pathways involved in induction of reactive oxygen intermediates or proinflammatory cytokines. Silent uptake using receptors for apoptotic, phosphatidyl serine positive neutrophils delivering viable promastigote has also beeen described. The transfer of surface lipophosphoglycan to the phagosome membrane results in delayed fusion with lysosomes. Following transformation to amastigotes, which are more hydrolase resistant due to an abundance of surface glycoinositolphopsholipids, phagosome maturation proceeds. Host cell phosphatases such as SHP-1 can be directly activated by Leishmania surface and released molecules, including GP63, to inactivate janus kinase 2 (JAK2) and inhibit interferon (IFN)γ-inducible macrophage functions. Toxoplasma actively invades by rapid discharge of adhesive proteins from secretory organelles called rhoptries, then by inserting and squeezing past a moving junction in the plasma membrane that acts as a molecular sieve, excluding from its vacuole host proteins required for acidification and fusion with the endosomal network. Various Toxoplasma-induced defects in IFNγ signaling have been described, including proteolysis of signal transducer and activator of transcription 1. T. cruzi trypomastigotes enter the macrophage by inducing the recruitment of lysosomes to the plasma membrane; they only transiently reside in the vacuole before escape into the cytoplasm via secretion of a pore-forming molecule. T. cruzi inhibits toll-like receptor–mediated macrophage activation by directly targetting nuclear factor-kappaB (NF-κB) p65 for cleavage by a released protease, cruzain.

may be important in protecting the host against the tissue damaging effects of excessive NK-cell–derived IFNγ and tumor necrosis factor (TNF)- α . NK-cell responses are further regulated by calibration of signals from activating and inhibitory receptors for major histocompatibility complex (MHC) molecules; moderation of NK responses to malariainfected RBCs by inhibitory receptors such as NKG2A/ CD94 and polymorphic killer-cell Ig-like receptors has been proposed.50 Although NK-cell–derived IFN γ can limit the

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initial phase of protozoal replication51 and may a play a role in the polarization and expansion of Th1 cells, in some situations, adaptive T-cell immunity is sufficient to control infection even in the absence of this early NK response.52,53 The role of NK-cell cytotoxicity in resistance to protozoan infection is less well understood; for murine malaria, it is not required for NK-mediated resistance to blood stages,52 but cytotoxic NK killing of malaria-infected liver cells has been reported.54

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Two other cell populations that may function to provide a rapid cytokine response to invading parasites are γ δ T cells and NK T cells. These “unconventional T lymphocytes” express T-cell receptor chains of limited diversity, which may be designed for innate recognition of microbial structures or self-components revealed by infection of host cells. Although the function of NK T cells in innate resistance to parasites is currently under debate, there is considerable evidence supporting a protective role for γδ T cells. Although representing a small percentage of lymphocytes in the periphery, γδ T cells are abundant in epithelial and mucosal tissues, the sites of initial host invasion by many parasites. Moreover, their numbers increase in peripheral blood in response to a number of protozoan infections55 where they can contribute effector cytokines or, in the case of extracellular P. falciparum merozoites, mediate direct granulysin-dependent killing.56 Nevertheless, rather than being essential for host resistance, it is likely that γ δ T lymphocytes (in common with NK cells) provide an adjunct to conventional α β CD4 + and CD8 + T cells in restricting parasite growth during the vulnerable period when the adaptive responses mediated by these lymphocyte subsets is emerging.57,58 A recently discovered group of effectors in the early cellular response to parasites are the innate lymphoid cells (ILCs). This cell population belongs to a heterogeneous family of innate non-T, non-B cells that are not antigen

TABLE

38.2

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restricted. However, as they express CD45 and are dependent on traditional T-cell growth factor signaling pathways, they have been called ILCs. In common with NK cells, which themselves have now been reclassified as ILC, ILC2 produce cytokines important in T-cell subset differentiation and amplification. In the case of ILC2, these cytokines are IL-4 and IL-13, which promote Th2 development and function. As outlined later in this chapter, ILC2 (which may comprise as many as four distinct cell populations) play an important role as a major source of IL-13 in the intestinal response to nematodes and in worm expulsion.59 Also as discussed subsequently, basophils can provide an innate source of IL-4, thereby driving Th2 differentiation, and have been proposed to do so while simultaneously serving as APCs for T-cell activation.

Role of Pattern Recognition Receptors in Innate Recognition of Parasites The innate immune system, in addition to providing a natural barrier that limits infection, also plays a critical role in the initial recognition of parasites and the triggering of adaptive immunity. Invading parasites, such as other pathogens, are sensed by host PRRs that recognize microbeassociated molecular patterns shared by different groups of organisms (Table 38.2). These PRRs are highly expressed

Parasite Molecular Patterns Recognized by Toll-Like Receptors

MAMPs

Parasite

Structure

TLR Stimulated

Reference

GPI anchors

Leishmania ssp. Trypanosoma cruzi

TLR2 TLR2

60

Trypanosoma brucei Plasmodium falciparum

LPG GPI anchors containing unsaturated alkylacylglycerol GIPLs containing ceramide GPI anchors of VSG GPI anchors of MSPs

Toxoplasma gondii

GIPLs and GPI anchors

T. brucei L. major L. braziliensis T. cruzi P. falciparum

Contain unmethylated CpG motifs Contain unmethylated CpG motifs Leishmania RNA virus-1 Contain unmethylated CpG motifs AT-rich stem-loop DNA motif

Hemazoin

P. falciparum

Protein Phospholipid

T. gondii (and related apicomplexa) Schistosoma mansoni

Polymerized heme from degradation of hemoglobin Profilin molecules

Phosphorylcholine

Filarial nematodes

RNA

S. mansoni

Genomic DNA

Lysophosphatidylserine in tegument Phosphorylcholine-containing glycoconjugates on ES-62 glycoprotein Double-stranded RNA in parasite ova

TLR4 Undefined TLR2 TLR4 TLR2 TLR4 TLR9 TLR9 TLR3 TLR9 STING, TBK1, and IRF3-IRF7 TLR9

118

472 64 65 61 80

TLR11

68

TLR2

73

TLR4

72

TLR3

74

DNA, deoxyribonucleic acid; ES-62, filarial excretory-secretory antigen; GIPL, glycoinositolphospholipid; GPI, glycosylphosphatidylinositol; LPG, lipophosphoglycan; MAMP, microbe-associated molecular pattern; MSP, merozoite surface protein; RNA, ribonucleic acid; TLR, toll-like receptor; VSG, variant surface glycoproteins. Adapted from Gazzinelli RT, Denkers EY. Protozoan encounters with Toll-like receptor signalling pathways: implications for host parasitism. Nat Rev Immunol. 2006;6:895–906.

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on both epithelial cells and APCs and, when ligated, trigger cytokine and costimulatory signals that initiate both innate and adaptive cellular responses. Toll-like receptors (TLR) are, so far, the major group of PRRs known to be triggered by parasites. The study of TLR involvement in parasitic infection began with the identification of parasite ligands that stimulate cytokine production from macrophages and DCs. In the case of protozoa, important classes of such ligands are the GPI lipid anchors present on many parasite surface proteins and phosphoglycans, and the membrane-associated glycoinositolphospholipids. Thus, GPIs from T. brucei, Leishmania, T. gondii, and P. falciparum can stimulate macrophages to upregulate inducible nitrogen oxide synthase expression and produce proinflammatory cytokines. Similarly, the GPI anchor fraction of mucin-like molecules from T. cruzi trypomastigotes triggers macrophage production of IL-12 and TNF. Glycoinositolphospholipids from T. cruzi and T. brucei possess similar agonist activities.60 Studies employing reporter cell lines transfected with specific TLRs, or TLR-specific knockouts, have demonstrated that the responses induced by these parasite glycolipids are due to stimulation of TLR2 and to a lesser extent TLR4 (preferentially triggered by glycoinositolphospholipids in the case of T. cruzi). Parasite nucleic acids also represent important ligands for TLR recognition. Thus, genomic DNAs from several protozoan species have been shown to stimulate host proinflammatory cytokine production presumably through the recognition of unmethylated CpG motifs by TLR9. A major role for nucleic acids in stimulation of host resistance to T. cruzi is evidenced by the diminished host resistance of both TLR9 and TLR2/TLR9 double knockout animals61 and by more recent studies on mice deficient in UNC93B1, a protein that mediates translocation of TLR3, 7, and 9 to endolysosomes. These animals showed greater susceptibility to infection than TLR9-deficient mice, a finding that points to an additional role for TLR7 recognition of parasite RNA in the innate response to T. cruzi.62 TLR9 signaling also contributes to host resistance to Leishmania apparently through DNAdriven activation of both DCs and NK cells, although there is debate as to whether this stimulation also plays a role in Th1 response development.63,64 Leishmania strains isolated from patients with mucocutaneous leishmaniasis were found to harbor high amounts of a ribonucleic acid virus that induced a TLR3-mediated hyperinflammatory response in mice that may explain the destructive metastatic lesions associated with mucosal disease.65 In the case of P. falciparum, hemazoin, a product of malaria-induced hemoglobin degradation is a TLR9 agonist,66 although at present there is controversy as to whether this results from contamination with immunostimulatory DNA fragments of parasite origin.67 A chemically different class of TLR ligands are the profi lin proteins expressed by apicomplexan protozoa. These molecules are unique to eukaryotes and are typically associated with intracellular actin. Profi lins from T. gondii, Eimeria, and C. parvum potently trigger IL-12 production from murine DCs as well as systemically following in vivo inoculation.68 Experiments in the murine T. gondii model established that this response is due to the stimulation of TLR11, a TLR that although present in mice and other small

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animals is not functionally expressed in primates. Recent studies have demonstrated that UNC93B1 is required for triggering of TLR11 activation and IL-12 production by T. gondii profi lin in mice, thus revealing an endosomal localization for this TLR.69 Nevertheless, whereas UNC93B1deficient mice are highly susceptible to T. gondii infection, animals deficient in TLR1168 (as well as TLR3, 7, and 9) show no or only partial impairment of resistance, indicating that UNC93B1 must serve additional functions in innate immunity to the parasite beyond its role in TLR11 localization and activation.70 Helminth parasites also express TLR ligands, although as discussed subsequently, their role in the immune response has been harder to defi ne than those characterized in protozoa. Moreover, in many studies, the possibility of contamination by bacterial or viral TLR ligands from symbionts has not been systematically ruled out. Indeed, Wolbachia symbionts confer strong TLR4 and TLR2 agonist activity on fi larial parasites due to their endotoxin-like components.71 Well-studied examples of helminth TLR microbe-associated molecular patterns include the phosphorylcholine containing moieties of the fi larial glycoprotein ES-6272 and the lysophosphatidylserine components of schistosome membranes73 that trigger TLR4 and TLR2, respectively. In addition, schistosome eggs possess double stranded ribonucleic acid molecules that stimulate TLR3 in DCs.74 The role played by TLRs in the host response to parasites is complex and not fully delineated in any of the host–parasite models studied. The main evidence for TLR involvement comes from experiments in mice deficient in myeloid differentiation primary response gene 88 (MyD88), a major adaptor protein required for signaling by most TLRs as well as by the IL-1 and IL-18 receptors. MyD88-deficient animals exhibit a loss in resistance to T. gondii, T. cruzi, L. major, T. brucei, and—in the case of Plasmodium berghei—decreased immunopathology60 that likely reflects the role of TLR signaling in accessory cells (eg, DCs, epithelial cells) in the initiation and maintenance of Th1 responses. However, the altered susceptibility of these mice could also involve non–TLR-related effects of MyD88 deficiency and/or T-cell intrinsic functions of the gene, as demonstrated in the T. gondii mouse model.75 Significantly, no major alterations in helminth-induced immune responses have as yet been described in MyD88deficient hosts on susceptible genetic backgrounds, arguing against a significant role for TLR signaling in Th2-dependent host resistance and pathology in worm infections. In contrast to the dramatically increased susceptibility often observed in MyD88 − / − mice following protozoan infection, mice deficient in single TLRs exposed to the same parasites rarely show pronounced changes in resistance, even when such animals display major immune response impairments. For example, while TLR11− / − mice infected with T. gondii develop severely blunted IL-12 responses both in vitro and in vivo, they nevertheless survive the acute-phase infection and show only a minor increase in parasite load in comparison to fully susceptible IL-12–deficient animals.68 Such findings may reflect redundant functions for different TLR or a requirement for multiple MyD88-dependent TLR (or IL-1/IL-18) signals in host resistance. An additional

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complexity is that mice deficient in single TLRs may show unaltered (or even enhanced) resistance because the TLR in question controls an immunopathologic response or downregulates host effector functions.76 Moreover, as exemplified by recent studies in the peroral T. gondii infection model, TLR signals triggered by host commensal flora (presumably as a result of intestinal barrier breakdown) can also influence the outcome of parasitic infection.77 A major challenge of current research in this area is to decipher such positive and negative TLR signals triggered by parasite, commensal, or host ligands and establish how their integration governs host resistance. There is also a dearth of knowledge concerning the function of PRR signaling pathways outside the TLR family. A topic of particular interest in this regard is the role of inflammasome activation in parasite-induced immunopathology. Two recent studies have demonstrated that malaria hemazoin is a potent activator of the NLRP3 inflammasome in vitro but have yielded conflicting results on the involvement of this pathway in cerebral malaria in vivo.78,79 Most recently, sensing of AT-rich malarial DNA by an an unknown receptor that signals via the stimulator of IFN gene (STING), TANK-binding kinase 1 (TBK1), and interferon regulatory factor (IRF)3 to IRF7 signaling pathway has emerged as an entirely novel pathway of pattern recognition.80 DCs play a major role in linking parasite pattern recognition signals to the induction of both NK-mediated innate responses and T-cell–dependent adaptive immunity. As discussed previously, in the case of many protozoa, DC-derived IL-12 provides a major stimulus for the initiation of host defense pathways. The critical role of DCs is underscored by the impaired IL-12 production as well as impaired control of protozoan (T. gondii) infection81 in mice in which CD11c + DC populations have been genetically depleted. Such DC– depleted mice are also unable to generate CD8 + cytotoxic T-lymphocyte responses against Plasmodium yoelli sporozites.82 The requirement for protozoan invasion in DC activation is complex and depends on the parasite species and DC subset in question. Indeed, as discussed subsequently, infection of DCs can result in suppressed responsiveness to activation stimuli. Nevertheless, infection of DCs with, for example, live Leishmania83 or T. gondii appears to be important for efficient priming of CD8 + T cells despite the sequestration of many of the protozoa in question within parasitophorous vacuoles physically removed from the class I MHC antigen presentation machinery of the host cell.84 In addition, it appears that under certain situations (eg, immunization with irradiated malarial sporozoites85) infected, apoptotic host cells are taken up by DCs as a mechanism of CD8 + T-cell priming. Thus, although live infection is clearly important for efficient T-cell response induction, direct infection of DCs (as opposed to other host cells) may not be required in vivo. The failure to observe parasite-containing DCs engaged in long-lived contact with T cells in in vivo imaging studies lends credence to this hypothesis but is subject to alternative interpretations.86,87 In addition to their role in initiating immune responses, DCs appear to be efficient vehicles for dissemination of parasites into different tissues as illustrated by studies in the T. gondii mouse model.88 Infection of DCs can also serve as a survival strategy for protozoa and as a means for maintaining

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cryptic infection, as suggested by recent studies in which DCs from mice with rodent malaria were found to be capable of initiating infection when inoculated into naïve hosts.89

Downregulation of Innate Signaling Pathways by Parasites: Role in Virulence Determination In addition to upregulating APC function, parasite products can also dampen their activity either as a mechanism of immune evasion or for the purpose of protecting the host against the pathology associated with an uncontrolled immune response. Leishmania, T. cruzi, and Toxoplasma have in common their ability to inhibit proinflammatory and IFNγ inducible responses in infected macrophages, such that the parasites might not only prevent or delay the induction of Th1 responses but also render infected macrophages unresponsive to activation signals during the effector phase of the immune response (see Fig. 38.1). In many cases, the parasites exploit the presence of host cell phosphatases whose normal role is to temper the magnitude and duration of the proinflammatory response. For Leishmania, the inhibition is due in part to activation by the Leishmania metalloproteinase gp63 of host cell protein tyrosine phosphatases, including SHP-1 and other nonreceptor protein tyrosine phosphatases, which inactivate Janus kinase (JAK) family members involved in the IFNγ inducible phosphorylation cascade.90 Other host cell phosphatases were found to be activated by Leishmania cysteine proteinases to regulate the Mitogen-activated protein kinase (MAPK) family members p38 and extracellular signal-regulated kinase (Erk)1/2, resulting in the upregulation of IL-10 and downregulation of nitrogen oxide (NO) and TNF-α production.91 T. cruzi trypomastigotes, via expression of GPI-anchored mucin-like molecules, also activate macrophage phosphatases that target downstream elements of the TLR pathway, including MAPK and nuclear factor-kappaB (NF-κB), to establish a state of tolerance in the infected cells.92 Furthermore, NF-κ B p65 was found to be targeted directly for cleavage by the T. cruzi protease cruzain, and cruzain-deficient parasites rapidly activated macrophages via NF-κ B p65 for IL-12 expression.93 A homologue of cruzain, cysteine peptidase B, is expressed in Leishmania mexicana and was likewise found to degrade NF-κ B p65 to inhibit IL-12 production by infected macrophages.94 The need to counterbalance the excessive inflammation that can be triggered by T. gondii infection extends to the modulation of IFNγ-induced responses, and in particular the signal transducer and activator of transcription (STAT)1 signaling cascade that is critical for resistance to T. gondii. Various defects in IFNγ-initiated STAT1 signaling have been described in T. gondii–infected cells, including proteolytic cleavage of STAT1, dephosphorylation, and prevention of nuclear translocation.95 Thus, although both T. cruzi and T. gondii initiate strong proinflammatory responses in host macrophages, these signaling pathways appear to be subsequently impaired to avoid reaching pathologic levels that may be detrimental to the host and/or lethal to the parasite during the adaptive phase of the immune response. The differential ability of parasites to dampen cellular activation signals appears to be a major factor in virulence

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determination. This has been elegantly studied in recent work on T. gondii examining the basis of the marked virulence differences in mice between Type I, Type II, and Type III strains of the parasite. By examining the progeny of genetic crosses between Type I and III or Type II and III parasites, two highly polymorphic genes ROP18 and ROP16 were identified as major virulence determinants.96,97 Both genes encode serine-threonine kinases associated with a parasite organelle known as the rhoptry. During the process of invasion, rhoptry proteins are discharged into the host cell. Studies with ROP18 indicate that in Type I strains the kinase inactivates effectors known as immunity-related GTPase (IRG) proteins (discussed subsquently) that mediate parasite killing by disrupting the parasitophorous vacuole.98 By contrast, the Type 1 variant of ROP16 transits to the host cell nucleus where it phosphorylates and activates STAT3 and STAT6, which in turn downregulate the proinflammatory IL-12, IL-6, and inducible NO synthase response to the parasite via their roles as positive regulators of silencers of cytokine signaling and/or IL-10.96 Recently, a third virulence factor has been identified, in part, through a new genetic cross between Types II and I. ROP5 is a pseudokinase and serves as a scaffold to enable ROP18 activity.99,100 The mechanism(s) by which these three rhoptry proteins interact in suppressing host cellular function is a major area of interest in the field. A current model of the function of the major T. gondii virulence determinants is presented in Figure 38.2. In addition to their suppressive effects on macrophages and other host cells, parasites or their products can negatively regulate DC function, both as a means to delay the onset of the adaptive response or to restrain an ongoing response to prevent immunopathology. For example, IL-12 production by splenic DCs is rapidly suppressed following initial in vivo stimulation with soluble T. gondii antigen and cannot be restimulated for approximately 1 week thereafter. The antiinflammatory outcome appears to result from the induction, by parasite products, of lipoxin A4, an arachadonic acid metabolite that upregulates silencers of cytokine signaling-2 to inhibit soluble T. gondii antigen–induced DC migration and IL-12 production.101 Similarly, in murine malaria, the initial burst of proinflammatory cytokines is down regulated as DCs become refractory to further TLR signaling.102 The ligands implicated in these interactions include a conserved domain of P. falciparum erythrocyte membrane antigen and hemozoin that has been shown to directly inhibit the maturation of human DCs.103 This TLR tolerance is similar to that induced by endotoxin and may explain the prevalence of “regulatory” CDllclowCD45RBhigh DCs later in infection that have been shown to induce IL-10– secreting T cells as a negative feedback mechanism to control immunopathology.104 The relevance of these findings to human disease is supported by the observation that the interethnic differences in malaria infection outcome in Malian children are associated with reduced expression of activation markers and reduced TLR-induced responses in DCs from P. falciparum –infected Dogon children who experience more severe disease.105 Also, the percentage of human leukocyte antigen–DR + DCs has been reported to be significantly lower in Kenyan children with severe or mild malaria compared to healthy controls,

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PO4

ATF6β

STAT3/6

ROP18I/II

IL-6,IL-4

ROP16I/III STAT3/6 PO4

IL-12

Nucleus

ROP5I/III

Parasitophorous Vacuole! GRA15II

NFκB

PO4 NFκB

Cytoplasm

IRGs TRAF6

FIG. 38.2. Proposed Cellular Roles for Toxoplasma gondii Secretory Proteins in Determining Acute Parasite Virulence. The diagram depicts a cell infected with T. gondii showing the major tachyzoite secretory proteins implicated as virulence factors and the host functional pathways they regulate. The rhoptry protein ROP16 is secreted into the host cell and accumulates in the nucleus. There, it is thought to phosphorylate signal transducer and activator of transcription (STAT)3 and STAT6 transcription factors that upregulate interleukin (IL)-4– and IL-6–dependent responses, leading in turn to impaired IL-12 production. The granule protein GRA15 is also released following invasion and associates with the parasitophorous vacuole membrane (PVM). In the case of Type II strains, this results in the induction of IL-12 through a TNF receptor associated factor 6 (TRAF6), NF-kB–dependent pathway. ROP18 is secreted into the host cell where it remains tethered to the PVM and can directly phosphorylate immunity-related guanine triphosphatase (GTPase), blocking their recruitment to vacuoles resulting in impaired parasite clearance. ROP18 has also been shown to phosphorylate activating transcription factor 6 beta (ATF6b), an endoplasmic reticulum (ER) stress response transcription factor involved in control of T. gondii–induced dendritic cell function. Recent genetic studies indicate that ROP5, a polymorphic, tandemly duplicated pseudokinase, also plays a major role in acute virulence, likely through interaction with ROP18. See Fentress and Sibley470 for a recent review. (Adapted from Sibley LD, Boothroyd JC. Genetic mapping of acute virulence in Toxoplasma gondii. In: Sibley LD, Howlett BJ, Heitman J, eds. Evolution of Virulence in Eukaryotic Microbes. Hoboken, NJ: Wiley Blackwell; 2012, with permission.)

and they also had increased frequencies of DCs expressing BDCA-3, a marker that is upregulated on IL-10 treated monocyte-derived dendritic cells (MDDC).106 In Chagas disease, compromised DC function has been linked to immune suppression in chronically infected mice. T. cruzi blood stages inhibited the lipopolysaccharide (LPS)-induced activation of mouse bone marrow–derived DCs, with both IL-10 and TGF-β important in the induction of the regulatory DC phenotype.107,108 Together, these studies involving T. gondii, malaria, and T. cruzi suggest that parasites drive DC activation and proinflammatory reactions during the acute stages of infection, followed by the emergence of regulatory DCs that modulate the adaptive response, limiting both immunopathology and pathogen clearance. By comparison, studies with Leishmania suggest that for most strains their initial encounters with DCs fail to activate these cells and, as a consequence, proinflammatory responses and cell-mediated immune mechanisms are effectively inhibited or delayed even during the acute stage of infection. In the case of Leishmania major, the infective

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promastigotes deposited in the skin by vector sand flies were found to be poorly taken up by DCs in vitro. However, later in infection, efficient uptake of amastigotes by DC in vitro and in vivo is dependent on parasite-reactive IgG binding to Fc γRI and Fc γRIII; this primes DCs for efficient production of IL-12.109 Furthermore, the initial encounter of other Leishmania species associated with nonhealing infections in mice not only failed to activate DC but also inhibited their subsequent responses to activation signals.110 For example, DCs with clear regulatory properties and bearing the phenotype of CD11clo CD45RB + CD11b + IL-10–producing cells, emerge as the predominant DC subset in the spleen of Leishmania donovani –infected mice and induce antigenspecific tolerance in vivo.111

Mechanisms Underlying Th1/Th2 Response Selection Because parasites often stimulate CD4 + T-cell responses that are highly polarized in either the Th1 or Th2 direction, parasitic infection models have become important tools for studying the cellular basis of Th1/Th2 response selection. DCs are thought to be an important source of the signals that determine CD4 + T-cell effector choice, and their role is best understood for Th1 responses. T. gondii, T. cruzi, and Plasmodium have been shown upon their initial encounter with DCs to upregulate expression of IL-12 and costimulatory molecules. The nature of these encounters has been extensively studied for T. gondii, whose possession of important Th1-inducing TLR ligand(s) has been inferred by the high susceptibility to infection of mice lacking MyD88112 and shown to be selectively acting on DCs.93 Importantly, the high susceptibility of MyD88 − / − mice is comparable to that observed in IL-12− / − mice and is not due to the absence of IL-1/IL-18 signaling.113 As noted previously, the stimulation of TLR11 by parasite profilin appears to be the major MyD88 pathway that triggers this IL-12 response in the murine model.56 T. cruzi induces a delayed, although ultimately strongly and persistently polarized, Th1 response in infected mice that is also MyD88 dependent and, as already mentioned, appears to be induced largely by nucleic acid ligands.91 The Th1 responses that contribute to immunopathology during blood-stage malaria infections are driven, at least in part, by TLR ligands as MyD88 − / − mice have decreased production of IL-12 and attenuated pathology,114 whereas—in acutely infected mice and humans—hyperresponsiveness has been linked to IFNγinduced enhancement of TLR expression on DCs.115 As previously discussed, hemozoin or a hemozoin-DNA complex hemozoin acting through TLR9, and GPI binding to TLR2, appear to be the main malaria component(s) that activate mouse and human blood DCs to secrete IL-12.116–118 Unlike the protozoan pathogens just described, Leishmania appears to trigger TLR signaling in DC poorly, and in most cases, their ability to activate these APCs—for upregulated expression of costimulatory molecules and especially for IL-12 production—requires additional signals that are host derived. For example, the interaction of CD40L on T cells with CD40 on infected DCs enhances IL-12p70 secretion in vitro119 and is essential for L. major –specific Th1 activation and immunity in vivo.120 In addition to IL-12,

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there are signals delivered by other cytokines, including IL18, IL-27, IFN .α , and IL-1, which have been shown to bias Leishmania-specific CD4 + T-cell priming toward a Th1 cell fate. Mouse strains with intrinsic deficiencies in host factors necessary to augment Th1-polarized responses against Leishmania are highly susceptible to infection, and as noted previously, the first direct demonstrations of the relevance of the Th1/Th2 balance to the regulation of disease outcome in vivo were based on studies of L. major infection outcome in different inbred mouse strains. The Th2 polarization that determines the extreme susceptibility of BALB/c mice to L. major is due, at least in part, to an intrinsically poor Th1-differentiating capacity, as even in the absence of IL-4 or IL-4 receptor signaling, IFNγ responses remain relatively weak.121,122 An additional strain difference that may influence Th subset development relates to the finding of very rapid dissemination of parasites from the site of inoculation to the draining lymph nodes and spleen in BALB/c mice, whereas early parasite containment is observed in resistant mice.123 These differences in parasite dissemination raise the possibility that distinct populations of DCs, with the capacity to induce preferential priming for either Th1 or Th2 cells, are present in different tissue environments and become differentially activated in resistant versus susceptible mice. In support of this hypothesis, targeting to selective DC subsets can be achieved by altering the site of antigen delivery; L. major parasites delivered intravenously, intranasally, or even to different skin environments can elicit Th2 responses and nonhealing infections in normally resistant mice.124,125 Although L. major infection in BALB/c mice remains an extraordinary tool for investigating the factors controlling Th2 response selection in vivo, the fact that the model reflects an aberrant response arising, at least in part, from inherent Th1 developmental defects suggests that these defects might not reflect the mechanisms underlying the Th2 polarization that is a hallmark of helminth infections in virtually all mammalian hosts. Furthermore, although Th2 immune deviation is clearly an inappropriate host response to an intracellular pathogen such as Leishmania, Th2 responses are an evolutionarily driven, integral aspect of acquired resistance to most parasitic worms or to the containment of the immunopathologic reactions that worms or their products can provoke. DCs conditioned by exposure to helminth products polarize naïve T cells toward a Th2 phenotype.126,127 Importantly, Th2 polarization driven by most helminth antigens is MyD88 independent,128 whereas Th1 differentiation is in most instances MyD88 dependent. Furthermore, activation of DCs by helminth antigens appears to be minimal as judged by the absence of upregulated MHC and costimulatory markers, cytokine production, and transcriptional or proteomic signatures.129 In the case of DCs exposed to schistosome egg antigens (SEAs) or Fasciola hepatica (liver fluke) tegumental antigen, their maturation and IL-12 p70 production in response to TLR ligands become severely impaired, associated with defective MAPK and NF-κ B activation.130–132 In human fi lariasis, live microfi lariae of B. malayi modulate DC function by altering TLR3 and TLR4 expression and function.132 The major component of Schistosoma mansoni eggs responsible for conditioning DCs for Th2 polarization

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is a secreted T2 ribonuclease omega-1 that is hypothesized to act by limiting conjugate formation between DCs and CD4 + T cells.133,134 N-glycans containing fucose and binding to DC-specific intercellular adhesion molecule 3-grabbing nonintegrin on DCs have also been implicated in the induction of Th2 responses by schistosomes.135 Many of these observations would seem consistent with a model of Th2 induction by DCs that, in the absence of a threshold of instructional, positive signals for Th1 priming occurs via a default pathway. However, defaulting to Th2 is not observed when IL-12–deficient mice are infected with either T. gondii or Mycobacterium avium.136 Furthermore, in the case of DCs exposed to SEA, there remains a requirement for NF-κ B signaling and costimulation (CD40 and OX40L) for the induction of Th2 responses.137 Therefore, it is more likely that helminth-conditioned DCs, rather than initiating a default pathway in naïve T cells, provide a set of active, instructive signals that result in Th2 priming. The exact nature of these instructive signals is, however, currently unclear. Surprisingly, IL-4 does not appear to provide an essential instructive signal for Th2 differentiation, as helminthconditioned DCs will polarize a Th2 response in vitro in the absence of IL-4,126 and mice deficient in IL-4, IL-4R, or STAT 6 develop diminished but still physiologically significant Th2 responses when infected with Nippostrongylus brasiliensis or S. mansoni.138,139 Using T-cell–specific gene ablation of Notch1 and 2 receptors, it was revealed that Notch is required for Th2 responses to Trichuris muris140 and also drives IL-4R/STAT 6 independent Th2 differentiation in vitro in response to SEAs.141 Although DC expression of the Notch ligand, Jagged-2, has been shown to be required for Th2 differentiation in response to SEA in vitro, it is apparently not required for Th2 polarization induced by SEA-pulsed DCs in vivo.142 Although IL-4R/STAT 6 signaling is not essential for priming of IL-4 + CD4 + T lymphocytes in many helminth infection models, it is clear that IL-4 plays a critical role in the maturation and stabilization of Th2 cells once their phenotype has been decided. In this context, Th2 cells need not be the only source of IL-4 for maturation of the Th2 response; basophils committed to express IL-4 are recruited to the liver and lungs of mice infected with N. brasiliensis,143 and a direct role for basophils in helminthinduced type 2 immunity was confirmed by the capacity of adoptively transferred IL-4 producing, adult-derived basophils to restore the ability of juvenile mice to expel N. brasiliensis.144 It has been argued that basophils rather than DCs are the critical cells responsible for helminth-induced Th2 polarization (see following discussion), but this hypothesis has been challenged and may be limited in validity to those experimental models where IL-4 appears necessary for Th2 priming.

EFFECTOR MECHANISMS OF HOST RESISTANCE Once parasites have successfully evaded innate host defenses and their antigens have been processed and presented by APCs, adaptive cellular and humoral immune responses

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are invariably induced, usually against a wide array of antigenic constituents of each pathogen. The problem is that because of the nature of the host–parasite adaptation, these responses are rarely orchestrated in a manner that will completely eliminate the parasite or restrict its growth. The design of successful immune intervention strategies depends on the identification of relevant target antigens but even more importantly on an understanding of the type of immune responses and protective mechanisms that must be induced. These effector mechanisms can be broadly classified based on the type of parasite (intracellular or extracellular) against which they are directed.

Intracellular Parasites Because of their primary habitat within host cells, intracellular parasites are thought to be particularly susceptible to cell-mediated immune effector mechanisms, often involving a mixture or succession of CD4 + and CD8 + T-cell responses. The extent of CD8 + T-cell involvement appears to be related to the degree of class II versus class I MHC expression on the host cells infected. CD8 + T cells are especially critical effector cells for the control of T. cruzi or T. gondii infections, as well as the liver stages of malaria, as these parasites infect nucleated cell types that express only MHC class I molecules. Although slow to develop during the early stages of T. cruzi infection, CD8 + T cells with specificity for immunodominant antigens encoded by the trans-sialidase gene family reach enormous numbers in mice (30% of the total CD8 + T-cell population) and following a contraction phase, persist throughout the chronic stage of infection.145 Unlike some chronic viral infections, T. cruzi –specific CD8 + T cells in chronically infected mice do not appear to become exhausted and bear the phenotype of effector memory cells that require persistent antigen to be maintained.146 In mice chronically infected with T. gondii, an effectormemory population of CD8 + T cells is especially critical for long-term resistance to toxoplasmic encephalitis.147 Even in Leishmania infection, where parasites reside almost exclusively in macrophages, CD8 + T cells can be highly protective against both primary infection and reexposure.148,149 In addition to their contribution of IFNγ to the effector response, CD8 + T cells might also control intracellular parasitic infection through the lysis of host cells. In every protozoan infection analyzed, however, including T. gondii,150 malaria,151 and T. cruzi,152 mice deficient in the lytic molecules perforin or granzyme B showed no or minimal loss of host resistance. In fact, perforin-mediated lysis of vascular endothelial cells was found to contribute not to protection but to the severity of experimental cerebral malaria in mice.153 These observations suggest that, as already noted for NK cells (see previous discussion), the protective functions of CD4 + and CD8 + T cells against intracellular parasites are mediated primarily through cytokine production rather than target cell lysis. IFNγ is the key cytokine involved in control of intracellular protozoan parasites, as demonstrated by the extreme susceptibility of IFNγ-deficient mouse strains to infections with Leishmania,154 T. cruzi,155 T. gondii,156 Plasmodium,157 and even C. parvum,158 which dwells in epithelial cells inside the

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gut. Its mechanism of action is perhaps clearest in the case of Leishmania, which replicate primarily in macrophages— a cell type readily activated by this cytokine. The major function of IFNγ in restricting parasite growth appears to be the induction of inducible nitrogen oxide synthase (also known as NOS2) and the subsequent generation of toxic reactive nitrogen intermediates (RNI) within infected macrophages. Thus, disruption of the NOS2 gene in a normally resistant strain leads to a susceptible phenotype, and macrophages from the same knockout strain show defective IFNγ-induced control of parasite growth.159 In addition to IFNγ, optimal production of RNIs is dependent on costimulation with TNF-α or triggering by alternative signals such as IFN- αβ or by CD40L produced or expressed by activated CD4 + T cells.160 The production of RNI by IFNγ-activated macrophages is inhibited by IL-4, IL-10, IL-13, and TGF-β,161 and this is likely to be a major mechanism by which the Th2 response prevents healing in Leishmaniasis. Cytokine-mediated control of intracellular T. gondii infection involves a more complicated mechanism than induction of RNIs. The immunity induced cannot be attributed solely to activated macrophages as originally thought, as the parasite infects multiple host cell types and host resistance requires IFNγ signaling in cells of both hemapoietic and nonhemapoietic origin.162 Accordingly, the role of RNIs in resistance has been shown to be limited, functioning predominantly in the chronic stage of infection, although the outcome may vary following oral infection with T. gondii. An important clue concerning the mechanism controlling acute infection came from studies in mice deficient for members of the p47 GTPase family, now referred to as IRG genes. The IRG proteins involved are induced by IFNγ in a variety of hemapoietic as well as nonhemapoietic cell types. Mice deficient in either Irgm3 (IGTP) or a second family member, Irgm1 (LRG-47), were found to be highly susceptible to infection with T. gondii while developing a normal IFNγ response.163 Upon IFNγ stimulation of T. gondii– infected cells, Irgm3 has been shown to traffic from the endoplasmic reticulum to the parasitophorous vacuole where it participates in a process involving disruption of the vacuole, stripping of the tachyzoite membrane, and autophagic elimination of the parasites in the host cell cytosol.164,165 Although Igrm1 is similarly required for effective IFNγ -dependent control of T. gondii, it is not recruited to the parasitophorous vacuole and is thought instead to serve as a negative regulator protecting immune cells from a variety of cytotoxic functions.166 In this regard, Irgm1 also plays a major role in IFNγ -dependent host resistance to T. cruzi where, in addition to regulating intracellular killing of the parasite, it is required for a normal hematopoietic response to the infection.143 As noted previously, IRG proteins (eg, Irgm3) are targeted by T. gondii virulence factors, and thus, the IRG system may be coevolving in response to pathogen pressure.98,166 Another IFNγ -dependent mechanism of intracellular parasite killing that limits T. gondii replication in human but not mouse nonhemapoietic cells is the induction of indolamine 2,3-dioxygenase, an enzyme that catabolizes tryptophan, an essential amino acid for growth

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of this protozoan.167 These examples underscore the complexity of the effector pathways triggered by IFNγ, which act against different parasites in different host cells. Although resistance to the erythrocytic stages of malaria is largely mediated by antibodies, they likely act in concert with T cells. Thus, even when the primary role of antibody is clear, as with the passive transfer of immune serum, the extent of protection is reduced by prior splenectomy or T-cell depletion. The relevant pathways seem to function through cytokine (eg, IFNγ, TNF-α) activation of macrophages that phagocytose and destroy infected RBCs in the spleen. This process is augmented when infected RBCs are opsonised by Fc receptor (FcR)-binding antibodies, providing an excellent example of cooperation between cellular and humoral arms of the immune response. In support of this concept, resistance to human malaria has been correlated with T-cell production of IFNγ168 and generation of NO169 in vitro and with FcR-binding antibody subclasses.170 Moreover, blood-stage immunity can be established in the absence of antibody, for example, after infection in B-cell–deficient mice, and can be transferred with defined CD4 + T cell lines and clones, indicating that cellmediated effector mechanisms can operate independently of antibodies.171 Experimental infection studies in humans further support this idea. Ultra-low dose challenge with P. falciparum–infected RBCs172 or sporozoites173 confer immunity to reinfection; Th1 cell responses are induced, but little or no malaria-specific antibody was detected. However, experimental vaccines designed to induce potent T-cell responses have been associated with high levels of immunopathology. For example, 60% of mice receiving a vaccine designed to induce CD4 + Th1 responses to an immunodominant blood stage antigen of Plasmodium yoelii died despite being able to effectively eliminate infected erythrocytes.174 Intracellular protozoa live briefly in the extracellular milieu during initial host infection and when they invade new cells during their in vivo multiplication. During this period, they are vulnerable to attack by antibody. In addition, while not directly killing free parasites, antibodies can block their invasion of new cells, thereby suppressing infection. These forms of humoral immunity are of special interest in vaccine development. After repeated infection, humans living in areas endemic for P. falciparum gradually develop immunity to asexual blood stages, preventing high-density parasitemia, and thereby preventing clinical disease. The contribution of antibody to this resistance was demonstrated in experiments in which serum from highly immune adults was transferred to acutely infected children, resulting in a temporary but highly significant reduction in parasitemia.175 The generally accepted explanation for slow acquisition of immunity to malaria is that it is strain specific, and that an individual becomes immune only after being exposed to the strains circulating in his or her community. Furthermore, humoral immunity to malaria is likely to depend upon an array of antibodies of differing antigen specificities and functions, including agglutination of sporozoites, merozoites, or parasitized RBCs; inhibition of parasitized RBC cytoadherence to small blood vessels; and/or blocking of hepatocyte or red cell invasion by sporozoites or free merozoites.176 For the latter mechanism, the fine specificity of the

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antibody is crucial as some antibody specificities are able to block merozoite invasion into erythrocytes, whereas others, with distinct but overlapping specificities, either have no effect or, in the worst case, impede the activity of invasion inhibitory specificities.177 A well-studied example of antibody-mediated protection is the response to the circumsporozoite (CS) protein present on preerythrocytic stages of malaria.178 Monoclonal antibodies directed against the CS protein prevent invasion and development of sporozoites in cultured hepatocytes and in vivo, in passive transfer studies, confer protective immunity against P. berghei, P. yoelii, Plasmodium vivax, or Plasmodium knowlesi sporozoite challenge.179 With the advent of intravital imaging techniques, it has become apparent that sporozoites are initially inoculated into dermal connective tissues where they may take up to 30 minutes to locate and invade a blood vessel. In immunized animals, or those that have been passively transfused with anti-CS antibodies, sporozoites become immobilized within minutes and fail to invade blood vessels (see Vanderberg and Frevert180 for excellent video footage of these effects). Although incomplete sporozoite neutralization or inhibition of hepatocyte invasion allows the development of forms that can infect red cells, reducing the number of developing hepatic schizonts can significantly reduce the size of the blood inoculum, delay the onset of patent parasitemia, and may allow the host more time to develop effective anti–blood-stage immunity. Sporozoite antigens can confer significant protection in their own right (as described subsequently, see vaccines) and may also prove to be very valuable components of a multivalent vaccine.

Extracellular Parasites Extracellular parasites are a highly diverse group of pathogens that include nematoda (round worms) and platyhelminthes (trematode and cestode flat worms) as well as some extracelluar protozoa such as Giardia spp. and African trypanosomes. Unlike bacteria, viruses and protozoa that replicate within their hosts and most helminth parasites require an intermediate host or a period outside the mammalian host to replicate and complete their life cycle. Extracellular parasites exhibit variability in size, tissue tropism, and mechanism of immune evasion; helminths, in particular, often live in their definitive host for several years, hiding out in the gut, blood, lymphatics, and a variety of other host tissues. Together, these life history traits likely explain why a distinct set of complementary immune effector mechanisms are required to combat these large, multicellular pathogens. A variety of specialized innate and adaptive immune cells and mediators are triggered during infection including ILCs, T cells, eosinophils, mast cells, basophils, macrophages, and antibodies, and together these effectors mount a multipronged attack. Thus, immunity is achieved through a variety of mechanisms that include antibody-dependent cellular cytotoxicity, mucus secretion, alterations in gut physiology, and exposure to toxic mediators produced by epithelial cells, eosinophils, and alternatively activated macrophages (AAMs). Intestinal microflora have also been shown to influence parasite fecundity181 and host immunity.182

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Most extracellular helminth parasites induce highly polarized CD4 + Th2 cell responses (IL-3, IL-4, IL-5, IL-9, IL-10, and IL-13) that promote immunity by triggering mast cell, eosinophil, giant cell, IgG1/IgE/IgA, and mucosal cell responses. Mice deficient in the IL-4 receptor alpha-chain (IL-4Rα), STAT6, or the transcription factor GATA-binding protein 3 (GATA3) display increased susceptibility to a wide variety of helminths, identifying a critical role for the IL-4 and IL-13 signaling cascade in antihelminth immunity.183,184 In addition to antigen-specific adaptive immune responses, it has become clear that type 2 cytokines secreted by ILCs are also involved in the development of resistance to some helminth parasites.59,185–188 Although the mechanisms that trigger the differentiation and development of Th1 and Th17 type responses are well defined,189,190 the mediators that drive Th2 development appear to be more complex, although DCs,191 select parasite antigens,133,134,192 basophil-derived IL-4,193,194 and epithelium-derived alarmins such as thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 released during parasite invasion188,195–198 are all involved to varying degrees depending on the particular helminth. Whereas basophils were recently reported to be the critical drivers of Th2 responses during helminth infection—by serving both as APCs and by producing IL-4199,200 — subsequent studies have questioned the ability of basophils to serve as APCs201 and instead identified DCs as the primary APCs and basophils as the dominant source of IL-4.144,191,194,202–206 Thus, conventional DCs and basophils are both involved in the development of helminth-induced Th2 immunity. Many helminths cause significant tissue damage as they migrate through tissues, triggering the release of alarmins by mucosal epithelial cells. One of the first epithelial-derived alarmins shown to play a role in helminth-induced Th2 immunity was epithelial cell-derived TSLP,197,207,208 with a recent study demonstrating that TSLP can elicit a unique population of basophils that promotes Th2 cytokine-mediated immunity.209 Nevertheless, other studies have suggested that the contribution of TSLP to Th2 response development may be limited to T. muris infection.196,198 Indeed, several Th2promoting helminth antigens have been shown to bypass the need for TSLP in the induction of Th2 immunity because they can directly inhibit the production of IL-12 p40 in DCs.196,210,211 IL-1R4 (T1/ST2) and its ligand IL-33, an alarmin released from the nucleus of necrotic epithelial and endothelial cells and fibroblasts, have also been shown to contribute to Th2 response development,212 again by suppressing the Th1 arm of immunity.213 A similar role has also been identified for the IL-17 family cytokine IL-25 (IL-17E).214–216 Studies conducted with IL-25 − / − mice showed that by controlling the downstream actions of IL-13,217 epithelial cell-derived IL-25 is required for the development of immunity to both T. muris and N. brasiliensis.215,216 Thus, multiple alarmins released by epithelial cells are involved in the generation of protective IL-4/IL-13 responses during helminth infection, with the relative contribution of each mediator being largely dictated by the pathogen and its site of infection. When these alarmins are released during parasite invasion, they induce the recruitment of ILCs called nuocytes or

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innate helper cells, which are poor producers of IL-4 but secrete large quantities of IL-13, thus quickly activating downstream protective type 2 effector responses.59,186–188 Their rapid production of type 2 cytokines also provides a feedforward mechanism to activate the adaptive immune response, further amplifying type 2 immunity. The combined innate and adaptive type 2 response activates a broad range of downstream antiparasite effector mechanisms. Epithelial cells and goblet cells in the gut express the type II IL-4 receptor,218 which binds IL-4 and IL-13, triggering goblet cell differentiation and mucus secretion.219–221 Indeed, the mucins Muc2 and Muc5AC were recently shown to be critically required for the development of resistance to several intestinal nematodes.222,223 Intestinal epithelial cells also secrete resistin-like molecule-β (Relmβ),224 which regulates both the spontaneous and IL-4–induced expulsion of the luminal dwelling parasites N. brasiliensis and Heligmosomoides bakeri (formerly called Heligmosomoides polygyrus).225 IL-4 and IL-13 also have effects on intestinal physiology, causing decreased peristalsis, increased mucosal permeability, reduced sodium-linked glucose absorption, and decreased chloride secretion in response to 5-hydroxytryptamine and acetylcholine,226,227 which facilitates the expulsion of nematodes from the gut. IL-4Rα signaling also stimulates intestinal smooth muscle contractility and epithelial cell proliferation and turnover,228,229 which operate together with epithelial secretions to promote parasite entrapment in mucus and ultimately expulsion from the gut via a combined “weep and sweep” mechanism.230,231 Intestinal permeability and smooth muscle contractility are also regulated by the actions of mast cell–derived proteases and protease-activated receptors that

are expressed throughout the small intestine. An overview of antihelminth effector mechanisms is shown in Table 38.3. Several worms have been studied in detail in this regard: T. muris, a natural parasite of the mouse and closely related to human whipworm, H. bakeri, Litomosoides sigmodontis, T. spiralis, N. brasiliensis (the rat hookworm), and Strongyloides stercoralis. Although Th2 cytokines are clearly involved in resistance to many of the intestinal nematodes, the importance of Th2 immunity is less certain with many of the filarial (B. malayi, Wuchereria bancrofti, L. sigmodontis)231–233 and schistosome species,234 as discussed in detail in the following text. T. muris and H. bakeri are both transmitted by the oral– fecal route independently of an intermediate host; in some strains of mice, they are capable of establishing chronic infections. In the case of T. muris, development of immunity is dependent on the genetic background and sex of the definitive host, with resistant animals rejecting the parasite shortly after exposure and susceptible animals developing chronic infections.235,236 With this parasite, resistant mice develop type 2 responses, whereas susceptible mice mount type 1 responses, with a variety of immunoregulatory cytokines and mediators influencing this decision. For example, in susceptible strains of mice, IFNγ, IL-12, MyD88, and TLR4 deficiencies235,237 can promote the expansion of type 2 cytokine responses and facilitate the clearance of the parasites. For N. brasiliensis, H. bakeri, and T. muris, primary immunity in most strains of mice depends on the development of a parasite-specific CD4 + T-cell response,238,239 with recent studies demonstrating that a broad T-cell receptor repertoire is needed for the development of efficient immunity to N. brasiliensis.240 However, exogenous IL-4 or IL-13 can cure

NO

Yes Yes NO

Yes

NO

NO

Yes Yes NO NO NO NO

Yes

Yes NO Yes NO Yes

NO

NO Yes NO

Yes

NO

Yes ? (fecundity)f

Yes NO NO NO

?

Yes NO ?

?

Yes

NO

?

NO NO

Yes

NO

NO

NO ?

?

?

?

?

?

?

Yes

Yes

?

NO Yes

Yes

NO

Yes

?

NO NO NO Yes

Yes

?

?

?

?

Yes

NO (Yesg NO (Yes NO human) human)

?

?

NO ?

NO Yes NO

NO NO NO NO (Yes ? human)

?

IFN-gamma

NO Yes Yes Yes Yes

IL-10

IL-9

Yes

IL-4/IL-13

NO NO NO

IL-25

Yes Yes ?

TSLP

IL-5

T1/ST2e -IL-33

Mucins goblet cells Relmb

NO

IgE

NO

M1 Mø/NOd

Basophils

Yes

M2 Møc

Mast Cells

Trichuris muris Nippostrongylus brasiliensis Heligmosomoides bakeri (Heligmosomoides polygyrus) Litomosoides sigmodontis Trichinella spiralis Schistosoma mansoni

ILC2b

Eosinophils

Neutrophils

Major Antihelminth Effector Mechanisms

CD4+ Th2 cells

38.3

923

Parasitea

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Yes Yes Yes NO (minor)h NO NO NO NO (Yes Yes NO NO NO human) (minor)

CD, cluster of differentiation; IFN, interferon; Ig, immunoglobulin; IL, interleukin; NO, not needed; TSLP, thymic stromal lymphopoietin; YES, required; ?, indicates not investigated. a Effector mechanisms involved in parasite clearance/expulsion determined from animal studies. b ILC2: innate lymphoid cells producing Th2 cytokines (IL-13). c M2 Mø: M2 alternatively activated macrophages. d M1 Mø: M1 classically activated nitric oxide–producing macrophages. e T1/ST2: IL-33 receptor. f Decreases parasite fecundity only. g No role in immunity in rodents but strong correlative evidence of a role in acquired immunity in humans. h Only a minor role observed in some animal studies.

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primary N. brasiliensis, T. muris, and H. bakeri infections in T-cell–deficient mice.241 Recent studies demonstrated that IL-25 and IL-33 induce IL-13–expressing nuocytes that are capable of mediating immunity independently of CD4 + T cells, suggesting that the production of IL-13 by these ILC may be sufficient, particularly during primary infections.59 The rapid immunity that develops during secondary infections, however, is likely facilitated by antigen-specific CD4 + T cells. Interestingly, the individual roles of IL-4 and IL-13 in these infections appear to be dependent on the genetic background of the host as C57BL/6 IL-4 knockout mice are susceptible to T. muris infection, whereas BALB/c IL-4 knockout mice are resistant.242,243 These latter studies and other comparative studies conducted in IL-4Rα –, STAT6-, and GATA3-deficient hosts revealed an IL-4–independent role for IL-13 in resistance,183,184 which was confirmed by treating BALB/c IL-4–deficient mice with a soluble IL-13 inhibitor. IL-13– and IL-13Rα1–deficient mice also develop chronic infections despite generating relatively normal IL-4 responses,244 further emphasizing the critical role of IL-13 in antinematode immunity.242 In contrast to T. muris where the requirement for IL-4 depends on the genetic background of the host, development of immunity to N. brasiliensis appears to be almost entirely IL-4 independent, as demonstrated in experiments with IL-4 − / − and anti–IL-4 monoclonal antibody (mAb)-treated mice.183 The IL-4–independent mechanism was, however, dependent upon IL-4 receptor and STAT6 signaling, again identifying a critical role for IL-13, which was confirmed in IL-13 blocking studies. IL-13– and IL-4/IL-13–deficient mice are also more susceptible to N. brasiliensis than IL-4 − / − mice, further emphasizing the dominant role of IL-13.245 Type 2 immunity was suggested to be controlled predominantly by an innate, IL-13– expressing, noneosinophil type cell of hematopoietic origin,185 which has subsequently been hypothesized to be one or more of the recently described lineage-negative (CD4 − CD127+IL1RL1+IL-17RB +RoRgt−) innate lymphoid cell populations: natural helper cells or fat-associated lymphoid clusters, nuocytes, innate natural helper type 2 cells, multipotent progenitor type 2 cells, and/or basophils.59,186–188,203,204 Studies are ongoing to decipher the relative contributions of each of these populations in antinematode immunity. Unlike infections with N. brasiliensis and T. muris, most mouse strains are susceptible to primary H. bakeri infections, but following drug clearance, the animals develop strong type 2 responses and become highly resistant to secondary infection. The maintenance of immunity during recall infections is crucially dependent on the presence of memory CD4 + Th2 cells at the site of infection,246 with the lung being an important location for the initial priming of memory CD4 + T cells.247 The Th2 response triggers the development of arginase-1–expressing AAMs, which help clear primary N. brasiliensis and secondary H. bakeri infections.218,248 The relative importance of AAMs in immunity to other nematode infections, however, remains less clear.249 Other cytokines, chemokines, and signaling pathways, including TNF, IL-18, IL-1, IL-10, IL-21, IL-27R (WSX-1), CCL2, Notch, and the NF-κB family have also been identified as important regulators of protective IL-13 responses during intestinal nematode infection. For example, mice treated with

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neutralizing antibodies to TNF develop relatively normal Th2 responses following infection with T. muris, yet they fail to expel their parasites, illustrating a critical downstream role for TNF in the mechanism of IL-13–mediated immunity.250 IL-1α and IL-1β are also protective, although unlike TNF deficiency, which does not affect the Th2 response, IL-1 deficiency has a profound inhibitory effect on IL-13 production, thus identifying a unique role for IL-1 in the development of Th2-dependent antinematode immunity.251,252 IL-21 also appears to play a critical role by augmenting Th2 cytokine production and the differentiation of AAMs.253–255 Resistance to T. muris is also highly dependent on IL-10, which actively downregulates expression of the antagonistic type 1 cytokines IL-12 and IFN-γ.220,256 IL-10–deficient mice also display marked morbidity and mortality following T. muris infection, identifying an additional role for IL-10 in the suppression of immune-mediated pathology during nematode infection. Interestingly, broad-spectrum antibiotics were found to partially protect T. muris–infected IL-10–deficient mice from morbidity and mortality, suggesting that an outgrowth of opportunistic bacteria in the gut exacerbates disease.220 Immunity to T. spiralis also requires STAT6-257 and IL-4R signaling but not IL-4,231 again identifying a dominant role for IL-13–mediated signaling in the development of immunity.258 The fact that exogenous IL-13 can promote resistance in immunodeficient hosts suggests that a non–bone marrow– derived cell in the gastrointestinal tract expressing the IL-4R is required for immunity, which was confirmed in elegant bone marrow chimera experiments.259 The contribution of IL-13–producing innate effector cells in immunity to T. spiralis, however, has not yet been examined. Mast cells, which have been linked with IL-4/IL-13–induced changes in intestinal physiology, also play an important role in immunity to T. spiralis, as mice treated with mAbs against stem cell factor (a non–T-cell–derived cytokine) or c-kit (both of which play a central role in mast cell development) are unable to expel the parasites.260 In these experiments, there was no inhibitory effect on the CD4 + Th2 cell response, and once the mAb treatment was stopped, the parasites were quickly expelled. These studies suggested that the CD4 + Th2 cell response cooperates with stem cell factor to promote mastocytosis, which in turn facilitates parasite expulsion by secreting proteases that degrade tight junctions, leading to increased fluid production in the gut.261 Several cytokines made by CD4 + T cells, including IL-3, IL-4, IL-9, and IL-10, have been implicated in the development of the protective mast cell response during T. spiralis infection.256,262–266 IL-9 is particularly important as high concentrations of IL-9 can accelerate the clearance of T. spiralis267 and T. muris,268 whereas anti–IL-9 mAb treatment significantly inhibits immunity to T. muris.269 IL-9–producing DCs also generate protective CD4 + Th2 responses,270 raising the possibility that IL-4 derived from mast cells operates in a feedback loop to enhance Th2 cell differentiation.268 Mast cell–derived tryptases (eg, mMCP-6) are also be involved in the development of anti–T. spiralis immunity.271 Eosinophils are frequently associated with helminth infections, and AAMs have been shown to play an important role in eosinophil recruitment to peripheral sites.272 Surprisingly, however, eosinophils do not appear to play

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a significant role in immunity to T. muris, H. bakeri, T. spiralis, or N. brasiliensis.273 Nevertheless, there is evidence from experiments with anti–IL-5 mAb treated, IL-5 transgenic, and IL-5Rα –chain knockout mice that these cells participate in immunity against tissue-invasive larval forms of Strongyloides spp. and Angiostrongylus cantonensis.274–277 One study suggested that eosinophils promote immunity by serving as APCs.277 There is also a large body of literature, primarily from studies in rats and humans, which suggests that eosinophils play a critical role in protection against (non–gut dwelling) schistosomes,278,279 although anti–IL-5–treated and eosinophil-deficient mice displayed similar parasite burdens as their wild-type counterparts, possibly suggesting host-specific roles for eosinophils in the development of immunity.278,280 In contrast to S. mansoni infections, immunity against primary T. spiralis infection in mice is impaired in IL-5–deficient mice and associated with decreased muscle hypercontractility.281 However, recent studies conducted with eosinophil-deficient mice suggested that eosinophils promote the survival of muscle stage larvae in chronic T. spiralis infection by suppressing IFNγ and NO production.282 Overall, despite the prevailing dogma, the majority of studies have failed to support a critical function for eosinophils as direct effector cells in immunity to intestinal helminths and instead suggest more of an immunoregulatory role for these cells.283–285 In addition, because tissue eosinophils have been shown to produce IL-4 and IL-13,280,286 one of their primary functions may be to participate in type 2-cytokine–mediated wound repair,287,288 which appears to be a critical determinant in the successful establishment of chronic infections by many tissue-destructive helminths.289 A similar Th2-inducing mechanism has also been proposed for tissue basophils, which were identified as an important source of IL-4 in schistosome, filarial, and hookworm infections in both mice and humans.143,290,291 In individuals infected with fi larial parasites, the presence of antigen-specific IgE appears to be critical for the secretion of IL-4 by basophils.291 Transgenic mice with constitutive and selective deletion of basophils are highly susceptible to secondary but not primary N. brasiliensis infection,203 suggesting that the activation of basophils by antigen-specific IgE is critically required for the development of secondary immunity. Thus, CD4 + Th2 cells, ILCs (eg, nuocytes), eosinophils, mast cells, IgE, and basophils all contribute to the generation of protective type 2 responses to varying degrees. Not withstanding the clear results in mice, the relative importance of the type 2 effector response in the development of immunity in humans remains uncertain. The most straight forward hypothesis, and one predicted for many years, that Th2-induced increases in IgE antibody production is protective against intestinal helminths, has either been refuted following intensive investigation using mouse models, or at least received little direct confirmation.273,292 Indeed, B cell– and Ig-deficient mice display only modest increases in susceptibility to most helminth infections,293 although parasite-specific IgG and IgA antibodies have been found to inhibit the fecundity of adult H. bakeri.294 There is, however, a great deal of epidemiologic evidence that type 2 antibody responses, particularly in the form of

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antigen-specific IgE, mediate the resistance that develops with age in endemic areas. The possibility exists that there are host species differences in this regard because in rats the rapid expulsion of a secondary T. spiralis infection is easily transferred to naïve animals with IgE.295 IgE − / − mice also expel adult T. spiralis worms at a much slower pace and develop nearly twice as many muscle larvae as their wild-type counterparts.296 Although the exact mechanism by which IgE mediates protection is unclear, it is possible that parasitespecific IgE cooperates with eosinophils or macrophages in an antibody-dependent cellular cytotoxicity–type antiworm effector mechanism.297–299 It may also play an important role in the production of IL-4 and IL-13 by mast cells and basophils. IgE-primed basophils secreting IL-4 may also enhance immunity by promoting the differentiation of CD4 + Th2 cells.143,290,291 Consistent with the potential protective role of IgE, carriage of the human leukocyte antigen-DRB1*13 class II allele is associated with increased posttreatment IgE levels against S. mansoni antigens and decreased reinfection levels.300 Further support for this mechanism comes from field studies in Brazil, where it was discovered that resistance to schistosome infection is controlled by a major gene that localizes to a region of chromosome 5, which encodes the type 2 cytokines.301 IgE and eosinophils are also both required for vaccine-mediated immunity against larval O. volvulus in mice immunized with irradiateiated larvae. Thus, in the case of secondary helminth infection, IgE, eosinophils, and basophils all collaborate to enhance immunity. Further work is required to elucidate the precise roles of these effector mechanisms in primary versus secondary infections and in different host species, with particular attention being paid to testing of hypotheses derived from model organisms in naturally infected hosts and vice versa. In contrast to the intestinal parasites, where immunity is clearly dependent on type 2 cytokines, immunity to filarial parasites appears to be dependent on both type 1- and type 2-associated effector mechanisms.302 In the Brugia mouse model of filariasis, B cells are also required for the development of immunity,303 with IgM antibodies participating in the clearance of both primary and secondary infections and IgE assisting in clearance of primary but not secondary infections.304 Parasite-specific IgA also plays a protective role in human bancroftan filariasis.305 Genetic polymorphisms in genes encoding endothelin-1 and TNF-RII have also been correlated with the development of chronic human disease,306 whereas studies in mouse models have identified important roles for IL-10 and possibly TGF-β1 in the establishment of chronic infections with B. malayi.307 Eosinophils and antibody are required for resistance to primary Brugia pahangi infection,308 suggesting an antibody-dependent cellular cytotoxicity–type effector mechanism is required for parasite clearance. However, studies with Onchocerca ochengi, a filarial parasite of cattle related to O. volvulus, have suggested that eosinophil degranulation on the parasite cuticle can in some cases actually promote worm viability.309 Worm viability is also increased by endosymbiotic Wolbachia bacteria, which play a critical role in the growth, development, and survival of B. malayi.310 Wolbachia also convert potentially deleterious eosinophil responses into neutrophildominated responses, which are incapable of contributing to

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antifilarial immunity.309 In the L. sigmodontis model of filariasis, IL-4 blocks nematode development in resistant hosts but not in susceptible strains of mice, further suggesting that Th2dependent and Th2-independent mechanisms are involved in resistance.311 Some studies have also identified important synergistic activity between IFNγ and IL-5 in antifilarial immunity, although the role of the Th1-associated cytokine IFNγ remains unclear.312 Finally, IL-10–expressing CD4 +Foxp3 + regulatory T cells have been shown to suppress immunity to L. sigmodontis, identifying a critical role for regulatory T cells in the establishment of chronic filarial infections.313 As with many of the extracellular parasites, resistance to protozoan trypanosomes requires elements of both cell-mediated and humoral immunity. The African trypanosomes are tsetse-transmitted parasites that inhabit the extracellular compartment of their host’s blood and avoid detection by the humoral immune system by switching among antigenically distinct variant surface glycoproteins (VSGs) (see following discussion). Trypanosome-infected hosts typically do not produce antibodies that destroy the parasite, other than those that are VSG specific. Parasitemias manifest as recurring waves, with each wave of parasites expressing a different VSG, and are cleared following development of VSG-specific antibodies. Before an effective antibody response can be generated, however, the host develops quite high parasitemia, severe trypanosomiasis-associated pathology, generalized immunosuppression, and, in some circumstances, debilitating secondary infections.314 It is clear that overproduction of IL12, IFNγ, and NO is the primary trigger for these deleterious side effects315 while also contributing to host resistance,316 as parasite control within the extravascular tissue compartment requires a parasite antigen-specific Th1 response, associated with IFNγ-dependent activation of macrophages.317,318 Type I IFNs have also been shown to play a role in early resistance to African trypansomes, although they may contribute to downregulation of IFNγ production and subsequent loss of host resistance later in infection.319 In humans, resistance to most African trypanosomes, including T. b. brucei, is mediated by the TLFs, as previously discussed.320 Interestingly, the higher rates of kidney disease in infected Africans compared to Americans of European descent appears to be linked to the expression of disease-associated variants of the apolipoprotein L (Apo L) components in TLF that are able to lyse T. b. rhodesiense in vitro.321 Another important extracellular protozoan, Giardia, is a flagellated intestinal parasite that causes both acute and chronic diarrheal disease. Despite its intestinal habitat, Giardia appears to be controlled by mechanisms distinct from those mediating resistance to most gastrointestinal nematodes, although recent studies suggest that mast cellderived IL-6 maybe important for the rapid elimination of Giardia in mice.322,323 While a T-cell– dependent mechanism involving TNF-α is essential for resistance to acute infections,324 numerous studies have suggested that antibodies, particularly the IgA isotype, are required to control chronic Giardia lamblia infections.325 Moreover, it has been proposed that neuronal NOS (NOS1) might facilitate clearance of Giardia from the gut by increasing gastrointestinal motility and parasite-induced diarrhea.326

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These examples clearly demonstrate that although intracellular and extracellular parasites often stimulate distinct immune responses, their immune control may involve overlapping immunologic effector arms.

MECHANISMS OF IMMUNE EVASION Pathogens that rely on an insect vector to complete their life cycle, or are only sporadically transmitted from one host to another, are under strong evolutionary pressure to prolong their survival within their host. As the adaptive immune response is the principal barrier to the persistence of pathogens in mammalian hosts, parasites have evolved diverse strategies to evade immune control, either by evading immune recognition or by suppressing immune effector mechanisms. The former strategy refers to the ability of some parasites to sequester within sites that are inaccessible to immune attack, to mask themselves with host antigens, to shed their own target antigens, or most notably, to undergo antigenic variation. The latter strategy refers to the active suppression of established, ongoing immune responses that may contribute to the state of equilibrium that is established between host and parasite in sites of chronic infection.

Evasion of Immune Recognition The asexual, blood stage of malaria would seem the most obvious example of a well-hidden parasite. Its ability to invade mature erythrocytes, which lack both class I and II histocompatibility molecules, in theory at least should protect it from recognition by antibodies or effector T cells. However, because parasitized erythrocytes are efficiently cleared by the spleen, additional immune evasion strategies (ie, antigenic variation) are required (described subsequently). Other intracellular protozoa appear to hide within immunologically privileged sites. The persistence of T. cruzi within heart or skeletal muscle, which is believed to underlie the pathogenesis of Chagas disease, occurs despite the fact that parasites are cleared from most other tissue.327 Infected muscle cells may be only poorly recognized as targets for cytotoxic T lymphocytes, poorly accessible to their homing, or they may have intrinsic defects in immune-mediated killing mechanisms. A similar form of sequestration has been proposed to explain the long-term persistence of Leishmania within fibroblasts and dendritic cells following their efficient killing by activated macrophages during the acute stage of infection.328 While persistent low-level infection of host cells has been proposed as an explanation for latency in T. gondii infection, the major parasite reservoir during chronic infection is undoubtedly provided by the tissue cyst, essentially a modified host cell carrying a specialized dormant parasite stage, the bradyzoite. Helminths (with the exception of Trichinella, which develop long-lived muscle stage larvae) do not invade host cells and, therefore, cannot use this strategy for evading immune recognition. Furthermore, because most multicellular helminth parasites do not replicate within their mammalian hosts, they are not equipped to evade immune recognition by undergoing antigenic variation. Instead, they employ alternative mechanisms

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such as disguising their surfaces with host molecules and rapidly shedding membrane (tegument)-bound immune complexes.329 In addition, helminths have evolved a series of elaborate processes for inactivating antibody, complement, and cellular effector elements that threaten the parasite surface.330 Interestingly, recent data suggest that helminths may take advantage of host T lymphocyte and cytokine signals as developmental triggers; if these signals are in low abundance or lacking in vivo, parasite growth may be aborted or severely attenuated.331–333 In the case of schistosomes, development of female parasites is not directly influenced by the adaptive immune system, whereas male development is.334 In this sequential model, adaptive immune signals trigger development of mature males, which subsequently stimulate development of mature females. Although the need for antigenic variation might be obvious for extracellular protozoa such as trypanosomes and Giardia, it is less obvious why malaria parasites—hiding inside RBCs—should have evolved a similar strategy of immune evasion.335 The most plausible explanation is as follows. Developing schizonts cause erythrocyte distortion, loss of flexibility, and abnormal surface exposure of various membrane components, all of which make infected erythrocytes vulnerable to clearance from the circulation during passage through the spleen in a manner very similar to that by which normally aged red cells are removed and destroyed. In order to avoid passage through the spleen, the parasite exports to the red cell surface a number of molecular anchors—of which the best characterized is P. falciparum erythrocyte membrane protein-1 (PfEMP-1)—that bind to endothelial receptors, allowing the parasite to sequester in peripheral tissues. Parasite sequestration contributes to malaria pathology, clogging blood vessels and triggering focal inflammation, giving rise to cerebral, respiratory, and renal symptoms; sequestration in the placenta gives rise to pregnancy-associated malaria. Because these molecular anchors are parasite-derived, they are recognized by the immune system; antibodies bind to them, prevent endothelial sequestration (and ameliorate disease), and allow parasitized RBCs to be cleared in the spleen.336 Thus, in order for the parasite to establish a chronic infection, a system of clonal antigenic variation is required. The importance of these molecular anchors as a parasite survival strategy is demonstrated by the fact that there are upward of 50 copies of the gene for PfEMP-1 (var genes) per parasite, more than 200 copies of other clonally variant surface protein genes,337 and innumerable allelic variants of each gene in the global parasite population.

Evasion by Immune Suppression Generalized immunodepression, which is a feature of many chronic parasitic infections, including malaria, African trypanosomiasis, and visceral leishmaniasis, appears in most instances to be secondary to other immune evasion strategies and results from the need to control inflammation (see the next section) or from a variety of immune dysfunctions that high-systemic parasite burdens can produce. These dysfunctions include disruption of normal lymphoid architecture, such as occurs in the mouse spleen during acute

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malaria infection338 and during chronic infection with L. donovani.339 In the case of T. cruzi, which can express and secrete multiple members of the highly polymorphic surface sialidase superfamily at one time, epitope-specific T-cell responses are suppressed either by altered peptide ligand inhibition or because immune recognition is impeded by a flood of competing targets.340 Similarly, lymphocyte polyclonal activation, which can result in depression of antigen-specific responses, is a feature of many parasitic protozoan infections, including blood and tissue trypanosomes, L. donovani, T. gondii, and rodent malarias, but again, a causal link between polyclonal activation and immune evasion has not been established. The original hypothesis—that these organisms possess mitogenic or superantigenic moieties341— has been substantiated only in the case of T. cruzi from which the B-cell mitogen has been cloned and characterized as a eukaryotic proline racemase.342

Regulatory T Cells and Parasite Persistence The modulation of ongoing immune responses in sites of chronic infection, such as the gut or the skin, may in many instances reflect not only parasite survival strategies but also normal mechanisms of immune homeostasis that operate to control immunopathology. Accumulating evidence has implicated a crucial role for regulatory T cells in the dynamic equilibrium that is established between parasites and their hosts. Regulatory T cell is the name given generally to the subsets of CD4 + T cells, and more recently CD8 + T cells, which negatively regulate multiple immune functions. Among the different subsets of CD4 + regulatory T cells that have been described, the best characterized are the so-called naturally occurring CD4 + CD25 + Foxp3 + T cells (nTreg) that are present in naive animals. T cells with suppressive function can also be generated from conventional naïve cells, and include induced regulatory T cells (iTreg), which are converted from activated CD4 + CD25 −Foxp3 − T cells to CD25 + Foxp3 + suppressor cells following encounter with antigen and TGF-β in the periphery, and adaptive regulatory T or Tr1 cells, also generated from antigen activated cells in the periphery but which remain Foxp3 − and secrete high levels of IL-10. There is data linking one or more of these populations to the suppression of immune responses to all of the major classes of pathogenic protozoa and helminths. The evidence that nTregs contribute to pathogen persistence was initially demonstrated in the L. major mouse model, in which CD4 + CD25 + T cells accumulated in the dermal lesion and promoted subclinical persistence of amastigotes following clinical cure in resistant mice.343 Similarly, in a model of mouse malaria characterized by extremely rapid parasite growth (P. yoelii YM or 17XL), very early induction of TGF-β and IL-10 appears to inhibit the development of responses that are required for parasite clearance. Neutralization of IL-10 and TGF-β 344 —or, in some studies, depletion of nTreg345 —at the onset of infection allows parasite replication to be contained and mice are able to resolve their infections and survive. A similar scenario has been observed in experimental human infections within the context of malaria vaccine trials.346 More recently, however, it has

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begun to be appreciated that classical Foxp3 + regulatory T cells and adaptive IL-10–producing T cells have rather different roles in malaria-infected hosts. Thus, the major role of Foxp3 + regulatory T cells seems to be in maintaining immune homeostasis during asymptomatic infection, whereas IL-10–secreting cells are required to restore immune homeostasis after acute clinical infection.347,348 The role of IL-10–producing T cells in controlling immunopathology and parasite persistence is especially striking in the case of T. gondii and Leishmania. IL-10 production by CD4 + CD25 −Foxp3 − T cells that also produced IFNγ was required for the control of Th1-driven immune pathology associated with T. gondii infection in mice349 (discussed in more detail in the following section). A similar phenotype of IL-10–producing Th1 cells was associated with the inability to heal infection with a substrain of L. major that induces a polarized Th1 response in C57Bl/6 mice.350 The clinical data has consistently supported an association of human visceral leishmaniasis with elevated levels of IL-10, which in the visceral leishmaniasis spleen was found to be expressed primarily by CD25 −Foxp3 − T cells.351 It is important to emphasize that even in patients with active visceral leishmaniasis in whom systemic infections go uncontrolled, their elevated levels of IL-10 are accompanied by augmented production of IFN γ, which in the visceral leishmaniasis spleen is also expressed primarily by CD25 −Foxp3 − T cells. Together, these data suggest that in sites of strong inflammation, IL-10– producing Th1 cells may be activated as a powerful mechanism of feedback control to prevent collateral tissue damage. The regulation of IL-10 expression in these inflammatory settings has recently been described. IL-27, an IL-12 family member, was shown to upregulate IL-10 expression by Th1 cells in T. gondii –infected mice in a STAT1- and STAT3dependent manner.352 Accordingly, IL-27ra− / − mice developed lethal inflammation that was associated with defective IL-10 production by Th1 cells isolated from the brains of infected mice. IL-27ra− / − mice efficiently control P. berghei NK65 infections but have reduced capacity to produce IL-10 and develop severe, frequently fatal, Th-1–mediated liver pathology.353 IL-27ra− / − mice infected with L. major also developed more severe pathology, which in the case of nonhealing L. major infections is associated with fewer numbers of T cells coexpressing IL-10 and IFNγ, and an increased frequency of Th17 cells.354 The L. major mouse model has also revealed a potential role for suppression mediated by IL-10 and TGF-β produced by macrophages following uptake of Ig-opsonized parasites.355,356 Thus, redundant mechanisms of homeostatic control, including innate cells, natural and adaptive regulatory T cells, appear to be activated to control persistent immune pathology associated with antimicrobial immune responses in tissues that are especially susceptible to injury (eg, the liver, brain, skin, and mucosa).

IMMUNOPATHOLOGIC MECHANISMS AND THEIR REGULATION If the appropriate protective response fails to develop or if the host is not able to achieve sterile immunity, then inflammation and other pathologic changes may be

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unavoidable consequences of persistent infection. This is the situation for many parasitic diseases. This does not mean that all infections with the same parasite species lead to the same immune pathology. One of the most striking features of human parasitic disease is the great variability in clinical outcome, ranging from asymptomatic infection to fatal disease. Esophageal disease due to T. cruzi, liver fibrosis, portal hypertension, and hepatosplenomegaly due to S. mansoni, and cerebral malaria due to P. falciparum are a few examples of the many immunopathologic complications that may occur in some individuals but not others. Part of this variability is determined by host genetics, whereas other potential determinants include parasite virulence factors, infection intensity, and the prior level of immunity. The picture is further complicated in individuals that are coinfected with other pathogens: For example, the severity of P. falciparum malaria is increased by concomitant bacteremia but reduced by concomitant P. vivax infection.357 Antiretroviral drugs also impair CD36mediated cytoadherence and nonopsonic phagocytosis of parasitized erythrocytes by human macrophages, which may lead to severe malaria disease outcomes in antiretroviral treated coinfected individuals.358 The pathology associated with chronic S. mansoni infections reduces the CD4 + T cell-count, which can exacerbate the effects of HIV-1 infection.359 S. mansoni infection also increases susceptibility to HIV infection, transmission, and replication in nonhuman primates, 360 and similar observations have been made in malaria-infected patients.361 Concomitant exposure to malaria can affect the regression of hepatosplenomegaly in drug-cured S. mansoni patients.362 Coinfections of T. gondii and S. mansoni in mice are also more lethal, 363 whereas preexisting infections with N. brasiliensis have been shown to inhibit innate pulmonary antituberculosis defense by activating the IL-4 receptor pathway in murine macrophages.364 Finally, although endosymbiotic Wolbachia bacteria are believed to increase the fitness of fi larial parasties, Wolbachia LPS is believed to be the principle driver of river blindness in chronic fi lariasis.365 It is difficult to do justice to the remarkably broad range of immunologic mechanisms that contribute to the pathology of parasitic disease. Twenty-five years ago, much of the research in this field concerned the role of immune complexes, complement, and anaphylaxis. These areas remain important, but the focus has shifted to the molecular basis of cellular processes such as inflammation, granuloma formation, wound repair, and immune regulation. An important issue that is the focus of intensive research is how the host maintains the fine balance between a protective immune response and one that causes pathologic complications. It is becoming increasingly clear that achieving balance in the immune response is the most critical determinant in the establishment of a long-term host–parasite relationship and, therefore, should be of considerable interest to vaccinologists. Interestingly, in many chronic parasitic infections, this balance appears to be regulated by the coordinated actions of several distinct immunoregulatory mediators, including regulatory T cells, IL-10–expressing regulatory B cells, and AAM, to name just a few.

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Achieving Balance between the Anti-infective Immune Response and Host Pathology Whereas Th1 responses are required to control intracellular infections, there is a need to balance these potentially tissue-destructive responses. The harmful effector molecules induced by Th1 responses include NO, reactive oxygen intermediates, and TNF, which operate in a synergistic fashion to promote antimicrobial immunity but are often associated with undesired inflammatory and toxic side effects. Proinflammatory Th17 responses have also been shown to contribute to the acute pathology seen in some schistosomeinfected mice.366,367 IL-10, TGF- β, arginase-1, and IL-4, produced by distinct subsets of regulatory cells including myeloid-derived suppressor cells, AAMs, regulatory T cells, IL-4/IL-13–producing Th2 cells, IL-10–producing regulatory T cells (Tr1), FoxP3-expressing conventional Th1, and IL-10 + IFNγ + Foxp3− CD4 + T cells, may all help prevent Th1 and Th17 immune responses from overshooting and becoming pathologic during infection with intracellular pathogens.349,368–371 There are also several examples in which persistent helminth-induced Th2 responses appear to be detrimental to the host. Strong antibody responses can lead to the formation of antigen–antibody complexes or complement activation resulting in bystander lysis.372 Eosinophils, typically associated with the Th2 response, are involved in hypersensitivity reactions to the fi larial worm O. volvulus and promote Th2 pathology in schistosomiasis.280 Th2 cytokines are also important drivers of hepatic fibrosis and morbidity in chronic schistosome infections.373 Similar to chronic Th1 and Th17 responses, a variety of regulatory mediators including IFN γ , IL-12, IL-10, the IL-13 decoy receptor, regulatory T cells, regulatory B cells, and silencers of cytokine signaling proteins have all been shown to contribute to the suppression of pathogenic Th2 responses.374–376 In summary, the successful resolution of infection typically requires precise titration of the specific T helper cell response induced by the pathogen. This is not just in terms of amount but also where, when, and for how long the polarized Th1, Th2, or Th17 response persists.

Pathogenesis of Chronic Th1 Responses As discussed previously, control of intracellular pathogens such as Leishmania spp., T. gondii, and T. cruzi requires the coordinated activation of both antigen-specific cells (T lymphocytes) and less specific responses (NK cells, neutrophils, and macrophages) with IFNγ and TNF playing critical roles by upregulating macrophage activation and nitric oxide production. Interestingly, IL-10– deficient mice inoculated with a normally avirulent T. gondii strain or with a virulent strain of T. cruzi succumb to infection within the fi rst 2 weeks of infection.377,378 In both of these infections, animals lacking IL-10 show increased suppression of parasite growth and, in the case of T. cruzi, inflammation and necrosis within the endocardium and interstitium of the myocardium is reduced. The increase in mortality is caused by systemically high levels of IL-12, IFNγ, and TNF produced in large part by activated CD4 + T lymphocytes and macrophages. The livers of

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both T. gondii – and T. cruzi –infected mice show numerous and prominent necrotic foci together with dramatically increased mononuclear cell infi ltration. Similarly, macrophages from the mutant mice activated in vitro or in vivo with T. gondii secrete higher levels of TNF, IL-12, and inducible NO than macrophages from IL-10– sufficient animals. The combined clinical manifestations suggest that the IL-10 − / − mice die in response to an overwhelming systemic immune response, resembling that observed during septic shock. In support of this conclusion, administration of anti-CD4, anti–IL-12, or anti-TNF mAbs reduces mortality in IL-10 − / − mice.377,378 Thus, in these models, IL-10 plays a major role in protecting the host against an excessive and lethal type 1 cytokine response. Much has been written about the protective versus pathologic consequences of proinflammatory cytokine production in malaria (Fig. 38.3379). During the preerythrocytic stage in the liver, when parasite burden is relatively low and the infection is clinically asymptomatic, there is evidence that IFNγ and NO each play an important role in preventing the infection from progressing further.380 Once the parasites invade erythrocytes and grow to large numbers, the riskbenefit equation is less clear. At this stage, the inflammatory cytokine response is systemic, and some pathologic side effects are inevitable. The most common clinical consequence in humans is fever, whereas life-threatening complications such as profound anemia and cerebral malaria occur in a proportion of infections. Depending on the specific host– parasite combination, mice may develop anemia, fatal neurologic symptoms, or multiorgan failure; IL-1, IL-6, TNF, and, more recently, lymphotoxin-α have been the cytokines most consistently associated with severe pathology in these models.381,382 Conversely, experimental studies suggest that IL-10 and TGF-β cooperate to downregulate potentially pathogenic proinflammatory cytokine responses in malaria.348 IL-10– deficient mice have increased mortality compared with normal littermates; this results not from fulminant parasitemia but from a sustained and enhanced proinflammatory cytokine response383 and severe liver necrosis. Immunopathology in IL-10 − / − mice can be prevented simply by adoptive transfer of wild-type (ie, IL-10 competent) CD4 + T cells, indicating that they are a sufficient source of IL-10 to prevent severe disease in the latter stages of acute infection.383 Similarly, treatment of infected mice with a neutralizing antibody to TGF-β exacerbates the virulence of P. berghei and Plasmodium chabaudi infection; it was concluded that the protective effects of this cytokine are also due to downregulation of inflammatory responses.384 However, as described previously, very early induction of TGF-β and IL-10 can inhibit the effector response that is required for parasite clearance344,345 ; thus, the outcome of malaria infections is determined, in part at least, by the sequence and timing of proinflammatory and anti-inflammatory (immune regulatory) responses. This is likely to depend upon sequential activation of innate immune cells, such as NK cells and γδ T cells, then Th-1 CD4 + T cells, and finally IL-10–secreting T cells. Preliminary evidence suggesting that CD4 + T cells that secrete both IFNγ and IL-10 may be a signature of

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Parasites engage PRR

Vascular occlusion NO Toxic radicals

IL-1 IL-6 TNF-α

iRBC sequestration Local inflammation

Mφ/DC

IL-12 IL-18 IFN-α

IL-2

Fever

IL-2

NK-T IL-10/TGF-β

Th1

IL-10/TGF-β

NK IFN-γ IFN-γ

IFN-γ

Mφ

IL-10/TGF-β

IL-1 IL- 6 TNF-α

Treg?

iRBC γδ TCR

Treg? CD1-lipid antigen

IFN-γ activated macrophages - phagocytose iRBC - secrete inflammatory cytokines

MHC-peptide αβ TCR PRR

FIG. 38.3. Immunopathogenesis of Malaria. Parasitized red blood cells (pRBC) and parasite products bind to pattern recognition receptors (PRRs; eg, toll-like receptor [TLR]2 and TLR9) on monocytes and dendritic cells, and induce a cascade of proinflammatory cytokines (most importantly interleukin [IL]-12, interferon-γ, and tumor necrosis factor [TNF]-α). These inflammatory signals initiate numerous pathologic processes. They cause upregulation of endothelial adhesion molecule expression on vascular endothelium, promoting vascular sequestration of pRBCs, which clogs the vessels, reduces blood flow, and simultaneously raises intracranial pressure. The pRBC products also bind to PRRs in tissue, initiating local inflammatory loops, which amplify pRBC sequestration. Vascular occlusion and subsequent tissue damage contribute to cerebral malaria, respiratory distress, and multiorgan failure. Other cytokines such as IL-1, TNF-α , and IL-6 induce the fever response, which includes elevated temperature, nausea, headache, prostration, and muscle pain. The actions of inflammatory cytokines are antagonized by anti-inflammatory cytokines, principally IL-10 and transforming growth factor-β, which inhibit both production of, and cellular responses to, inflammatory signals.

protection against severe malaria in African children clearly needs to be explored further.385

Pathogenesis of Chronic Th2 Responses During nematode infection, the protective Th2 response can cause significant pathologic changes in the intestine including inflammation, epithelial cell damage, excess mucus production, and diarrhea. However, these effects are often transient and resolve quickly once the parasite is expelled from the gut. If the parasites are not expelled, the immune response typically shifts to a more Th1/Th17-polarized reaction,386 which can lead to an even more severe inflammation with characteristics similar to Crohn disease.387 Thus, there are only a few chronic helminth infections where persistent Th2 reactions are established. Perhaps the most widely studied experimental model in this regard is the mouse model of schistosomiasis. Upon infection, adult parasites of S. mansoni migrate to the mesenteric veins where they live up to 10 years or more, laying hundreds of eggs per day. Some of the eggs become entrapped in the microvasculature

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of the liver and once there, they induce a granulomatous response.388 Subsequently, hepatic fibrosis, portal hypertension, hepatosplenomegaly, and bleeding from esophageal and gastric varices may develop, which in some cases may ultimately lead to the death of the individual. Consequently, much of the symptomatology of schistosomiasis is attributed to the egg-induced inflammatory response and associated fibrotic pathology.389,390 Granulomas are pathogenic, not because they cause hepatic failure in the short-term, but rather because they induce hepatosplenomegaly and contribute to liver scarring.390,391 CD4 + Th2 cells are essential for granuloma formation, whereas many other lymphocytes appear less critical including B cells, CD8 cells, NK T cells, and γδ T cells. B cell–deficient (μMT) mice, however, fail to downmodulate granuloma formation in the latter stages of the disease, suggesting that B cells and/or antibody might play an important immunoregulatory role in chronic infections.392 In support of regulatory role for B cells and antibody, perinatal exposure to specific anti-SEA idiotypes has been shown to regulate survival, pathology, and immune response patterns in mice

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subsequently infected with S. mansoni.393 Cross-reactive regulatory idiotypes can also been used to predict clinical outcomes in chronically infected mice.394 Th2 cells also play an important role in the pathogenesis of schistosomiasis.283 Indeed, a critical role for Th2 cells was confirmed in experiments in which mice vaccinated with egg antigen plus IL-12 to induce an egg-specific Th1 response upon subsequent infection developed smaller granulomas and less severe fibrosis than did nonvaccinated infected controls.395 The decreased pathology was associated with a diminished Th2 and increased Th1 response. IL-4 − / −, IL-4Rα− / −, and IL-13Rα1− / − mice as well as anti–IL-4 mAb-treated wild type (WT) mice were also shown to develop smaller granulomas and less fibrosis than similarly infected WT mice, further emphasizing the critical role of Th2 immunity in the pathogenesis of chronic schistosomiasis.138,244,396,397 Although the Th2-driven granulomatous response is widely believed to be detrimental in chronic infections, it is clear that IL-4–driven granuloma formation also serves an important host-protective role, particularly during acute infections with S. mansoni. During infection, the schistosome eggs that are produced in large quantities by adult parasites provide a continuous antigenic stimulus for the immune response. If these antigens are not sequestered or neutralized effectively, toxic components produced by the eggs are known to damage hepatocytes and reduce barrier immunity in the intestine.396,398 Indeed, T cell–deprived, nude, severe combined immunodeficiency defect, and egg-tolerized mice infected with S. mansoni all die earlier than comparably infected immunologically intact mice because they are incapable of mounting a protective CD4 + Th2 cell–driven granulomatous response.399 Widespread microvesicular hepatic and intestinal damage induced by toxic egg products contributes to the poorer survival of the infected immunosuppressed mice. IL-4R signaling is also required for the efficient passage of eggs through the intestine into the lumen.398 Consequently, increased numbers of eggs are trapped in the intestinal wall when IL-4 is deficient, causing localized inflammation and increased systemic exposure to bacterial toxins such as LPS. This response combined with the decreased Th2- and enhanced Th1-type response results in increased proinflammatory cytokine production that contributes to weight loss in and death of IL-4–deficient animals.388 Therefore, although Th2-driven granuloma formation and fibrosis are detrimental in the long-term, they are also critically important in the short-term because they allow the establishment of a successful host–parasite relationship.400 Many of the pathologic complications associated with chronic helminth infections (portal hypertension, bleeding collateral vessels, anemia, mucosal barrier dysfunction, lymphatic blockage, etc.) do, however, result from the persistent expression of type 2 cytokines.283 Whereas IL-4 was identified as the principle inducer of the protective Th2 response during acute schistosome infection,396 IL-13 has been identified as the key driver of hepatic fibrosis and the fibrosis-associated complications observed in chronically infected mice.374,398,401 In mice, the progression of hepatic fibrosis correlates with the intensity of the type 2 cytokine response,402 and immunologic interventions that impair IL-13 activity have been

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shown to reduce collagen deposition and improve survival during chronic infection374,398,401 as long as IL-4 production is preserved. Mediators produced during polarized type 1 cytokine responses have been shown to inhibit IL-13–dependent fibrosis, including TNF-α , IL-12, and NO.403,404 Because macrophages and DCs are important sources of these mediators and have been shown to be important drivers of Th2 immunity in vivo, a major effort has been undertaken to elucidate the roles of distinct myeloid cell populations in the pathogenesis of schistosomiasis and other helminth infections.191,400 Numerous studies have shown that IL-4/IL-13–induced macrophages are found in many helminth infections. Gordon405 first noted that macrophages treated with IL-4 and IL-13 develop an alternative activation state that is distinct from “classically activated macrophages” exposed to IFNγ. In vitro, the AAM or M2 phenotype405 is characterized by elevated MHC class II, mannose receptor (CD206), YM1, Fizz1/Relm-α, and arginase (Arg) activity.406–409 Studies of murine schistosomiasis have found that 20% to 30% of the cells in granulomas are AAMs,410 and studies conducted with IL-4Rα conditional knockout mice have suggested that AAMs actively suppress the development of IL-12/IL-23p40–driven intestinal pathology during acute infection with S. mansoni.371,411 Related studies conducted in conditional Arg1-deficient mice have also identified an important role for Arg1-expressing macrophages in the suppression of hepatic fibrosis during chronic S. mansoni infection.407 These findings were supported in studies with mice deficient in cationic amino transporter-2, which imports L-arginine for use in NO production in macrophages. Cationic amino transporter-2− / − mice develop exacerbated liver fibrosis following schistosome infection associated with decreased NO production and increased Arg1 activity in AAMs and fibroblasts.412 Together, these studies support the hypothesis that the phenotype of the macrophage plays a critical role in the pathogenesis of schistosomiasis. Similar studies are now underway to dissect the role of AAMs in other helminth infections.

PARASITE VACCINE STRATEGIES Aside from the recent encouraging results involving the RTS,S malaria vaccine (discussed subsequently), there is still no safe, uniformly effective vaccine against any human parasitic infection. The lack of progress in this field is due to many factors, including the low priority that has historically been given to development of vaccines against diseases confined mainly to the developing world. By contrast, the perceived economic benefit to the agricultural industry of vaccines against livestock parasites has led to the licensing of several antihelminthic vaccines for veterinary use.413 The greater impediments, however, may be related to the nature of parasitic infections themselves. In contrast to those bacterial and viral infections for which highly effective vaccines exist, and for which there is complete immunity induced by primary infection, most antiparasite vaccines will need to outperform the immune response to natural infection. Further, virtually all bacterial and viral vaccines that are currently in use mediate their protection by inducing a strong, long-lived,

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humoral response that inhibits attachment or invasion, promotes clearance, or neutralizes released toxins. By contrast, there are no vaccines that are uniformly effective against diseases caused by intracellular pathogens that require cellular immunity to mediate protection. Thus, the manner in which potentially protective antigens can be administered to generate and maintain appropriate T-cell responses has yet to be proven in a clinical setting. Consequently, for the development of vaccines against intracellular protozoa (eg, malaria, Leishmania, T. cruzi, Toxoplasma), it will not be sufficient to simply identify target antigens; novel and rational approaches to vaccine design and delivery will need to be explored. In fact, from the examples discussed in the following, it is clear that ample numbers of potentially protective antigens have been identified and cloned from most of the major human parasitic disease agents, and the completion of their genomic sequences has already led to the identification of additional vaccine candidates based on their predicted developmental stage specificity, surface expression, secretion, or virulence associations.414,415 The vaccination strategies that are currently being explored to meet the challenge of both antigen selection and delivery will be considered in the general context of B- and T-cell antiparasite vaccines. Note that the examples provided are by no means exhaustive but reflect general principles of vaccination against extracellular and intracellular targets.

B-Cell Vaccines Vaccination Against Intestinal Protozoa Parasitic protozoa that have an exclusive extracellular lifestyle in their mammalian hosts and are sensitive to antibody-mediated control include the intestinal pathogens E. histolytica and G. lamblia. Most deaths from E. histolytica arise from amebic liver abscess, the major extraintestinal manifestation of disease. Clinical studies suggest that the presence of mucosal antibodies to the surface Gal/GalNAc lectin of E. histolytica capable of blocking amebic adherence to intestinal epithelial cells, correlate with reduced risk of recolonization.416 Furthermore, active immunization with the Gal/GalNAc lectin can induce IgA antilectin antibodies and provide protection against intestinal amebiasis in a mouse model of disease.417 An amebic serine-rich protein (SREHP) and an alkyl hydroperoxide reductase (Eh29) are also highly immunogenic surface antigens of E. histolytica that can protect against amebic liver abscess in animal models via induction of intestinal IgA antibodies and together with the Gal/GalNAc lectin, represent the most promising candidates for oral vaccines against amoebiasis.418 Specific serum and mucosal antibodies targeting surface antigens are also known to be important in elimination of Giardia from the host intestine. Giardia vaccines containing whole trophozoite preparations protected animals even when challenged with heterologous strains,419 suggesting that an immune response to variant surface proteins, which are known to be targets of cytotoxic antibodies, are not essential for control of acute infection. However, by interfering with the mechanism controlling variant surface protein switching in Giardia, it was more recently shown that primary infection

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with trophozoites expressing many variant surface proteins was necessary to protect gerbils from subsequent infection.420 More importantly, such genetically manipulated parasites might be used as cross-protective vaccine. Genes coding for Giardia cyst wall proteins, which could be used for developing a transmission-blocking vaccine, have been cloned, and a recombinant protein induced IgA antibodies such that immunized mice shed fewer cysts following challenge with live cysts.421

Vaccines Targeting Extracellular Stages of Malaria Because both preerythrocytic- and erythrocytic-stage malaria parasites are at least transiently exposed to humoral antibody, vaccine strategies based on eliciting high-titered antibodies that can inhibit their invasion of RBCs or hepatocytes have long been favored (Fig. 38.4). As discussed previously, antibodies to sporozoite surface proteins can immobilize invading parasites, preventing them from reaching or invading hepatocytes; such antibodies can be protective, and the dominant antibody epitope is represented by the CS central repeat sequences (NANPn in P. falciparum). These observations have made the CS protein the most extensively studied of all the malaria vaccine candidates.422 The latest version of the vaccine—RTS,S—comprises a recombinant CS polypeptide fused to the surface protein of hepatitis B virus and administered together with an adjuvant containing LPS (monophosphoryl lipid A) and a water-soluble glycoside obtained from tree bark (Quillaja saponaria).423 The vaccine has been designed to induce both antibodies and T cell–mediated effector mechanisms, and it is not clear which of these mechanisms is most important in conferring immunity. This vaccine is discussed in more detail in a following section. Antibodies that inhibit the invasion of erythrocytes by the extracellular merozoite stage of malaria in vitro are found in many, but not all, individuals living in malaria endemic regions. Although the significance of these inhibitory antibodies to naturally acquired resistance remains unclear, their target antigens nonetheless remain prime candidates for asexual malaria vaccines422 (see Fig. 38.4). Numerous antigens that form the surface coat of the merozoite or that are essential components of the parasite’s cell invasion mechanism (apical complex and rhoptry proteins) have been evaluated as potential vaccine candidates. Although antibodies to several of these antigens have been found to be able to inhibit parasite development in vitro, or are associated with clinical immunity in vivo, the results of these studies tend to be rather inconsistent,424 and the results of vaccine trials have been disappointing.422 One of the problems seems to be that when one pathway of merozoite invasion into an erythrocyte is blocked by antibody, the parasite is able to switch to an alternative pathway using entirely different proteins. Recently, however, a novel receptor-ligand interaction has been identified that seems to be absolutely essential for merozoite invasion. P. falciparum rhoptry protein-5 (PfRh5) binds to the blood group antigen basigin on the red cell surface; blocking of this interaction with antibasigin antibody completely prevents parasite invasion and replication.425 Just as importantly, antibodies to PfRh5 also block invasion and PfRh5 seems to

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• Antibodies to circumsporozoite protein (CSP) trap sporozoites in the skin or block their invasion of liver cells

• IFNg+ T cells targeting CSP and other liver stage expressed antigens (e.g. LSA-1) inhibit parasite development within hepatocytes

gamete zygote

ookinete

SPOROZOITES

LIVER

MEROZOITES

RBC

IN MOSQUITO GUT

• Antibodies to merozoite surface proteins (e.g. MSP-1) opsonize free merozoites or, together with antibodies to apical complex antigens (such as AMA-1 and PfRH5), block merozoite invasion of RBCs. Antibodies to variant surface proteins (e.g. PfEMP-1) prevent sequestration of infected RBC in brain, lung, kidney, placenta, etc.

GAMETOCYTES

Complement fixing antibodies binding to gamete-specific antigens (Pfs230, Pfs48/45) prevent fertilization of gametes; antibodies to Pfs25 prevent development of the zygote

• IFN-g+ NK cells and/or CD4+ T cells recognizing internal merozoite proteins activate macrophages to phagocytose intraerythrocytic parasites and produce toxic molecules such as free radicals and NO

FIG. 38.4. Stage-Specific Vaccine Targets in Malaria Parasites. The left figure depicts the life cycle of malaria and the immune effector mechanisms that target the different developmental forms of the parasite (reproduced from Malaria Vaccine Initiative Web Site [www. malariavaccine.org/ mal-what_is_malaria.htm]). (Adapted from Richie TL, Saul A. Progress and challenges for malaria vaccines. Nature . 2002;415:694–701, with permission.).

be a remarkably conserved protein among different parasite isolates, raising hopes that PfRh5 might be a credible new candidate for a blood stage malaria vaccine. Despite their extreme polymorphism, the variant surface antigens present on infected erythrocytes that mediate adhesion to endothelial cells are potential vaccine candidates.426 Epidemiologic data suggest that the risk of severe manifestations of the disease is reduced after only a very few clinical episodes,427 and that parasites causing severe disease tend to express a subset of variant surface antigens.428 Thus, a finite number of variant antigens might be sufficient to elicit broad immunity against severe disease. Similarly, relatively few PfEMP-1 variants appear to mediate parasite sequestration in the placenta, and a pregnancy malaria vaccine might work by targeting the domains that bind to chondroitin sulphate, the major placental ligand for parasitized red blood cell (pRBC) sequestration. Antimalarial transmission-blocking immunity works primarily by antibody-mediated, complement-dependent lysis of extracellular sexual stages of the parasite within the midgut of a blood-feeding mosquito429 (see Fig. 38.4). Transmission-blocking immunity has been induced in vivo by immunization with gametes of avian, rodent, and monkey malarias. Several potential transmission-blocking vaccine candidates have been identified, and the genes encoding these surface proteins have been isolated and sequenced, but their production as recombinant proteins is hampered by failure to recreate the highly complex tertiary structures that are the targets of inhibitory antibodies. Furthermore, for those antigens expressed only by invertebrate stages of

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the parasite, lack of natural boosting may prevent maintenance of sufficiently high antibody titers. Clearly, an optimal vaccine against malaria would need to target multiple antigens and induce immunity against all stages. However, by targeting certain antigens confined to asexual blood stages, the induction of an adult-like immune status among high-risk infants in sub-Saharan Africa could greatly diminish severe disease and death caused by P. falciparum.

T-Cell Vaccines Vaccination Against Leishmaniasis Vaccines against intracellular parasites will need to induce long-lived cellular immune responses. As already discussed, for diseases such as Leishmaniasis, Chagas disease, and toxoplasmosis, Th1 and/or CD8 + T-cell responses are the effector mechanisms required for protective immunity. An inherent problem with most nonliving vaccines is their relative inefficiency in generating and/or sustaining these sorts of cellular responses. A major advance in T-cell vaccine development was the demonstration that proteins derived from L. major could elicit a powerful Th1 response and protective immunity if given with recombinant IL-12 as adjuvant.430 IL-12 or IL-12–inducing adjuvants such as BCG, CpG-oligodinucleotides, or CD40L have since been used extensively in animal models to potentiate the efficacy of whole cell killed or a diversity of recombinant protein Leishmania vaccines.431 A polyprotein containing several

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Leishmania antigens (TSA, LmSTI1, and LeIF), formulated with the TLR4 agonist MPL®, a detoxified derivative of 4′-monophosphoryl lipid A of LPS, is the only defi ned, subunit vaccine against leishmaniasis currently being tested in human trials. The gold standard of the protection that can be achieved by vaccination in mice, and the only vaccination strategy against leishmaniasis that has worked so far in humans is “ leishmanization,” which is based on the lifelong convalescent immunity that is acquired following induction of a lesion at a selected site with a cutaneous strain of L. major. The nature of the acquired resistance that develops following healing of a primary lesion seems especially important to consider as only healed mice were protected against L. major transmitted by sand fly bites, whereas mice vaccinated with killed parasites plus CpG-oligodinucleotides were protected against needle but not sand fly challenge.432 Importantly, the strong and durable protection against secondary challenge conferred by live vaccination in mice and humans is associated with the persistence of parasites long after clinical cure. The presence at the time of the challenge exposure of a population of effector memory cells that are rapidly tissue homing and readily secrete IFNγ upon encounter with antigen in the challenge site appears to be critical for the full expression of acquired immunity.432,433 The maintenance of effector memory T cells in live vaccinated hosts, and the loss of these cells following antigen clearance in antigen vaccinated hosts, likely explains the failures of a large number of human trials involving whole cell killed vaccines to significantly reduce the incidence of cutaneous leishmaniasis in individuals living in areas of relatively low transmission, where natural boosting is unlikely to have occurred.434 The requirement for persistent antigens reinforces the rationale for live attenuated Leishmania vaccines, a number of which have been generated by targeted deletion of genes involved in parasite survival or virulence. Although the efficacy of live attenuated vaccines against needle challenge in mice has been shown,431 none have been evaluated using infected sand flies and more generally pose greater difficulties in standardization and delivery in field settings. Similar to Leishmania, a diversity of protein-, DNA-, and viral vector–based vaccines have been developed that successfully induce protective immunity against an experimental T. cruzi infection in mice, typically measured as a CD8 + T-cell– dependent reduction in acute-phase parasitemia or associated mortality.435 In contrast to an anti-Leishmania vaccine, however, a prophylactic vaccine for human Chagas disease would almost certainly have to provide sterile immunity in order to be effective, as the cardiac pathologies are associated with persistent infection. Whereas a number of experimental vaccines were shown to reduce the tissue inflammation and parasitism associated with late chronic phase infections, it does not seem that infection itself, or even infection and drug cure, confers sterilizing immunity against reinfection,146 and there is so far no evidence that a vaccine can achieve a better result. Furthermore, the immune response seems to be focused on epitopes encoded by genes of the large and strain variant trans-sialidase gene family,436 which would require that a

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massive number of target epitopes be included in an effective vaccine. Thus, the rationale for the development of a safe and effective vaccine against human Chagas disease remains suspect, particularly as vector control methods have proven to be so highly effective. Vaccines to prevent toxoplasma infection are needed primarily to protect livestock and prevent transmission to humans from felines. As with T. cruzi, CD8 +T cells working in conjunction with IFNγ-producing CD4 + T cells appear to provide optimal immunity to challenge infection and so far this type of protection is best induced by live attenuated strains of the parasite.437 Recently, modern tetramer sceening and bioinformatic approaches have been used to identify dominant CD8 + epitopes for both mice438 and humans439 and in the latter case have been tested for their vaccine potential in transgenic mice expressing supertype human leukocyte antigen-A molecules. These findings offer new promise to peptide-based approaches for vaccination against T. gondii.

Vaccination Against Malaria Liver Stages Because malaria parasites infect and replicate in hepatocytes, which express MHC class I, infected hepatocytes are potential targets of CD8 + T-cell responses (see Fig. 38.4). Irradiated or genetically attenuated Plasmodium sporozoites, which can i nfect hepatocytes but do not progress to a blood-stage infection, have been shown to protect rodents, monkeys, and humans against malaria and are believed to work by inducing IFNγ -producing CD8 + T cells specific for preerythrocytic antigen.440 Overirradiation of sporozoites, or attenuation of sporozoites very early in their development inside the hepatocyte, negates their ability to immunize, suggesting that the targets of protective mechanisms are novel antigens expressed only if sporozoites are to undergo partial differentiation within hepatocytes. Efforts to mimic the protection generated by irradiated sporozoites using nonliving protein- and DNA-based vaccines have yielded encouraging results. A recombinant polypeptide representing the central repeat and C-terminal portions of the P. falciparum CS protein and fused to hepatitis B surface antigen (HBsAg) elicits IL-2–secreting CD4 + T-cell responses as well as antisporozoite antibodies in human volunteers, particularly when given with the GSK proprietary adjuvant AS01 (containing liposomes, MPL and QS21). The vaccine, RTS,S, induced antibody and T-cell responses in adults already primed to CS by preexposure to malaria. Most significantly, in this fi rst phase II trial, vaccine efficacy during the fi rst 9 weeks of follow-up was 71% but decreased to 0% over the next 6 weeks.441 In the intervening decade, more than 50 clinical trials of RTS,S have been undertaken—many of them in Africa—refi ning the dose, the adjuvant, the immunization schedule, and age of vaccination,442 and a phase III trial (the very fi rst for a malaria vaccine) was begun in 2009. The preliminary results of this trial confi rm that the vaccine has approximately 50% efficacy against clinical malaria attacks and against malaria-related hospital admissions,443 but the duration of protection is still an area of concern. Moreover, as there are still no clear immunologic

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CHAPTER 38

correlates of vaccine-induced immunity, refi nement of the vaccine to enhance its efficacy may mean that clinical trials need to continue for many years to come. Despite initial enthusiasm that DNA vaccination strategies might improve the durability and potency of the cellular response, clinical trials have been disappointing, and the use of sequential immunizations using various heterologous prime/boost protocols are being tested to enhance the effectiveness of preerythrocytic vaccines.178 Priming with plasmid DNA encoding CS and various liver stage–specific proteins and boosting with recombinant adenovirus or pox virus, such as modified vaccinia virus Ankara or fowlpox, has induced complete protection and very high levels of IFNγ-secreting CD8 + T cells in mice. Although protection in human trials has been less convincing, delays in time to emergence of blood-stage parasites have been consistently obtained, suggesting that the vaccination substantially reduces, but does not eliminate, liver stage parasites.

Vaccination to Prevent Pathology In many parasitic infections, disease is a consequence of the host immune response. Because these pathogens are so well adapted to their hosts, it may be easier and more efficient to design immune interventions that prevent parasite-induced immunopathology rather than eliminate the infection itself. Although this approach will not lead to eradication of the parasite, it would likely reduce or alleviate the health consequences of infection. The feasibility of antipathology vaccines was demonstrated in a murine models of schistosomiasis.395 Because disease in schistosomiasis is largely due to the granulomatous pathology that develops around parasite eggs trapped in target host tissues, a valid approach toward immunoprophylaxis for schistosomiasis is to vaccinate to minimize granulomatous pathology395 or reduce parasite fecundity.444,445 In mice, granuloma size and collagen deposition are correlated with the intensity of the type 2 response, and immunologic interventions, such as the administration of IL-4 and IL-13 antagonists, reduce both the size of granulomas and magnitude of fibrosis (discussed previously).401,446 In extensions of these studies, mice immunized with parasite egg antigens plus IL-12447 or CpGoligonucleotides448 to induce an egg antigen-specific type 1 response, upon subsequent infection, exhibited far less severe egg-associated liver disease than did infected nonimmunized controls. Importantly, several immunodominant egg antigens have been described; 449,450 thus, it may be possible to design recombinant antipathology vaccines that duplicate the promising results produced with crude parasite extracts. Parasite-derived GPI has been implicated in much of the pathology of malaria,451 binding to TLRs on DCs and macrophages, and inducing release of TNF-α and other proinflammatory cytokines. As a proof of principle that GPI might serve as a target of an antidisease vaccine, mice immunized with synthetic GPI were protected against the acute immune pathology associated with P. berghei infection; however, as the mice were not immune to the parasite itself, they were unable to control parasite replication and eventually died

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of overwhelming parasitemia.452 These observations suggest that although malaria vaccines that prevent immune pathology might reduce some aspects of morbidity, they are unlikely to be deployed in the absence of vaccine components that also limit parasite burden.

Vaccines Against Helminths Infection with helminthic parasites remains a significant health problem in many tropical countries. Whereas control measures are available in some areas, in most cases, patients living in endemic regions are quickly reinfected. Therefore, vaccines that reduce parasite and/or egg burdens would be a valuable tool to complement existing disease prevention programs and could represent a less costly and more practical approach than repeated chemotherapy. Although many subunit vaccines have been described and tested in various animal models, suboptimal levels of protection have hindered the development of all but a few of these candidate vaccines.453 Significant advances in vaccination technology over the past decade have made it possible to identify novel targets using immunomics454 and engineer vaccines that elicit strong cellular and humoral immunity.455 Novel DNA vectors, improved delivery systems, new adjuvants, and immunomodulatory cytokines allow significant augmentation of the immune response to vaccines and preferential induction of specific effector mechanisms, including antibody isotypes, T helper cell subsets and cytotoxic T cells. However, in order to effectively harness and implement these advances, it will be necessary to fully understand the mechanisms of resistance to helminth parasites. Vaccine models using radiation-attenuated larval parasites have provided the best examples of successful immunization against helminths. With the irradiated schistosome vaccine, although complete sterilizing immunity appears to be an unachievable goal, immunity approaching 60% to 80% is possible with the addition of adjuvants such as IL-12.456 This model has served as the gold standard for schistosome vaccine development.457 The cumulative evidence from vaccine studies conducted in numerous gene knockout mice suggests that irradiated parasites induce protection via both Th1- and Th2-dependent pathways,234 and that both humoral and cellular mechanisms will be required for the generation of optimal immunity against most helminth parasites.458,459 While there has been extensive research on defined vaccines against helminth infection,460,461 none of the candidate antigens have been shown to induce protective immunity in humans, although a few are actively being tested in clinical trials.462 At least one of the antigens—glutathione-S-transferase (P28/GST)—has moved through a phase I trial, and phase II trials are now underway in Africa. Other antigens of interest include paramyosin (Sm97), IrV5 (myosin-like 62kDa protein), triose phosphate isomerase, Sm23, the integral membrane protein tetraspanin-2, and Sm14, a fatty acidbinding protein.463,464 With the recent advances in schistosome genomics, proteomics, and immunomics,, a new panel of vaccine antigens is being identified, and these antigens will warrant further investigation in animal models.465,466

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A number of fi larial vaccine candidates have also been described and are being tested, including chitinase, tropomyosin, paramyosin, and several larval antigens called the “abundant larval transcript family.”460 Finally, several experimental antihookworm vaccines are also being investigated.467 While these accomplishments represent a significant advance for the field of helminthology, it is important to note that all of the candidate antihelminth vaccine antigens will at best provide only partial protection. It is hoped, however, that ongoing improvements in vaccination technology, combined with greater knowledge of the mechanisms controlling resistance, will allow development of more efficacious and better defined vaccines for these complex organisms.

CONCLUSION In the 4 years since the publication of the last edition of Fundamental Immunology, studies on the host–parasite interaction have contributed important advances to our understanding of the immune response and its regulation. Interestingly, many of these discoveries emerged from the study of worm infection models and led to new insights into the mechanism of Th2 polarization, the orchestration of mucosal immune responses, and the function of ILCs, basophils, and AAMs in promoting adaptive immunity. Indeed, although once regarded as a less sophisticated topic than protozoan immunology, the study of immunity to helminths has undergone a major resurgence in part because of the growing appreciation of the phylogenetic uniqueness of worm pathogens and their host interaction. Research on the immune response to helminths has also been stimulated by the growing interest in neglected tropical diseases, where worm infections account for the most affected individuals. Finally, there has been a growing awareness of the important evolutionary role played by helminth infection in conditioning the mammalian immune system, in the maintenance of immunologic homeostasis at mucosal tissue barriers, and of the likely impact of the loss of this symbiotic relationship on immune function due to modern improvements in human hygiene.468 A second trend in immunoparasitology in recent years has been the widespread use of genetic and genomic screening technologies to identify genes with functional roles in the host–parasite relationship. Interestingly, many of these projects98,438,439 have brought together molecular and immunoparasitologists with the common goal of simultaneously identifying both parasite epitopes/virulence determinants and their host receptors and signaling pathway targets. The findings gained from such studies should provide important groundwork for future systems biology approaches that seek to develop a broader vision of the interaction of parasites with the immune system. Another important development since the publication of the last version of this chapter is the increased emphasis on the study of the human immune response to parasitic

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infection. The most notable advances have occurred in the field of malaria, which benefits from a wealth of clinical material and the ability to use peripheral blood as a physiologically relevant source of immune cells. The study of human malaria is yielding novel insights into the nature of B-cell effector and memory responses to the parasite as well as the role of innate cellular function in regulating disease outcome. Similarly, as noted previously in this chapter, human visceral leismaniasis has proved a fertile ground for the study of the role of T cells and cytokines in immunosuppression and, in the case of the disease itself, implicated IL-10 production as target for immunotherapeutic intervention. Perhaps the most heralded recent advance in the field of immunoparasitology has been the success of the field trials of the RTS,S vaccine for falciparum malaria.442 While still clearly limited as a tool for controlling malaria because of its partial efficacy, important lessons have been learned from the experience of bringing the RTS,S vaccine to its current stage of development. Ironically, although RTS,S was originally designed to simultaneously trigger humoral and cell responses to the circumsporozoite protein (CSP) Ag, it is not all clear that the protection induced by the vaccine depends on this multipronged response. Instead, its improved efficacy appears to have stemmed from changes to the GlaxoSmithKline (GSK) adjuvant formulations and dosing regimen employed.441 Indeed, the vaccine construct itself has not changed significantly from the time of the earlier unsuccessful trials. Thus, the RTS,S trials have both affirmed the importance of antigen delivery and presentation in the induction of parasite immunity while emphasizing our continued ignorance of the rules linking these parameters to the induction of host resistance. Nevertheless, having a defined antigen vaccine in which at least a proportion of the immunized individuals become protected offers a rare opportunity to identify correlates of immunity to malaria triggered by a single immunogen through the use of a “systems vaccinology” approach.469 Such studies, in addition to providing valuable information needed for the design of a more effective RTS,S vaccine, could supply an important precedent and platform for analyzing human immunity to parasitic pathogens at a wider level. It is hoped that exciting new approaches and opportunities of this kind will bring us closer to our ultimate goal of protecting human populations against the scourge of parasitic disease.

ACKNOWLEDGMENTS Thomas A. Wynn, David Sacks, and Alan Sher are all supported by the intramural research program, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. Eleanor Riley is supported by the London School of Hygiene and Tropical Medicine, the UK Medical Research Council and the Commission of the European Community. We thank Tom Nutman, Dragana Jankovic, and Thirumalai Ramalingam for helpful discussions.

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39

Immunity to Viruses Hildegund C.J. Ertl

INTRODUCTION Veni, vidi, vici —I came, I saw, I conquered. If viruses could talk or see, this would be their hendiatris. Although viruses are minute particles composed only of a genome surrounded by a few proteins, they have a fiendish way of wreaking havoc not only on humans, as everyone who ever had the flu knows, but also on animals, plants, and even bacteria. Each year, more people die of viral infections than of natural disasters such as hurricanes, earthquakes, and tsunamis combined, or even manmade tragedies such as war. For example, it is estimated that variola major, the causative agent for smallpox, killed nearly half of the population of Native Americans after the virus was introduced into the western hemisphere by European colonizers. The Spanish Flu caused by an H1N1 influenza A virus caused the death of 50 to 100 million humans between 1918 and 1919, which is well in excess of the 16 million casualties of World War I. Human immunodeficiency virus (HIV)-1 has killed more than 25 million humans since 1981 and continues to spread, threatening the already frail economic structures of the most afflicted countries in sub-Saharan Africa, where more than 30% of the adult population carries the virus. Not only do viruses cause acute or chronic infections with potentially fatal outcome, but they also contribute to other diseases. Viruses are associated with 20% of cancers, they have been implicated in the pathogenesis of human arteriosclerosis and autoimmune diseases, and they are linked to an overall reduction in life expectancy. How can something so small be so deadly? Viruses, which range in size from 10 to 300 nm in diameter with genomes of minimally 2 kilobases to over 1.2 megabases, are unable to propagate themselves, but require a host cell to replicate. Once a cell becomes infected, viruses hijack its transcription and translation machinery to promote their own replication. The physiologic functions of the infected cell are disrupted as it is being turned into a virus production facility. However, the cell fights back as soon as it senses the virus. This fight initially takes place intrinsically within the infected cells but then rapidly spreads extrinsically once the immune system has been alerted. In turn, many viruses mount defenses against the attack from their host by encoding proteins that actively subvert innate and adaptive immune responses. In acute virus infections, the fight between virus and host literally lasts until the death of one of the adversaries. In chronic infections, a truce is eventually reached where virus and host coexist, generally at the expense of the well-being of the latter.

Fewer than 200 viruses are known to cause disease in humans. Over the last 50 years, on average two new species of human viruses have been discovered annually; one can expect that this number will continue to rise.1 Where does this ever-increasing number of viruses come from? We are not certain about the origin of viruses, although we know from ancient texts as well as more modern data-driven genomic analyses that viruses have been around for a very long time.2 Whether they originated from cells, concomitantly with cells, or even primordially from some genetic soup remains debated.3 No matter how they evolved, their lack of common genes argues for a polyphyletic rather than monophyletic evolution. The constant discovery of new viruses may simply reflect improvements of detection technologies that traditionally were based on cell culture and that are now being replaced with high-throughput genomics. Notwithstanding, many of the newly discovered viruses seem to evolve from animal reservoirs through mutations that allow for an extension of their host range. Of importance is that viral genomes have far higher mutation rates than, for example, mammalian genomes as they fail to correct errors during replication; such errors most commonly lead to loss of viral fitness but occasionally benefit the virus in its quest for replication. One example of a stable host range altering mutation that caught global attention was that of a coronavirus, which caused the outbreak of severe acute respiratory syndrome (SARS) in 2003 and 2004. This virus, termed SARS-CoV, which in its wild-type form infects civets, a cat-like carnivore, mutated and became infectious to humans and then within this host rapidly underwent further positive selection.4 Other viruses such as pathogenic H5N1 avian influenza viruses have been isolated since the late 1990s repeatedly from humans, who commonly died as sequela of the infection. Pathogenic H5N1 viruses, which were, and by some still are, feared to evolve into pandemic viruses, have thus far failed to mutate to achieve sustained human-to-human transmission, while concomitantly, another influenza A virus arose from a triple reassortment between viruses that naturally infect humans, avians, and swine, and caused the 2007 influenza pandemic. Considering that our knowledge of animal viruses remains limited, the sudden emergence of new and potentially deadly viruses from other species continues to threaten global health. While most other deadly disease can be treated with drugs such as antibiotics to resolve bacterial infections or can be prevented by lifestyle choices, our arsenal to combat viral infections remains limited. Vaccines, which can be effective

937

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in preventing viral infections, and even achieved the eradication of small poxvirus, are only available for 15 viruses. Drugs to specifically treat infections are only effective for some viruses such as HIV-1, herpes, hepatitis B and C, and influenza viruses. Our main defense thus remains the immune system. Over eons, it has evolved to sense viruses as pathogens, to produce factors that stop viral replication, and to develop lymphocytes that destroy those cells that serve as viral production factories. Like an elephant, the immune system never forgets. Nevertheless, this defense like that in every war, even if victorious, comes at a price: a runny nose at best and death due to insufficient or overwhelming responses at worst.

BASICS IN VIROLOGY Virus Classification According to the International Committee of Taxometry of Viruses, all viruses are classified into order, family, subfamily, genus, and species. Names of serotypes, genotypes, strains, variants, or isolates of virus species or artificial viruses are not ruled by the International Committee of Taxometry of Viruses. A species is defined as a polythetic class of viruses that constitutes a replicating lineage and occupies a particular ecologic niche. A genus defines a group of species that share common characteristics, while subfamilies and families defi ne a group of genera with common characteristics. An order, in turn, defines families with shared characteristics. Currently, 6 orders, 87 families, 19 subfamilies, 348 genera, and 2288 species of virus have been defined.5 The Baltimore classification differs and divides viruses according to their genome or their mode of replication. Viruses contain either a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) genome, which can be single stranded (ss) or double stranded (ds). SsRNA viruses carry either a negative- or positive-stranded RNA. During their lifecycle, some DNA and RNA viruses undergo an intermediate step in which the RNA genome is converted by reverse transcription (RT) into DNA or vice versa their DNA genome into an RNA genome. Accordingly, viruses are classified into dsDNA viruses (eg, adeno or poxviruses), ssDNA viruses (eg, parvoviruses), dsRNA viruses (eg, reovirus), positive-sense ssRNA viruses (eg, picornaviruses), negativesense ssRNA viruses (eg, rhabdoviruses), positive-sense ssRNA-RT viruses (eg, retroviruses), or dsDNA-RT viruses (eg, hepadnaviruses). Other classifications are based on host range (Holmes classification) or structural characteristics (Lworff-Horne-Tournier [LHT] system). The viral genome can be linear (eg, poxvirus), circular (eg, papillomaviruses), or segmented (eg, influenza virus). Viruses are further classified into enveloped (eg, rhabdoviruses) or naked (eg, picornaviruses) viruses. Viruses can also be divided according to their morphology, which can be polymorphic or structured, the latter having either icosahedral or helical symmetry. To give an example of the taxonomic division of viruses: the 2007 pandemic H1N1 virus belongs to the species of influenza A virus, the genus of influenza virus, the family of Orthomyxoviridae with a negative-stranded ssRNA genome

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covered by a helical envelope. A list of common human viruses and additional viruses repeatedly referred to in this text and their classification is shown in Table 39.1. Other characteristics of virus families, such as their genomes and surface structures, are shown in Table 39.2.

Virus Transmission Viruses have a single-track mind; their only goal is to replicate. The goal of their unwilling hosts is to get rid of them by mounting an immune response. Viruses have evolved a multitude of mechanisms to evade this immune response, and their hosts have adapted countermeasures accordingly, as only those that survived the onslaught of infections reached reproductive age. Highly contagious viruses can afford to not be overly concerned if their hosts, which are essential for their replication, survive, die, or mount a rapid and successful immune response because their ease of transmission ensures their continued existence. A typical example for such a virus is influenza virus, which is transmitted by aerosols before its host becomes sufficiently ill to seek solace in bed, which would limit contact with others and thus reduce the chance for the virus to spread. An example for a virus that is not overly contagious is rabies virus; it is transmitted by the bite of an infected animal, and it ensures its transmission by literally driving its host into an insane rage so that it will randomly attack and bite everyone in sight. Other viruses evolved to ensure the continued survival of their hosts, which enables their own continued replications without necessitating rapid transmission to a new individual. Such viruses are usually more complex, as much of their genome is devoted to combat immune responses. Many viruses can only replicate in one species and therefore human infections require human-to-human contact. For viruses that are heat labile, such contact needs to be close, while more stable viruses can remain on surfaces or in water until the opportunity for infection arises. Viruses that only replicate in humans can potentially be eradicated once a vaccine becomes available as exemplified by smallpox virus. Other viruses are less discriminatory, and they replicate in multiple species, not necessarily only mammals but also birds or invertebrates. For example, influenza viruses can infect aquatic birds, chicken, swine, horses, humans, and even cats. Although they are most commonly transmitted to humans from other humans, spread from infected animals can occur, such as infections of humans with pathogenic H5N1 from chickens or ducks. Some viruses, such as rabies virus, infect all warm-blooded mammals, while other viruses alternate between mosquitoes and vertebrate animals. An example for the latter is Japanese encephalitis virus, which, in addition to mosquitoes of the Culex tritaeniorhynchus species, can infect humans, birds, most domestic animals, snakes, and frogs. The host range of a specific virus affects the mode of transmission, which is further influenced by the tissue tropism of the virus and its resistance to environmental factors like temperature or water. One of the main protections against virus invasion is healthy skin; the upper layer of the keratinized epidermis effectively prevents entry of viruses. Mucosal surfaces,

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CHAPTER 39

TABLE

39.1

IMMUNITY TO VIRUSES

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Taxonomy of Viruses

Order

Family

Subfamily

Genus

Species (Alternative Names)

Abbreviation

Herpes virales

Herpes virideae

Alphaherpes virinae

Simplex virus

Human herpesvirus 1 (Herpes simplex virus-1) Human herpesvirus 2 (Herpes simplex virus-2) Saimiriine herpes virus 1 Human herpesvirus 3 Human herpesvirus 5 Human herpesvirus 6 Human herpes virus 7 Human herpesvirus 4 Epstein-Barr virus Human herpesvirus 8 Saimiriine herpes virus 2 Zaire ebolavirus Measles virus Human parainfluenza virus 1 Sendai virus Mumps virus Human respiratory syncytial virus Rabies virus Vesicular stomatitis New Jersey virus Severe acute respiratory syndrome–related coronavirus Human rhinovirus A Hepatitis A virus Human adenovirus C Lymphocytic choriomeningitis virus Dengue virus Japanese encephalitis virus Tick-borne encephalitis virus West Nile virus Yellow fever virus Hepatitis C virus Hepatitis B virus

HHV1

Betaherpes virinae Gamma-herpes virinae

Mononega virales

Filoviridae Paramyxo viridae

Paramyxo virinae

Pneumo virinae Rhabdo viridae Nidovirales

Corona viridae

Picorna virales Unassigned

Picorna viridae

Corona virinae

Varicellovirus Cytomegalovirus Roseolavirus Lymphocrypti virus Rhadinovirus Ebolavirus Morbillivirus Respirovirus Rubulavirus Pneumovirus Lyssavirus Vesiculovirus Betacoronavirus

Adeno viridae Arena viridae

Enterovirus Hepatovirus Mastadenovirus Arenavirus

Flaviviridae

Flavivirus

Hepacivirus Orthohepadna virus Influenzavirus A

Hepadna viridae Orthomyxo viridae Papilloma viridae Parvo viridae Poxviridae

Reoviridae Retroviridae Togaviridae

Alphapapilloma virus Parvo virinae Chordopox virinae

Sedoreovirinae Orthoretrovirinae

Dependovirus Avipoxvirus Leporipoxvirus Molluscipoxvirus Orthopoxvirus

Rotavirus Deltaretrovirus Lentivirus Alphavirus Rubivirus

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HHV2 SHV1 HHV3 HHV5 HHV6 HHV7 HHV4 EBV HHV8

RSV VSV SARS-CoV HAV Ad virus LVMV

WNV HCV HBV

Influenza A virus

Flu

Human papillomavirus 16

HPV16

Human papillomavirus 18 Adeno-associated virus-2 Fowlpox virus Myxoma virus Molluscum contagiosum virus Cowpox virus Ectromelia virus Vaccinia virus Variola virus Rotavirus A Primate T-lymphotropic virus 1 Human immunodeficiency virus 1 Simian immunodeficiency virus Sindbis virus Venezuelan equine encephalitis virus Rubella virus

HPV18 AAV2 MCV VV HTLV-1 HIV-1 VEE virus

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TABLE

39.2

IMMUNITY TO INFECTIOUS AGENTS

Characteristics of Viruses

Family

Genome

Virion

Capsid

Herpesvirideae Filoviridae Paramyxoviridae Coronaviridae Adenoviridae Arenaviridae Flaviviridae Hepadnaviridae Orthomyxoviridae Papillomaviridae Parvoviridae Poxviridae Reoviridae Retroviridae Togaviridae

dsDNA Negative-sense ssRNA Negative-sense ssRNA Positive-sense ssRNA dsDNA Negative-sense ssRNA Positive-sense ssRNA ds-DNA-RT Negative-sense ssRNA dsDNA, circular ssDNA dsDNA dsRNA RT-positive-sense ssRNA Positive-sense ssRNA

Enveloped Enveloped Enveloped Enveloped Enveloped Enveloped Enveloped Enveloped Enveloped Naked Naked Complex Naked Enveloped Enveloped

Isocahedral Helical Helical Helical Isocahedral Complex Isocahedral Isocahedral Isocahedral Isocahedral Isocahedral Complex Isocahedral Isocahedral Isocahedral

DNA, deoxyribonucleic acid; RNA, ribonucleic acid.

although they are commonly bathed in antiviral proteins present in saliva and tears, in acids found in the female outer genital tract or the stomach, or in destructive enzymes such as those present in the upper intestinal tract, provide a more permissive port of entry for viruses and, consequently, most viruses are transmitted through the mucosal surfaces of either the airways, the intestines, the genital tract, or the eye. Influenza viruses, parainfluenza viruses, some types of adenoviruses, and rhinoviruses spread through the airways and are transmitted by aerosolized droplets expelled by coughing or sneezing. Viruses that spread through aerosols tend to be highly contagious, such as influenza viruses, which cause annual epidemics and occasional pandemics that within a few months can spread throughout the world as was shown in the 2009 swine flu pandemic. The new pandemic influenza virus was first identified in Mexico on March 18th, 2009; reached California by March 28th; was detected in Canada, New Zealand, the United Kingdom, Israel, and Spain by April 28th; in Germany by the 29th; in Austria, Switzerland, and the Netherlands by April 30th; in other European countries, as well as in Asia, by May 2nd; in South America by May 5th; and on June 11th was officially declared as a pandemic virus by the World Health Organization. The total death toll of this pandemic was rather modest with approximately 5700 reported deaths by August 10th, 2010, when the World Health Organization announced the official end of the pandemic. Another highly contagious virus is varicella virus, which causes chickenpox. This virus is also spread by droplets from person to person, but, unlike influenza virus, which is fairly stable and can thus infect individuals that touch an infected surface and then their nose, varicella virus is very heat labile and, as a rule, requires direct person-toperson contact. Other viruses are transmitted by oral ingestion and are then spread by shedding into feces. These viruses are generally stable, allowing them to resist the acidic environment of the stomach or the digestive enzymes of the intestinal tract.

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Many of them, such as influenza viruses that predominantly infect aquatic birds through the oral route or rotaviruses that cause severe diarrheal disease in children, can also survive for a prolonged time in water. Improperly treated drinking water can spread a number of other viruses, such as enteric adenovirus, calicivirus, astrovirus, poliovirus, or hepatitis A virus. Sexually transmitted viral infections include herpes simplex virus (HSV) type 2, HIV-1, and several types of human papilloma viruses (HPV). Interestingly, all of these viruses establish sustained infections. HSV-2, after a replicative phase, persists latently in root ganglia from where it is periodically reactivated causing local sores that shed virus. HIV-1 first causes an acute flu-like infection and then persists mainly in CD4 + T cells while constantly dodging a vigorous antiviral immune response through mutations and immune evasion strategies. Although oncogenic types of HPV such as HPV-16 or -18 are commonly eliminated after genital infection, their persistence can over time cause transformation of the infected cells due to the activity of the two viral oncoproteins E6 and E7, which disrupt key cell cycle checkpoints and then lead to cervical cancer in women or penile or anal cancer in men. Although some of the sexually transmitted viruses (eg, HSV-2) are highly contagious, others (eg, HIV-1) transmit poorly and the average rate of HIV transmission has been estimated at 0.0082 per coital act in humans without comorbidities.6 Some viruses literally need to be injected into the body to cause an infection. These viruses are either transmitted by blood sucking insects or animal bites. Three flaviviruses, Dengue virus, West Nile virus, and Japanese encephalitis virus, are spread by mosquitoes, whereas Kyasanur forest disease virus, another flavivirus, is spread by ticks. Rabies virus, another vector-borne virus, is generally transmitted by the bite of an infected animal, most often a dog. The virus replicates in the central nervous system and is then transported to peripheral organs such as the salivary glands from where it is secreted into the saliva ready to spread to its

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next victim. Although the vast majority of rabies infections are caused by bites, mucosal transmission7 and transmission by transplantation of tissues from an infected individual have been reported.8

Virus Cell Entry and Replication Most viruses enter cells upon binding to a receptor, some of which are broadly expressed while others are specific for a certain cell type. In some instances, viruses bind with high avidity to one receptor but are also capable of infecting cells that lack expression of the high-affinity receptor through low avidity binding to an alternative molecule. Other viruses require binding to a receptor and a coreceptor. Receptor usage determines tissue tropism of many viruses and in some cases it also influences their host range. For example, the hemagglutinin (HA) of influenza A viruses that can spread in humans binds to sialyated glycan receptors with a terminal α2-6 linked N-acetylneuraminic acid. In contrast, α2-3 linked sugar residues are used as receptors for influenza A viruses that circulate in birds. Once the HA has bound to its receptor, it is cleaved. A trypsin-like enzyme present only in the lung cleaves HA into two subunits, which allows the virus envelope to fuse into the cell membrane. Some of the more pathogenic strains, such as the 1918 H1N1 virus or pathogenic 2006 H5N1 viruses, activate HA through a trypsin-independent mechanism.9 These strains have a multibasic cleavage site that can be digested by furin and furin-like proteases, which are more ubiquitously present in human tissues than the trypsin-like enzymes, allowing these viruses to infect tissues other than lung. The fiber knob of adenoviruses preferentially binds the coxsackie adenovirus receptor, which is expressed on epithelial cells. In addition, an Arg-Gly-Asp (RGD) motif present within the viral penton can bind α (v)-integrins with lower avidity. The fiber of adenoviruses of subfamily B2, on the other hand, binds CD46, a ubiquitously expressed complement component that also facilitates entry of measles virus and human herpesvirus (HHV)6. The herpes virus mediator (HVEM) is a bimodal switch that can provide both immunostimulatory and immunoinhibitory signals to the immune system. Upon binding to LIGHT (lymphotoxin [LT]-like, exhibits inducible expression and competes with HSV-1 glycoprotein D [gD] for HVEM), HVEM submits stimulatory signals. Upon binding to the B- and T-lymphocyte attenuator (BTLA), it acts as an immunoinhibitor. HVEM also binds HSV-1 gD, thus facilitating entry of this virus. Binding of gD to HVEM takes place on a site that overlaps with the BTLA binding site; therefore, gD can be used to inhibit an immunoinhibitory pathway. HSV-1 may have evolved to block such a pathway, as activation of NF-κ B promotes viral replication and assists in transcription of some of the early viral genes.10 The envelope protein of HIV-1 binds cluster of differentiation (CD)4 expressed mainly on T-helper cells. Upon binding, the protein undergoes structural changes that allow for its binding to a coreceptor, which for transmitting virions is CCR5, but following mutations, viruses circulating in an

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organism can also use CXCR4.11 A mutant allele of CCR5 termed CCR5d32, which results in lack of CCR5 expression on the cell surface and which is found in 10% of Caucasians of European descent, provides resistance to infections with HIV-1.12 Other viral receptors include the nicotinic acetylcholine receptor for rabies virus, heparan sulfate for dengue virus, adeno-associated viruses, and some of the herpes viruses, CD155 for poliovirus, CD81 for hepatitis C virus, CD21 for Epstein-Barr virus (EBV), C-type lectins, such as dendritic cell (DC)-specific intercellular adhesion molecule3-grabbing nonintegrin (DC-SIGN) and liver/lymph node– specific intercellular adhesion molecule-3-grabbing integrin for Ebola virus, integrin-β3 for Hantan virus or intercellular adhesion molecule (ICAM)-1 for rhinoviruses. Examples for viral receptors including their physiologic functions are listed in Table 39.3. Upon binding to a receptor, viruses not only need to gain access into the cell, but most of them then have to traverse to the nucleus to initiate their replication. Viruses enter the cell either through endocytosis31 or fusion.32 Clathrin-mediated endocytosis is used by enveloped as well as nonenveloped viruses including adenoviruses, influenza viruses, poxviruses, or rabies virus. In cadherin-mediated endocytosis, the virus-receptor complexes cluster into a cadherin-coated pit on the cell membrane that becomes invaginated, eventually closes, and detaches from the cell membrane. The clathrin-coated vesicles then deliver their cargo to early endosomes from where it travels to late endosomes. Other viruses such as coxsackie B virus, respiratory syncytial virus (RSV), and others enter cells by caveolar endocytosis. Caveolae are invaginations in the plasma membrane that are rich in cholesterol, glycosphingolipids, and claveolin, which are used for uptake of macromolecules into endosomes. In addition, caveolar endocytosis allows for transcytosis of molecules from the basal to the apical side of a cell or vice versa. Human enterovirus has been described to enter cells through a lipid raft dependent pathway, rotavirus infects through a cholesterol- and dynamin-dependent but clathrin- and caveolae-independent pathway, while other viruses enter cells by micropinocytosis or phagocytosis. Enveloped viruses such as paramyxoviruses, some herpes viruses, or HIV-1 invade cells by direct fusion of the virus envelope with the cell membrane. Fusion is promoted by hydrophobic sequences within a viral surface protein and causes release of the viral genome into the cytoplasm. Viruses that enter cells through endocytosis end up in endosomes. Mechanisms of escape from endosomes differ for enveloped and nonenveloped viruses. The decrease in pH between early and late endosomes favors conformational changes of viral surface proteins by exposing their hydrophobic residues, which allow for fusion of the viral envelope with the endosomal membrane. This in turn permits escape of the viral core or the genome into the cytoplasm. Nonenveloped viruses disrupt the endosomal membrane either by a pathway called carpet mechanism or by forming pores. In carpet-like disruption of endosomal membranes, viral peptides act like a detergent and thus interrupt the hydrophobic interactions between membrane lipids allowing for the development of micelles and for transient formation

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TABLE

39.3

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Viral Receptors

Receptor

Virus

Viral Antigen

Physiological Function

References

Herpes virus entry mediator

Herpes simplex virus-1

Glycoprotein D

13,14

Coxsackie adenovirus receptor

Adenovirus C

Fiber

Receptor for costimualtors/ coinhibitors Cell adhesion molecxule

Fiber Hemagglutinin Hemagglutinin Virus proteins 1–3 Glycoprotein 160

Complement regulatory protein

16

Complement regulatory protein Cell adhesion molecule T-cell receptor coreceptor

18

Glycoprotein

Forms ion channels in neuronal membranes Signal transduction Cell adhesion molecule Complement component

21

C-type lectin, adhesion molecule

25

CD46 CD55 CD155 CD4 Nicotinic acetylcholine receptor CD81 ICAM-1 CD21 DC-SIGN

P-selectin glycoprotein ligand-1 SLAM/CD150 Transferrin receptor 1

Coxsackie B virus Adenovirus B2 Measles virus Enteroviruses Poliovirus Human immunodeficiency virus-1 Rabies virus Hepatitis C virus rhinovirus Epstein-Barr virus Ebola virus

Glycoprotein E2 Viral proteins 1–4 Glycoprotein 350/220 Glycoprotein

Dengue virus Hepatitis C virus Enterovirus 71

Glycoprotein E Glycoprotein E2

Measles virus Lassa fever virus

Hemagglutinin Glycoprotein

15

17

19 20

22 23 24

26 27

Selectin receptor; mediates leukocyte rolling Signal transduction Import of iron

28

29 30

CD, cluster of differentiation; DC-SIGN, dendritic cell–specific intercellular adhesion molecule-3-grabbing non-integrin; ICAM, intercellular adhesion molecule; SLAM, signaling lymphocytic activation molecule.

of holes within the membrane.33 Other viruses carry proteins, which form amphipathic α-helices that assemble into a pore within the lipid membrane of the endosome where the hydrophobic parts interact with the lipid bilayer while the hydrophilic parts of the coils form the inner wall of the pore.34 Picornaviruses, parvoviruses, and reoviruses utilize this strategy. Once within the cell, viruses may be broken down by autophagy,35 a catabolic process involving the degradation of a cell’s own components to convert unneeded pieces into nutrients. During this process, so-called autophagosomes form from membrane structures containing autophagiarelated gene products (Atg), such as the ubiquitin-like Atg8, the Atg4 protease, and the Atg12-Atg5-Atg16 complex. The outer membrane of the autophagosome fuses with a lysosome to allow for degradation of its contents. Formation of autophagosomes is initiated by PI3K and Beclin-1. Most viruses block this pathway by inhibiting PI3K activation, but rhinoviruses and poliovirus sponsor formation of early autophagosomes but block their fusion with lysosomes and then use the structures to egress the cells.36 Once a virus has reached the cytoplasm, it must deliver its genome to the nucleus. Many viruses such as herpesviruses and adenoviruses use microtubules to reach nuclear pores. Very small genomes can diffuse passively though pores into the nucleus, while larger genomes or particles require an energy-dependent process. Some viruses use viral proteins to facilitate nuclear entry. For example,

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cytomegalovirus (CMV) encodes two proteins, pUL69 and pUL84, that facilitate the transport of its genome to the nucleus. pUL69 binds to UAP56, which facilitates nuclear export of unspliced RNA; pUL84 binds to importin-alpha proteins,37 which can dock to nuclear pores and then be transported through it. Most viruses initiate their replication in the nucleus as they depend on nuclear enzymes for transcription. Poxviruses and some of the RNA viruses are independent of such enzymes and can replicate their genome in the cytoplasm. HIV-1 replicates in the nucleus after it reverse transcribes its RNA in the cytoplasm. Replication of different types of virus can be exemplified using the following viruses: adenovirus, a dsDNA virus; adeno-associated virus, an ssDNA virus; reovirus, a dsRNA virus; poliovirus, a positive-sense ssRNA viruses; influenza A virus, a negative-sense ssRNA virus; HIV-1, a positive-sense ssRNA-RT virus; and hepatitis B virus, a dsDNA-RT virus. Adenovirus transcription is typical for that of some of the larger DNA viruses as it proceeds in stages. Initially, the immediate early gene is transcribed. The resulting gene products alter the host cell to provide a more favorable environment for viral replication and initiate transcription of early viral genes, which have regulatory functions and serve to modify host cell functions or subvert immune responses. Thereafter, the viral genome replicates concomitantly with transcription of the late viral genes that encode

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structural proteins. Specifically, the replication cycle of adenovirus starts with expression of E1A, which encodes two polypeptides that bind to cellular proteins, including cellular transcription factors, which in turn changes the cell’s gene expression profi le and allows for transcription of the other early viral genes E1B, E2, E3, and E4. E1A promotes apoptosis, while E1B proteins are antiapoptotic. E1B polypeptides turn off host cell protein synthesis and help to stabilize, transport, and selectively translate viral RNA. E2 encodes DNA-binding proteins and a polymerase. E3 gene products are nonessential for virus replication but serve to evade immune responses. E4 encodes seven polypeptides, which collaborate with E1 gene products in promoting viral transcription and modulating host cell functions. E4 gene products are also essential for nuclear export of viral RNA. Adenoviruses also encode one or two virus-associated (VA)-RNA species, which form short hairpin loop structure of approximately 200 bases, are transcribed by polymerase III and stimulate translation of viral genes. VA-RNA can be processed into shorter RNAs and act as microRNA,38 inhibiting activation of protein kinase R, which inhibits further messenger RNA (mRNA) synthesis through phosphorylation of the translation initiation factor EIF2A. Transcription and translation of early gene products is followed by DNA replication, which is initiated by a terminal protein that is covalently bound to the 5′ ends of the long terminal repeats. Once DNA replication is initiated, the late gene products, which form the viral capsid, are produced from the L1-L4 domains. Viral assembly begins in the cytoplasm and is completed in the nucleus. Adeno-associated viruses are ssDNA viruses that cause no known disease in humans. They are dependoviruses and require coinfection with another virus, most commonly an adenovirus, to complete their lifecycle. The approximately 4.7 bp genome is flanked by terminal repeats that contain a multipalindromic terminus that forms a loop and thereby promotes priming for DNA replication. The genome contains only two genes; one, the rep gene, encodes four regulatory proteins needed for DNA replication and conversion of the dsDNA intermediate into the final ssDNA. The viral capsid is composed of three virus proteins derived from the cap gene by transcript splicing. Initiation of transcription of the rep gene requires proteins from a helper virus such as gene products from E1, E2, E4, as well as VA-RNA from adenovirus. Final assembly of the adeno-associated virus takes place in the nucleolus. Reovirus infections are asymptomatic in humans but cause disease in newborn mice. This virus, which contains 10 to 12 segments of dsRNA, replicates in the cytoplasm of infected cells without completely uncoating. RNA is transcribed from the negative strand of the genomic RNA and leaves the capsid to be translated. Secondary transcription occurs later followed by assembly within the cytoplasm. The viral genome of positive-sense ssRNA viruses, such as poliovirus, a picornavirus, can directly serve as mRNA. Poliovirus RNA lacks the methylated cap structure that is typical for mammalian mRNA but rather has an internal ribosomal entry site. To avoid competition with translation of mammalian mRNA, poliovirus interferes with

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recognition of the host’s methylated cap, thus inhibiting host cell protein synthesis.39 The poliovirus RNA is translated into a single polypeptide that is cleaved into a replicase, proteases, and structural proteins. The polymerase transcribes the positive-stranded RNA into a minus-sense RNA to serve as template for new positive-stranded RNA. The latter can either be translated, serve as template for minus stranded RNA, or be packaged into new virions. Replication as well as assembly occurs in the cytoplasm. Influenza viruses are segmented negative-sense ssRNA viruses, which replicate in the nucleus. The RNA-dependent RNA polymerase transcribes positive-stranded RNA segments that are either transported to the cytoplasm for translation or remain in the nucleus to serve as templates for negative-stranded RNA synthesis. Newly produced internal proteins are transported into the nucleus where they, together with RNA segments, form new virus particles. The two viral surface proteins, the HA and the neuraminidase are secreted through the Golgi apparatus to the cell surface where they are then picked up by the envelope once the virus leaves the cell. HIV-1 is initially reversed transcribed in the cytoplasm by the viral reverse transcriptase into an RNA/negativestranded DNA hybrid. This process is error prone and contributes to the high mutation rate of HIV-1. The RNA is degraded, and a positive-stranded DNA is synthesized allowing for the formation of a dsDNA, which, together with some enzymes, enters the nucleus; there the viral genomes integrates with the help of the viral integrase and serves as a template for synthesis of viral transcripts. Two newly produced viral proteins, Tat and Rev, are essential for efficient protein production: Tat by enhancing transcription and Rev by supporting export of unspliced mRNA from the nucleus, which allows for production of the structural proteins Gag and Env. The full-length viral RNA binds initially to Gag and is then packaged into new virus particles. Env is transported to the cell surface after it is cleaved into two subunits and, with the help of cellular chaperone proteins, folded into a trimer. Assembly of mature virions takes place at the plasma membrane. Hepatitis B virus (HBV) carries a circular partially dsDNA genome that encodes four structural and two nonstructural proteins through overlapping open reading frames (ORFs). Within the nucleus of an infected cell, the genome is converted into a full dsDNA, which serves as a template for the viral transcripts. The largest mRNA, which is longer than the viral genome, is called the RNA pregenome and is packaged into core particles within the cytoplasm. Within these particles, the pregenomic RNA is reverse transcribed into viral DNA genomes. Upon synthesis, the viral surface protein is transported to the cell membrane and complete assembly of the virion takes place during budding of the virus. Once replication is completed and full virions have been assembled, viruses need to leave the cells. This again can occur through several pathways. Some viruses, such as HIV-1 or influenza virus, assemble their newly synthesized viral surface proteins on the cell surface and then bud through this part of the cell membrane, picking up not only their own surface proteins but also membrane proteins belonging to

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the host cell. Budding eventually destroys the cell membrane and leads to cell death. Other viruses, especially those that are nonenveloped, instruct the infected cell to undergo apoptosis and virus released from dying cells is encapsidated into apoptotic bodies, which are taken up by neighboring cells, thus facilitating infection of new cells. Some viruses are released by exocytosis, a process that resembles reversed pinocytosis in which virus particles are encapsidated into small vesicles that enter the secretory pathways. This form of exit does not kill the cells and is used by so-called nonlytic viruses.

Viral Persistence Some viruses such as poxviruses or influenza viruses are lytic, which means they inevitably kill the cells they infect. Such viruses cause acute infections in immunocompetent hosts. Other viruses can replicate within a cell without causing its demise, or they can switch between a lytic and a nonlytic infection. These viruses can persist and cause chronic or latent infections, which pose unique challenges to the immune system. In chronic infections, some viruses, such as HIV-1 or hepatitis C virus (HCV), replicate constantly, dodging destruction by the immune system. Other viruses replicate and then persist by turning off synthesis of most of their viral proteins, causing so-called latent infections. Herpesviruses can switch to a latent phase from which they are periodically reactivated to undergo renewed lytic cycles of replication. Adenoviruses persist at low levels in activated T cells, presumably as episomes that remain transcriptionally active.40 Yet other viruses, such as HIV-1 or HPVs, integrate into the host cell genome and thus become an integral part of the cell. In general, DNA viruses with a nuclear replication cycle are able to persist, which may be favored due to the complete lack of DNA degrading enzymes within the nucleus. Rabies virus, a negative-sense ssRNA virus, does not cause chronic infections but kills within days after causing symptoms. Nevertheless, in some individuals years pass between viral transmission and onset of symptoms,41 and it is unknown where and how the virus persists during this long incubation time. Measles virus, another negative-sense ssRNA virus of the paramyxovirus family can cause subacute sclerosing panencephalitis in about 1 out of 100,000 infected individuals within 5 to 15 years after primary infection. Subacute sclerosing panencephalitis is most common in children who are infected early in life, and it has been speculated that the relative immaturity of their immune system allows for the development of a chronic central nervous system infection.42

INNATE ANTIVIRAL IMMUNE RESPONSES Both innate and adaptive immune responses are essential to wards off pathogens, and individuals with inherited or acquired immunodeficiencies rapidly succumb to virus infections. Even individuals with weakened immunity, such as the very young whose immune system is still immature, the elderly undergoing immunosenescence, or pregnant women whose immune system is transiently suppressed, show markedly increased susceptibility to many viruses.

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Immune responses to viruses can roughly be divided into four stages. At first, the immune system has to recognize the threat, then an immediate early response is mounted by cells of the innate immune system, which is followed a few days later by a response from the adaptive immune system. Once the virus is eliminated, the immune response contracts and adaptive immunity enters a stage of immunologic memory. Memory T and B cells, upon reexposure to the same pathogen, mount a response that is more potent and comes up faster than a primary response. In cases where virus persists, the acute phase of the immune response is followed by a chronic immune response.

Early Recognition of a Virus Viruses like other microbes carry so-called pathogenassociated molecular patterns (PAMPs) that are recognized by pattern recognition receptors (PRRs) expressed on many cell types including cells of the innate and adaptive immune system.43 This recognition system is not as specific as the antigen-recognition receptors of T and B cells, but rather it responds to motifs that are commonly found on pathogens but not within mammals or it identifies molecules present in the wrong compartment within the cell. PRRs can be subdivided into four main types (ie, toll-like receptors [TLRs], retinoic acid inducible gene [RIG]-I–like receptors, nucleotide oligomerization domain [NOD]-like receptors [NLRs], and the interferon-inducible p200 family member absent in melanoma 2). Ten TLRs (TLR1–10) have been defi ned in humans and 9 in mice.44 Some TLRs are widely expressed on many different cell types such as TLR1 or 4, while others are expressed mainly on cells of the immune system such as TLR5, antigen-presenting cells (TLR8), or subsets of antigen-presenting cells, such as TLR7 and TLR9, which are primarily expressed in plasmacytoid DCs. Expression patterns of TLRs in humans do not always mirror those in mice. Expression levels of most of the virus-sensing TLRs are upregulated by inflammatory cytokines mainly interferon (IFN)- γ. but for TLR3 that is modulated upon cell differentiation. Viruses are sensed by five TLRs. TLR4, which is best known for its response to lipopolysaccharide, also reacts to the fusion protein of RSV45 and to a surface glycoprotein of Ebola virus.46 TLR3 senses double-stranded RNA, TLR9 senses viral and bacterial CpG sequences, and TLR7 (in humans only) and TLR8 (in both humans and mice) reacts to ssRNA. It was initially debated if indeed TLRs directly recognized their PAMP or became instead activated by an intermediate host cell protein. More recent evidence has shown direct binding between TRLs and their ligands. TLR4, which can recognize viral surface proteins, is expressed on the cell membrane where an encounter with such an antigen is most likely. TLR3, 7/8, and 9 are within endosomes where many viruses uncoat, which in turn leads to exposure of their genomes. Viruses such as herpesviruses,47 West Nile virus,48 or influenza virus49 signal through TLR3; RSV50 and Ebola virus46 can signal through TLR4; influenza virus,51 HIV-1,52

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and herpesviruses53 can signal through TLR7 or 8; DNA viruses such as herpesviruses signal through TLR9.54 All TLRs carry an intracellular toll-IL-1 receptor– resistance (TIR) domain, which interacts with TIR domains on intracellular adaptor molecules. TLRs, with the exception of TLR3, signal through myeloid differentiation primary response gene (MyD)88, which in turn interacts with interleukin-1 receptor associated kinases (IRAKs)1, 2, and 4, leading to activation of tumor necrosis factor receptor–associated factor (TRAF)6 and upon additional steps to activation of NF-κ B, mitogen-activated protein (MAP) kinases, and Jun-terminal kinases (JNKs). TLR3 signals through TIR-domain–containing adapter-inducing interferon-β (TRIF), which binds to TANK-binding kinase 1 (TBK1), thus activating interferon regulatory factor (IRF)3. In addition, TRIF interacts with receptor-interacting protein 1, which can activate NF-κ B. TLR4, in addition to signaling through MyD88, can also bind the TRIF-related adaptor molecule, which recruits TRIF, allowing for signaling to IRF3. Activation of IRF3 results in production of type I IFN, whereas activation of NF-κ B induces production of a number of proinflammatory cytokines, such as interleukin (IL)-6, tumor necrosis factor (TNF)- α , and IL-12 (Fig. 39.1). Deficiencies in TLRs can change an individual’s susceptibility to viral infections. For example, TLR3 deficiency in humans is associated with increased susceptibility to HSV-1 infections,47 whereas mice that lack TLR3 are more resistant

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to West Nile virus.55 West Nile virus triggers TLR3, and the resulting cytokine response opens the blood–brain barrier, which allows the virus to establish an infection within the central nervous system; in absence of TLR3, the virus remains excluded from the brain. TLR signaling and the resulting production of cytokines can be extremely toxic. TLR signaling is therefore tightly regulated.56 Some negative regulators target specific TLRs while others affect common downstream adaptor molecules. Regulators that affect TLRs, which react to viruses, include IRAKM, an IRAK homolog that inhibits IRAK1 and blocks TLR4 and 9, suppressor of cytokine signaling 1, which also suppresses IRAK. Others include phosphoinositide 3-kinase (PI3K), a key regulator of T-cell differentiation, which inhibits JNK and NF-κ B functions, toll-interacting protein, which phosphorylates IRAK1, A20 which deubiquitylates TRAF6 and thus affects TLR3, 4, and 9, ST2L which sequesters MyD88, single immunoglobulin (Ig) IL-1R–related molecule, which binds to TRAF6 and IRAK and TRIAD3A, and E3 ubiquitin-protein ligase, which initiates degradation of TLRs (see Fig. 39.1). Some viruses neither carry PAMPs on their surface for recognition by membrane-bound TLRs nor enter cells through endosomes, and their genomes thus fail to become accessible for recognition by TLRs. Such viruses can be recognized by RIG-I–like receptors, which are located in the cytoplasm.57,58 RIG-I–like receptors are RNA helicases, which respond to ss or dsRNA. Three RIG-I–like receptors

FIG. 39.1. Toll-like Receptor Signaling Pathways.

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have been identified to data (ie, RIG-I, melanoma differentiation-associated gene 5 [Mda5], and LGP2). RIG-I and Mda5 carry N-terminal caspase activation and recruitment domain (CARD)-like regions, which are involved in downstream signaling and C-terminal RNA helicase domains, which bind RNA and can distinguish between viral and cellular RNA species. LPG2 lacks the CARD domain and is assumed to negatively regulate RNA virus-induced inflammatory responses by blocking binding of RNA to RIG-I. RIG-I recognizes a number of ssRNA viruses such as paraand orthomyxoviruses,59 rotavirus,60 fi loviruses,61 and flaviviruses,62 while Mda5 recognizes picornaviruses.63 RIG-I and Mda5 signaling involves binding of their C-terminal CARD domains to the CARD domain on IPS-1, which then through kinases receptor interacting protein 1 or Fasassociated protein with death domain activates NF-κ B; they also activate IRF3 and IRF7 through TRAF3/TBK1, which results in production of IFNs and other proinflammatory cytokines (Fig. 39.2). NLRs are a very large family of PRRs that respond to viral RNA in the cytoplasm.64,65 They contain N-terminal domains for protein-protein interactions such as CARD, pyrin or inhibitor of apoptosis domains, NOD domains for nucleotide binding, and C-terminal leucin-rich repeat domains. NLRs are subdivided according to their N-termini into CARD (CIITA, NOD1, 2, NLRC3-5), pyrin domain (PYD, NLRO1-14), or pyrin or inhibitor of apoptosis domain (NAIP) members. Most NLRs activate cytokine responses, although some are inhibitory and dampen innate immune responses. To give some examples of the specificity of NLRs,

NOD-2 senses ssRNA and interacts with paramyxoviruses and myxoviruses.66 NLRC5 interacts with Poly(I:C) and responds to some paramyxoviruses and herpesviruses.67,68 NLRX1, an inhibitory NLR, signals upon recognition of Sendai or Sindbis virus and blocks activating signals through RIG-1 like helicases.69 Signaling pathways have not yet been fully characterized for NLRs. NOD-1 and -2, the best known NLRs, bind receptor-interacting serine-threonine kinase 2 resulting in NF-κ B and MAP kinase signaling. They also induce autophagy and activate the mitochondrial antiviral signaling protein for induction of type I IFN. Several NLR members containing CARD or PYD domains can assemble into inflammasomes. Inflammasomes are multiprotein complexes that recruit and activate inflammatory caspases.70 Inflammasomes include PRRs and, as such, are divided into NLRP3 inflammasomes, RIG-I inflammasomes, and absent in melanoma 2 inflammasomes. In these complexes, the PRR activates caspase I through an adaptor, which cleaves the immature forms of IL-1β and IL-18 resulting in biologically active cytokines. NLRP3 inflammasomes were shown to react with influenza virus, rhabdoviruses, and picornaviruses.71,72 Viral RNA can directly activate the caspase activities. The M2 protein of influenza virus, which has ion channel activity, localizes to the trans-Golgi network and reduces the H + content; the acidity of the Golgi then results in NLRP3 activation.73 Absent in melanoma 2 inflammasomes recognize DNA within the cytoplasm and, as such, play a role in responses to DNA viruses (eg, poxviruses and herpesviruses).74

FIG. 39.2. Signaling through Retinoic AcidInducible Gene I Receptors.

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The Early Inflammatory Response: Cytokines and Chemokines Interaction of a PRR with a PAMP initiates a cellular response. Signaling through most of the PRRs results in activation of NF-κ B. In resting cells, NF-κ B is retained in the cytoplasm through binding to Iκ B. Upon activation of the Iκ B kinase (IΚΚ), Iκ B becomes phosphorylated, then ubiquinated and finally degraded. This in turn releases NF-κ B and allows for its entry into the nucleus where it initiates gene expression. NF-κ B thus does not require de novo synthesis, which accelerates its activity. A very large number of genes have NF-κ B binding sites including genes involved in antigen processing and presentation, lymphocyte effector functions and motility, and cell metabolism. Cytokine genes induced by NF-κ B include those for type I IFN, IL-1A and B, IL-2, IL-6, IL-9, IL-11, IL-15, TNF-α , colony stimulating factor (CSF)1 to 3, lymphotoxin B, and the chemokine genes IL-8, CCL2, 5, 11, 15, and CXCL5. In addition, several cytokine-inducing transcription factors, such as IRF1, 2, and 4, are produced in response to NF-κ B. IRF3 activated by TLR3, TLR4, or cytosolic PRR signaling is a transcription factor that promotes production of type I IFN and the chemokine RANTES. The MAP kinase activated AP-1 transcription factors also have binding sites specific for regulatory sequences of multiple genes. Most pertinent for early immune responses is probably induction of signal transducers and activators of transcription (Stat)1 and 3, which upon formation of homo- or heterodimers bind to the IFN-gamma activated sequences. Thousands of such motifs can be found in the human genome, and their products affect most cell functions. Most cells can produce cytokines and chemokines, although, during the initial phase of an immune response, many are synthesized by cells of the innate immune system. The early cytokines have a multitude of functions, which are in part antiviral and in part designed to promote further activation of immune responses. Type I IFNs, which can be produced at capacious amounts by plasmacytoid DCs, bind to the IFN-α receptor. This causes activation of tyrosine kinase (Tyk)2 and Janus kinase (Jak)1, which in turn causes tyrosine phosphorylation and then nuclear translocation of Stat1 and Stat2 proteins. In addition, type I IFN has strong antiviral activity75 and has been licensed for treatment of chronic infections with HBV76 and HCV77 and for treatment of HPV-associated genital warts (Condyloma acuminata).78 IFN-1s downregulate viral promoters. They dampen expression of viral receptors and thus reduce viral entry. They induce expression of the dsRNA-activated protein kinase PKR, which phosphorylates translation initiation factor 2α (eIF2α) causing inhibition of translation of both viral and cellular transcripts. IFN-1s also trigger expression of 2′5′-oligoadenylsynthases that upon binding dsRNA generate AMP-oligomers, which activate RNAseL to cleave both cellular and viral RNAs. IFN-1s induce dsRNA kinase, which inhibits production of viral progeny. IFN-1s lead to synthesis of MxA.79 This protein binds to the nuclear membrane and inhibits trafficking of viral nuclear capsids. In addition, MxA can bind and inhibit the RNA polymerase of influenza virus. APO-Bec3G and F are IFN-induced

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deoxycytidine deaminases that interfere with the replication of retroviruses.80 In addition to their antiviral activity, IFNs and other early cytokines play a dominant role in initiating both innate and adaptive immunity by causing activation of macrophages, natural killer cells, and DCs. The early cytokines promote proliferation and differentiation of lymphocytes, granulocytes, and antigen-presenting cells, whereas chemokines recruit such cells. IL-6 should be noted, as this cytokine, due to its immunostimulatory effects on B cells, has been implicated in the pathobiology of EBV-associated lymphoproliferative disorders and HHV-8–associated lymphomas in patients with acquired immunodeficiency syndrome.81,82 The apparent redundancy of some of the molecules that contribute to the initial inflammatory response is remarkable. Viruses commonly trigger multiple PRRs, some of which are expressed on or in the same cells, while other are carried by different subsets of cells. For example, influenza viruses thus far are known to signal through TLR3, 7/8, NOD-2, RIG-1, and NLPR3 inflammasomes. Most of the PRRs in the end initiate transcription through NF-κ B or through members of the IRF family. Nevertheless, the immune response that is very much guided by the initial inflammatory reaction is unique for each virus, suggesting a finely orchestrated series of events during which the type, strength, and location of PRR signaling elicits for each virus a special mixture of cytokines and chemokines that results in signature immune responses. This is exemplified by recent studies with a vaccine, in which antigen-containing nanoparticles were mixed with ligands for TLR4 and 7.83 This vaccine did not induce a strong effector cell response but drove differentiation of T cells toward memory and B cells toward long-lived plasma or memory cells. Although the slow release of antigens from the nanoparticles may have contributed to this, both TLR ligands were needed to maximize the vaccine’s immunogenicty.

Cells of the Innate Immune System Cells of the innate immune system, which include DCs, neutrophils, eosinophils, basophils, mast cells, γ/δ T cells, macrophages, and natural killer cells, provide a first layer of defense against virus infections and promote activation of adaptive immunity. Unlike T and B cells, which carry antigen-specific receptors, cells of the innate immune system are, as described previously, activated by PRRs. They act rapidly without undergoing the massive proliferation of T and B cells and then, in general, with the potential exception of natural killer cells,84 fail to establish long-lasting memory.

Granulocytes: Neutrophils, Basophils, Eosinophils, and Mast Cells Granulocytes play major roles in controlling bacterial and parasitic infections but also influence viral infections. Neutrophils, which are abundant and contribute to more than 50% of circulating leukocytes, release hydrogen peroxide, free oxygen radicals, and hypochlorite, and have been described to limit replication of HSV-1.85 They may also

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contribute to the immunopathology, such as of pulmonary RSV infections.86 Basophils release histamine, which increases blood vessel permeability and thus allows for lymphocyte trafficking into inflamed tissues. Basophils are recruited and activated by a number of viruses, such as influenza viruses or RSV.87 Eosinophils, which accumulate in lungs during RSV infection,86 release enzymes, such as ribonuclease, deoxyribonucleases, lipase, as well as plasminogen and peroxidase. Mast cells release heparin, histamine, and chemokines, and have been reported to respond to Dengue virus infections.88

Gamma/delta T cells

T cells that carry the γ/δ T-cell receptor (TCR) exhibit characteristics of both innate and adaptive immune cells. They are mainly located in the gut and skin but can also be found at low frequencies in blood and lymphatic tissues. Although they are educated in the thymus, their receptor recognizes pathogen patterns or cellular stress molecules independent of major histocompatibility complex (MHC) molecules, and they can expand without the intricate antigen presentation pathways that dictate differentiation of α/β T cells. γ/δ T cells, which evolutionary predate α/β T cells, can secrete cytokines such as IFN-γ, IL-4, or IL-17,89 chemokines such as macrophage inflammatory protein (MIP)-1α ; MIP-1β ; regulated upon activation, normal T cell expressed and secreted; lymphotoxin (LT)90 ; and lytic enzymes (ie, perforin and serine esterases for target cell lysis). These T cells play diverse roles in virus infections. To name a few, they promote Th1 responses following infection with coxsackievirus B391; they can kill cells infected with HSV-192 ; they have been described to lyse cells infected with influenza virus93 ; and they provide resistance to humans against HIV-1 infections94 and to mice against infections with West Nile virus95 or vaccinia virus.96

Macrophages Monocytes upon activation by proinflammatory cytokines, specifically IL-6 and macrophage-CSF (M-CSF), differentiate into macrophages. These cells, as their name “large eaters” implies, are phagocytic. Phagocytosis is a specialized form of endocytosis during which the engulfed particles are transported to lysosomes where they are degraded by a toxic and acidic soup composed of oxygen radicals, nitric oxide, proteases, and defensins. Macrophages, in addition, release cytokines, such as IL-1, 6, 10, and 12, type I IFN, and TNF-α , as well as chemokines, such as MIP-2, IL-8, and cytokine-induced neutrophil chemoattractant–1. Some viruses replicate in macrophages (eg, HIV-1, influenza viruses, rhinoviruses, and Ebola viruses).

Dendritic Cells A crucial role of DCs is their ability to present antigen to naïve T cells. A few years ago, DCs were divided into myeloid DCs, plasmacytoid DCs, and lymphoid DCs, although it is now accepted that lymphoid DCs do not belong to a separate lineage. Viral antigens are mainly presented by myeloid DCs. Myeloid DCs, which differentiate from Lin-CD115 +Flt3 + CD117lo precursors, can further be divided into subsets according to phenotypic markers, functions, and anatomic site of origin.97

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Three subsets of myeloid DCs have been identified in human skin. Langerhans cells, which are CD1hiCD14 − CD207+, are located in the epidermis. They look and act like DCs, and they are originally derived from bone marrow precursors but unlike other DC subsets, numbers of Langerhans cells are maintained by local proliferation. They only express discreet amounts of TLRs, induce CD8 + T cell responses in vitro and trigger proliferation of naïve CD4 + T cells. Interestingly, CD4 + T cells induced by Langerhans cells do not produce the typical Th1, Th2, or Th17 cytokines but rather produce IL-22,98 which causes skin inflammation as well as wound healing through induction of keratinocyte proliferation. The dermal layer of skin contains CD14 + CD11bhi DCs, which differentiate from CD34 + precursors and express TLR 1, 2, 4, 5, 6, 8, and 10. They produce a multitude of proinflammatory cytokines including IL-6 and IL-12, which allows them to induce differentiation of naïve B cells into plasma cells. They also prime CD4 + T cells that can promote B-cell differentiation. The other DC subset in the dermis is CD103 + CD11blow, which can cross-present antigen and drive proliferation and differentiation of CD4 + T cells into Th1 cells. Mouse spleen contains two subsets: CD8 + CD205 + DCs and CD8 +33D1+ CD11b + DCs; whereas the former can cross-present antigen in association with MHC class I and thus drive activation of CD8 + T cells, the latter is more efficient at processing antigen for MHC class II presentation for CD4 + T cell activation. Lymph nodes also contain these two subsets and in addition migratory DCs such as Langerhans cells, which transport antigen from other locations to lymph nodes. Lymphatic tissues in addition contain follicular DCs.99 These cells are not derived from hematopoietic progenitors but are of mesenchymal origin. They are an integral part of B-cell follicles, where they present antigen to naïve B cells and maintain long-lived plasma cell responses by initially capturing and then slowly release antigen-antibody complexes. The intestine has three populations of DCs. CD103 + CD1blow DCs can be found in Peyer patches and CD103 + CD11bhi and CD103 − CD11bhi DCs in the lamina propria. The latter subset is known to transport antigen from the intestine to draining lymph nodes. Other tissues such as lung, kidney, and spleen contain similar subsets. It was initially thought the DCs are terminally differentiated, nondividing cells. Recent studies have shown that DCs can divide and that this division is driven by fms-related tyrosine kinase (Flt)-3, which is also a key factor for DCs differentiation from bone marrow precursors.97 Plasmacytoid DCs were originally though to originate from lymphoid progenitors as they share many of the characteristics of lymphocytes: they lack the typical dendrites of other DC subsets, but look like lymphocytes; they express B-cell markers, such as B220 and T cell markers (eg, CD4), as well as transcription factors involved in lymphocyte development, such as terminal deoxynucleotidyltransferase, recombination activating genes 1 and 2, Ig supergene family members, and the pre–T-cell antigen receptor alpha chain. But plasmacytoid DCs also have characteristics of myeloid

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DCs and can even differentiate into myeloid DCs.100 It is assumed that plasmacytoid DCs can arise from both lymphoid and myeloid bone marrow precursors, although the latter probably provide the dominant source. Plasmacytoid DCs, which can be distinguished from myeloid DCs by expression of plasmacytoid DC antigen-1 (CD317) in mouse or blood DC antigen-2 (CD303) or -4 (CD304) in human, circulate in blood and are present in lymphatic tissues. Unlike myeloid DCs, they express CD62L and CCR7, which allows them to cross high-endothelial venules to enter T-cell–rich areas in lymphatic tissues. While myeloid DCs take up antigen to present it to cells of the adaptive immune system, plasmacytoid DCs do not phagocytose antigen. Their primary role might be regulatory by producing cytokines most notably type I IFNs, which assist maturation of myeloid DCs. DCs present in tissues are immature. They take up antigen, but they do not produce cytokines, nor are they able to present antigen efficiently to naïve lymphocytes. Their maturation starts either through interactions of a PRR with a PAMP, through cytokines, ligation of CD40, receptor activator of NF-κ B (RANK) or even, as has been observed in vitro, vigorous shaking.101 DC maturation is associated with marked changes in their gene expression profi les102 and their biological functions. Maturing DCs stop phagocytosis and endocytosis. They start secreting chemokines and thus initiate an inflammatory response at the site of infection. They upregulate expression of CCR7 and migrate from tissue to the T-cell–rich zones of draining lymph nodes. They increase synthesis of molecules that are involved in antigen

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processing and presentation, and translocate MHC class II molecules that are preformed and stored within intracellular vesicles to the cell surface. They also, in a presumably orchestrated but yet ill-defined pattern, start to increase expression of cell surface molecules that regulate immune responses (Fig. 39.3). Such molecules include CD80, CD86, CD40, inducible T-cell costimulator (ICOS) ligand, ligands 1 and 2 for programmed death 1 (PDL1, PDL2), HVEM, and others that are less well defined thus far. Interactions of T and B cells not just with their cognate antigen, but also with these ligands, is crucial for the induction of an adaptive immune response that is of sufficient magnitude to combat the threat without being overwhelming and thus causing undue damage. These interactions are also important to determine the ultimate differentiation fate of responding T and B cells. Signaling through CD40, which is expressed at low levels on immature DCs, facilitates full maturation of DCs; its expression increases upon activation of TLR pathways. CD40 is upregulated on myeloid DCs upon interactions with TLR4 agonists103 and on both plasmacytoid and myeloid DCs upon TLR7 and 9 signaling.104 The CD40 ligand (CD40L) is induced upon CD40 stimulation.105 Ligation of CD40 causes signaling through TRAF6, which results in activation of MAP and Jun kinases and in production of CD86 and cytokines, such as IL-6 and IL-12. CD40 ligation also results in activation of NF-κ B and in many aspects seems to complement signaling through TLRs.106 Ligation of CD40L upregulates expression of RANK (also called TNF-related activation-induced cytokine [TRANCE]

FIG. 39.3. Costimulatory (blue lines) and coinhibitory (red lines) T cell (green)–dendritic cell (yellow) interactions.

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receptor), which is a member of the TNF-receptor family. RANK interacts with RANK ligand (also called TRANCE), which is induced on T cells upon their TCR ligation. Signaling through TRANCE induces B-cell lymphoma-extra large expression, which in turn promotes DC survival.107 Signaling between DCs and cells of the adaptive immune system involves a number of B7 family members of costimulatory molecules that are expressed by mature DCs.108 CD80 and CD86 on DCs interact with the activating CD28 and inhibitory cytotoxic T-lymphocyte antigen (CTLA)-4 molecules on T cells. Ligation of CD28 activates Akt/PI3K and thus mammalian target of rapamycin (mTOR) while CTLA-4 inhibits this pathway. Activation of these pathways is needed to adjust the increased bioenergetic needs of differentiating lymphocytes by augmenting glucose uptake and glycolysis through the Krebs cycle and inhibiting forkhead box O, thus allowing for cell cycle entry. Another means by which CD80 and CD86 may influence immune responses is through reverse signaling into B7-expressing DCs.109 The ICOS ligand pathway has some overlapping functions with CD28. Ligation of ICOS on T and B cells upregulates PI3K and thereby influences cellular metabolism.110 ICOS through PI3k/Akt and Rho family members also affects lymphocyte polarization and migration.111 ICOS also plays a role in controlling T-helper-cell development and function.112 ICOS also plays a critical role in the development of both Th17 cells and follicular T-helper cells by inducing the transcription factor c-Maf and the cytokine IL-21.113,114 Patients with a defect in ICOS expression exhibit a profound defect in B-cell maturation and Ig isotype switching.115 PDL1 and PDL2 are coinhibitors expressed on DCs and some other cells that interact with PD1 expressed on T cells, B cells, macrophages, and some types of DCs. PD1 ligation induces cell cycle arrest.116 PDL1 regulates development, maintenance, and function of regulatory T cells (Tregs) through downregulation of Akt and mTOR, and upregulation of phosphatase and tensin homolog.117 PD1 expressed

on CD4 + T cells regulates selection and survival of PDL2 + B cells in germinal centers, and affects the magnitude and the quality of the plasma cell response.118 Another inhibitory pathway involves the BTLA13 expressed by B and T cells. BTLA provides inhibitory signals upon binding to HVEM expressed on DCs. HVEM also interacts with glycoprotein gD of herpes virus and with the immunoinhibitory receptor CD160.13,119 Distinct regions of HVEM interact with two immunoactivators of the TNF family members (ie, LIGHT and lymphotoxin-A) (Fig. 39.4). BTLA, unlike other immunoinhibitory molecules, is expressed on naïve T cells, whereas CD160 is induced upon their activation. Blockade of the HVEM-BTLA/CD160 pathways during T-cell stimulation results in enhanced primary T-cell responses both in young and aged mice.120–122 Signaling pathways initiated by BTLA ligation remain poorly understood. It is known thus far that the cytoplasmic tail of BTLA binds growth factor receptor-bound protein (Grb)-2, which in turn recruits p85 of PI3K.123 HVEM is also expressed on Tregs, which through ligation of BTLA suppress effector T cells.124 Another inhibitory molecule on DCs is CD48, which interacts with 2B4 on T and natural killer cells.125 A number of additional inhibitory molecules have been identified on T and B cells and include LAG3, PIR-B, GP49, KLRG1, NKG2A, and NKG2D,125–128 but their matching ligands remain elusive. Through the expression of costimulatory and coinhibitory receptors, DCs not only activate but also terminate adaptive immune responses. DCs thus play a dominant role in maintaining tolerance through a number of pathways. Thymic DCs negatively select for T cells with receptors that have high reactivity to self-proteins, and they promote thymic Treg selection. Presentation of antigen by immature DCs induces T-cell anergy and promotes the activity of Treg. Immature as well as mature CD123 + DCs can express indoleamine dioxygenase (IDO),129 which catalyzes the degradation of the L-tryptophan, an amino acid that is

FIG. 39.4. Stimulatory (+) and Inhibitory (−) Signals through Herpes Virus Mediator Interactions.

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essential for T cells, into N-formylkynurenine. Production of IDO is initiated by CD80/CTLA-4 interactions, TGF-β, or IL-10. IDO inhibits generation of effector T cells but instead promotes activation of Tregs. DCs influence the homing behavior of T cells. DCs within the intestine activate T cells that preferentially home to gut-associated lymphoid tissues. Such homing is mediated by expression of CCR9 and α4β7 on T cells. Skin or lamina propria–derived DCs, unlike DCs in the spleen or in central lymph nodes, express the integrin chain of CD103, which induces T cells with gut or skin homing preference.130

Natural Killer Cells Although it was initially assumed that natural killer cells originate from bone-marrow precursors, it is now understood that most of them develop in lymph nodes and tonsils. Natural killer cells express TLR2, 3, 4, and 9, but not 7 or 8, and can be activated by PAMPs. They can also be activated by proinflammatory cytokines and chemokines such as IFNs, IL-12, IL-15, IL-18, IL-2, and CCL5. Numerous activating and inhibiting receptors, some of which are constitutively expressed, while others are induced, regulate effector functions of natural killer cells. Natural killer cell receptors belong to the family of killer cell Iglike receptors in humans or C-type lectin receptors, such as CD94/NKGD2 and Ly49, in mice.127–129 CD244, also known as 2B4, can also serve as a natural killer receptor. Receptors

TABLE

39.4

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encoded by the same gene families can be activating or inhibitory (Table 39.4). All of the inhibitory natural killer cell receptors carry an immunoreceptor tyrosine-based inhibitory motif in the cytoplasm, which upon phosphorylation recruits the lipid phosphatase SHIP-1 or the tyrosine phosphatases SHP-1 or SHP-2. This in turn results in dephosphorylation of proteins bound to activating natural killer cell receptors. Activating receptors thus largely function through lack of signaling through inhibitory receptors. Activating receptors belong to the same families. The cytoplasmic tails of activating receptors are shorter than those of inhibitory receptors, and they do not carry an immunoreceptor tyrosine-based inhibitory motif. Signaling through combinations of activating receptors is needed to elicit natural killer cell functions. The inhibitory natural killer cell receptors respond to a variety of ligands, including classical MHC class I molecules, nonclassical MHC molecules (Qa-1 in mouse and HLA-E in human), adhesion molecules, cadherins, lectins, CD markers (CD44, 99, and 66), and sugars such as a-2,8-linked disialic acid. Activating receptors respond to some of the same ligands as inhibitory receptors, such as classical and nonclassical MHC determinants. Some also respond to Igs, CD112, CD155, themselves (ie, NTB-A and CD319 encoded by Slam6/7),130 or C-type lectins, such as AICL. They also recognize viral proteins or stress proteins released by cells in response to an infection. Ly49H, an activating receptor encoded by Kira8,

Natural Killer Cell Receptors

Gene

Protein

Superfamily

Ligand

Function

KIR2DL1 KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B KIR3DL1, KIR3DL3 LILRB1 LILRB1 PILRalpha KLRD1/KLRC KLRG1 KIR2DS1, KIR2DS2 KIR2DS4 KIR3DS1 PILRbeta NCR1 NCR2 NCR3 KLRK1 KLRC4 SLAM6 SLAM7 SLAMF4

CD158a CD158b1 CD158b2 CD158d

Ig superfamily

HLA-A, -Bw, Cw, G

Inhibitory

Ig superfamily

HLA-G

Inhibitory

Ig superfamily C-type lection C-type lectin Ig superfamily

Herpes virus, CD99-like protein HLA-E MIC and MHC class I–like proteins HLA-A, -Bw, Cw, G

Inhibitory Inhibitory Activating

Ig superfamily Ig superfamily

CD99-like protein Pathogens

Activating Activating

C-type lectin

MIC and MHC class I–like proteins

Activating

CD2-like molecule

NTBA CD319 CD48

Activating

CD158e1 CD158z LIR1 LIR2 CD94/NKG2 Mafa CD158e2 CD158j CD158i CD158e2 NKp46, CD335 NKp44, CD336 NKp40, CD337 NKG2D NKG2F Ly108, NTBA CD319 CD244

CD, cluster of differentiation; HLA, human leukocyte antigen; Ig, immunoglobulin; MHC, major histocompatibility complex; MIC, monolayer immune complex; NTBA, NK-T-B antigen; SLAM, signaling lymphocytic activation molecule.

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recognizes the m157 gene product of mouse CMV, while NKp46 recognizes influenza virus HA in humans.131 NKG2D recognizes the stress-induced MHC class I–related MICA and MICB proteins released in response to CMV infection.132 Some activating receptors contain immunoreceptor tyrosine-based activation motifs, which through Syk result in activation of the Ras/Raf/Erk and PI3K/Akt pathways leading to cytokine production and degranulation.133 Others, such as NKG2D, signal through DAP10, a PI3K adaptor protein that contains a small Tyr-Ile-Asn-Met (YINM) motif, which binds either PI3K or Grb2.134 Ligation of both is needed for cytolysis of NKG2D targets. Interestingly, IL-15 also promotes NKG2D-dependent lysis by causing Jak3mediated phosphorylation of DAP10.135 Signaling through CD244, a signaling lymphocyte activation molecule (SLAM) family member, which depending on circumstances can be activating or inhibitory, involves an immunoreceptor tyrosine-based switch motif, which upon recruitment of SLAM-associated protein causes activation through PI3K, while recruitment of EAT2 or ERT may cause inhibition.136 Natural killer cells contribute to early viral control through direct lysis of virus-infected cells and through the production of cytokines such as IFN-γ, IL-10, IL-18, TNF- α , MIP-1α , and MIP-β. Interestingly, activated natural killer cells can not only lyse virus-infected cells, but they can also kill immature DCs.137 It is assumes that this type of natural killer cell–mediated DC editing prevents presentation of viral antigens by not yet fully matured and hence tolerogenic DCs to T cell. Natural killer cells are clearly crucial for the early defense again viral infections and their lack has been shown to result in enhanced susceptibility to infections with a number of different viruses, such as Sendai virus, influenza virus, or CMV, a finding that was confirmed in human patients with natural killer cell deficiency.138,139 Recent publications have shown that natural killer cells upon stimulation with antigens such a murine CMV undergo expansion, persist for months, and mount a recall response upon reexposure with the same antigen, suggesting that they cells can differentiate into memory cells, a function that thus far had been reserved for cells of the adaptive immune system.140,141

Natural Killer T Cells Natural killer T (NKT) cells are a subset of natural killer cells that share characteristics of T and natural killer cells. NKT cells referred to as type 1 or iNKT cells express an invariant TCR that recognizes glycolipids presented by CD1d.142 Other NKT cells, referred to as type 2 NKT cells express a more variant TCR that does not recognize CD1d-associated ligands. NKT cells express T-cell lineage markers and, for example, in humans, iNKT cells can be CD4 +, CD8αβ +, or CD8αα +, but they also express natural killer cell receptors. NKT cells release cytokines, mainly IL-4 and IFN-γ, that affect the differentiation fate of CD4 + T cells, and they can be lytic through secretion of perforin and granzyme or through the Fas/FasL pathway. Upon activation, NKT cells upregulate CD40L and can thus contribute to DC maturation through CD40. In turn, IL-12 secreted by DCs can drive activation of NKT cells. NKT cells are involved in resistance

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to a number of viruses including HSV-1 and -2, CMV, HIV1, HBV, HCV, lymphocytic choriomeningitis virus (LCMV), and encephalomyocarditis virus,142 but they may also contribute to virus-associated immunopathology, such as upon infections with RSV.143

ANTIGEN-SPECIFIC IMMUNE RESPONSES The innate immune system, which can cause extensive symptoms or even death through massive release of cytokines, is our main defense system during the early phase of a viral infection until cells of the adaptive immune system (ie, T and B cells) differentiate into effector cells. This takes time and in a primary infection requires at least 4 to 5 days. Antigen-driven activation of T and B cells depends on signals from the innate immune system and is very much shaped in its flavor by characteristics of the initial inflammatory reaction and the antigen-presenting cells. Although cells of the adaptive immune system can express PRRs, their activation is driven by recognition of antigens with exquisitely specific, clonally expressed receptors, which in the case of B cells have a virtually unlimited repertoire. Prior to an infection, cells with a specific receptor for a given pathogen are only present at very low numbers. Therefore, upon activation, B and T cells first proliferate extensively with an approximate doubling time of 4 to 6 hours to accumulate to numbers suited to eliminate viruses that, if unchecked, can replicate by far more efficiently than lymphocytes.

T Cells T cells based on surface markers are divided into CD4 + T cells, which have primarily regulatory functions, and CD8 + T cells, which have mainly effector functions. T cells express an antigen-specific receptor that unlike B-cell receptors (BCRs) does not recognize soluble antigen, but rather peptides derived from degraded viral proteins that associate with MHC molecules. This ensures that T cells only respond to cell-associated antigens, which makes them uniquely suited to respond to intracellular parasites such as viruses.

Cluster of Differentiation 4+ T Cells

CD4 + T cells can be divided into Th cells, which promote activation of immune responses, and Treg cells, which have inhibitory functions. Cluster of Differentation 4 + T-Helper Cells: Subsets. CD4 + Th cells are divided into Th1, Th2, Th17, and follicular Th (Tfh) cells144–146 (Fig. 39.5). Th cells originate from a common precursor in the periphery, the naïve CD4 T cell. The inflammatory cytokine milieu during antigen-driven activation dictates its differentiation into any of these subsets. Th1 cells are induced when antigen activation occurs in presence of IL-12 or IL-18. Naive cells differentiate into Th2 cells in a milieu that contains IL-4. Th17 cells were discovered more recently. Naive cells develop into Th17 cells in presence of transforming growth factor (TGF)-β and IL-6. IL-1β and IL-23 enhance/stabilize their differentiation. Once activated, Th subsets block differentiation of uncommitted naïve cells into other subsets. Th1 cells

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FIG. 39.5. Cluster of differenation 4+ T-helper cells subsets: cytokines driving their induction. Lineage defining transcription factors and effector functions.

prevent Th2 differentiation through IL-12, Th2 cells block Th1 development through IL-4, and Th1 derived IFN-γ and Th2 derived IL-4 both inhibit Th17 formation. Tfh cells are only found in B-cell follicles in spleens, lymph nodes, and Peyer patches, where they are instrumental in germinal center formation by secretion of IL-21 and IL-4. Th cell subsets are characterized by lineage defining transcription factors.147 Th1 cells express T-bet, a T-box transcription factor that controls expression of IFN-γ. Th2 cells express trans-acting T-cell–specific transcription factor (GATA3), which promotes production of IL-4, IL-5, and IL-13. The transcription factors Stat3, the orphan nuclear receptors (ROR) gamma-t, and RORalpha are involved in Th17 differentiation. B-cell lymphoma (Bcl)-6 is required for differentiation of Tfh cells, which are distinguished from other subsets by the expression of CXCR5 that enforces their homing to lymph node follicles. Other Th cell subsets have been suggested, such as Th3 and Th9 cells, but as unique transcription factor signatures have not yet been defi ned for these proposed subsets, it remains unclear if they indeed represent distinct lineages. It should also be noted that there is a certain degree of plasticity between defined Th subsets; Th1 cells can differentiate into Th2 cells and vice versa, Th17 cells can differentiate into Th1 or Th2 cells and Th1, and Th2 and Th17 cells can turn into Tregs. Most crucial for control of viral infections are Th1 cells, which are induced by antigen-presenting DCs that upon virus-driven maturation typically produce IL-12. Some viruses produce IL-10 homologs148 or induce cellular IL-10

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mimetics,149 which support the development of Th2 cells, while suppressing Th1 cell differentiation. Induction of Th17 cells together with Th1 cells was reported in severe influenza A virus infections.150 Th17 cells are also generated during viral infections and have been reported to promote coxsackie virus–associated myocarditis151 and persistent infections with Theiler murine encephalomyelitis virus.152 They have been implicated in promoting liver injury in chronic HBV infections in humans.153 Tfh cells are presumably crucial for effective antiviral antibody responses, but, thus far rather little is known about their fate during viral infections. It is known that exceptionally strong TCR signaling through high-avidity interaction with MHC class II peptide complexes supports differentiation of naive cells into Thf cells.154 Cluster of Differentiation 4 + T-Helper Cells: Antigen Processing and Presentation. CD4 + T cells recognize MHC class II–bound peptides, which originate from pathogens that are broken down by proteolysis in the endosomes.155 Expression of MHC class II molecules is restricted to macrophages, B cells, and DCs in mice, and in humans is also expressed on CD4 + T cells. Expression of MHC class II on the cell surface increases upon cell activation, in part through translocation of preformed molecules from intracellular compartments to the cell surface, and in part through increased synthesis. MHC class II molecules are heterodimers composed of α and β chains. A number of chaperone proteins ensure transport of MHC class II molecules

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to the endosomes. First upon their synthesis, the two MHC class II chains assemble in the endoplasmic reticulum (ER) into a nonameric complex composed of three chains of the α and β molecules and three invariant molecules. The invariant molecules contain a peptide sequence, termed class II linked invariant peptide, that promiscuously binds to the groove of MHC class II α/β chain dimers, thus preventing binding of other peptides. It also contains an ectodomain, which mediates trimerization and interferes with DM/ MHC class II binding. The MHC class II-invariant chain complexes are translocated to endosomes/lysosomes, where the invariant chain is degraded by cathepsin S and dissociates from MHC class II molecules, which then bind the DM accessory protein. DM binds close to the peptide groove inducing conformational changes that allow for loading of antigenic polypeptides. Polypeptides that fit poorly are forced to disconnect again through DM-mediated conformational changes, while peptides that fit well are insensitive to DM-mediated dissociation. Long peptides that bind the MHC class II groove are clipped at this stage; the portions of the polypeptide that overhangs the groove are cleaved off by peptidases. The MHC class II-peptide complexes are then transported to the cell surface, where they can be recognized by the TCR of CD4 + T cells. Cluster of Differentiation 4 + T-Helper Cells: Activation. Naïve CD4 + T cells circulate between blood and secondary lymphoid tissues. Their activation requires presentation of antigen by MHC class II molecules carried by professional antigen-presenting cells, such as mature DCs. Once a CD4 + T cell recognizes its specific antigen, the DC-CD4 + T cell interaction is stabilized through integrins such as ICAM-1 or DC-SIGN on DCs, which bind lymphocyte function-associated antigen (LFA)-1 or ICAM-3 on CD4 + T cells, respectively.156 Full activation of CD4 + T cells requires then a second signal in form of interactions with costimulatory molecules.157 In the initial activation of CD4 + T cells, CD80 and CD86, which are both expressed on mature DCs and signal through CD28, provide costimulation. Although costimulation through CD28 is dominant, other molecules, such as CD2, Ox40, ICOS, CD40, or SLAM (CD150) can provide or complement this function. CD2 upon interaction with LFA-3 induces Jun kinases, which in turn results in IL-2 production.158 Ligation of Ox40, which is expressed 2 to 3 days after T-cell activation, with Ox40L recruits TRAF2 and causes activation of NF-κ B, PI3K, and Erk pathways and augments CD4 + T-cell functions, such as cytokine production, proliferation, and survival at a later stage than CD28-mediated activation and may involve cells other than antigen-presenting DCs.159 Ox40 has also been implicated to be essential for Tfh development.160 ICOS has overlapping functions with CD28 and signals through PI3K.161 It has been reported that myeloid DCs mainly induce CD4 + T cell activation through CD28, while plasmacytoid DCs, which express high levels of ICOS-L, may costimulate through ICOS. CD40L is induced upon CD4 + T-cell activation and upon interaction with CD40 on DCs or B cells promotes CD4 + T-cell proliferation. SLAM, which is also induced upon CD4 + T-cell activation, results in a SLAM-associated

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protein/proto-oncogene tyrosine-protein kinase (Fyn) signaling cascade, which in turn enhances CD4 + T-cell proliferation and cytokine production.162 Upon activation, CD4 + T cells start to produce IL-2. They also increase cell surface expression of CD25, the receptor for IL-2, which promotes their own proliferation. CD4 + T cells do not expand as extensively as CD8 + T cells but they do undergo approximately 8 to 10 cycles following activation. Cluster of Differentiation 4 + T-Helper Cells: Effector Functions. CD4 + effector cells provide helper functions mainly through the secretion of cytokines and chemokines.163 All Th cells can produce IL-2, which promotes their own proliferation as well as proliferation of CD8 + T cells and Tregs. The Th1-derived cytokines TNF-α and IFN-γ activate natural killer cells, macrophages, and B cells. They are also essential for activation of CD8 + T cells, although some viruses, such as LCMV, sendai virus, influenza virus, or ectromelia virus (a mouse poxvirus) can induce CD8 + effector T cells without CD4 + T help. Nevertheless, T helper– independent CD8 + T cells are fundamentally defective and fail to differentiate efficiently into memory CD8 + T cells.164 Th cells can lyse virus-infected target cells that express MHC class II molecules, such as EBV-infected B cells165 or HSV-1– infected macrophages.166 Th2 cell secrete cytokines, such as IL-3, IL-4, IL-5, and IL-10, that drive B-cell proliferation and differentiation and activate mast cells and eosinophils. Th17 cells produce IL-17, IL-21, and IL-22 and induce strong inflammatory responses mainly through IL-17–mediated recruitment and activation of neutrophils. Tfh cells, upon initial stimulation through the TCR and CD28 followed by interactions with Ox40, upregulate CXCR5 and home to B-cell follicles, where they assist in B-cell proliferation and differentiation through CD40L interactions and production of cytokines, such as CD21 and IL-4, which promote germinal center formation, class switching, and plasma cell differentiation. Cluster of Differentiation 4 + T-Helper Cells: Memory Differentiation. Upon removal of antigen, effector CD4 + Th-cell numbers contract over a period of weeks and the remaining antigen-experienced CD4 + T cells differentiate intro long-lived memory cells, which preferentially home to the bone marrow. The overall size of the CD4 + T-cell memory pool depends on signals present during activation such as antigenic load, duration of antigen presentation, and presence and type of costimulators. Transition of CD4 + effector T cells into long-lived CD4 + memory Th cells depends on HVEM-LIGHT interactions.167 Such interactions do not require classical antigen-presenting cells but can happen between adjacent T cells, which express both HVEM and LIGHT. Memory CD4 + T cells, upon reencounter of their antigen, differentiate into effector Th cells and they assume effector functions more rapidly than naïve T cells, in part through storage of RNA for key cytokines and chemokines, which are available for immediate translation once the TCR has been engaged. Although this reactivation is less dependent on costimulation compared to activation of naive CD4 +

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T cells, it is nevertheless optimized by interactions between CD28–CD80/86, OX40–OX40L, or ICOS-ICOSL.168 As a rule, the Th cell’s fate decision during primary activation remains unchallenged during recall responses (ie, memory Th1 cells will become effector Th1 cells, memory Th2 cells will become effector Th2 cells, etc.). Regulatory Cluster of Differentiation 4 + T Cells. Two subsets of CD4 + T cells have immunoinhibitory functions169 ; one subset is constitutively present and is termed natural Tregs (nTregs) while the other is induced by antigen (iTregs). CD4 + Treg subsets are characterized by expression of the lineage defining transcription factor forkhead box P (FoxP)3 in mice, although in humans other T-cell subsets can transiently become positive for FoxP3. Tregs constitutively carry surface markers, such as CD25, CTLA-4, GITR, and Lag3, that are also transiently expressed by recently activated CD4 + Th cells. Tregs suppress immune responses and are essential to maintain tolerance and limit immunopathology following infections. Lack of Tregs, such as in scurfy mice, which have a mutation in the foxP3 gene, rapidly succumb to a fatal lymphoproliferative disease.170 nTregs, which comprise approximately 5% to 10% of the circulating pool of CD4 + T cells, are educated in the thymus and recognize self-peptides, while iTregs develop in the periphery from FoxP3 − CD4 + T cells. This conversion seems to by driven by DCs that provide a metabolic microenvironment that does not meet the high energetic demands of activating effector T cells. Specifically, DCs that produce enzymes that cannibalize essential amino acids such as IDO, which targets tryptophane; IL-4i1, which converts aromatic amino acids; arginase, which depletes arginine; or histidine ammonia lyase, which destroys histidine and promotes iTreg development.171 Central to this conversion is the mTOR pathway, which is activated by signals from nutrient receptors, growth factor receptors, and presumably branched amino acids, and then initiates translation and mitochondrial energy production. TCR signaling in absence of mTOR activation or under conditions of artificial mTOR inhibition, such as through rapamycin, induces FoxP3 and conversion of CD4 + T cells into iTregs.172 Tregs suppress effector immune responses through several mechanisms. They inhibit IL-2 production and through expression of high levels of CD25 consume large amounts of IL-2, thus causing depletion of this cytokine, which is essential for T-cell proliferation. They produce IL-10, which downregulates the expression of Th1 cytokines, MHC class II, and costimulatory molecule expression by inhibiting NFκ B and Jak/Stat signaling pathways. Tregs produce TGF-β, which upon signaling through similar to mothers against decapentaplegic 2 and 3 modulates gene expression. They also produce IL-35, a heterodimeric protein composed of IL-12α and IL-27β chains, which promotes proliferation of Tregs and inhibits Th17 development.173 Tregs can form very stable clusters with DCs, and it is assumed that this limits access of other T-cell subsets. Through CTLA-4 or PDL1, Tregs downregulate expression of CD80 and CD86 on DCs, thus reducing their ability to provide costimulatory signals.174 Tregs can directly bind B and T cells, such as through

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interactions between HVEM on Tregs and BTLA on T and B cells,175 and cause lysis of cells through the release of granzyme and perforin.176 Tregs play a role in infectious diseases by reducing the magnitude of primary immune responses, by limiting effector T-cell functions, and by reducing secondary immune responses. Mice that lack Tregs are better able to control viral infections, but they are also prone to more damaging immune responses.169 This is exemplified by ocular infections of mice with HSV, which causes severe blinding infections upon depletion of Tregs. Dampening of potentially pathologic immune responses upon viral infections not only benefits the host but also the virus by allowing for its persistence. For example, in chronic HCV infections, the severity of liver damage as well as viral loads are inversely correlated with numbers of Tregs in liver and blood.177 CD8 + T-cell responses to HIV-1/simian immunodeficiency virus (SIV) are dampened by Tregs.178 The chronic phase of such infections is associated with a loss of Tregs, which may contribute to T-cell hyperactivation in response to microbial translocation from the gut.179 Chronic infection of mice with Friend leukemia virus is associated with an increase in Tregs and a suppression of CD8 + T-cell function.180 Depletion of Tregs in this model rescues cytokine production and lysis by CD8 + T cells resulting in sustained lowering of viral loads.

Cluster of Differentiation 8+ T Cells

CD8 + T cells are crucial to resolve virus infections through direct lysis of virus-producing cells and through the release of antiviral cytokines. Cluster of Differentiation 8 + T Cells: Antigen Processing and Presentation. CD8 + T cells respond to peptides displayed by MHC class I determinants.181 The pathway for peptide-MHC class I loading differs from that of MHC class II loading. While MHC class II molecules associate with peptides derived from degraded proteins in endosomes, MHC class I peptides largely originate from misfolded newly synthesized proteins. MHC class I molecules, which are expressed on nearly all cells are thus uniquely suited to detect cells that upon infection are producing new viral progeny. Proteins that are not faithfully translated or that are incorrectly folded are degraded in the cytoplasm into peptides by proteolytic enzymes that form the proteasome complex.182 The resulting peptides are then transported into the ER though the transporter associated with antigen presentation (TAP).183 Newly synthesized MHC class I heavy chains are also transported into the ER, where they bind to calnexin, which promotes appropriate folding of the protein. The correct folding of the MHC class I heavy chain is further facilitated by calreticulin and the oxidoreductase ERp57, which breaks up incorrectly folded MHC molecules. Once the heavy chains are folded, calnexin is released and the heavy chains bind β2 microglobulin. The resulting complex is linked to TAP by interactions with tapasin. Peptides, once they reach the ER, may be digested further by peptidases, until they get to a size that permits their association with the MHC class I groove, which can accommodate peptides of 8 to 10 amino acids in length. Binding of peptides to MHC

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class I molecules causes release of calreticulin and tapasin, and is followed by transport of the MHC-peptide complex through the Golgi apparatus to the cell surface (Fig. 39.6). Synthesis of molecules needed for MHC class I–mediated antigen presentation is upregulated by proinflammatory cytokines, such as IFNs. Naïve CD8 + T cells generally require antigen presentation by DCs in presence of costimulatory molecules. Proteins from viruses that directly infect DCs readily provide peptides that enter the MHC class I presentation pathways. Antigens from viruses that do not infect DCs are processed and presented by alternative mechanisms, called cross-presentation or cross-priming.184 In cross-priming, DCs can gather antigen from the outside by a number of pathways (see Fig. 39.6). DCs can pinocytose or phagocytose antigen released by other cells. They can actively chop off parts of viral surface proteins displayed on membranes of infected cells, or they can acquire preformed MHC class I antigen by trogocytosis, which involves conjugation of DCs with antigen-carrying cells through immunologic synapses, followed by ingestion of membrane fragments of one cell by the other. DCs that express connexin-34 can form gap junctions with other connexin-34 + cells, which allows for

influx of small molecules, such as peptides, from the cytoplasm. A number of receptors facilitate antigen uptake by DCs, such as mannose receptors, DC-SIGN, DEC205, and Fc receptors, and contribute to cross-presentation. MHC class I molecules are normally formed in the ER, but they can be recycled from the cell surface into early endosomes. TLR signaling increases MHC class I recycling and recruits TAP to endosomes. Proteins that are internalized into endosomes initially exit into the cytoplasm, where, still in close proximity to endosomes, they are degraded by proteasomes. The resulting peptides then reenter the endosomes through TAP and now can bind to empty, recycled MHC class I molecules. Although MHC class I molecules can, in theory, bind thousands of different peptides that have fitting anchoring residues and, vice versa, viruses, especially those that are large, carry proteins with hundreds of putative MHC class I–binding motifs, antiviral immune responses are commonly dominated by CD8 + T-cell responses to one or two epitopes, with rapidly mutating viruses allowing for their escape.185 Immunodominance of some peptides and immunosubdominance of others is dictated mainly by affi nity between peptide and the MHC class I molecules, and most of the defined immunodominant peptides have an

FIG. 39.6. Major histocompatibility complex class I processing and presentation pathways: endogenous pathway (green) and crosspresentation (pink).

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affinity constant of 500 nm or higher. It is also affected by processing as epitopes can be lost through proteolytic cleavage or they are not generated by lack of flanking sequences that permit such cleavage. Immunodominance is further affected by location of the peptide within the viral protein. Translation is initiated at the 5′ end of an mRNA, which for surface proteins encodes the signal sequence. Incomplete protein synthesis is common, especially for larger proteins or mRNAs with mutations that incorporate a stop codon. Truncated proteins do not fold correctly and are degraded in the cytoplasm. Peptides encoded by the 5′ part of gene and thus located toward the N-terminus of a protein are more likely to be present in incomplete proteins, and as such more likely to be immunodominant compared to epitopic peptides within the C-terminus. The timing of viral protein synthesis affects which epitopes are predominantly recognized. CD8 + T cells often respond preferentially to early viral proteins, which is highly advantageous for the host, as such proteins are produced well ahead of viral replication, so that destruction of cells expressing early viral protein proceeds assembly of infectious viral progeny.186 As viruses commonly interfere with cellular protein synthesis, the favored recognition of early viral proteins may in part be a default pathway, as such proteins are produced at a time when the cell is still relatively intact and thus able to process and present the antigen efficiently. Viral antigens are not produced in equal quantities. The most abundant structural protein of HIV-1 is gag, which is also the major target antigen for CD8 + T cells. Enzymes such as the polymerase of rhabdoviruses are produced at minute quantities that are unlikely to be immunogenic. Some viral proteins are extremely stable. For example, EBNA1, which is produced in cells latently infected with EBV, carries a glycine-alanine repeat sequence that is resistant to proteolytic degradation.187 Last but not least, the TCR repertoire, which has holes to ensure tolerance, influences immunodominance; epitopes that can bind with very high affinity to MHC class I molecules will not elicit a response if they resemble self peptides. The TCR of CD8 + T cells only needs a few MHC class I peptide complexes to mount an immune response. Although some viral infection can result in the display of hundreds of peptide-MHC class I molecules on the surface of infected cells, others only express a dozen or so, which still suffices for CD8 + T-cell activation and effector functions. Cluster of Differentiation 8 + T Cells: Activation. Naive CD8 + T cells are activated within the T-cell–rich zones of lymphatic tissue, which provide a sufficient density of antigen-presenting DCs and Th cells. During activation, T cells interact repeatedly with DCs until they form stable connections, which are sustained by adhesion molecules, such as LFA-1 and CD2 on T cells and ICAM-1 and CD48/ CD59 on DCs, respectively, and by costimulatory molecules that can function as adhesion molecules. Binding between T cells and DCs leads to the formation of an immunologic synapse,188 in which the TCR-MHC-peptide complexes are central, surrounded by interacting costimulators and their ligands. Interacting adhesion molecules form the next ring

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and the outer ring contains CD43, CD44, and CD45 molecules.189 Formation of synapses presumably allows for activation of a CD8 + T cell by low density of its cognate antigen. Costimulation is essential for CD8 + T-cell activation and the predominant pathway signals through the constitutively expressed CD28 upon its interaction with CD80 or CD86. Ligation of CD28 initiates a number of signaling cascades that result in activation of NF-κ B and Jun/Fos transcription factors. It also activates the PI3K/Akt/mTor pathway, which augments energy production and protein synthesis by increasing amino acid transport, ribosome functions, and translation.190 In addition, mTOR affects expression of the transcription factors T-bet and eomesodermin, which are crucial for lineage decisions in T cells.191 Later during CD8 + T effector cell differentiation, CTLA-4 expression is induced, which binds to the same molecules that CD28 recognizes, but serves as a coinhibitor, by for example blocking CD28induced Akt activation.192 As CTLA-4 has higher affinity than CD28 to CD80 and CD86, coinhibition prevails if both are expressed. Other costimulators and conihibitors play a role in initiating, sustaining, or dampening CD8 + T-cell activation. Interactions between HVEM-lymphotoxin/ LIGHT, Ox40–Ox40L, or 4-1BB-4-1BBL provide costimulatory signals by activation of NF-κ B, Jun kinase, and Ap1. Costimulation through different pathways is functionally, spatially, and temporarily segregated. T cells during or shortly after activation also receive inhibitory signals through PD1, BTLA, CD160, or LAG-3, which are either induced or upregulated during T-cell activation. While such inhibitory signals dampen primary immune responses, they may be crucial to allow for fate-decisions of antigenexperienced CD8 + T cells.193 As a third signal, CD8 + T-cell activation requires cytokines such as type I IFN or IL-12. Activating CD8 + T cells requires T help in form of IL-2 for sustained proliferation. Upon activation, CD8 + T cells proliferate extensively in a 4- to 6-hour cycle. Naïve cells, which are present at frequencies below 0.00001% of all CD8 + T cells prior to the infection, can reach frequencies of 10% or more within 5 to 7 days. Early after activation, CD8 + T cells transiently express CD25 and CD69. In addition, they start to express CD44, an adhesion molecule, and CD27, a TNF-receptor family member. T cells destined to become central memory cells increase expression of CD127. Upon activation and proliferation, CD8 + T cells downregulate expression of CCR7, the receptor for CCL19, which is primarily produced in lymphatic tissues and CD62L, an adhesion molecule, which facilitates entry into lymph nodes and then they can enter the bloodstream to migrate to sites of infection following gradients of chemokines, such as CXCL9 or 10. Proinflammatory cytokines produced in response to a virus increase expression of the adhesion molecules P- and E-selectins on adjacent blood vessels, which slows down the flow of CD8 + T cells; they first attach loosely to the blood vessel wall and then assume a rolling motion, which is replace by tight adhesions of T cells allowing them to transmigrate between the endothelial cells to the infected tissue,194 where, upon encounter of their antigen, they commence effector function.

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Cluster of Differentiation 8 + T Cells: Effector Functions. CD8 + T cells can release a number of cytokines and chemokines. Antiviral CD8 + T cells predominantly produce IFNγ, IL-2, TNF-α , MIP-1-α and MIP-1-β, or regulated upon activation, normal T cell expressed and secreted. CD8 + T cells can secrete single or several cytokines/chemokines, and in infected individuals multiple subsets, defi ned by their cytokine/chemokine production profi le, arise.195 Although the profi le of cytokine/chemokine may be predictive for effectiveness of the CD8 + T cells in limiting viral spread, the highly dynamic patterns that evolve during the course of an infection suggest that they may readily assume new functions or let go of others. IFN-γ upregulates the antigen processing machinery and MHC class I expression, thus facilitating interactions between infected cells and activated CD8 + T cells. IFN-γ also activates natural killer cells and macrophages, and blocks viral spread, similar to IFN-1s. TNF-α can inhibit replication of the viral genome or assembly of viral capsids.196,197 Another important function of activated CD8 + T cells is their ability to kill infected target cells. CD8 + T cells predominantly lyse through the release of two lytic enzymes, granzyme, and perforin.198 These two enzymes are present in granules that, once CD8 + T cells encounter their target, are released by exocytosis. Perforin molecules insert themselves into the plasma membrane of target cells and form a pore. Granzyme molecules enter through the pore into the cells where granzyme A activates DNase and inhibits the DNA repair machinery. Granzyme B activates caspase 3, which in turn activates caspase 7, while cleaving the proapoptotic molecule BID, thus causing cells to apoptose. Alternatively, CD8 + T cells can lyse target cells through interactions between CD95 expressed by all activated T cells and Fas expressed by some target cells. Interactions between CD95 and Fas trigger activation of caspase 8 and apoptotic cell death. CD8 + T-cell–mediated lysis is rather inefficient, as each CD8 + T cells can only lyse a limited number of targets; nevertheless, it is crucial for rapid viral clearance as mice lacking lytic enzymes showed increased susceptibility to a number of viruses.199,200 Cluster of Differentiation 8 + T Cells: Memory Differentiation. Four transcription factors play a role in determining the fate of an activating CD8 + T cell: T-bet and eomesodermin (Eomes), which are both required for effector and memory formation, the transcriptional repressor B-lymphocyte–induced maturation protein (Blimp)-1 which dampens and Bcl-6 which sponsors differentiation into memory.201 The differentiation fate of CD8 + T cells is at least in part dictated by the conditions of their stimulation. CD8 + T cells can develop into short-lived effector cells, and this is supported by expression of the transcriptional repressor Blimp-1.202 Alternatively, CD8 + T cells can transition into the memory T cells’ pool. Strong TCR signaling in presence of excessive amounts of antigen seems to favor effector cell differentiation, while limited amounts of antigen promote development of memory T cells.203 IL-2 through binding to CD25 similar to CD28 activates PI3K, which in turn initiates downstream phosphorylation of Akt/PKB. Activated Akt/PKB inhibits forkhead box O and

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thus expression of the IL7 receptor CD127, which favors memory formation.204 Signaling through CD25 downregulates CD127 expression, 205 inhibits expression of Bcl-6, a memory marker,206 and induces T-bet, which, if present at high levels, induces generation of CD8 + effector T cells. Memory CD8 + T-cell development is promoted by low levels of T-bet and by proinflammatory cytokines such as IFN-1s, IL-12, and IL-15.207 In acute viral infections, the immune system eliminates the infected cells within 1 or 2 weeks. Once their cognate antigen is gone, most of the activated CD8 + effector T cells undergo apoptotic cell death and only 5% to 10% survive and differentiate into memory cells, leaving the organism with a 10- to 1000-fold increase in numbers of CD8 + T cells able to react against a given virus. Two major types of memory cells have been identified: effector memory T cells, which remain comparatively active and circulate in the periphery, where upon reencounter of their antigen then can assume immediate effector functions; and central memory T cells, which express increased levels of CD62L and CCR7 and return to lymphatic tissues, where they become reactivated upon reinfection.208,209 Effector memory T cells, although longer lived than effector T cells, decline in numbers over time, while numbers of central memory T cells remain at constant levels through homeostatic antigen-independent proliferation driven by IL-7, which provides survival signals, and IL-15, which promotes proliferation.210 There is ample evidence to suggest that during activation T-cell differentiation is rapidly imprinted, potentially already during the first division of activating T cells.211 Within a week after the initial activation, T cells that are terminally differentiated can be distinguished from those that will evolve into memory T cells; those that express low levels of CD127 and high levels of KLRG1 die once the effector phase is terminated, while those that are CD127high and KLRG1low become central memory T cells.212 Lineage relationships between effector, effector memory, and central memory CD8 + T cells remain debated; some studies suggest a linear relationship, in which effector T cells differentiate into effector memory CD8 + T cells and then central memory CD8 + T cells,213 while others argue for independent development of these populations.214 The importance of effector/effector memory CD8 + T cells versus central memory CD8 + T cells in providing protection to viral infections remains debated as well. While some argue that the more activated effector-like CD8 + T cells, which are present at the port of viral entry, are crucial,215,216 others provide evidence that central memory CD8 + T cells with their higher proliferative potential are essential.213 For example, in a low-dose SIV infection model of rhesus macaques, effector memory CD8 + T cells were shown to correlate with protection.217 Similarly, in an influenza virus challenge model of mice, activated CD8 + T cells present in the lung at the time of challenge were required for protection.218 In contrast, central memory CD8 + T cells were shown to provide superior protection to LCMV.213 One could argue that mode of infection dictates which T-cell subset is indeed more suited to eliminate virus-infected cells before extensive viral replication takes place. Viruses such as HIV-1 and SIV invade through mucosal surfaces

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and a productive infection is established by very small numbers of founder viruses. Activated effector memory CD8 + T cells present within the mucosa might be superior to eliminate the few cells that initially become infected compared to central memory CD8 + T cells, which reside in far away lymph nodes, where they most likely remain ignorant of the incoming few viral particles. In an infection where substantial amounts of virus are implanted directly into a body cavity, central memory CD8 + T cells with their higher proliferative capacity may be better suited to eliminate the virus. Cluster of Differentiation 8 + T Cells: The Effect of Persistent or Repeated Infections on Cluster of Differentiation 8 + T Cells. Many viruses, especially DNA viruses, are not completely cleared from an organism, but rather establish long-lasting infections. Such infections can be chronic when the virus continues to replicate extensively, such as HIV-1 or HCV in humans or some strains of LCMV in mice. Herpesviruses establish latent infections by shutting off most viral protein synthesis; they periodically become reactivated and resume replication. Other viruses, such as adenoviruses, persist at very low levels and apparently remain transcriptionally active. In this case, the immune system is continuously stimulated by low amounts of antigen, which maintain high frequencies of activated effector-like CD8 + T cells. T cells may encounter the same viral antigen repeatedly due to repeated infections. Although neutralizing antibodies induced by a viral infection may prevent a subsequent infection with this particular virus, they are not effective against other viruses from the same family, which may not share neutralizing antibody binding sites, but, nevertheless, carry conserved T cell epitopes, such as different serotypes of adenoviruses. T cells may thus encounter the same epitope presented by related viruses over and over again. Repeated stimulation in turn affects the quality of memory CD8 + T-cell responses. Chronic infections overwhelm the immune system. The continued high presence of antigen prevents differentiation of effector CD8 + T cells into central memory CD8 + T cells and instead induces, in a hierarchical fashion, loss of effector cell functions217; first, T cells lose the ability to proliferate and then they cease to produce cytokines or lyse target cells. Loss of function is accompanied by upregulation of coinhibitory receptors such as PD1, LAG-3, CD160, 2B4, CTLA-4, PIR-B, and Gp49 in a process that is controlled by Blimp-1.218 Signaling through coinhibitors such as PD1 inhibits the PI3K/Akt/mTor pathway and thus the T cell’s ability to meet its demand for energy. The degree of exhaustion is correlated with the numbers of coinhibitory receptors on the CD8 + T cell’s surface, as numbers of upregulated coinhibitors correlate with the severity of the T cell’s impairment.219 Partially, but not fully, exhausted CD8 + T cells can be rescued by treatment with antagonists to immunoinhibitory pathways.220 Reactivation of latent viruses causes expansion of specific memory CD8 + T cells, which once activated rapidly control the infection. The profi le of memory CD8 + T cell changes upon repeated recall; they become more activated and their

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numbers increase.221 This can become rather extreme, and in elderly humans up to 30% of peripheral CD8 + T cells show specificity to antigens of herpesviruses, most notably (HCMV).222 High frequencies of Hhuman CMV CMVspecific CD8 + T cells are inversely correlated with life expectancy,223 presumably as a consequence of increased disease susceptibility due to irreversible loss of immunologic space and repertoire. Frequencies of circulating activated CD8 + T cells to adenoviruses can also be high.224 It is unknown if this relates to the ability of the virus to persist at low levels causing continuous activation of CD8 + T cells or if it rather reflects repeated infections with serologically distinct adenoviruses. The former pathways seems more likely as other viruses, such as influenza A viruses, which also infect repeatedly, fail to establish high frequencies of specific CD8 + T cells. Regulatory Cluster of Differentiation 8 + T cells. CD8 + T cells that express FoxP3 and exert immunosuppression through the release of IL-10 or TGF-β have been identified in acute SIV infection of nonhuman primates, where they correlate with viral load and inversely correlate with effector CD8 + T-cell responses.225 They also may play a role in limiting liver damage during chronic HCV infection.226

B Cells The primary task of B cells is to produce antibodies that can limit viral spread. In addition, they serve as antigenpresenting cells and produce cytokines.

B Cells: Subsets In mice, B cells are divided into three distinct subsets: marginal zone (MZ) B cells, follicular B cells, and B1-B cells, which are further divided into B1a- and B1b-B cells.227 B1-B cells, which have not yet been identified in humans, reside mainly in the pleural and peritoneal cavities, although, upon activation, they can migrate to lymph nodes. B1-B cells are activated by PAMPs and can mount early IgM or IgA responses without requiring T-cell help. MZ B cells, which, as their name indicates, reside mainly in the MZ of the spleen, can also rapidly, without T-cell help, differentiate into shortlived plasma cells that produce IgM. MZ B cells, which express high levels of MHC class II antigens and costimulatory molecules, can also present antigen to Th cells. Unlike MZ or B1-B cells, follicular B cells, which circulate between blood and B-cell follicles in lymphatic tissues, are not directly activated by antigen ligation of the BCR and concomitant PRR stimulation, but, in addition, require T-cell help through CD40–CD40L interactions. B Cells: Activation and Differentiation. Mature naïve B cells derive from bone marrow precursors. They express IgD and IgM molecules as antigen receptors. B cells with high affinity to self are deleted or edited first in the bone marrow and then again in the periphery. B cells that pass these checkpoints then circulate from blood through lymphatic tissues where they are guided toward follicles by a network of reticular fibers and follicular DC dendrites. Small antigens, such as peptides, reach lymph nodes rapidly by passive diffusion;

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once there, they are taken up by macrophages that line the lymph node sinuses or complexed onto follicular DCs through complement and Ig Fc receptors. Larger antigens, such as virions, are engulfed by peripheral DCs, which then migrate to lymph nodes, a process that takes about 12 to 18 hours. B cells through their Ig receptors can acquire antigen from the surface of follicular DCs or macrophages.228 Once the BCRs are cross-linked by complex antigens, which carry repeat sequences of the same antigen, or are oligomerized by soluble antigen, a conformational change occurs in the cytoplasmic domain of the BCR. This first activates Lyn. Lyn in turn phosphorylates the Ig chains leading to the attachment of Syk and recruitment of CD19.229 These initial signals promote the formation of larger clusters, which form an immunologic synapse with the BCRs in the center surrounded by LFA-1. Formation of BCR clusters, which are needed to initiate a signaling cascade, is favored by high-affinity BCRantigen interactions and may thus serve as an early selection process for the best fitting antibodies. Full BCR-CD19 signaling activates a number of cascades that result in activation of the NF-κ B, Pi3K/Akt, Erk, and JNK/p38 pathways. This in turn initiates proliferation and maturation of B cells, which at this stage can differentiate into short-lived plasma cells or enter follicles to form germinal centers and differentiate into long-lived plasma cells and memory B cells. Shortlived plasma cells produce antibodies rapidly, although such antibodies are of low affinity compared to those produced by long-lived plasma cells. The fate decision between shortlived versus long-lived plasma cells or memory cells is in part governed by cytokines such as IL-12, which favors the development of short-lived plasma cells, as well as by the affi nity between antigen and the BCR with very-high-affi nity interactions apparently also favoring the development of shortlived plasma cells. It may also be influenced by local PAMPs and the resulting PRR signaling cascades.230 For example, vaccines providing antigen together with TLR4 and TLR7 ligands favor the development of long-lived plasma cells and memory B cells.83 Germinal center formation requires Tfh cells, which promote B-cell responses through cytokines, such as IL-4, IL-6, IL-10, and IL-21, through direct cell-to-cell interactions and through costimulatory signals, such as CD40, ICOS, or lymphotoxin.230 Coinhibitory signals also shape the fate of activated B cells. For example, upon activation, B cells rapidly adjust their metabolic needs by increasing glucose uptake and glycolysis in an Akt/PI3K dependant fashion. This in turn is inhibited by signaling through Fc γRIIB.231 PD1 on Tfh cells through interactions with PDL2 and to a lesser extend PDL1 reduce rates of cell death and thus increases numbers of surviving cells with lower-affinity BCR.118 During proliferation, B cells undergo hypermutation leading to the development of plasma cells producing antibodies with increased affinity. They also undergo isotype switching through recombination, and this process is influenced by the surrounding cytokine milieu; in mice, IL-4 favors switching to IgG1 and IgE, IL-5 promotes switching to IgA, and IFN-γ supports switching to IgG2a. Competition for T-cell help and T-cell–derived cytokines favors continued proliferation of B cells with the highest affinity receptors.

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Differentiation of naïve or memory B cells into plasma cells causes changes in key transcription factors with a reduction of paired box protein-5 and Bcl-6 and an induction of Blimp-1, which turns off germinal cell functions and allows for a switch to antibody secretion.232 It is not yet fully understood what drives B cells within the germinal center to differentiate into resting memory B cells or longlived plasma cells. B cells with lower-affinity receptors may be outcompeted by those that have higher-affinity receptors and thus be forced to differentiate into memory cells, whereas B cells with the highest-affinity receptors continue to proliferate and eventually form the pool of long-lived plasma cells. The finding that the memory B-cell pool forms earlier than the pool of long-lived plasma cells supports this idea.230 Plasma cells home preferentially to bone marrow, while memory B cells home to spleen or bone marrow. Upon reexposure to antigen, memory B cells differentiate into antibody-secreting plasma cells, which requires proliferation accompanied by another round of hypermutations and affinity maturation. Chronic viral infections can lead to B-cell exhaustion.233 During this process, B cells express increasing levels of the inhibitory Fc-receptor-like-4 molecule, which is accompanied by a loss of their proliferative capacity. Chronic infection can also cause a pathologic accumulation on antigen-antibody complexes causing vasculitis or glomerulonephritis. B Cells: Effector Functions. B cells protect against pathogens primarily through the secretion of antibodies. The rapid production of low-affinity antibodies by B1-B cells and MZ B cells can reduce the spread of viral pathogens by forming complexes that are retained within lymphatic tissues, where they may aid activation of other adaptive immune responses. Plasma cells derived from B2-B cells can produce IgM, IgD, IgG, IgA, and IgE. Of those, IgA, IgG, and IgM are important to control viral infections. IgM is produced first and is then over time replaced by IgG or IgA upon isotype switching. IgGs, which are further divided into IgG1 to 4 circulate in blood and tissues. IgA can be transported actively across epithelial surfaces, allowing for its secretion at mucosal surfaces. IgA is thus crucial for protection against pathogens that invade through the mucosal epithelium. All antibody isotypes can block viral infections by neutralization. Virus-neutralizing antibodies are directed against parts of viral surface proteins that are exposed and commonly nonessential for viral fitness. For example, humans can be infected with more than 40 different serotypes of adenoviruses that belong to six different families; chimpanzees are infected with viruses that are phylogenetically related. The hexon of adenoviruses, which is the main target of neutralizing antibodies, forms trimers on the surface of virions. The stalk of the trimers, which is poorly accessible by antibodies, is highly conserved. The tower region contains a number of flexible loops that are highly variable. Neutralizing antibodies bind to these loops.234 As these loops do not contribute to the structural integrity of the protein, they can readily be mutated, thus allowing the virus to escape neutralization. Antibodies of the IgG or IgM isotypes can lyse viruses or virus-infected cells through activation of complement.

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They can complex viruses, thus facilitating phagocytosis by macrophages. They can lyse virus-infected cells through antibody-dependent cell-mediated cytotoxicity, during which natural killer cells through Fc-receptors bind to an IgG molecule on an infected cell and then release perforin and granzyme. Although preexisting natural antibodies provide an immediate layer of defense, which is rapidly joined by antibodies secreted by B1 and MZ B cells, production of affinity-matured antibodies takes up to 2 weeks and, by then, an acute viral infection has been resolved, while in chronic infections the virus, such as HIV-1 or HCV, has mutated extensively, rendering antibodies directed against the incoming virus rather useless. Affinity-matured antibodies are thus unlikely to play a dominant role in limiting a primary infection, but they are crucial to ward off secondary infections. Neutralizing antibodies can completely prevent a secondary infection or they can reduce the amount of incoming virus to levels that can rapidly be controlled. Protection by neutralizing antibodies against reinfection has been documented for numerous viruses such as rabies virus, influenza A virus, poliovirus, HPV, HIV-1, and many others, and is the main correlate of protection for most antiviral vaccines. Nonneutralizing antibodies can also dampen infections without necessarily providing sterilizing immunity. For examples, antibodies to the ectodomain of the matrix protein of influenza A virus lack neutralizing activity, but nevertheless can prevent fatal infections with influenza A viruses in animal models.235 In some infections, preexisting antibodies can exacerbate disease as exemplified by Dengue virus. Dengue virus replicates preferentially in macrophages, monocytes, and DCs. A first infection causes relatively mild symptoms and induces antibodies that cross-react between any of the four strains of Dengue virus. Following a second infection with a serologically distinct virus, circulating nonneutralizing antibodies form complexes with the virus. Such complexes facilitate uptake by macrophages, which in response produce IL-10 rather than IFN-γ. This allows for more vigorous replication of the virus, a skewing toward a Th2 immune response leading to hemorrhagic fever, which is characterized by impaired clotting and vascular leakage.236 Antibodies play an additional altruistic role. Children are born with an immature immune system and are thus potentially hypersusceptible to viral infections. Antibodies of the IgG isotype are transferred from mothers to their offspring across the placenta and through breast milk. Maternal antibodies are crucial to protect an infant against the onslaught of pathogens that will attack as soon as it has left its mother’s womb. Maternal antibodies do not necessarily provide sterilizing immunity, but may attenuate inflections and thus allow for active immunization under conditions that do not threaten the life of the infant. The argument has been made that vaccination reduces transfer of maternal antibodies as natural infections of the mother as a rule elicit higher and more sustained levels of antibodies than most vaccines. This, combined with improved hygienic standards, which delays exposure to common pathogens, may in the end harm the offspring by delaying their first exposure to pathogens

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until such a time when maternal antibodies have declined from their circulation, which may happen before their own immune system is fully developed. Exposure to viruses at this susceptible stage, in turn, may favor the induction of pathological or self-reactive immune responses.237 Although there is no direct proof for this ominous side effect of mass vaccination, it might be supported by recent increases in the incidence of allergies and autoimmune diseases.

THE MUCOSAL IMMUNE SYSTEM The mucosa of the airways, the intestines, and the urogenital tract, which in adult humans covers a total surface area of approximately 400 m2 are the most common ports of viral entry. Mucosal surfaces are also bathed in antigens that are harmless or useful such as the 1014 commensal bacteria that live in the intestine, where they are essential to outcompete pathogenic bacteria and promote normal intestinal functions. The immune system that controls mucosal surfaces, the so-called mucosal immune system, must be able to distinguish between dangerous and nondangerous antigens and has thus unique characteristics. The mucosal immune system consists anatomically of local inductive sites, called organized mucosa associated lymphoid tissue, such as tonsils in the oral cavity, Peyer patches along the wall of the small intestine, the appendix close to the junction between small and large interesting, and bronchus associated-lymphoid tissues in the respiratory tract. Although these sites resemble lymph nodes, they are not encapsidated nor are they connected by lymphatic vessels.238,239 The mucosa of the intestine is covered by the lamina propria (LP) and a single layer glanular epithelium. Both activated and resting B- and T-lymphocytes can be found within the LP, while the mucosal intestine is densely seeded with highly activated T cells. The vaginal surface is covered by a multilayered squamous epithelium that contains both B and T cells originating from iliac lymph nodes. T and B cells can also be found in the epithelium of the airways. B cells in the intestine and airways are skewed toward those that produce IgA. Most T cells in the mucosa of the genital tract and the airways carry the α/β TCR, unlike T cells in the intestinal part, many of which carry a γ/δ TCR. In newborn mice, nearly all if the intraepithelial lymphocytes that initially populate the intestine express the γ/δ receptor, which has a less diverse repertoire than the α/β receptor. T cells with a γδ receptor develop independently of the thymus and do not recognize MHC class I peptide complexes but rather respond to self-antigens and microbial phosphorylated metabolites. Their response does not involve extensive proliferation and differentiation; upon encounter of antigen, they commence production of cytokines within a few hours.240 T cells expressing the α/β TCR appear later in the intestine under the influence of antigenic stimulation. T cells within the intestine can also be double positive for CD4 and CD8, or they can carry two CD8α chains rather than heterodimers of CD8α and CD8β. DCs acquire antigen in the intestine with the help of M cells, which sample luminal antigen and then pass it on to

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the underlying DCs.241 DCs in the mucosa predominantly secrete IL-6, IL-21, and TGF-β, thus favoring induction of Th17 cells, the latter in turn produce IL-17 and IL-22.242 IL-17 increases tight junction formation in the gut thus reducing permeability, while IL-17 and IL-22 induce granulopoiesis through granulocyte-CSF and recruit neutrophils through CXC chemokines. Infections with HIV-1 or SIV cause rapid depletion of mucosal Th17 cells, which in turn increases gut permeability and causes microbial dissemination and chronic immune activation.243 Viral infections induce CD8 + T cells that can be isolated from the LP and the gut epithelium. These CD8 + T cells can produce IFN-γ and kill infected target cells. Antigeninduced CD8 + T cells in the epithelium and the LP were found to contract more rapidly than antigen-specific CD8 + T cell in spleen. Interestingly, antigen-specific CD8 + T cells from the LP and the overlaying mucosa were shown to be distinct in their TCR usage; T cells from epithelium were more oligoclonal and had lower affi nity to their antigen, suggesting limited exchange of T cells between these two adjacent compartments.244 Effector T cells of the central immune system can migrate to the intestinal mucosa, and this process requires expression of α 4β7. Strength of the initial activation seems to control expression of α 4β7. For example, CD8 + T cells induced by LCMV, which proliferates extensively in mice, express α 4β7, 245 while those induce by an E1-deleted adenoviral vector only express this homing marker very transiently and consequently fail to migrate to the intestinal mucosa.246 Homing of activated T cells is also influenced by characteristics of the antigenpresenting cells; CD103 + DCs derived from skin, lung, and intestine drive T cells to increase expression of α4β7 integrin.247 Within the female genital tract, primary immune responses are induced in draining lymph nodes. Upon clearance of a virus, as was shown in mice and humans infected with HSV-2 or HIV-1, respectively, clusters of memory T cells, B cells, and DCs remain underneath the epithelium of the vagina and the cervix, potentially providing an immediate barrier against infection.248,249 Systemically induced effector CD8 + T cell can readily migrate to the mucosa of the genital tract250 ; it remains unknown if this requires a specialized homing receptor. Within the airways, an infection, such as with influenza virus, can result in organized lymphoid structures within the lungs composed of B-cell follicles and surrounded by T cells. These structures called inducible bronchus associated-lymphoid tissue can recruit naïve lymphocytes and provide a first-line defense against subsequent pulmonary infections.251

THE AGED IMMUNE SYSTEM Immune responses become impaired during aging, resulting in an increased susceptibility to infectious agents, as exemplified by influenza virus that mainly kills the very young or the aged. Immunosenescence affects multiple aspects of the immune system. DCs derived from blood or bone marrow from the elderly develop defects in their

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responses to inflammatory cytokines and their ability to present antigen and activate adaptive immune responses.252 B-cell lymphopoiesis is reduced with aging, leading to a decline of naïve B cells and an increase of antigen-experienced B cells with an extended lifespan. Primary B-cell responses in the elderly are commonly both low and short-lived, resulting in antibodies with low affinity.253 Formation of germinal centers decreases, antigen transport is impaired, and follicular DCs have reduced capacity to form antigen depots.254,255 Autoantibodies are more common, and the B-cell repertoire becomes more restricted. The E2A-encoded transcription factor E47 is downregulated in old splenic B cells, which causes a reduction in the activation-induced cytidine deaminase, needed for class switch recombination and Ig somatic hypermutation.256 Some of the defects of B-cell responses are secondary to an age-related decline of helper functions from CD4 + T cells, which show reduced expression of critical costimulatory receptors that are essential for activation of B cells, germinal center formation and rearrangement, and hypermutation of Ig genes.257–259 T cells show clonal senescence, their potential for expansion decreases, and their ability to produce certain cytokines or to respond to cytokines may become impaired. The proportion of T cells with a memory cell phenotype increases, while numbers of naïve T cells decrease, in part reflecting loss of thymic output following thymic involution, and in part resulting from chronic stimulation. Expression of activating molecules, such as CD40L and CD28 decreases,260,261 while that of inhibitory pathways increase.262,263 Stimulations with new antigens or previously encountered antigens result in CD8 + T-cell responses that are delayed and show defects in transition into memory. The delay in T-cell expansion reflects an intrinsic defect of aged T cells and may well be one of the reasons for increased susceptibility of the aged to rapidly replicating pathogens. Upon aging, the T-cell repertoire loses diversity.264,265 Chronic antigenic stimulation leads to continued clonal expansion of some T cells. The effect of aging on cytokine production remains controversial.264–267 The number of Treg cells appears to increase with age,268 while autoimmunity is more common, suggesting that inhibitory pathways may become dysregulated during aging.

AUTOIMMUNITY AND DEGENERATIVE DISEASE Viral infection can lead to the development of autoimmunity. Some viruses carry antigens with superantigen activity, such as the nucleoprotein of rabies virus or nef of HIV-1.269,270 Superantigens trigger polyclonal activation of T cells and can thus lead to stimulation of self-reactive T cells. Some viruses carry sequences that resemble those of their host. For example, a sequence in coxsackie B virus is homologous to one in glutamic acid decarboxylase, a protein expressed by insulin-producing pancreatic islet cells. Coxsackie B virus infections can trigger in animals antibodies to glutamic acid decarboxylase, which can cause diabetes.271 The polymerase of HBV mimics myelin basic protein, 272 and may thus, in theory, stimulate an immune response that could cause multiple sclerosis.

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Nevertheless, thus far antigenic mimicry of a self-protein has not been confi rmed to be causative for human autoimmune diseases. Strong inflammatory responses elicited by a virus can cause concomitant activation of immune responses to selfproteins. For example, infections of mice with Theiler virus can elicit T cells recognizing myelin basic protein or proteolipid protein causing neurologic damage.273 Coxsackievirus B3 can infect the heart muscle and inflict initial damage; this can induce autoreactive lymphocytes against cardiac myosin,274 which cause chronic myocarditis resulting in permanent heart damage and even death. Herpesviruses have been implicated as etiologic factors in the pathogenesis of human arteriosclerosis by accumulating saturated cholesteryl esters and triacylglycerols to the endothelial walls of blood vessels.275 This can be prevented by cytokines such as TNF-α or IL-1.275

CANCER Viruses can cause cancer. Oncogenic types of HPV such as HPV-16 or -18 are causative for cervical 276 and head and neck cancer through the activity of two oncoproteins, E7 and E6, which disrupt the retinoblastoma (Rb) and p53 pathways, respectively. Chronic infections with HCV and HBV can lead to hepatocellular carcinoma, primate T-lymphotropic virus 1 can cause T-cell leukemia, EBV can result in lymphoma and nasopharyngeal carcinoma, Merkel cell polyoma virus is associated with Merkel cell carcinoma, and HHV-8 is associated with with Kaposi sarcoma.277 Some types of viral cancers are common in the general population, while others arise primarily in immunocompromised individuals. The immune system, although able to prevent the establishment of some types of viral cancers, is in general ill suited to halt progression of an already established advanced cancer even if its cells express viral antigen, as tumors establish an immunosuppressive microenvironment that is effective at suppressing immune responses.278 Virus-specific T cells have found utility in treatment of cancer. EBV-specific T cells isolated from children with neuroblastoma were expanded in vitro and transduced with an artificial TCR composed of an antibody variable region with specificity to a surface antigen on the cancer linked to the intracellular signaling domain of the TCR. Upon transfer of these modified T cells back into the children, remission of some of the advanced cancers was achieved.279

IMMUNOEVASION BY VIRUSES While cellular organisms evolved over the millennia to combat virus infections, first with a primitive innate immune system and then, about 410 million years ago, once jawed vertebrates developed with the more sophisticated adaptive immune system, viruses evolved in tandem to subvert their hosts’ defense mechanisms. While small RNA viruses, which replicate and spread rapidly and, as a rule, do not establish chronic infections, attack mainly the early defense

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mechanisms, the more complex DNA viruses, especially those with large genomes, also subvert adaptive immune responses.

Escape by Hiding The central nervous system is an immunologically privileged site hidden behind the blood–brain barrier that cannot be penetrated by antibodies or resting lymphocytes. It also contains cells such as neurons that lack MHC class I expression and can thus not be attacked by CD8 + T cells. Viruses, exemplified by rabies virus, take advantage of this safe haven. Herpesviruses after an acute lytic infection assume a stage of latency where most viral synthesis is shut off. HSV-1 establishes latent infections in neurons, where it is sheltered from attack by CD8 + T cells, while EBV remains more exposed in B cells. The only protein of EBV that is expressed during deep latency is EBNA-1, which contains a glycine-alanine repeat domain that appears to protect the antigen from proteasomal breakdown and thus from the display of antigenic peptides on MHC molecules.280

Escape through Mutation Viruses can evade by rapid mutations caused by the infidelity of their replication machinery that lacks the proofreading capacity of mammalian polymerases. Although lack of faithful genome replication is costly and many of the resulting viruses lack the fitness of the parent virus, it serves the virus population as a whole, which produces thousands of new infectious viruses from each original virus and can thus afford to lose some due to defective genomes. Mutations are primarily driven by neutralizing antibodies through selection of variants with mutations of their surface molecules. Most binding sites for neutralizing antibodies are on exposed areas on the very top of viral surface proteins, which do not contribute to the assembly of the virus and as such are not essential for the virus. Nevertheless, such mutations can affect viral fitness. Mutations of the HA of influenza virus can increase the affinity to its receptor, which makes it difficult for the virus to gain release from the cell where it replicated. Influenza viruses compensate by additional mutations that readjust the affinity between HA and its receptor.281 Some neutralization sites are located in domains that are essential for the virus and that cannot be mutated without devastating consequences. Neutralizing antibodies can bind to the stalk of the HA of influenza virus.282 This antibody-binding site is highly conserved in several strains, indicating that it is crucial for some aspect of the life cycle of the virus. Nevertheless, this site is so well hidden that most humans fail to produce antibodies against it. HIV-1 is a master in escaping neutralizing antibodies. HIV-1 originated from SIVcpz, which is endemic in common chimpanzees, Pan troglodytes troglodytes, although it can spill over into gorillas. It has been calculated that humans acquired this virus sometimes at the beginning of the last century around 1908 on three occasions from chimpanzees and in one event potentially from a gorilla, resulting in infections with three groups of HIV-1 (ie, M, N, and O).283 Group M was the most

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successful and within a mere hundred years evolved into several clades and multiple subspecies. Even in a new host, where a few founder viruses establish an infection, viral variants evolve so rapidly that the humeral immune system is hard pressed to keep up. T cells can select for mutations in the generally more conserved internal proteins. This is typically found during choric infections such as with HIV-1 or HCV,283,284 but can also happen in acute infection, as exemplified by influenza virus.285 Antiviral cytokines, such as IFNs, can select for mutants. The only approved treatment for chronic infections of HCV is IFN-α , which results in complete viral clearance in only a fraction of patients. In many patients, the virus escapes through mutations that increase its resistance to IFN- α.286

Escape by Destruction of Immune Cells An efficient way to stage a hostile takeover is to eliminate a crucial component of the opponent’s armor. HIV-1 infects and eventually kills CD4 + T cells, which are essential to drive B and CD8 + T cell responses. HIV-1 preferentially replicates in activated CD4 + T cells as resting CD4 + T cells express the antiretroviral deoxycytidine deaminase APOBEC3G, 287 which through editing C to U nucleotide base exchanges inhibits elongation of the HIV-1 genome. To ensure an ample supply of activated CD4 + T cells, HIV-1 rapidly depletes gut-residing CD4 + T cells, causing intestinal hyperpermeability and leakage of bacterial products into the organism, which in turn nonspecifically activates more CD4 + T cells.

Escape from Early Innate Immune Responses Inhibition of Pattern Recognition Receptor Signaling The invention of stealth technology should be credited to viruses, which long ago figured out that flying under the radar (ie, dodging initial recognition by PRRs) promotes their own ability to propagate. Many viruses antagonize recognition by PRRs either by sheltering products that can be sensed or by directly interfering with components of PRR signaling cascades. An example of sheltering recognizable patterns is the Ebola virus vp35. It binds viral dsRNA in a rather peculiar manner; one molecule binds the backbone of the RNA, whereas the other caps the terminus, thus effectively blocking dsRNA binding to TLR3, MDA5, RIG-I, or PKR.288 Similar dsRNA hiding mechanisms are employed by vaccinia virus E3L, influenza virus NS1, reovirus σ protein, and HSV-1 US11.289 Several viruses interfere with TLR signaling. Vaccinia virus A46R and HCV NS5A bind to the TIR domain of MyD88, thus preventing activation of most TLRs. Vaccinia virus A46R, HCV NS4A, and ORFI329L of African swine fever virus290 bind TRIF thus blocking stimulation through the alternative TLR4 pathway. Hantavirus G1 and MC159 from molluscum contagiosum virus (MCV) block TRAF3.291 Vaccinia virus A52R binds IRAK2 thereby inhibiting signaling to TRAF6. Of note, IRAK2 operates redundantly with IRAK1 (see Fig. 39.1) in the initial response but continues to function after IRAK1 disappears (Fig. 39.7). A number of viral proteins target sensors in the cytoplasm (Fig. 39.8). Influenza virus NS1 binds and blocks RIG-I.

FIG. 39.7. Viral evasion of toll-like receptor signaling: (i ) to (ix) indicate points of interactions between viral antigens and components of the pathways.

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FIG. 39.8. Inhibition of retinoic acid-inducible gene I signaling by viral antigens: (i ) to (v) indicate points of interactions between viral antigens and components of the pathways.

Protein V of paramyxoviruses binds MDA5 and inhibits access of ssRNA. Picornaviruses, such as poliovirus, cause proteasome-mediated degradation of MDA5. IPS1, which is downstream of RIG-I or MDA5, is cleaved by HCV NS3-4A or hepatitis A virus 3ABC. The ATP-dependent DEAD box RNA helicase, DDX3, is part of the RIG-I signaling complex. By binding to RIG-I–associated IPS-1, DDX3 can bind viral RNA and as such enhance its recognition by RIG-I. Vaccinia virus K7, HCV core protein, or HBV polymerase can bind DDX3 and inhibit its association with cellular proteins.289 Kaposi sarcoma–associated herpesvirus (KSHV) Orf63 is an inhibitory homolog of NLRP1.291 Multiple proteins of vaccinia virus interfere with NF-κB or IRF3 activation. Vaccinia virus N1L associates with several components of the multisubunit IΚΚ-B kinase complex, including the TBK1 and thus inhibits activation of both NF-κB and IRF3 pathways.292 B14R of vaccinia virus binds the IΚΚ complex and prevents phosphorylation of Iκκβ,293 K1L inhibits NF-κB activation by blocking degradation of IκBα,294 and M2L downregulates ERK-mediated NF-κB induction.295 Another poxvirus, MCV, encodes MC160, which induces IΚΚ1 degradation296 and M159L, which inhibits PKR signaling.297 Measles virus V protein can bind Iκκα and thus prevent phosphorylation of IRF7. It can also prevent phosphorylation of IRF3 by acting as an IRF3 mimetic298 (see Fig. 39.7). KSHV encodes an immediate-early nuclear transcription factor that promotes the ubiquitination and degradation of

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IRF7299 and another protein K-bZIP, which binds DNA and competes with IRF3 binding.300 ICP10 from bovine herpesvirus, protease N from flaviviruses, E6 of HPV-16, HIV-1 Vpr, and Vif cause degradation of IRF3. NSP1 of rotavirus causes destruction of IRF5 and 7.289 Rabies virus protein P prevents phosphorylation of IRF3.301 Papain-like protease of SARS-CoV blocks the nuclear transport of IRF3.302 PKR, once activated by dsRNA, inhibits viral protein synthesis and as such is another favorite target for viruses in their zest to optimize their chance for replication. The VA-RNA of adenoviruses can serve as a decoy; it binds PKR but does not trigger signaling.303 Similarly, the TAR RNA of HIV-1’s long terminal repeat, which initiates tat-mediated activation of transcription, can block PKR activation.304 Tat is phosphorylated by PKR and outcompetes phosphorylation of eIF2α. HCV NS5A prevents dimerization of PKR.305 Vaccinia virus K3L306 and HCV E2307 prevent interactions between PKR and eIF2α . Poliovirus activates a protease that destroys PKR,308 while influenza virus NS-1 activates the cellular PKR inhibitor p38 to prevent PKR dimerization.309 HSV-1 protein IPC34.5 activates a cellular phosphatase that dephosphorylates PKR and eIF2α .310 Some viruses manipulate PRR signaling pathway to create a more favorable environment. Vaccinia virus A52R blocks IRAK2 but allows signaling through TRAF6, which activates MAPKp38 and JNK leading to the induction of IL-10, which in turn suppresses Th1-type immune

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responses.311 The ATP-dependent DEAD box RNA helicase DDX3 interacts with rev of HIV-1 and enhances nuclear export of viral RNA. HCV utilizes DDX3 for its replication possibly by taking advantage of DDX3’s ability to unwind dsRNA.312

Inhibition of Apoptosis Inhibition of NF-κ B, activation of PKR, or signaling through TNF receptors can induce apoptotic pathways. Apoptosis can also be initiated in response to cell damage. Although many viruses eventually cause death of the infected cells, premature apoptosis before the virus replication has been completed would be counterproductive and viruses therefore devised mechanisms to block apoptosis pathways (Fig. 39.9). Apoptosis can be triggered through members of the TNF-receptor family. TNF receptors carry an intracellular receptor domain that contains a death effector domain (DED), which binds to corresponding DEDs on adaptor proteins such as Fas-associated protein with death domain. Upon ligation of the extracellular part of the receptor, the cytoplasmic tail binds to the death inducing signal complex containing procaspase 8, which is then cleaved into caspase 8. Caspase 8 activates caspase 3, which induce cells death. Cellular FLICE-like inhibitors can inhibit caspase 8 activation by competitive binding to the death inducing signal complex. Inhibition of the AKT/PI3K pathways or p53 can lead to dephosphorylation of the Bcl2-associated death promoter, which forms a heterodimer with Bcl-2-associated X protein. This complex can increase the permeability of mitochondrial walls. Consequently, cytochrome C is released from mitochondria, which forms a complex with apoptotic protease activating factor 1 (APAF1) and procaspase 9. Procaspase 9 is cleaved into

an active form and then activates caspase 3. This apoptosis pathway can be blocked by Bcl-2. Poxviruses encode proteins that subvert TNF-receptor signaling. MT2 of rabbit myxomavirus sequesters TNF-α , and vaccinia virus encodes a number of TNF-receptor homologs, which can be secreted or expressed on cell surfaces.313 The adenovirus E3 gene product RID induces internalization and degradation of TNF-receptor family members.314 Several herpesviruses and MCV prevent caspase 8 activation through a viral FLICE inhibitory protein.315,316 Some poxviruses produce serine protease inhibitor-like molecules called crmA (cowpox) or SPI-2 (ectromelia virus), which contain DED domains and inhibit activation of caspases.316,317 The adenovirus E3 14.7 kDa protein inhibits caspase 8 through a yet-to-be-identified pathway upon binding to the host protein FIP-2.318 A number of herpesviruses and adenoviruses increase antiapoptotic pathways by encoding a Bcl-2 homolog.319 Cells infected with viruses can undergo a stress response with accumulation of reactive oxygen species, which in turn increase mitochondrial permeability and induce apoptotic cell death. Cellular enzymes, such as glutathione peroxidase, a selenocysteine containing protein, can reduce H2O2. Poxviruses encode a homologous protein.320 The p28 of ectromelia virus (mousepox) and the N1R of Shope fibroma virus have a RING finger motif that inhibits apoptosis induced by UV light.321

Inhibition of Cytokines/Chemokines Manipulation of PRR signaling reduces production of proinflammatory cytokines. Viruses have developed additional strategies to inhibit production and effector activities of cytokines and chemokines, which may interfere with their replication.

FIG. 39.9. Viral interference with apoptotic pathways: (i) to (vii) indicate points of interactions between viral antigens and components of the pathways. Cyt c, cytochrome C.

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Interferons. IFNs and IFN-induced proteins have potent antiviral activity and are thus targeted by numerous viruses. IFN-1, upon binding to its receptor, activates Jak1 and Tyk2. This causes phosphorylation of Stat1 and Stat2, which form heterodimers, translocate to the nucleus, and associate with IRF9 to form the transcription factor complex ISGF3. This complex initiates transcription of IFN-stimulated genes (ISGs). The binding of IFN-γ to its receptor causes complex-formation between Jak1 and Jak2. This causes phosphorylation of Stat1, which forms homodimers, which then translocates to the nucleus to interact with gamma activated sequence elements in promoters. Poxviruses encode proteins that are secreted from infected cells and bind with high-affinity type I or II IFN receptors, thus preventing those neighboring, not-yet-infected cells from becoming resistant. They also produce molecules that mimic the receptors for IFNs and thus soak up IFN molecules to prevent their binding to cellular receptors.322 Rabies virus P protein blocks translocation of phosphorylated Stat1 to the nucleus, and in the nucleus binds to Stat1/2-IRF9 complexes thus preventing activation of transcription.323 SARS-CoV ORF6 binds to nuclear transport proteins thus blocking transport of Stat1.324 Stat2 is targeted for destruction by Dengue virus NS5325 or RSV NS1.326 Stat1 can be degraded by a cellular ubiquinase induced by paramyxoviruses.327 IFN induces synthesis of over 300 proteins, many of which have antiviral or immunomodulatory functions. One of those is tetherin, which can prevent the cellular egress of viruses such as lyssaviruses, KSHV, HIV-1, or Ebola virus. KSHV K5 or HIV-2 Vpu ubiquinate tetherin and thereby initiate its degradation.328 ISG15 is an IFN-induced ubiquitin-like protein, which upon binding to proteins causes their degradation through an ubiquitin-independent pathway in a process called ISGylation. As ISG15 has broad antiviral activity and increases resistance to numerous viruses, many have devised avenues to subvert its activity (341). Influenza B virus NS1 sequesters ISG15,329 Crimean Congo hemorrhagic fever virus, a member of the Bunyaviridae family, encodes proteins with de-ISGylating activity, which removes ISG15 from its target.330 MX-1 is another IFN-induced protein that in mice is critical for recovery from influenza A virus infection. A human homolog MxA has broad antiviral activity against RNA viruses and DNA viruses with an RNA intermediate.331 The functions of Mx1 or MxA are not yet fully understood, but the proteins seem to bind capsid or nucleoproteins early after infection, thus somehow blocking virus replication. A rather old-fashioned mechanism allows some influenza A viruses to escape control by Mx1; they grow so fast that they can outrun the IFN-induced production of Mx1. Other Cytokines. Parapoxvirus, which infects sheep and goats, as well as EBV, encodes an IL-10–like protein that inhibits synthesis of IL-12 and thus generation of Th1 immune responses.332,333 Similarly, the UL111A ORF of HCMV has sequence homology with IL-10.334 IL-18-binding proteins are encoded by poxviruses, such as MCV, ectromelia virus, cowpox, or vaccinia viruses.335 KSHV encodes an

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IL-6–like protein.336 The poxvirus-encoded GIF binds to both granulocyte M-CSF and IL-2.337 EBV encodes BARF1, a M-CSF–binding protein.338 Vaccinia virus encodes two proteins (ie, B15R and B18R), which bind and inhibit IL1β.339 The C10L protein of vaccinia virus encodes a protein that blocks the IL-1 receptor.340 IL-1β is synthesized as a precursor that only gains functional activity upon cleavage by caspase 1. The same caspase also cleaves the precursor molecule of IL-18. Cowpoxvirus encodes a serine protease inhibitor called crmA that inhibits the function of caspase 1.341 Several viruses encode molecules that resemble those of the mammalian TNF-receptor family. HCMV encodes UL144, which is related to HVEM.342 Shope fibroma virus and myxoma virus encode a single TNF-receptor homolog called T2,343,344 whereas orthopoxviruses encode one to three different TNF-receptor homologues called crmB, crmC, crmD, and CrmE.345 T2, crmB, and cmrD bind TNF-α and lymphotoxin A, while crmC binds only TNF- α . T2, crmC, and crmD TNF-α –mediated cell lysis. The E13 of ectromelia virus resembles CD30, another TNF-receptor family member, and can bind and inhibit CD30L signaling, which under physiologic conditions can support DC maturation.346 Chemokines Chemokines direct trafficking of leukocytes and are essential to initiate an inflammatory response by attracting a cellular inf iltrate. Chemokines are divided into four classes depending on the spacing of their first two cysteine residues (ie, C CC, CXC, and CX3C). Chemokines are referred to as ligands (L), which bind to correspondingly named receptors (R). There are a total of 27 CC chemokines, 17 CXC chemokines, 2 C chemokines, and 1 CX3C chemokine. Viruses manipulate chemokine functions by producing decoy chemokines or chemokine receptors or by secreting chemokine-binding proteins. Viral chemokines can be agonistic or antagonistic. Examples of agonists are tat of retroviruses, which binds CCR2/3,347 herpesvirus UL146, a CXCR2 binder,348 and murine CMV m131. MC148R of molluscipox virus and K4 of HHV8 are CCR antagonists.349,350 Marek disease virus produces a viral IL-8, which recruits granulocytes.351 UL146 and 147 of HCMV also encode an IL-8 like molecule.352 Herpes and poxviruses encode numerous members of the chemokine receptor family that serve as decoys for chemokines. UL33, UL78, US27, and US28 of HCMV,353 U12 and U51 of HHV6/7,354 and M78 and M33 of murine CMV show homology with CCR receptors.355 ORF74 of several herpesvirus strains mimics CXCR2.356 K2R of is a homolog of a CXCR 357 whereas MC148 protein of molluscipox virus shows similarity with CCR8.358 Poxviruses and herpesviruses produce secreted viral chemokine proteins, which prevent binding of chemokines to their receptors. MT-1 of myxoma, B29R of vaccinia virus, and a 35 kDa protein of Shope fibroma virus inhibit CC chemokines.359–361 M3 of murine gamma-herpesvirus 68 inhibits all four classes of chemokines.362 Viruses, by enhancing the effects of some chemokines while inhibiting others, presumably create a microenvironment that is best suited for their propagation. They bias the immune response so that it is only marginally harmful to

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virus-producing cells, by, for example, recruiting eosinophiles and Th2 cells and excluding Th1 cells, or by secreting IL-8, which can block the activity of type I IFN. They may also selectively recruit cells that are permissive for viral replication, such as CMV, which through production of viral IL-8 recruits neutrophils so that they can then become infected.363

Inhibition of Complement Activation The complement system is composed of more than 25 proteins that are sequentially activated through three distinct pathways called the classical, alternative, and lectin pathways. The classical pathway is activated once a pathogen or an antigen-antibody complex binds to C1q, which forms the C1 complex with C1r and C1s. Conformational changes of C1q activate C1r, which cleaves C1s into a protease, which in turn cleaves C2 and C4 into a and b subunits. C4b and C2a bind and form C3 convertase, which cleaves C3 into a and b. C3b binds the C3 convertase forming C5 convertase, which cleaves C5. C5b, C6, C7, C8, and C9 form the membrane attack complex, which initiates cell lysis. In the alternative complement pathway, C3 is spontaneously split into C3a and C3b. C3b can bind to a cell surface–expressed antigen from a pathogen and then also to factor B, which, in presence of factor D, is cleaved into Ba and Bb. The C3bBb complex can act as a C3 convertase. In the lectin pathway, mannan-binding lectin on the

cell surface binds mannose on the surface of pathogens. This activates mannan-binding lectin–associated serine protease-1 and -2, which can cleave C4 and C2 thus allowing formation of C3 convertase. The complement pathways, which can be very damaging once fully activated, are tightly controlled by regulators of complement activation, which acts at three key points. C1 inhibitors block spontaneous activation of C1 and bind C1 in serum. Decay accelerating factor (CD55) and CR1 destroy C3 convertase. C4 binding protein and factor H promote dissociation of C4 or C3 subunits from complement complexes. They can also activate factor I, which together with its cofactor CD46 cleaves C3b and C4b. Polymerization of C9 into the final membrane attack complex is blocked CD59 and homologous restriction factor. Complement can alter the structure of viruses, together with antibodies lyse viruses or virus-infected cells, and promote phagocytosis. Viruses have evolved three strategies to avoid destruction by complement: 1) they can interfere with activation of the classical pathway by shedding antigenantibody complexes from the surface of infected cells or by expressing Fc-receptor–like structures that complex antibodies that are bound to viral surface proteins; 2) they can encode complement inhibitors; and 3) they can incorporate host-derived complement inhibitors into their envelope (Fig. 39.10).

FIG. 39.10. Virus-Mediated Inhibition of Complement Pathways. The classical complement pathway is shown in green, the alternative complement pathway is shown in blue, and the lectin pathway is shown in pink; physiological inhibitors are shown in orange; Ig, virus-specific antibody. (i) to (vii) indicate compenents of the pathway that are inhibited or enhanced by viral antigens.

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Herpesviruses and coronaviruses avoid recognition of antigen-antibody complexes by shedding such complexes from the surface of infected cells, by internalizing them, or by expressing Fc-receptor–like molecules. More specifically, gB and gD of pseudorabies virus are internalized once they bind antibodies.364 The gE and gI of a number of herpesviruses,365 the gE of varicella zoster virrs,366 and the spike protein of some coronaviruses367 resemble mammalian Fc receptors that nonspecifically bind Igs, thus sterically hindering binding of antibodies to their antigen on infected cells. Fc receptors present on an infected cell force antibodies bound with their variable region to an antigen to loop over and bind with their constant region to the Fc receptors, which prevents complement activation. Viral complement inhibitors interfere with complement activation. HSV-1, HSV-2,368 and herpesvirus saimiri369 encode gC1, gC2, or CCPH, respectively, which bind C3b and inhibits formation or accelerates decay of C3 convertases. VCP of vaccinia virus370 and SPICE of smallpoxvirus371 bind C4b and C3b and serve as cofactors for factor I. Enveloped viruses bud through the cell membrane at lipid rafts that are rich in cholesterol and other lipids and also contain glycosyl phosphatidyl inositol–anchored complement control proteins such as CD46, CD55, and CD59, which are incorporated into the membrane of the extracellular enveloped form of poxviruses372 and HIV-1.373 Furthermore, some viruses directly increase numbers of complement regulatory proteins; HCMV augments cellular expression of CD55 and CD46,374 while herpesvirus saimiri encodes a CD59 homolog.375

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Modulation of Natural Killer Cell Activity Natural killer cells have antiviral functions through secretion IFN, which also promotes adaptive immune responses, and they can lyse virus-infected cells directly, especially if these cells downregulate MHC class I expression or if they bind antibody-dependent cellular cytotoxicity–inducing antibodies. Viruses thus evolved to kill natural killer cells, strengthen signals through the natural killer cell’s inhibitory receptors, or weaken those through activating receptors (Fig. 39.11). Influenza virus can infect natural killer cells and induce their apoptosis.376 HCV E2 uses CD81, which is expressed on natural killer cells, as a receptor and attenuates its signaling.377 Herpesviruses reduce MHC class I expression, which would allow for activation of natural killer cells through loss of inhibitory signals. Herpesviruses solve this problem by ensuring expression of HLA-E, an MHC class I–like molecule that only presents the leader sequence of other MHC class I molecules and as such is not recognized by traditional CD8 + T cells, but by inhibitory receptors on natural killer cells. The signal sequence of UL40, an HCMV-encoded protein, carries a sequence that associates with HLA-E, thus allowing its translocation to the cell surface.378 In addition, UL18 of HCMV is an MHC class I analog and binds inhibitory ILT2 (LIR1), but not the TCR of CD8 + T cells.379 Flaviviruses take the opposite approach and augment MHC class I expression.380 Cellular stress responses can lead to expression of MHC class I chain-related genes, such as MICA/MICB and ULBP proteins in humans, and Rae-1, a likely RNA export protein,

FIG. 39.11. Subversion of Natural Killer Cell Responses by Viruses. Activating receptors are shown in pink, inhibitory receptors in blue, and ligands in green and orange, respectively. (i) to (vi) indicate interactions with viral antigens and components of natural killer cells or their ligands.

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H60, a minor histocompatibility antigen, and mouse UL16binding protein-like transcript 1, an MHC class I–like molecule, which can be spotted by activating natural killer cell receptors, especially NKG2D.381,382 Both HCMV and murine CMV encode proteins that downregulate the cellular ligands for NKG2D. Murine CMV proteins m145, m152, and m155 interfere with the expression of all known NKG2D ligands, whereas m138 affects the expression of mouse UL16-binding protein-like transcript 1 and H60. HCMV UL16 causes intracellular retention of MICB and ULBP, UL142 downregulates MICA, micro-RNA UL122 interferes with the translation of MICB, as does the EBV encoded micro-RNA BART2-5b, and the KSHV encoded micro-RNA K12-7. KSHV also produces K5, which causes ubiquitination of MICA and MICB. HIV-1 nef down-regulates expression of MICA and ULBP.383 Orthopoxviruses produce a secreted NKG2D ligand thus saturating the receptor.384 Ly49s are inhibitory receptors that recognize MHC class I molecules. MCMV encodes two proteins, m157 and m04, that can serve as ligands for inhibitory Ly49 receptors, specifically in murine CMV–resistant strains of mice; m157 is a ligand for the natural killer–activating Ly49H receptor; in susceptible strains of mice it binds the inhibitory Ly49A receptor.385 NCR1-encoded NKp46 recognizes the HA of influenza virus. NKp46 and NKp30 receptors associate with the CD3 ζ-chain for signaling. Free HA can induce lysosomal ζ chain degradation thus blocking signaling.386 HCMV UL83 uses a similar pathway and dissociates the ζ. chain to block its recognition by NKp30.387 Viruses not only increase the strength of inhibitory signals to natural killer cells, but also interfere with activating receptors. K5 of KSHV downregulates AICL, a target for NKp80.388 UL141 of HCMV sequesters CD155, the poliovirus receptor that is a target for DNAM-1.389 UL16 of HCMV reduces expression of LFA-3, an adhesion molecule that binds to CD2 on natural killer cells and by unknown mechanisms increases the infected cells’s resistance to natural killer cell-mediated lysis.390

Escape from Adaptive Immune Responses Subversion of Antigen Presentation by Major Histocompatibility Complex Class II Stimulation of CD4 + T cells, which requires presentation of epitopes on MHC class II, can be reduced by limiting the amount of antigen available for processing and presentation of by decreasing expression of MHC class II molecules on the cell surface. Autophagy serves the cells to eliminate unwanted proteins from the cytoplasm by delivering them to lysosomes for degradation. This pathway is also used to destroy viruses and deliver viral peptides to compartments, where they associate with MHC class II molecules. The importance of autophagy in antiviral defense is underscored by its induction through signals from viral sensors, such as TLR4. Viruses, as described previously, interfere with such signals. Alpha- and gamma-herpesviruses through ICP34.5 and viral BcL-2, respectively, can directly block Beclin-1, a molecule needed for formation of autophagosomes.391,392

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Herpesviruses, poxviruses, and HCV interfere with MHC class II expression. For example, gB of HSV-1 binds HLA-DR complexes in a post-Golgi department and then shuttles them into exosomes rather than allowing for their transfer to the cell surface.393 A mutation of the precore protein of HBV has been linked to decreased levels of MHC class II on antigen-presenting cells, suggesting a potential evasion pathway,394 and a variant in the HCV NS3 proteins has been shown to serve as an epitope antagonist by binding MHC class II molecules and blocking binding of the CD4 + T-cell epitope.395 HCMV can impair MHC class II expression through two pathways. MHC class II transcription is tightly regulated, mainly through the class II transactivator (CIITA), a non–DNA-binding protein that interacts with transcription factors, which then associate with the MHC class II promoter. CIITA is rate-limiting factor for MHC class II production and thus determines the expression pattern of these molecules. Four promoters, two of which sponsor constitutive expression of CIITA in antigen-presenting cells, regulate CIITA production. One is activated by IFN-γ through the Jak/Stat1 pathway. Proteins of HCMV inhibit induction of CIITA by disrupting this pathway.396

Subversion of Antigen Presentation by Major Histocompatibility Complex Class I Many viruses subvert presentation of their antigens by the MHC class I pathway, thus reducing activation and effector functions of antigen-specific CD8 + T cells, again stressing the importance of this cell subset for antiviral defense. Nearly every step of the MHC class I presentation pathway can be affected, and some viruses encode multiple proteins that act at different levels of the MHC class I processing/ presentation pathway (see Fig. 39.12). Major Histocompatibility Complex Class I Synthesis. Viral gene products such as tat of HIV-1 can suppress transcription of MHC class I genes.397 Protein Degradation. EBNA-1 of EBV and LANA1 of KSHV carry leucine-alanine repeats that appear to block their degradation by proteasomes.398 Once proteins are degraded, the aminopeptidases CD10 and CD13 trim them to a size that allows their binding to the groove of MHC class I molecules. HCMV blocks synthesis of CD10 and retains CD13 within the ER.399 Peptide Transport. Loading of MHC class I molecules depends of active transport of peptides from the cytoplasm to the ER with the help of TAP. The E7 of HPV-18 and the E1A protein of adenovirus human serotype 12 inhibit the promoter controlling TAP production. EBV encodes a viral IL-10 receptor homolog that reduces expression of TAP. The mK3 protein of mouse gamma-herpesviruses binds to TAP and tapasin resulting in their ubiquination and degradation. The 19K polypeptide encoded by the E3 domain of adenoviruses binds to TAP and prevents association between TAP and tapasin. US6, a protein encoded by HCMV, binds to TAP and prevents binding of ATP, which is needed for peptide transport. The ICP47 polypeptide from HSV inhibits TAP by blocking its peptide binding. EBV encodes

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FIG. 39.12. Inhibition of Major Histocompatibility Complex Class I Processing and Presentation of Endogenously Produced Proteins by Viral Antigens. (i) to (xi) indicate at what points the pathway is attacked by viral antigens.

a protein that weakly binds the IL-10 receptor. Similar to cellular IL-10, the EBV protein reduces expression of TAP.400 BNLF2a, a lytic phase protein of EBV is anchored by its C-terminal tail to the ER membrane, while its cytosolic N terminus inhibits TAP.401 Major Histocompatibility Complex Class I Peptide Loading. Loading of peptides to MHC class I is supported by tapasin, which stabilizes the molecule and prevents binding of self-peptides. US3 of HCMV binds and inhibits tapasin.401 E3-19K of adenovirus blocks the formation of the TAPtapasin complex.402 Major Histocompatibility Complex Class I Transport. Once MHC class I molecules have bound peptides, they are transported to the cell surface. A number of viral products interfere with this transport by either retaining MHC

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class I molecules within the ER, by inducing their export into the cytoplasm, or by interfering with passage through the Golgi apparatus. The E3-19K protein of adenovirus402 and proteins of HCMV and murine CMV prevent the exit of MHC class I from the ER and from pre-Golgi compartments.403 US11 and US2 of HCMV dislocate the MHC class I molecules from the ER to the cytoplasm, where they are rapidly degraded. The m6/gp48 protein of MCMV binds to MHC class I-β2-microglobulin in the ER and during transport to the cell surface, targets the MHC class I complexes to lysosomes, where they are proteolytically digested.404 Nef of HIV-1 reduces cell-surface expression of MHC class I by redirecting the molecules from the trans-Golgi network to the endosomes.405 The K3 and K4 proteins of HHV8 lower expression of cell surface MHC class I molecules by enhancing their endocytosis into clathrin-coated pits.406

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Escape from Antibodies To summarize, viruses escape antibody-mediated neutralization mainly by mutations; they evade destruction by antibody and complement by interfering with activation of complement, and they dodge antibody-dependent cellular cytotoxicity by masking antigen display on cell surfaces and by inhibiting natural killer cell functions. All of these pathways have been described previously. EBV directly subverts B cells, where it resides during latency and where it can be transformed through the viral oncoprotein LMP1, a molecule that has functional homology with CD40 and can activate the NF-κ B pathway. Unlike CD40 signaling, which requires ligation with CD40L, LMP1 is constitutively active, although one would assume that it is negatively regulated by yet unknown pathways as it would otherwise inevitably lead to B-cell transformation.

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CONCLUSION Viruses continue to raise havoc. Each year, millions of humans die prematurely due to viral infections and many more become temporarily incapacitated. Vaccines licensed for a handful of viruses and antiviral drugs available for even fewer have lessened the impact of some viral infections, but for most the immune system remains our only defense. Our knowledge in basic immunology has exploded over that last century. Although early vaccines were developed without knowing anything about immunology, but as it is stated in the “Míshlê Shlomoh,” the Book of Proverbs: ‘scientia potentia est,” knowledge is power. A better understanding of the intricate pathways that regulate the interactions between viruses and their hosts’ immune system should empower us to eventually win this eternal battle and lessen human suffering caused by something as small but as deadly as a bit of invasive genome with a few proteins around it.

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40

Immunity to Intracellular Bacteria Anca Dorhoi • Stephen T. Reece • Stefan H. E. Kaufmann

INTRODUCTION This chapter focuses on infections by intracellular bacteria with emphasis on both pathogenicity and general immune mechanisms underlying protection. Intracellular bacteria comprise numerous pathogens, some of which are of utmost medical importance. Ancient (but still existent), as well as newly emerging, diseases are caused by intracellular bacteria, with tuberculosis (TB), typhoid, leprosy, and trachoma as the most relevant etiologically associated infections. Intracellular bacteria live inside host cells for most of their lives. This coexistence must allow survival of the infected cell; therefore, intracellular bacteria generally lack toxicity. Instead, they have evolved multiple strategies to interfere with key host cell biologic processes to promote replication or persistence. This has direct consequences for the immune response evoked by the host attempting to resolve intracellular infection. In fact, acquired resistance against intracellular bacterial infections depends on activation of multiple arms of the immune system. In this chapter, we start by examining the complex molecular crosstalk between pathogen and host. We then consider how these molecular events translate into the development of adaptive immunity. Finally, we examine effects of both adaptive and innate immune mechanisms on long-term effects of intracellular bacterial infection. Such long-term effects include tissue remodelling, nonresolved inflammation, and chronic immune responses, which for the most part are tolerated by the host. We hope this chapter will stimulate an interest in intracellular infection and the unique immunologic insights it can provide.

and listeriae1,2) or secretion apparatus (type III secretion system [T3SS], type IV secretion system (T4SS), type VII secretion system [T7SS] in Salmonella,3 Legionella,4 and Mycobacteria,5 respectively), and metabolic characteristics that dictate replication time (fast- versus slow-growing, Salmonella versus Mycobacterium). This diversity is reflected most often in differences related to cell biology of the pathogen. However, major patterns of the immune response are shared, as detailed in the following. Of paramount significance for humans are Mycobacterium tuberculosis, Mycobacterium leprae, Salmonella enterica serovar Typhi, Chlamydia trachomatis, and the etiologic agents of TB, leprosy, typhoid, and trachoma, respectively, which together afflict more than 200 million people globally. Some opportunistic pathogens such as Mycobacterium avium/Mycobacterium intracellulare are gaining increasing significance particularly for immunodeficient patients, such as acquired immunodeficiency syndrome (AIDS) sufferers. Many zoonotic agents are intracellular bacteria and include Chlamydia psittaci (psittacosis), Brucella (brucellosis, Malta fever/Bang disease), Coxiella burnetii (Q fever), Rickettsia (Rocky Mountain/Mediterranean spotted fever), Francisella tularensis (tularemia), Burkholderia mallei (glanders), Yersinia pseudotuberculosis (yersiniosis), Yersinia pestis (plague), Bartonella (cat scratch disease), and Listeria monocytogenes (listeriosis). Other opportunistic pathogens with an intracellular lifestyle that are relevant for human health include Legionella pneumophila (Legionnaires disease) and Ehrlichia (ehrlichiosis).

Epidemiology and Pathogenesis

FEATURES OF INFECTIONS WITH INTRACELLULAR BACTERIA Intracellular Bacteria/Public Health Relevance Bacterial pathogens are prokaryotic microorganisms that cause disease in a given host species. They are single cells, typically micrometers in length, and present a specific spectrum of interactions with their human host. Infectious disease is the direct and invariable consequence of an encounter between host and pathogen. Often, it is the eventual outcome of complex interactions between them. There is a marked degree of diversity among bacterial species that are able to induce intracellular infections. This encompasses structural organization of the cell wall (ie, gram-negative versus -positive, salmonellae versus listeriae), presence of motility organelles (eg, fimbriae and flagella, of salmonellae

Some intracellular bacteria, in particular Rickettsia, are introduced directly into the bloodstream by insect bites from where they have ready access to internal tissues. Most intracellular bacteria, however, enter the host through the mucosa.6 Major ports of entry are the lung for airborne pathogens, such as M. tuberculosis and L. pneumophila, and the intestine for foodborne pathogens, such as S. enterica and L. monocytogenes. Subsequently, intracellular bacteria pass through the epithelial layers. Either they actively induce transcytosis (ie, endo- and exocytosis) through the epithelial cells or they are passively translocated within phagocytes. Bacteria may be removed by nonspecific defense mechanisms such as mucociliary movements and gut peristalsis, or they may be destroyed by professional phagocytes without necessitating the specific attention of the immune system. Cells that survive these nonspecific defense reactions

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BOX 40.1. CENTRAL FEATURES OF INFECTIONS WITH INTRACELLULAR BACTERIA • • •

•

Infection is separated from disease, and the immune response is already induced at the stage of infection. Infection persists latently in the face of dynamic interactions between pathogen and immune mechanisms. The host–pathogen relationship represents a highly sophisticated form of parasitism that does not necessarily lead to disease but rather allows for long-lasting coexistence. Infection includes the potential to harm the host severely at a later stage, and pathogenesis is strongly influenced by the immune response.

colonize deeper tissue sites and stably infect a suitable niche. At this stage, the host generally develops a specific acquired immune response. Infection is unsuccessful when the immune system succeeds in eliminating the pathogen before overt clinical disease develops. Alternatively, tissue damage increases to a significant level before the immune system succeeds in controlling the pathogen effectively and clinical disease develops. This is the case with many extracellular bacteria and is due to their cell lytic strategies (toxins) that cause diseases of acute type, but is less common for intracellular bacteria. Finally, it is possible that the immune response restrains the infectious agent but fails to completely eradicate it. Under these conditions, a long-lasting equilibrium between microbial persistence and the immune response unfolds. This balance, however, remains unstable and can be tipped in favor of the pathogen at a later time, converting infection into disease. The time lapse between host entry and expression of clinical disease is termed incubation time and from what has been said previously, it follows that in many intracellular bacterial infections the incubation times are long-lasting to lifelong. By improving the immune response or by impairing bacterial growth (typically accomplished by chemotherapy), or both, disease can be overcome. Ideally, bacterial eradication is achieved; alternatively, some dormant bacteria continue to persist in niches poorly accessible to the immune response (Box 40.1). There is a close correlation between the cell biology and cell tropism of the pathogen and disease signs and pathology in, for instance, enteric versus lung conditions. However, even bacteria that use the same niches as habitat show major clinical differences. This is due to pathogenintrinsic biology (ie, metabolism) and peculiarities of each species to subvert host elimination. For instance, M. tuberculosis, L. pneumophila, F. tularensis, and C. burnetii cause aerogenic infections with Coxiella being the most successful in establishing infection (one bacterium is sufficient to induce disease, probably the most infectious bacterium). These pathogens successfully infect lung macrophages and parenchymal cells to establish infection. However, TB has

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a protracted course and insidious clinical signs while francisellae and coxiellae induce acute and subacute pneumonias with tissue changes substantially different from TB. Disease pathogenesis is principally the outcome of the crosstalk between host and pathogen at the cellular level with strong influences from bacteria-specific behavior, while clinical signs and pathology are often induced by host responses.

“Idealized” Intracellular Bacterium Although this chapter focuses on general mechanisms underlying immunity to intracellular bacteria, it is important to emphasize that this group is extremely heterogeneous. Therefore, the major hallmarks of intracellular bacterial infections will first be described for a nonexistent “idealized” intracellular bacterium (Table 40.1). Subsequently, characteristics of selected intracellular bacteria will be specified.

Facultative and Obligate Intracellular Bacteria With respect to preferred habitat, intracellular bacteria can be divided into two groups: First, those pathogens that do not essentially depend on the intracellular habitat, including M. tuberculosis, Mycobacterium bovis, M. leprae, S. enterica, Brucella, L. pneumophila, L. monocytogenes, and F. tularensis (Table 40.2).7–15 Although these pathogens

TABLE Hallmark 1 Hallmark 2 Hallmark 3

Hallmark 4

Hallmark 5

Hallmark 6

40.1

Hallmarks of Intracellular Bacterial Infections

The intracellular lifestyle represents the distinguishing feature of intracellular bacteria. T cells are the central mediators of protection against intracellular bacterial infections. In contrast, antibodies play a facilitating role. Infections with intracellular bacteria are accompanied by delayed-type hypersensitivity, which expresses itself after local administration of soluble antigens as a delayed tissue reaction mediated by T cells and effected by macrophages. Tissue reactions against intracellular bacteria are granulomatous. Protection against, as well as pathology caused by, intracellular bacteria are centered on these lesions. Rupture of a granuloma promotes bacterial dissemination. Intracellular bacteria express little or no toxicity for host cells by themselves, and pathology is primarily a result of immune reactions, particularly those mediated by T-lymphocytes. Intracellular bacteria coexist with their cellular habitat for long periods of time. A labile balance develops between persistent infection and protective immunity, resulting in long incubation time and in chronic disease. Accordingly, infection is dissociated from disease.

Hallmarks 1 to 4 should be considered essential, and Hallmarks 5 and 6 conditional, criteria for defining intracellular bacteria.

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40.2

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Major Infections of Humans Caused by Facultative Intracellular Bacteria

Pathogen

Disease

Preferred Target Cell

Preferred Location in Host Cell

Preferred Port of Entry

Mycobacterium tuberculosis Mycobacterium leprae

Tuberculosis

Macrophages

Early phagosome

Lung

Leprosy

Macrophages, Schwann cells, other cells Macrophages Macrophages

Phagolysosome, cytosol

Nasopharyngeal mucosa

Autophagosome Late phagosome, spacious phagosome Cytosol Early phagosome Endoplasmic reticulum–derived phagosome Cytosol

Gut, skin Gut

Yersinia pestis Salmonella enterica serovar Typhi Shigella spp. Brucella spp. Legionella spp.

Plague Typhoid fever

Listeria monocytogenes

Shigellosis Brucellosis Legionnaires’ disease Listeriosis

Francisella tularensis

Tularemia

Macrophages Macrophages Macrophages Macrophages, hepatocytes Macrophages

favor mononuclear phagocytes (MPs) as their habitat, other types of host cells are infected as well. M. leprae, for example, lives in numerous host cell types, notably in Schwann cells and hepatocytes serve as an important reservoir for L. monocytogenes. Although M. tuberculosis can infect a variety of mammalian cells in vitro, in vivo it seems to restrict itself to phagocytes and perhaps epithelial cells. The second group includes so-called obligate intracellular bacteria, which fail to survive outside host cells. Most of these bacteria prefer nonprofessional phagocytes as their habitat—for example, endothelial and epithelial cells. Rickettsiae, chlamydiae, and ehrlichiae are representatives of this group. They include Rickettsia prowazekii, Rickettsia

TABLE

40.3

Late endosome, cytosol

Gut Skin, lung, mucosa

rickettsii, Rickettsia typhi, and Orientia tsutsugamushi (Rickettsia tsutsugamushi until 1995), the etiologic agents of louse-borne typhus, Rocky Mountain spotted fever, typhus, and scrub typhus, respectively.16–19 Various biovars of Chlamydia trachomatis, which are responsible for trachoma,20 conjunctivitis, urogenital infections, and lymphogranuloma venerum,21 as well as C. psittaci and Chlamydia pneumoniae, causative agents of psittacosis or rare types of pneumonia,22 respectively, also belong to this group (Table 40.3). Certain obligate intracellular bacteria, such as Ehrlichia and Anaplasma phagocytophilum, parasitize blood cells.23,24 C. burnetii, the causative agent of Q fever, resides in macrophages and lung parenchymal cells.25,26

Major Infections of Humans Caused by Obligate Intracellular Bacteria

Pathogen

Disease

Preferred Target Cell

Rickettsia rickettsii

Rocky Mountain spotted fever Endemic typhus Typhus Scrub typhus Q fever

Endothelial cells, smooth muscle cells Endothelial cells Endothelial cells Endothelial cells Macrophages, lung parenchymal cells Endothelial cells

Rickettsia prowazekii Rickettsia typhi Rickettsia tsutsugamushi Coxiella burnetii Chlamydia trachomatis

Chlamydia psittaci

Chlamydia pneumoniae Ehrlichia ewigii Ehrlichia chaffeensis Anaplasma phygocytophilum

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Gut Mucosa Lung

Urogenital infection, conjunctivitis, trachoma, lymphogranuloma, venereum (different serovars) Psittacosis

Pneumonia Ehrlichiosis Ehrlichiosis Anaplasmosis

Macrophages, lung parenchymal cells

Preferred Location in Host Cell

Preferred Port of Entry

Cytosol

Blood vessel (tick bite)

Cytosol Cytosol Cytosol Late phagosome

Broken skin, mucosa Blood vessel (flea bite) Blood vessel (mite bite) Lung

Phagosome/ nonacidified inclusion

Eye, urogenital mucosa

Phagosome/ nonacidified inclusion Lung parenchymal cells Phagosome Granulocytes Cytosol Monocytes, macrophages Cytosol Granulocytes Cytosol

Lung

Lung Blood vessel (tick bite) Blood vessel (tick bite) Blood vessel (tick bite)

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Preferential living in macrophages does not depend on specific invasion mechanisms but rather on highly sophisticated intracellular survival strategies. Yet, most facultative intracellular bacteria express unique invasion factors, if only to cross epithelial layers. Selection of nonprofessional phagocytes as habitat essentially depends on invasion molecules whereas survival inside these cells is generally less hazardous.

CELL BIOLOGY For intracellular bacteria, entry into host cells represents the central requirement for survival in, as well as elimination by, the host. Host cell–directed uptake, called phagocytosis, is a feature of the so-called professional phagocytes that comprise polymorphonuclear granulocytes (PNGs) and MPs. Examples of bacteria that are engulfed by phagocytosis include M. tuberculosis, L. pneumophila, and C. burnetii. Entry induced by the pathogen is termed invasion, which allows entry into nonphagocytic cells (nonprofessional phagocytes). Salmonellae, shigellae, and listeriae are paradigms of enteroinvasive pathogens.27 Contact between host cells and pathogens proceeds either directly via receptor–ligand interactions or indirectly via deposition on the surface of the pathogen of host molecules for which physiologic receptors exist on the target cell. Depending on the cellular target, the final outcome of host cell entry varies markedly. 1. Nonprofessional phagocytes are nonphagocytic, and hence entry depends on expression of surface receptors that can be exploited for invasion. Because of their low antibacterial activities, they primarily serve as a habitat. 2. PNGs are short-lived. Because they are highly phagocytic and express potent antibacterial activities constitutively, uptake by PNGs is often fatal for the pathogen. 3. MPs are phagocytic and express medium to high antibacterial activities depending on their activation status. Accordingly, they serve both as habitat and as effector cell. Following entry, bacterial pathogens begin intravacuolar life.28 Two main strategies are followed to avoid killing: 1) avoidance of delivery to degradative lysosomes, either by blocking phagosome maturation, divergence from the endocytic pathway to establish a vacuole with unique features, or by escape into the cytosol; and 2) development of strategies to survive within acidic degradative organelles. Certain bacteria have developed mechanisms that allow them to impede nutrient flow inside the infected cell for their own benefit, to modulate generation of antimicrobial molecules, or to alter cell death pathways.29,30 In the following, the major steps from uptake to bacterial elimination by, or survival in, host cells will be described (Fig. 40.1).

Adhesion and Invasion Adhesion to mammalian cells is a prerequisite for extracellular colonization and for host cell invasion. Bacterial adhesins that solely expedite adhesion to host cells are expressed by numerous extracellular bacteria. In contrast, invasion-inducing molecules are a feature of bacteria that

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permanently or transiently enter host cells. Adherence to the cell membrane is based on protein–protein interactions mediated by adhesins, such as internalins (bind E-cadherin) of L. monocytogens or invasins (bind β1 integrins) from Yersinia. Adhesins may be located on the bacterial surface or on pili. In addition, adhesion may be induced by bacterial virulence factors, which recruit fibronectin to attach to host cells by binding to integrins.29,31 Although induced by the bacterium, invasion is ultimately a function of the host cell. Following adhesion, invasion can be induced in two ways. First, cell signaling by host cell receptors that serve as targets of adhesion induces uptake; second, uptake is induced independently from the molecules that mediate adhesion.31,32 The term “zipper mechanism” refers to the first process. Bacterial proteins interact with host cell surface proteins to mediate internalization. This term has been suggested for the highly selective receptor-mediated bacterial entry, whereas the term “trigger mechanism” has been proposed for indiscriminate, apparently adhesion-independent uptake. Bacterial effectors are delivered to the host cytosol via a secretion system to induce bacterial entry.27

Entry by Zipper Mechanisms Host cell invasion by Yersinia and L. monocytogenes are examples of invasion via the “zipper mechanism.” Receptor binding induces phagocytic mechanisms in nonprofessional phagocytes similar to those that are constitutively operative in MPs. The eukaryotic cell membrane tightly enwraps the bacterium and a cascade of events, including protein phosphorylation, ubiquitination, and phospholipid modifications then contribute to vacuole genesis.28 Host entry of L. monocytogenes through the intestinal epithelia is mediated by internalin on the surface of this pathogen and E-cadherin on human epithelial cells.33 Murine E-cadherin does not serve as a receptor for internalin due to an amino acid substitution in position 16.34 Schwann cells, a major target of M. leprae, are shielded by a basal lamina composed of laminin, collagen, and proteoglycans. The unique tropism of M. leprae for peripheral nerves appears to be due to bacterial binding to laminin. This molecule, which serves as natural ligand for integrins, thus provides a link between pathogen and Schwann cell.35 Caveolin and lipid rafts serve as entry portals for Brucella and certain strains of Chlamydia. More recently, evidence was provided that clathrin-mediated endocytosis contributes to entry of L. monocytogenes and Rickettsia in a similar zipper mechanism.36,37 Septins, which are small guanine triphosphatases (GTPases), are able to form fi laments and interact with actin to facilitate bacterial entry as well.38

Entry by Trigger Mechanisms Different molecules and mechanisms participate in host cell entry by S. enterica. Interactions between S. enterica and host cells causes large “membrane ruffling” at the site of attachment followed by bacterial entry. Ruffling induces indiscriminate uptake even of other particles in the vicinity of S. enterica. This process has been termed macropinocytosis. S. enterica triggers its uptake by exploiting the signaling machinery of

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FIG. 40.1. The Multiple Encounters Between Phagocytes and Intracellular Bacteria. Bacterial uptake is both pathogen-induced and receptormediated. Multiple opsonic receptors, including complement receptor (CR) and receptors for immunoglobulins (FcγR), facilitate phagocytosis. In addition, certain pattern recognition receptors (PRRs) may contribute to bacterial internalization. Macrophages and neutrophils direct their bacterial cargo to the endosomal pathway, endosomes become acidic and progressively accumulate reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs). The phagosome maturation process culminates in fusion between late endosomes and lysosomes, which leads to bacterial killing but intracellular bacteria can interfere with phagosomal killing. Certain bacteria modulate early uptake events to prevent endosomal maturation. Other species prevent acidification of the phagosome or nullify ROIs and RNIs. Escape from the phagosome into the host cytosol is a strategy used by pathogenic bacteria and can occur at multiple stages of phagosome maturation. Certain bacteria exploit PRRs to enable self-replication in modified endosomes or modulate iron (Fe) abundance. Inhibition of phagolysosome formation, apoptosis, and autophagy also contribute to establishment of infection with intracellular bacteria. DAMP, danger-associated molecular pattern; NADPH, nicotinamide adenine dinucleotide phosphate-oxidase.

the host cell, thus inducing cytoskeletal rearrangements. In certain mouse cells, S. enterica induces phosphorylation of the receptor for the epidermal growth factor.39 Yet, S. enterica can also enter cells that do not express the epidermal growth factor receptor. This pathogen possesses two T3SS that allow it to directly manipulate intracellular molecules within host cells.40 Salmonella outer proteins are secreted into the host cells rapidly after contact. Salmonella outer proteins activate the small GTP-binding protein cell division control (CDC)42 of the Ras superfamily, which, in turn, induces the reorganization of the actin cytoskeleton, thus promoting bacterial invasion through membrane ruffling. A homolog of Salmonella outer protein (termed Salmonella outer protein 2) performs similar functions, and hence the two molecules may partly

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compensate each other’s functions. The transiently intracellular pathogen Shigella utilizes similar mechanisms for uptake via membrane ruffling.41,42 More recently, filopodia were shown to trap and direct shigellae to target cells in a process that involves bacterial T3SS.43 Generally, modulators of the eukaryotic cell cytoskeleton are T3SS or T4SS products, which modulate GTPase cycling of proteins of the Rho, Rab, and Arf families.44

Invasion of Nonprofessional Phagocytes Microbe-directed uptake allows entry into nonphagocytic cells and hence can be seen as an evasion mechanism of phagocytosis by professional phagocytes. The target spectrum of intracellular bacteria ranges from very broad to

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highly specific. M. leprae is found in a large variety of host cells and hence shows a broad target cell spectrum. L. monocytogenes enters the host through the gut epithelium and its major target besides MPs is the hepatocyte; M. tuberculosis is almost, if not exclusively, restricted to MPs, although pneumocytes have been proposed as a safe niche in the lung. It is noteworthy that intracellular bacteria are often capable of entering a variety of cell lines in vitro. These in vitro experiments do not necessarily reflect an in vivo situation, and care should be taken in extrapolating conclusions from them. For obligate intracellular bacteria, nonprofessional phagocytes rather than MPs represent the preferred habitat. These bacteria are primarily found in endothelial and epithelial cells.17,21

Recognition and Downstream Events Microbes are composed of various molecules that are structurally different from host cell/tissue composition (ie, lipopolysaccharide [LPS], peptidoglycan [PGN], cytosinphosphatidyl-guanosin [CpG] deoxyribonucleic acid [DNA]). These microbe-specific molecules encompass various biochemical entities and have been named pathogenassociated molecular patterns (PAMPs).45 Once pathogens assault host tissues, PAMPs are recognized by pattern recognition receptors (PRRs). PRRs are nonclonally distributed on various cell types; they are germline encoded and their activation, following PAMP ligation, is an essential event for initiation of the immune response and disposal of the intruder.46–49 Microbial ligand diversity parallels the receptor repertoire, and currently there are several classes of PRRs known (Fig. 40.2), which may be classified based on chemical structure as detailed in the following: • Toll-like receptors (TLRs) are type-I transmembrane proteins. Currently, 10 TLR members in humans and 12 in mice have been described.50 TLRs 1, 2, 4, 5, and 6 are cell surface–associated, and TLRs 3, 7, 8, and 9 are associated with vesicles of the endoplasmic reticulum (ER), endosomes, and lysosomes (see Fig. 40.2).51 TLRs are signaling receptors and recruit single or a combination of toll-interleukin (IL)-1 receptor–resistance (TIR)-containing adaptors, including myeloid differentiation primary response gene 88 (MyD88), TIR domain–conining adaptor protein, TIR domain– containing adaptor-inducing interferon-beta (IFN-β, and TIR domain–containing adaptor-inducing IFNβ-related adaptor molecules.46 Timing (sequential versus simultaneously) and space (vacuole characteristics, early versus late phagosome) are essential for TLR downstream effects. TLR-4 senses LPS and thus detects extracellular gram-negative bacteria. TLR-4 first initiates nuclear factor kappa-beta (NF-κB) and secondly IFN I pathway activation via MyD88 and TIR domain–containing adaptor-inducing IFN-β, respectively. Once bacteria are phagocytosed, TLR-9 senses CpG DNA and signals via MyD88 to NF-κB in early endosomes while in lysosomes Traf3/IRF3 is assembled to enable IFN I responses.50 TLR activation may impact the microbicidal capacity of the infected cell.52

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• C-type lectin receptors (CLRs) belong to a large superfamily of membrane or soluble proteins, which have one or more calcium-dependent carbohydrate-binding lectin domains (see Fig. 40.2).53,54 CLRs have high avidity for carbohydrates/glycans. CLRs mediate endocytic uptake, a feature distinct from that of TLRs. Signaling is mediated by immune-receptor tyrosine-based activation motif/spleen tyrosine kinase (Dectins; macrophage-inducible C-type lectin, Mincle), hemITAM (Dectin-1), immune-receptor tyrosine-based inhibition motif, and the kinase Raf1 (Dectin-1, dendritic cell [DC]-specific intercellular adhesion molecule-3grabbing nonintegrin [SIGN]) to activate mitogenactivated protein kinase (MAPK) and NF-κB. CLRs are involved in generation of reactive oxygen species via the syk pathway in myeloid cells, modulate adaptive immune responses, and seem to be indispensable elements for T helper (Th)17 responses. Other CLR, notably DC-SIGN, induce inhibitory signals. • Nucleotide oligomerization domain (NOD)-like receptors (NLRs) are cytosolic proteins (see Fig. 40.2). This family comprises more than 20 members, which are structurally diverse and are involved in pathogen sensing (NOD1, NOD2) and initiation of the inflammatory response (nucleotide-binding domain and leucine-rich repeat with pyrin domain-containing [NLRP]1, NLRP3, nucleotide-binding domain with leucine rich repeat and caspase recruitment domain [NLRC]4; NLRC5).55–57 NOD1 and 2 use the receptorinteracting protein 2 adaptor to signal and are involved in PGN recognition. Thus, they are essential for sensing cytosolic bacteria in professional and nonprofessional phagocytes. NRLP and NLRC members, as well as NOD2, are essential components of inflammasomes, which are cytosolic platforms responsible for secretion of IL-1β, as detailed later. • Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are cytosolic ribonucleic acid helicases (RIG-I, MDA5, and LGP2) involved in antiviral immunity.58 • Scavenger receptors (SRs) are cell membrane proteins that recognize modified host molecules, as well as microbial structures.59 These receptors are mainly involved in uptake of particles including intracellular bacteria (eg, macrophage receptor with collagenous structure, MARCO; SR-A, cluster of differentiation [CD]36) (see Fig. 40.2). • Pyrin- and HIN-containing proteins (PYHINs) have been recently associated with pathogen sensing (see Fig. 40.2). Absent in melanoma 2 (AIM2) and gammaIFN-inducible protein 16 are the most prominent receptors involved in DNA sensing and consequently modulation of IFN secretion and inflammation.60 AIM2 is associated with inflammasome activation and senses host and bacterial cytosolic DNA. A key issue for host defense is the receptor profi le at particular time and tissue site of infection. PRR expression in phagocytes is influenced by cell type (resident tissue macrophage versus inflammatory monocytes), tissue (lung versus

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FIG. 40.2. The Pattern Recognition Receptors. Bacterial recognition is paramount in the crosstalk between host and pathogen. Several classes of receptors contribute to this process. Toll-like receptors are present at the surface membrane or within ensodomal compartments and recognize lipids, carbohydrates or proteins. C-type lectin receptors and scavenger receptors are expressed at the cell surface and sense glycolipids and lipoproteins, respectively. Surveillance of the cytosol is mainly performed by nucleotide oligomerization domain–like receptors (NLRs), RIG I-helicase receptors (RLRs), and pyrin- and HIN-containing proteins (PYHINs). RLRs are exclusively involved in viral detection, whereas NLRs recognize a wide range of structures. PYHIN detect deoxyribonucleic acid from various sources. AIM2, absent in melanoma 2; CpG, cytosinphosphatidyl-guanosin; CPS, capsular polysaccharide; DC-SIGN, dendritic cell–specific intercellular adhesion molecule-3-grabbing nonintegrin; ds, double stranded; FI16, gamma-interferon-inducible protein 16; LAM, lipoarabinomannan; LGP2, laboratory of genetics and physiology 2; Lox1, lectin-type oxidized LDL receptor 1; LPS, lipopolysaccharide; LTA, lipoteichoic acid; ManLAM, mannosylated lipoarabinomannan; MARCO, macrophage receptor with collagenous structure; MDA5, melanoma differentiation-associated gene 5; MDP, muramyl dipeptide; Mincle, macrophage-inducible C-type lectin; MR, mannose receptor; NAIP, baculoviral IAP repeat-containing protein; NLRP, NACHT, LRR and PYD domaincontaining protein; NLRC, NLR family CARD domain-containing protein 4; PGN, peptidoglycan; RIG-I, retinoic acid-inducible gene I; ss, singlestranded; TDM, trehalosedimycolate; SAP130, spliceosome-associated protein 130.

gut), milieu, and activation status (certain cytokines up- or downregulate receptors).61 Importantly, multiple PAMPs engage multiple PRRs simultaneously or sequentially and therefore, a given pathogen by means of specific combinations of PAMPs can tailor the host response in a highly specific way. TLRs can form dimers (eg, TRL-1/2 or TLR-2/6), and may collaborate with distinct CLRs (TLR-2/Dectin-1; TLR-2/MARCO; TLR-4/CD14). On the other hand, a given PAMP may be sensed by multiple PRRs (glucans sensed by Dectin-1; complement receptor [CR]; CD36). Thus, PRRs orchestrate pathogen- and cell type-specific host immune responses. Pathogens able to alter molecules or modulate host cell death add an additional layer to this recognition process by inducing activation of PRRs via recognition of

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pathogens in combination with sensing of danger signals. Certain PRRs are involved in sensing both microbial components and danger signals. For instance, Mincle recognizes mycobacterial trehalose dimycolate (TDM) and the ribonucleoprotein spliceosome-associated protein 130, a protein associated with eukaryotic cell death. Despite their name, PAMPs are not restricted to pathogens but are also expressed by nonpathogenic microbes. Hence, the host probably needs to sense pathogen-specific signals in addition. In the case of intracellular bacteria, such signals could emanate from bacterial persistence, that is, from duration of PAMP expression. More recently, bacterial messenger ribonucleic acid was suggested as a signal from viable bacteria (vitaPAMP), which alerts the immune system.62

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PRRs may be classified according to pathogen-binding propensity and impact on uptake as: • Opsonic receptors: Fc receptors (FcRs), CRs, pentraxins, surfactant proteins, ficollins • Nonopsonic receptors: TLRs, NLRs, RLRs Based on their effects on pathogen internalization, PRRs may be classified as: • Phagocytic receptors: CRs, FcRs, mannose receptors (MRs), and DC-SIGN (CLRs), CD36, MARCO, and SR-A (SRs) • Signaling receptors: TLRs, NLRs, RLRs, CLRs Bacterial cell wall composition, secretion products, as well as intracellular location are fundamental aspects for recognition of intracellular bacteria. M. tuberculosis is rich in several classes of PAMPs. This pathogen interferes with the endocytic pathway and resides in an early phagosome. Phagocytosis is mediated by CLRs (MRs, DC-SIGN) and SRs (CD36), which primarily recognize mannosylated cell wall mannans and by opsonic receptors, FcRs, and CRs. Another CLR, namely Langerin, seems to have a role in M. leprae recognition. Lung surfactant proteins mediate the recognition of tubercle bacilli in the alveolar space. Sensing of the lipopeptides and phosphatidylinositol mannans is performed by TLR-2, while TLR-4 involvement is still debated. Multiple CLRs sense mycobacterial glycans or lipoglycans. TDM, a specific component of mycobacteria, is recognized by Mincle, while DC-SIGN senses mannosylated lipoarabinomannan. SIGNR3 and Dectin-1 and -2 are also involved in recognition. However, to date no specific structure from mycobacteria has been identified as a ligand for these lectins. Once in the phagosome, bacteria may be sensed by TLR-9. Bacterial PGN, which accesses the cytosol, is recognized by NOD2. Notably, there is a degree of redundancy between receptors belonging to different classes, as revealed by studies with mice with single or multiple deficiencies in TLRs, NLRs, and SRs.63 It appears that adaptors able to gather signals from various PRRs, such as MyD8864 and caspase recruitment domain-containing protein 9 (Card9) 65 are key in controlling susceptibility to TB. Recognition of Salmonella, which lives in a late phagosome, is dominated by TLR members. LPS and the lipopeptides from the cell wall are sensed by TLR-2 and -4 before internalization. Ablation of these TLR renders mice highly susceptible to disease.66 Vacuolar bacteria are recognized by TLR-9 via CpG DNA. Interestingly, triple-deficient (TLR2, -4, -9) animals are resistant as simultaneous TLR activation is necessary for acidification of the vacuole, which in turn induces Salmonella pathogenicity island 2 (SPI2) genes. Virulence effectors are translocated into the cytosol transforming the phagosome into a replicative niche for Salmonella.67 Flagella are sensed by TLR-5 and NLRC4. Recognition of L. monocytogenes, which escapes from phagosome into cytosol, is complex. Cell wall lipopeptides are sensed by TLR-2, while PGN is recognized by NOD1 and NOD2. MyD88 seems to be essential for defense against listeriae.68 Bacterial DNA is monitored by multiple sensors in the cytosol, including AIM269–73 and leucine-rich repeat

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flightless-interacting protein 1.74 Francisella, which also egresses into the cytosol, is recognized in a similar way by multiple receptors, including AIM270,75and NLRC4. By sensing the broad molecular spectrum of pathogens, PRRs induce cellular programs as the fi rst line of defense, including antimicrobial effector functions and maturation of DCs for instruction of the adaptive immune response. However, PRRs and their adaptors may be exploited by intracellular pathogens to escape killing and establish stable infection. Mycobacteria use CRs to ensure a safe entry into macrophages and limit maturation of the phagosome. They also use the TLR/MyD88 pathway to induce cytokines (IL-6 and IL-10), which through signal transducer and activator of transcription (STAT)3 signalling bias arginine metabolism to arginase-1 over nitric oxide production by inducible nitric oxide synthase (iNOS).76,77 In addition, polymorphonuclear leukocyte encounters with mycobacteria results in MyD88/Card9-dependent IL-10 release to dampen immune responses.65,78 Similarly Yersinia uses TLR-2 to induce IL-10.79

Phagocytosis and Phagosome Dynamics Phagocytosis of inert particles initiates a series of events that ultimately lead to the formation of a phagolysosome (see Fig. 40.1).80,81 Phagosome maturation is a strictly coordinated sequence of fusion and fission events, which involves defined compartments of the endocytic pathway.81,82 Immediately after or even during phagosome sealing, phagosome maturation proceeds. • The early phagosome is characterized by a slightly acidic to neutral pH (6.0 to 6.5) and membrane markers, such as MR, the tryptophane aspartate-containing coat protein, and the transferrin receptor (TfR) with its ligand transferrin, small GTPase (Rab5), early endosomal antigen 1, phosphatidylinositol 3-phosphate, and Syntaxin13. • The late phagosome is characterized by a pH between 5.0 and 6.0 and the acquisition of the vacuolar adenosine triphosphatase (ATPase) proton pump (V-H+ATPase), mannose-6 phosphate receptor, Rab-interacting lysosomal protein, and Rab7. • The phagolysosome results from the fusion between phagosomes and lysosomes, characterized by a pH between 4.0 and 5.5, high density of lysosome-associated membrane proteins, and typical lysosomal enzymes (such as cathepsins). The three stages form a continuum involving the sorting of membrane proteins, as well as budding of, and fusion with, other vesicles. During this dynamic process, the phagosomes successively interact with the corresponding endosomes and subsequently with lysosomes.81 Characterization of the Rab family of GTPases on vacuoles harboring pathogens facilitates identification of the host membrane transport pathways, which are turned on during infection.83 M. tuberculosis and S. enterica interfere with the endocytic pathway by retaining Rab5 and Rab7, respectively, on their vacuoles.84 L. pneumophila, Brucella abortus, and C. trachomatis interact with the secretory pathway as revealed

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by the presence of Rab1, Rab2, and Rab6, respectively, on their phagosomes.85,86 Acquisition of a vacuolar ATPase proton pump plays a central role in acidification of the phagosome.81,87 Immediately after phagocytosis, the phagosome becomes alkaline for a short time before acidification is initiated. The basic milieu is optimal for the activity of defensins and basic proteins, whereas the acidic pH is optimal for lysosomal enzymes. Defensins are small (3.5 to 4.0 kD) peptides rich in arginine and cysteine.88,89 They are abundant in PNG and present in some, though not all, MPs (depending on species and tissue location). Purified defensins are microbicidal for certain intracellular bacteria, such as S. enterica and L. monocytogenes. The contribution of lysosomal enzymes to bacterial killing is likely minor. Their major task is the degradation of already killed bacteria. These enzymes reside in the lysosome and are delivered into the phagosome during maturation through several independent waves, and they reach their optimum activity during later stages, that is, in the phagolysosome. Most intracellular bacteria interfere with phagosome maturation and alter the phagosome in order to facilitate their own survival (see Fig. 40.1).80,90,91 These include L. pneumophila, M. tuberculosis, S. enterica, C. burnetii, and Chlamydia. Although the specific mechanisms are incompletely understood, mycobacterial sulfatides and some mycobacterial glycolipids, most notably mannosylated lipoarabinomannans, impede phagolysosome fusion. Mycobacterial products, such as SapM and MptpB, contribute as well to the arrest of maturation of the early endosome. Antibody-coated M. tuberculosis organisms lose their capacity to block discharge of lysosomal enzymes, suggesting an auxiliary function of antibodies in cell-mediated protection against TB.92 Finally, the robust, lipid-rich cell wall of mycobacteria renders them highly resistant against enzymatic attack. M. tuberculosis, as well as M. avium, arrest phagosome maturation at an early stage. They restrict phagosome acidification via the exclusion of the V-H +ATPase proton pump from the phagosome. Additional mechanisms may contribute to this event, such as NH4 + production by M. tuberculosis. Consistent with intraphagosomal NH4 + production, the urease of M. tuberculosis is active at low pH. It has been known for some time that NH4 + also interferes with phagosome–lysosome fusion. Exogenous adenosine triphosphate (ATP) has been shown to promote phagolysosome fusion resulting in concomitant death of macrophages and killing of M. bovis bacillus Calmette–Guérin (BCG).93,94 Phagosome maturation is arrested somewhere between the early and late stages by M. tuberculosis, M. bovis BCG, L. pneumophila, S. enterica, and C. trachomatis, all of which replicate in nonacidified vacuoles. Phagosomes containing S. enterica, M. bovis BCG, or C. trachomatis appear uncoupled from the maturation process through which phagosomes containing inert particles proceed.80,90 S. enterica remains in the spacious membrane-bound phagosome, which is formed after uptake by the trigger mechanism. Moreover, the bacteria manipulate the cytoskeleton via kinesin and tether the vacuole to membranes of the Golgi apparatus.95 The vacuole containing C. trachomatis, which lacks any

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specific phagosome markers, is loaded with ATP, which is required by the pathogen, by an unknown mechanism. In addition, elements of the cytoskeleton (actin and fi lamin) are used to stabilize the vacuole.96 L. pneumophila prevents fusion of the vacuole with the endosomal compartment and recruits vesicles derived from Golgi and ER97 by use of the T4SS. Moreover, Chlamydia employ mimics of soluble NSF attachment protein receptor to modulate membrane transport. Legionella uses an array of proteins to control Rab1 activity in the vacuole.98,99 Bacterial DrrA (SidM protein) is a highly efficient guanine nucleotide exchange factor for Rab1A.44 C. burnetii have evolved to live in the acidic late phagosome.82,100 Recent in vitro studies confirmed Coxiella’s requirement for low pH and oxygen tension. The fusogenic properties of the vacuole are tightly regulated by the T4SS apparatus, through ankirin proteins.101

Egression into the Cytoplasm Egress from the phagosomal into the cytoplasmic compartment represents a highly successful microbial survival strategy because bacterial killing is focused on the phagolysosome to limit self-damage of MPs. This egression has been extensively studied in L. monocytogenes, but is known to be utilized by other intracellular pathogens, including shigellae, rickettsiae, francisellae, and mycobacteria (see Fig. 40.1).102 Cytoplasmic invasion by L. monocytogenes depends on listeriolysin (LLO), an SH-activated cholesterol-dependent cytolysin. LLO requires activation by a host factor (gamma-IFN-inducible lysosomal thiol reductase), a thiol reductase.103 In the cytosol, LLO is degraded, thus avoiding killing of host cells.104 Deletion of the LLO gene (hly) renders L. monocytogenes avirulent. LLO is also required for replication of L. monocytogenes in spacious listeria-containing phagosomes. These compartments are nonacidic and allow slow replication of the pathogen.105 Other molecules, such as phospholipase and lecithinase, are likely involved in membrane transition but are insufficient on their own. Invasion of L. monocytogenes into the cytoplasmic compartment is markedly reduced in IFNγ-activated macrophages in which the microbe, entrapped in the phagosome, rapidly succumbs to attack by toxic oxygen and nitrogen species and/ or defensins. Cytosol evasion of shigellae is mediated by factors, which are also involved in bacterial entry (eg, IpaB, product of T3SS). Listeria simultaneously activates caspase-1 and consequently modulates death of infected cells via danger signals represented by remnant vacuolar membranes.106 M. tuberculosis uses proteins encoded by the T7SS, located in the region of difference 1 gene region, to successfully translocate to the cytosol.5,107

Cell-to-Cell Spreading L. monocytogenes is cleared from the blood by Kupffer cells and then spreads to adjacent hepatocytes without reentering the extracellular milieu. This mechanism of cell-to-cell spreading has been carefully studied in vitro.108 Having entered the cytoplasm, L. monocytogenes induces a tail of host

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actin fi laments, which push the bacterium forward to the outer region of the cell, where it induces pseudopod formation. Intracellular movement is achieved by coordinated actin polymerization at, and polarized release from, the bacterial surface. The ActA gene encodes a 90-kDa protein located on the bacterial surface, which is responsible for these actin-based movements.109 A host cytosolic complex composed of eight polypeptides has been identified which, on binding Act A, induces actin polymerization.110 The pseudopod-containing L. monocytogenes is engulfed by the adjacent cell, and the microbe reaches the phagosome of the recipient cell, which is still enclosed by cytoskeletal material from the donor cell. The two plasma membranes of the host and recipient cells apparently fuse, thereby allowing the introduction of L. monocytogenes into the cytoplasm of the recipient cell. Thus, L. monocytogenes can infect numerous cells without contacting extracellular defense mechanisms. Shigella use similar mechanisms for evasion and intracellular movement, and a similar spreading mechanism seems to be employed by S. enterica and by R. rickettsii, but not by R. prowazekii and R. typhi. A role for motility and manipulation of host actin-based structures was recently demonstrated for virulent mycobacteria.111 Bacterial T7SS mediates ejection from the infected host cell and facilitates spreading through actin structures coined “ejectosomes.”

Cell Death Patterns Death of mammalian cells occurs by accidental or programmed cell death, which were once thought to be associated exclusively with necrosis or apoptosis, respectively.112 Increasing evidence, however, suggests that necrosis can progress in a programmed sequence. Moreover, in addition to necrosis and apoptosis, additional modes of cell death have been described recently, namely autophagic cell death, pyroptosis, pyronecrosis, necroptosis, mitotic catastrophe, NETosis, and lysosomal membrane permeability.113–115 Although autophagy is a complex process aimed at preventing cell death, it has emerged as relevant mechanism to control infection and hence will be described in this section. Generally, intracellular pathogens often counteract host cell death in order to maintain their habitat. Thus, ability of microbes to modulate eukaryotic cell survival evolved as an essential pathogenicity feature.

Apoptosis and Necrosis Apoptosis is a tightly controlled process that is initiated by intrinsic mechanisms within the dying cell. It involves a series of tightly controlled enzymatic events, notably intracellular caspases. Necrosis is the result of cell destruction caused by various exogenous effector mechanisms, including those mediated by complement and cytolytic T-lymphocytes. In contrast to necrosis, apoptosis is generally noninflammatory and thus associated with tissue repair rather than destruction. However, cell death associated with bacterial infection frequently results in release of microbial PAMPs, which serve as inflammatory signals. Intracellular bacteria interfere with apoptosis in various ways to delay or even block this process and thus sustain their preferred

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habitat. C. burnetii antagonizes the intrinsic apoptotic death of macrophages by means of ankirin proteins related to T4SS.116,117 A. phagocytophilum blocks PNG apoptosis by secreting the Ats1 protein via T4SS across the phagosomal membrane. Ats1 enters the mitochondria and subverts apoptotic signaling.118 Chlamydia prevents immature cell death and uses autophagy in addition.113 L. pneumophila induces rapid apoptosis in DCs,119 but prevents programmed cell death in other cell types.120 Moreover, this pathogen limits multiple pathways leading to cell death, including necrosis in macrophages.121 Mycobacteria offer a good example of how virulence is associated with modulation of death of infected cells. Avirulent mycobacteria induce apoptosis, while M. tuberculosis preferentially causes necrosis.122 Tubercle bacilli interfere with eicosanoid-regulated cell death to facilitate necrosis. Generally, death of infected cells impacts on the acquired immune responses, pathology, and ultimately disease manifestation.

Autophagy Autophagy is a catabolic process that controls the integrity of eukaryotic cells.123 This mode of “self-digestion” is paramount for the disposal of protein complexes that cannot be degraded via the proteasomal route and for the elimination of damaged organelles. Three different autophagic processes have been described: chaperone-mediated autophagy, microautophagy, and macroautophagy (canonical autophagy).115 A unique feature of autophagy is the direct sequestration of the cargo into autophagosomes, which are surrounded by a double membrane and delivered to the lysosomal compartment. Autophagy is tightly orchestrated by over 30 autophagic components. This cell-autonomous housekeeping process is also an efficient system for the elimination of intracellular pathogens. Direct autophagy of intracellular pathogens occurs in any cell type and has been named xenophagy.124 As a process of cytosol surveillance, autophagy is directed primarily against pathogens that egress into the cytosol. However, most bacteria, which successfully adapted to the intracellular milieu, have developed mechanisms to protect themselves against autophagy. Some species even harness the autophagic machinery to their advantage. Autophagosome-like structures were first described in PNG infected with Rickettsia,125 and a role for autophagy in control of intracellular bacteria was first demonstrated for M. tuberculosis.126 Subsequently, the protective functions of autophagy were established in infections with M. tuberculosis,127,128 S. enterica,129 and L. monocytogenes.130–132 Microbe-derived PAMPs stimulate autophagy131,133 as do cytokines like IFNγ, most likely through induction of GTP/ guanylate-binding protein (GBP) molecules.134,135 Lipids were also found critical for autophagy in intracellular bacterial infections. Thus, the signaling lipid diacylglycerol is required for autophagy induced by S. typhimurium.136 Recently the term sequestosome-like receptors was coined to define cytosolic innate receptors (p62, NBR1, NDP52), which target intracellular pathogens to the autophagic machinery115 including salmonellae,137,138 shigellae,106 listeriae,139 and mycobacteria.140

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Pathogens interfere with autophagy through multiple strategies: by blocking induction or maturation of autophagosomes into autolysosomes, by evading recognition by the autophagic machinery, and by misusing autophagy for their own benefit. Microarray studies revealed that F. tularensis downregulates autophagy-related genes.141 Similarly, whole genome-wide profiling of macrophages infected with M. tuberculosis suggests that host factors that regulate bacterial replication are regulators of autophagy.142 S. enterica blocks autophagy by interference with the ubiquitination machinery,137,138 and L. pneumophila induces autophagy, but delays fusion with lysosomes.143 A similar scenario was reported for C. burnetii144 and A. phagocytophilum.145 A different strategy is employed by bacteria that egress into the cytosol. The shigella protein VirG targets bacteria to the autophagic pathway by binding to the autophagic protein Atg5. However, once in the cytosol, the pathogen uses the T3SS IcsB to competitively bind to Atg5 and thus renders it unavailable for VirG.146 Similarly, ActA impedes autophagic targeting of listeriae by recruiting actin and protecting it.139,147 Some intracellular bacteria not only affect autophagy but may also exploit it. Thus, chlamydiae gain access to nutrients by stimulating autophagy,148 and listeriae exploit this process for establishing chronic infection.105 F. tularensis reenters the endocytic pathway following its egression into the cytosol through autophagosomes.149 Autophagy is implicated in the biogenesis of vacuoles and persistence of C. burnetii therein.150

Pyroptosis Pyroptosis is a form of programmed cell death that is controlled by caspase-1. It is an inflammatory process that results in rapid lysis of infected cells, mostly macrophages.114 This process was first described for shigellae151 and subsequently for salmonellae,152,153 legionellae, and burkholderiae.154 Mouse studies showed that pyroptosis is an innate immune effector mechanism during infection with S. typhimurium.154 It still needs to be clarified how intracellular bacteria escape or modulate pyroptosis.

Intracellular Iron Iron is required by most organisms and is a cofactor for enzymes involved in many essential biologic processes. The same divalent cation is toxic at high concentration and therefore is tightly controlled by multiple elements.155 As both host and intracellular pathogens require iron, microbes have evolved ways to interfere with iron homeostasis. Generally, the strategy of the mammalian host is to deprive the pathogen of iron. However, depending on microbial habitat (extracellular versus intracellular bacteria), a given mechanism may be beneficial or detrimental. While systemic iron withdrawal and hypoferremia contribute to the control of extracellular bacteria, these strategies are counterproductive for intracellular microbes. Rather, downregulation of iron uptake and augmentation of iron export are advantageous for the host in defense against intracellular pathogens.156,157 Intracellular bacteria require iron, and production of reactive oxygen intermediates (ROIs) and reactive nitrogen

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intermediates (RNIs) also depends on iron. Thus, competition for the intracellular iron pool between the intracellular pathogen and the host cell markedly influences the outcome of their relationship.158 To improve their iron supply, mammalian cells utilize specific molecules. In the extracellular host milieu, iron is tightly bound to transferrin and lactoferrin, and the transferrin–iron complex is taken up by host cells via TfRs. The lactoferrin–iron complex does not enter the cell. Iron is released from the transferrin–TfR complex under the reducing conditions of the early phagosome. This event is controlled by Hfe (the product of the hereditary hemachromatosis gene).155 Hfe reduces iron uptake either by inhibiting TfR internalization or by blocking iron release from transferrin in the early phagosome. Macrophages can also acquire iron via the divalent metal transporter-1. Iron may also bind to haptoglobin, haemopexin, and lipocalin-1 and -2 in the extracellular space. Within the endocytic pathway, the Nramp system is involved in iron transport from the phagosome to the cytosol, where iron is bound to ferritin. Ferroportin (FPN) is one of the best-characterized iron export molecules in eukaryotic cells. Hepcidin, a product not only of liver cells but also macrophages, regulates FPN activity by inducing FPN internalization and proteasomal degradation. Accordingly, iron availability is controlled in multiple ways, including lactoferrin concentration in the extracellular space, intracytosolic ferritin concentration, hepcidin levels and abundance of TfRs, divalent metal transporter-1, and FPN on the cell surface. Many intracellular bacteria, including M. tuberculosis, L. pneumophila, and S. enterica, accommodate themselves in the early phagosome, where the abundance of iron-loaded transferrin guarantees a high availability of iron. Moreover, these bacteria, as well as Chlamydia and F. tularensis, induce divalent metal transporter-1 expression to support their replication.159 Hepcidin is induced in mycobacterial phagosomes160 and limits iron export by degrading FPN. Hepcidin also promotes Salmonella’s growth.161 Similar observations, suggesting that iron efflux is detrimental for intracellular pathogens, were reported for C. psittaci and L. pneumophila.162 To successfully compete for iron, bacteria possess a variety of iron-binding proteins. These include iron chelators (siderophores), transferrin-binding proteins, hemelike proteins, and ATP-binding cassette transporters.163–165 Expression of genes involved in iron uptake is controlled by a conserved mechanism involving the Fur protein. However, lipocalins, which are host proteins produced by professional phagocytes and epithelial cells, bind siderophores from mycobacteria and salmonellae and thus minimize iron usage by microbes. Moreover, deletion of lipocalin-2 impacts on invasion of epithelial cells by tubercle bacilli.161,166 Importantly, there are many crossregulatory interactions linking iron homeostasis and immune responses. Th1 immunity and iron availability are interconnected. IFNγactivated MPs downmodulate TfR expression and intracellular ferritin, resulting in reduced iron availability within the phagosome. Overall, IFNγ induces iron sequestration by downregulating FPN, while IL-4 favors iron release.156,167 IFNγ, on the other hand, induces Nramp1, whose relevance for protection against salmonellae and mycobacteria has

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been established.168 The iron content of the phagosome in the resting MP seems to be sufficient for L. pneumophila. However, available iron is markedly reduced in IFNγactivated MPs and, as a consequence, L. pneumophila, which lacks efficient iron uptake mechanisms, starves from iron deprivation in activated macrophages. In contrast, M. tuberculosis possesses a potent iron acquisition system comprising exochelins and mycobactins. The exochelins successfully compete for iron under limiting conditions and transfer it to mycobactins in the cell wall.169,170 Similarly, SPI2 are upregulated as a response to Nramp-1induced iron starvation and modify the vacuole to ensure bacterial replication.

Toxic Effector Molecules Killing of intracellular bacteria by MPs and/or PNGs is primarily accomplished by highly reactive toxic molecules, notably ROIs and RNIs.171–174 Within infected cells, these molecules are bactericidal. However, both ROIs and RNIs have a broader functional spectrum. They are also involved in signaling, regulation of vascular tone, tissue injury, and control of inflammation. Thus, ROIs and RNIs can both alleviate and promote tissue damage.171 Most if not all bacteria are susceptible to ROIs in vitro. Yet, contribution of ROIs to killing of intracellular bacteria by MPs is less clear; in murine macrophages, RNIs are more important. On the contrary, in PNGs, the role of ROIs seems to prevail.175 In the mouse, ROIs and RNIs act consecutively in defence against S. enterica infection.176,177 Production of RNIs by human MPs at concentrations sufficiently high for bacterial killing remains controversial.178–180 However, evidence is accumulating that human MPs from sites of intracellular bacterial infection produce adequate concentrations of RNIs. Thus, using antibodies with exquisite specificity for human iNOS, this enzyme could be detected in a large proportion of lung macrophages from patients with TB.181–184 iNOS can be induced by different mechanisms in different species as suggested, for example, by divergence of the iNOS promoter in mouse and human.171,185,186 ROI production is initiated by a membrane-bound nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) complex composed of six enzyme units (Rho guanosine triphosphatase, gp91PHOX also named NOX2, p22phox, p40phox, p47phox, and p67phox). NADPH oxidase is activated by IFNγ and by immunoglobulin (Ig)G– FcR binding: O2 + NADPH

NADPH oxidase

In addition, O2− is transformed into 1O2. The 1O2 and • OH radicals are short-lived powerful oxidants with high bactericidal activity causing damage to DNA, membrane lipids, and proteins. (Note: O2−, hyperoxide anion; • OH, hydroxyl radical containing a free electron; 1O2, singlet oxygen, a highly reactive form of O2.) Granulocytes, blood monocytes, and certain populations of tissue macrophages possess myeloperoxidase, thus allowing halogenation of microbial proteins172 : H2O2 + C1−

MPO

OC1− + H2O

In addition to hypochlorous acid, chloramines are formed and both agents further increase the bactericidal power of the ROI system by destroying biologically important proteins through chlorination. Nitric oxide is exclusively derived from the terminal guanidino nitrogen atom of L-arginine (Fig. 40.3). This reaction is catalyzed by the iNOS, which leads to the formation of L-citrulline and NO • . NO • can act as oxidizing agent alone or interact with O2− to form the unstable peroxynitrite (ONOO −). This may then be transformed to the more stable anions, NO2− and NO3 −, or decomposed to NO • : O2− + NO • ONOO− + H+ NO2− + • OH ONOO− + H+

ONOO− NO2− + • OH NO3− + H+ •

OH + NO •

NO • and ONOO − are highly reactive antimicrobial agents. NO • may be transformed to nitrosothiol, which expresses the most potent antimicrobial activity. In contrast, NO2− and NO3 − are without notable effects on microorganisms. Three distinct nitric oxide synthase isoenzymes are known. The two constitutive nitric oxide synthases (neuronal nitric oxide synthase and endothelial nitric oxide synthase) exist in various host cells and account for basal nitric oxide synthesis, whereas iNOS is primarily found in professional phagocytes and is responsible for microbial killing. Its induction is controlled by exogenous stimuli such as IFNγ, agonist of PRRs, and inflammatory cytokines. This iNOS stimulation results in a burst of high RNI concentrations required for microbial killing, whereas the low nitric oxide levels produced by neuronal nitric oxide synthase

NADP + O2− H+

O2− is further metabolized by superoxide dismutase: 2O2− + 2H+

SOD

O2 + H2O2

In the presence of appropriate iron catalysts, the HaberWeiss reaction takes place: O2− + Fe3+ H2O2 + Fe2+

O2 + Fe2+ •

OH + OH− + Fe3+ (Fenton reaction)

Net reaction: O2− + H2O2

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•

OH + OH− + O2

FIG. 40.3. Generation of Nitric Oxide from L-Arginine. L-Arginine is metabolized to the toxic molecules nitric oxide and L-citrulline by inducible nitric oxide synthase.

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and endothelial nitric oxide synthase perform physiologic functions. The RNIs exert their bactericidal activities by directly inactivating iron–sulfur-containing enzymes, by S-nitrosylating proteins, by damaging DNA, or by synergizing with ROIs. Both ROIs and RNIs are relevant for killing of Salmonella, while most other intracellular pathogens are primarily controlled by iNOS products. In chlamydial infections, insufficient as well as excessive production of nitric oxide can be immunosuppressive.187 RNIs prevail as antimicrobial molecules against L. monocytogenes,188 C. burnetii,189 C. trachomatis, F. tularensis, Brucella, and L. pneumophila.190 The pivotal role of iNOS in control of M. tuberculosis infection has been demonstrated in the mouse system.191 Despite resistance of NOX2-ablated mice against TB, recent data supporting a role of macrophage ROIs in humans have been reported.192 Thus, nitrosative and oxidative processes under the control of iNOS and NADPH oxidase together are paramount for protection against intracellular bacteria.

Evasion of Killing by Reactive Oxygen Intermediates and Reactive Nitrogen Intermediates Microbes use multiple strategies to nullify ROI and RNI attack: evasion, inhibition, enzymatic inactivation, generation of scavenger molecules, as well as stress and repair mechanisms (see Fig. 40.1).171 SPI2 enables S. enterica to exclude NADPH oxidase from the phagosomal membrane, thus interfering with ROI release into the Salmonella phagosome. S. enterica mutants deficient in SPI2 are susceptible to ROIs.193 Similarly, proteins translocated into the cytosol interfere with iNOS activity.194 F. tularensis undergoes phase variation to switch to a phenotype that is less stimulatory for iNOS.195 A. phagocytophilum inhibits ROI production in PNGs after an initial ROI induction process.196 Many intracellular bacteria produce superoxide dismutase and superoxide catalase that detoxify O2 and H2O2, respectively.171,197 Production of ROIdetoxifying molecules by intracellular bacteria is not constitutive; rather, expression of these enzymes is controlled by regulators such as soxR or oxyR that sense for concentrations of O2 or H2O2, respectively. Accordingly, transposon mutants of S. enterica that fail to survive inside murine MPs are highly sensitive to ROI in vitro. Although less is known about specific mechanisms by which intracellular bacteria interfere with killing by RNIs, catalase and other antioxidative enzymes may indirectly inhibit RNI functions. ROIand RNI-detoxifying gene products have been identified in M. tuberculosis.198 KatG,199 SodA, and SodC are involved in these processes. Recently, mycobacterial nicotinamide adenine dinucleotide dehydrogenase and enhanced intracellular survival (eis) gene products were reported to be relevant for inflammatory responses by controlling infected cell death as a consequence of ROI generation.200,201 These mechanisms are separated from the direct antimicrobial effects of toxic radicals and will be discussed in the following. Intracellular pathogens may generate scavenger molecules to dispose of toxic radicals. Low molecular mass thiols, such as mycothiols from M. tuberculosis197 or homocysteine in Salmonella,202 are examples of ROI/RNI scavengers. In addition, both oxidative and nitrosative stresses are reflected in transcriptional changes in bacteria and initiation of DNA

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repair processes. Proteasome involvement in RNI resistance was reported for M. tuberculosis.203 Further, certain macrophage receptors interfere with NADPH oxidase function. Binding to CR1/CR3 does not induce respiratory burst and ROI production.204 The CRs, therefore, provide a relatively safe way of entry for intracellular bacteria.

Antimicrobial Peptides Antimicrobial peptides (AMPs) encompass diverse groups of molecules that can be divided in subgroups based on their amino acid composition and structure. They are present systemically, at mucosal sites, or packed in granules of phagocytes.205 AMPs active against intracellular microbes belong to different classes. Cathelicidin (LL-37) is a cationic peptide with bactericidal functions against M. tuberculosis.206 Cationic and anionic peptides, including human neutrophilic peptides and defensins, kill microbes inside PNGs. PR-39 promotes fi lamentation of S. typhymurium. Most AMPs need to attach to the bacterial cell wall. Next, the peptides are inserted into the bacterial membrane rendering it permeable. AMPs are also generated upon infection-induced autophagy. During mycobacterial infection, ubiquitin generates peptides with lytic activity within autophagosomes.207 Importantly, microbes develop mechanisms of resistance against AMPs.

Cell Autonomous Defense: Guanine Triphosphatases The dominant role of IFNγ in defense against intracellular pathogens is beyond doubt. Besides ROI and RNI generation (detailed previously), a role for small GTPase family cognates was recently established. Three families of GTPases are regulated by IFN: 65kDa GBPs, Mx family, and p47 kDa GTPases or immune-related GTPases.208 Mx proteins are critical for antiviral responses and are mostly induced by type I IFN. In their promoters, GBPs and immune-related GTPases possess both gamma-activated site and IFNstimulated response element binding sites. Accordingly, although the main inducer is IFNγ, type I IFN may also initiate their transcription.209 Studies with mice ablated of proteins from these GTPases revealed that distinct members are paramount for protection against a range of intracellular bacteria, while other GTPases are redundant. Irgm1 (formerly Lrg-47) is essential for resistance against infection with M. tuberculosis, M. avium, L. monocytogenes, S. typhymurium, and C. psittaci.210–215 Irga6 (formerly Iigp1), on the other hand, restricts C. thrachomatis replication only. More recently, GBP7 and GBP1 were found to be involved in control of infection with L. monocytogenes and M. bovis BCG.134,135 Interestingly, GBPs and immune-related GTPases seem to trigger multiple pathways, as members of both families are essential for defense against intracellular bacteria residing in different cellular compartments. Members of these GTPase families localize to the ER, Golgi, plasma membrane, or intracellular vesicles. They may be recruited to the bacterial phagosome early upon infection, as demonstrated for Irgm1 in infections with mycobacteria or listeriae. However, the antimicrobial mechanisms are

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diverse and encompass enhancement of phagosome fusion with lysosomes, interference with the autophagy machinery, regulation of oxidative responses, or induction of bacteriolytic peptides. Irgm-1 regulates T-cell dynamics and macrophage motility, suggesting cellular responses rather than effector functions are controlled by this and perhaps other GTPases.216 In contrast to the multitude of p65 and p47 kDa GTPases in mice, only few cognates exist in humans. Accordingly, the relevance of these molecules in human diseases remains to be clarified.

INNATE IMMUNITY: PROFESSIONAL PHAGOCYTES The innate immune system comprises various cell types of different hematopoietic lineages. Besides phagocytes (macrophages, monocytes, DCs, PNGs) and other cells of myeloid origin (eosinophils, basophils, mast cells), certain lymphoid cells (natural killer [NK], NKT, γδ T lymphocytes, B1 lymphocytes), as well as platelets and the nuocytes act in concert to limit replication of the pathogens. Many intracellular bacteria infect professional phagocytes, and this strategy contributes to their evolutionary success. The professional phagocytes are represented by mononuclear cells, including monocytes, macrophages, and DCs, and by polymorphonuclear cells, namely the PNGs.

Mononuclear Phagocytes Metchnikoff 217 was the first to realize the importance of professional phagocytes in resistance against bacterial infections. He observed that leukocytes accumulated at the site of inflammation and bacterial growth, and were heavily engaged in microbial engulfment and destruction. Metchnikoff distinguished two types of phagocytes: 1) the early appearing and short-lived microphages now referred to as PNGs, and 2) the late-appearing long-lived macrophages still known under the same name. The preferential localization of tubercle bacilli inside macrophages discovered at the time of Koch218 and Metchnikoff 217 pointed to the central role of these MPs in defense against intracellular bacteria. Metchnikoff also observed that during infection, macrophages are nonspecifically activated. Macrophage activation as an important factor of acquired resistance against bacterial infections was further substantiated by Lurie219 and shown to be under the control of lymphocytes by Mackaness.220 Later, cytokines were identified as the mediator of macrophage activation.221,222 Ontology studies and careful characterization of MP populations revealed that they show a tremendous degree of diversity and plasticity.223 Tissue cues modulate the MP mode of response to bacterial insult (eg, lung versus liver, alveolar macrophages in the lung are mostly antiinflammatory, whereas liver Kupffer cells are mostly proinflammatory). Moreover, developmental branching of the mononuclear progenitor enables differential responses to pathogens. Thus, monocytes are specialized in their response to pathogens: certain populations (CD14dim) primarily react to viral infections while others (CD14 +) respond to a broad range of microbes.224

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Many of the antibacterial activities of the MPs are not constitutively expressed. Rather, expression of full antibacterial activities by MPs depends on appropriate stimulation by cytokines, with IFNγ being of paramount importance.225 Furthermore, significant differences exist among MPs of different maturation states or from different species. For example, higher RNI levels are produced by murine MPs as compared to human MPs. IFNγ activation of MPs coincides with increased phagocytosis, elevated CRs, reduced FcR expression, and a higher overall metabolic rate. Most importantly, during macrophage activation, iNOS and NADPH oxidase, which initiate RNI or ROI production, respectively, are stimulated. Moreover, this type of activation augments autophagy, which in turn contributes to microbial destruction. In other words, activation by cytokines results in transition of MPs from habitat-supporting microbial replication into an effector cell capable of terminating, or at least restricting, microbial survival.185,226–228

Polymorphonuclear Granulocytes Although the role of PNGs in intracellular bacterial infections has often been neglected, their high antibacterial potential allows them to kill many intracellular bacteria. Pathogen disposal is the synergistic outcome of toxic molecules (mainly ROIs), PNG granular enzymes, and antimicrobial peptides.229 PNGs are short-lived, however longer than previously appreciated, and thus may interact not only directly with bacteria, but modulate the function of other immune cells.230 Generally, intracellular bacteria are sequestered in intracellular niches; hence, the overall contribution of PNGs to defense against chronic infections seems to be hindered to a certain extent due to the location of the pathogen. Nevertheless, PNGs may exclusively harbor certain intracellular bacteria (A. phagocytophilum) despite their broad arsenal of antimicrobial factors. M. tuberculosis, although it infects primarily MPs, has been found in PNGs in sputum from patients with TB.231 It is likely that during the early acute inflammatory response, PNGs can help to reduce initial bacterial load. During experimental listeriosis, which is an acute disease, the first day of infection is characterized by extensive PNG infi ltration at sites of listerial growth.232 Elimination of PNGs and inflammatory monocytes/macrophages by monoclonal antibody treatment remarkably exacerbates listeriosis.233,234 However, recent studies using specific depletion of PNGs have questioned the central role of these cells during systemic listeriosis.235 Depletion of PNG apparently does not affect experimental TB in resistant mice, despite modulation of T-cell responsiveness.236,237 However, susceptible mouse strains can be rendered resistant following PNG depletion.63,238 The detrimental effect of PNGs is largely due to their tissue-damaging propensity. PNGs are potent secretors of hypochlorous acid as well as of proteolytic enzymes, such as elastase (a serine proteinase), collagenase, and gelatinase.172,239,240 Such proteases express potent microbicidal activity but can also be deleterious and induce tissue damage. These secretion products of PNGs act as mediators of tissue destruction. At the same time, neutrophil elastase has been shown to specifically destroy the virulence

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proteins of Salmonella organisms.239,240 In the extracellular milieu, protease inhibitors are normally present, preventing tissue damage by these proteases. However, the concomitant secretion of hypochlorous acid inactivates these proteinase inhibitors, thus promoting cell lysis. Accordingly, PNGs have been shown to cause inflammatory liver damage by destroying infected hepatocytes during early listeriosis.232 MPs are less potent secretors of proteinases and fail to produce the major inactivator of proteinase inhibitors, hypochlorous acid. Thus, solid granulomas in chronic TB are dominated by MPs and are characterized by necrosis and fibrosis and lack signs of tissue liquefaction. During reactivation, however, PNGs may eventually be recruited to tuberculous granulomas and then contribute to granuloma caseation and liquefaction. In PNGs, pathogen sensing or exposure to cytokines or chemokines rapidly causes transcriptional events that result in release of soluble mediators. PNGs are important mediators and regulators of the Th17 pathway. They release proinflammatory cytokines (eg, IL-1α , IL-1β, tumor necrosis factor-α [TNF]-α) and chemokines,230 thus amplifying inflammation. Recent studies suggest that PNGs may participate in the resolution of inflammatory processes and thus may have a role in nonresolving inflammation. Mycobacteria stimulate production of IL-10 from PNGs via MyD8878 and Card965 pathways. The capacity to manifest diverse and probably context-dependent functions suggests that PNGs can polarize into distinct phenotypes.230 This concept has been developed for certain cancers and awaits clarification for infectious diseases. PNGs are able to kill microbes via a unique process coined neutrophil extracellular trap formation.241 This mechanism is characterized by entrapment of pathogens in fibrillar structures composed of chromatin and specific proteins from PNG granules, NADPH and myeloperoxidase dependence, and by tight coordination.242,243 Neutrophil extracellular trap killing has thus far been reported for shigellae and salmonellae, and may be relevant for other intracellular microbes.

ACQUIRED IMMUNITY Acquisition of immunity against intracellular bacteria crucially depends on T-lymphocytes that, ideally, accomplish sterile bacterial eradication. Bacterial clearance is rapidly achieved in the case of bacteria such as L. monocytogenes. In the case of persistent pathogens such as M. tuberculosis, clearance frequently remains incomplete and is arrested at the stage of bacterial containment. Bacterial containment and eradication occur in granulomatous lesions. The longer the struggle between host and microbial pathogen continues, the more essential the granuloma becomes. The T-cell requisite is probably best exemplified by the high incidence of TB and other intracellular bacterial infections in patients suffering from T-cell deficiencies, particularly AIDS.244–247 At the same time, T-lymphocytes are an unavoidable element of the pathogenesis of intracellular bacterial infections. First, granulomas impair tissue functions by occupying space and affecting surrounding cells.

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Second, the physiologic functioning of host cells may be affected by specific T-lymphocytes and their cytokines.

Dendritic Cells MPs not only serve as major habitat for intracellular bacteria, they are also potent antigen-presenting cells (APCs). However, MPs are not the most efficacious APCs, and their capacity may even be reduced during infection. DCs are the most proficient APCs.248–250 DCs, like MPs, are present in all tissues (resident DCs), and they rapidly accumulate at sites of pathogen entry. PRR composition in DC populations is variable, according to origin and location. The “maturation” status of DCs, which relates to their propensity to stimulate naïve T cells, anticorrelates with their phagocytic potential. Immature DCs, exposed to pathogens for the first time, are phagocytic, while upon microbial encounter they augment their T-cell stimulatory capacities and minimize their phagocytic propensities. Following pathogen encounter in peripheral tissues, DCs migrate to draining lymph nodes (dLNs), where stimulation of T cells and antigen presentation takes places. Microbial killing is limited in DCs. Thus, DCs are specialized in linking innate immunity to adaptive immunity rather than in pathogen elimination.251 Antigen presentation requires controlled proteolytic processing of microbial antigens. In DCs, phagosomal degradation252 and acidification253 is limited compared to MPs, thus conserving antigenic peptides for presentation via major histocompatibility complex (MHC)-class I and -II molecules. In addition, the superior antigen-presenting capacity of DCs is promoted by their copious expression of 1) MHC and CD1 molecules for antigen presentation; 2) PRRs to rapidly sense infection47; and 3) costimulatory molecules to regulate T-cell stimulation. Moreover, they produce cytokines that influence T-cell activation and differentiation. DCs have critical roles in infections with intracellular bacteria. First, DCs can be infected by these bacteria, and can thus readily present accessible antigens from microbes they harbor. For instance, tubercle bacilli infect lung-resident and -recruited DCs.254 Second, the transfer of microbial antigens from infected MPs to bystander DCs can combine the high phagocytic and degradative capacity of MPs with the high antigen-presenting capacity of DCs (“cross presentation,” see following discussion).

Induction and Modulation of T-Cell Responses The peripheral T-cell system comprises several phenotypically distinct and stable populations. T-lymphocytes expressing the αβ T-cell receptor (TCR) make up > 90% of all T cells in secondary lymphoid organs and peripheral blood of humans and experimental mice. They are further subdivided into CD4 αβ T cells that recognize antigenic peptides presented by gene products of the MHC class II, and CD8 αβ T cells that interact with antigenic peptides in the context of MHC class I molecules. Undoubtedly, these conventional αβ T cells are of primary importance for antibacterial resistance, although evidence exists that unconventional T cells also participate in the control of intracellular bacteria.226,255–257

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Generally, antigen presentation and activation of distinct T-cell populations is highly dependent on the route of antigen acquisition. Most intracellular bacteria reside in the phagosomal compartment of APCs and hence pathogenderived peptides have ready access to the MHC-II presentation pathway to stimulate CD4 T cells (Fig. 40.4). Cytosolic antigens, potentially derived from microbes, which access the cytosol, are processed by the proteasomes and loaded to MHC-I to stimulate CD8 T cells. Certain intracellular bacteria are capable of egressing into the cytosol (eg, listeriae, shigellae, and mycobacteria). Obviously, antigens from cytosolic pathogens can be readily introduced into the MHC-I processing pathway, thus promoting activation of CD8 T cells. Yet, CD8 T cells have also been isolated from mice infected with phagosomal pathogens, such as S. enterica. Thus, bacteria unable to egress into cytosol may induce potent CD8 T-cell immunity. This is achieved by a process coined cross presentation, namely presentation of exogenous antigens by MHC-I molecules. Antigen cross presentation may occur directly and multiple pathways likely lead to this phenomenon. Another way of accomplishing cross presentation is indirect and is a consequence of engulfment of vesicles derived from infected cells undergoing infection-induced apoptosis, by bystander APCs (see Fig. 40.4). During mycobacterial infection, the direct and indirect modes of cross presentation coexist. M. tuberculosis can access the cytosol107 and induce death of infected cells.258,259

Intracellular bacteria also invade nonprofessional phagocytes, some of which do not express MHC-II molecules constitutively. Consequently, such cells remain unrecognized by CD4 T-lymphocytes and provide a niche for persistent bacteria—a situation that has consequences for the course of disease. Because MHC-I molecules are expressed by almost every cell, CD8 T-lymphocytes have the potential to survey the whole body. This is particularly important for intracellular bacteria that hide in MHC-IIdeficient host cells. Obviously, recognition of these cells depends on CD8 T-lymphocytes (and perhaps unconventional T cells). L. monocytogenes, on the one hand, resides in nonprofessional phagocytes with low antibacterial potential and, on the other hand, promotes MHC-I presentation of its antigens. This may explain the predominance of MHC-I-restricted CD8 T-lymphocytes in defense against experimental listeriosis both by number and by biologic relevance. In contrast, S. enterica is primarily restricted to MPs and remains in the phagosome. This is compatible with its preferential control by MHC-II-restricted CD4 T-lymphocytes. Intracellular bacteria interfere at various levels with antigen presentation. Tubercle bacilli affect activation of CD4 T cells by interfering with MHC-II expression.260 Autophagy can contribute to T-cell activation261 and numerous intracellular bacteria impair autophagy thus reducing adaptive immune responses.

FIG. 40.4. Multiple Antigen-Processing Pathways for Stimulation of T Cells during Bacterial Infections. Infected antigen-presenting cells (APCs) directly present phagocytosed bacterial antigens to naïve cluster of differentiation (CD)4 T cells through the major histocompatibility complex (MHC)-II pathway. In addition, bacterial glycolipids are processed for presentation by CD1 molecules to stimulate double negative CD4/CD8 lymphocytes or natural killer T (NKT) cells. Antigen cross presentation occurs either following the direct way, as a consequence of cytosolic access of the bacterial antigens in infected APCs, or following a detour pathway. In the latter case, apoptotic bodies from infected cells carry antigens to bystander APCs, which activate MHC-I-restricted CD8 T cells. Recognition of infected cell death also stimulates CD4 and NKT cells. β2m, β2 microglobulin; DN, double negative; ER, endoplasmic reticulum; TAP, transporter associated with antigen processing.

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T cells may be polarized to various phenotypes, as discussed in the following, and this has a tremendous impact on the ensuing immune response. Certain factors are pivotal for this process. For instance, microbes rich in CLR ligands (eg, mycobacterial TDM) polarize T cells toward a Th17 phenotype.262 Abundance and composition of PRRs in various APC populations contribute to this process by modulating cytokine production. Susceptibility of resident tissue (eg, epithelia) to infection and their capacity to release cytokines/chemokines influences T-cell differentiation. Finally, death of infected cells and subsequent recognition of pathogens in context of inflammatory signals from APCs impacts on T-cell polarization.263

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against pulmonary TB in mice resulting in enhanced protection.268 Therefore, induction of the Th17 subset could be important in vaccination strategies against TB. Recent work has also demonstrated that IL-17 is required for optimally protective Th1 responses during primary infection of mice with F. tularensis.269 In this model, IL-17A produced by Th17 cells directly induced DCs to produce IL-12, which in turn enhanced Th1 subset differentiation. IL-17A also acted directly on F. tularensis-infected MPs to produce both IL-12 and IFNγ, which enhanced killing of intracellular bacteria indicating a prominent role for this T-cell subset in control of intracellular bacterial infection.

Cluster of Differentiation 8 T Cells Cluster of Differentiation 4 T Cells Overwhelming evidence, including experimental animal studies, and their abundance in “protective” granulomas of patients suffering from bacterial infections clearly demonstrate a critical role of CD4 T cells in immune defense against intracellular bacteria. Further evidence for this is high prevalence of disease caused by intracellular bacteria in patients with AIDS suffering low CD4 T-cell counts.264 CD4 T cells recognize peptide epitopes in the context of MHC-II molecules, which gain access to peptides present within the endosomal system. Thus, antigens from all intracellular bacteria, even those that evade the phagosome at later stages, are accessible to processing and presentation through the MHC-II pathway. However, cells that do not express the MHC-II machinery consistently, namely endothelial cells, epithelial cells, hepatocytes, and Schwann cells, are invisible for CD4 T cells and thus potential targets of Rickettsia, C. trachomatis, L. monocytogenes, and M. leprae, respectively. The CD4 T-cell population has been subdivided into distinct subsets, according to the pattern of cytokine production. Th1 cells that overwhelmingly produce IFNγ, TNF-α , and IL-2, and Th2 cells which produce IL-4, IL-5, and IL-13 were the first subsets to be defined in both mice and humans. More recently, these subsets have been categorized based on expression of transcription factors, which mediate characteristic patterns of gene expression. The Th1 subset typically expresses the T-bet transcription factor, while the Th2 subset is consistent with expression of the transcription factor GATA-3.265 More recently, a further distinct Th-cell population, termed Th17, was identified that produces the cytokines IL-17, IL-22, and granulocyte macrophage-colony stimulating factor. In addition, Th17 cells are also characterized by expression of the transcription factor RORγ t.266 Cytokines of the IL-17 family strongly induce mobilization of granulocytes during infection. This occurs by abundant production of proinflammatory mediators such as IL-6, and more specifically by increased secretion of the chemokines CXCL1, CXCL8, and CXCL6. These chemokines both attract neutrophils and eosinophils into infected tissue but also act as prosurvival factors to prolong the activity of these cells.267 Th17 cells appear to have limited importance for protection in murine models of primary infection with mycobacteria, salmonellae, and listeriae. Despite this, Th17 cells are instrumental in driving more rapid Th1 responses

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In humans, the major function of CD8 T cells is believed to be via cell contact-mediated target cell lysis. CD8 T cells recognize peptide epitopes in the context of MHC-I gene products, which acquire peptides for presentation from the cell cytosol. Intracellular bacteria are under some circumstances able to enter the cytoplasm, where antigen becomes available to MHC-I processing and presentation in a manner similar to newly synthesized host or viral proteins.107,270 Upon target cell recognition by CD8 T cells, perforin, granzymes, and granulysin are transferred to the infected host cell to allow direct killing of intracellular bacteria in addition to host cell lysis.271 Perforin oligomerizes to form a pore in the target cell, through which granules containing granzymes and granulysin are conveyed. Granzymes are a family of serine proteases that include granzymes A and B.272 Granzyme B induces apoptosis of target cells by both caspase-dependent and -independent mechanisms, while granzyme A induces apoptosis by a caspase-independent mechanism. In addition, CD8 T cells also function by producing TH1 cytokines including IFNγ and TNF- α , and directly activating infected macrophages to control intracellular bacteria in mice, which lack granulysin. Perforin- or granzyme B-deficient mice are able to control M. tuberculosis equivalently to wild-type mice.273,274 Moreover, protection against primary infection in mouse models of C. pneumonia and C. trachomatis is perforin-independent.275,276 Despite this, perforin is critical for protection of mice against secondary L. monocytogenes infection.277 Granulysin is a saponin-like protein that has been shown to directly kill intracellular S. typhimurium, M. tuberculosis, and L. monocytogenes.278 Conventional CD8 T cells recognize peptide in the context of polymorhphic MHC-I. A group of CD8 T cells also recognize MHC-I of limited polymorphism and present short formylated peptides, which are often characteristic of intracellular bacterial infection in mice.279 In humans, this group includes human leukocyte antigen (HLA)-E– restricted T cells, which respond to M. tuberculosis antigens, and lung mucosal-associated invariant T cells, which recognize antigen in the context of MHC-related protein 1.280 Protection conferred by CD8 T cells is likely to be complementary to that conferred by CD4 T cells as unlike MHC-II expression, which is restricted to professional phagocytes, virtually all nucleated cells express MHC-I molecules.

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Unconventional T Cells

B Cells

Unconventional T cells express nonpolymorphic receptors for antigen recognition and make an important contribution to protection during the early stages of infection, shaping the response mediated by conventional αβ T cells. These comprise T cells that express the gamma-delta γδ TCRs, named γδ T cells and T cells that express a low-varient form of the αβ TCRs.281 Similar to CD8 T cells, γδ T cells both produce inflammatory cytokines and are cytolytic. In mice, γδ T cells recognize heat shock protein-derived peptides presented by nonpolymorphic MHC-I-like molecules.281 Human γδ T cells can respond to nonpeptidic phosphorylated metabolites, for example, of the isoprenoid pathway of bacterial and host origin.281 In experimental murine listeriosis, γδ T cells participate in granuloma development in the liver with liver abcesses developing in their absence.282 Recently, it was also shown that in the early stages of M. tuberculosis infection of mice, γδ T cells constitute a major source of IL-17, perhaps until canonical Th17 αβ T cells can take over production of this cytokine during the ensuing adaptive response.283 Unconventional T cells that express the αβ TCR also respond to CD1, a group of nonpolymorphic MHC-related molecules. In humans, T cells that respond to CD1a, b, and c (group I CD1) are either CD4 − /CD8– or CD8 +. These T cells recognize a variety of microbial glycolipids, including lipoarabinomannan, phosphatidylinositol mannans, mycolic acids, sulfatides, sulfoglycolipids, and lipopeptides.257 Their TCR repertoire shows broad variability, and these group I CD1restricted T cells appear to perform similar biologic functions as canonical αβ T cells. In contrast, CD1d (group II CD1) is expressed by both humans and mice, and controls development of invariant NKT (iNKT) cells that express both the NK cell marker NK1.1 and an evolutionarily conserved TCR with restricted variability.284 iNKT cells can recognize phosphatidylinositol mannans from mycobacteria and glycosphingolipids from Ehrlichia and Sphingomonas.285 Recent findings suggest that iNKT-cell activation may also be mediated by altered host cell metabolic activity. TLR engagement by bacterial ligands results in a temporary downregulation of the enzyme α-galactosidase A, which in turn leads to accumulation of lipid metabolites as α-galactosidase A processes these lipids during homeostatic metabolism. iNKT cells can respond to these lipids loaded onto CD1d.286 In addition, an abundant host glycolipid, β-D-glucopyranosylceramide can act as a potent iNKT stimulator, with activity dependent on composition of the N-acyl chain.287 Upon antigen activation, iNKT cells rapidly produce cytokines and are capable of secreting both IL-4 and IFNγ, and could be critical in early responses that drive later differentiation of canonical antigen-specific T cells during development of adaptive immune responses. iNKT cells can also provide cognate antigen-specific help to B cells that express CD1d, driving primary B-cell responses, but not B-cell memory.288 Therefore, iNKT not only rapidly produce cytokines to drive T-cell responses, they also stimulate rapid antibody production by both noncognate and antigen-cognate mechanisms. As a result, iNKT cells might play central roles in development of protective immunity against intracellular bacteria.

B cells and antibodies clearly play a role in infections with intracellular bacteria. Accumulating evidence suggests that IgG and IgA are important in preventing intracellular bacteria from gaining entry via mucosal surfaces.289 Furthermore, salmonellae are often present outside cells during infection where antibody is important in neutralizing them. B cells are also potent APCs for soluble antigens including lipids presented by CD1c257 and secrete many cytokines otherwise associated with T cells, DCs, and MPs. Recently, B cells have been shown to perform regulatory functions during development of host immunity, which may benefit intracellular bacteria. B cell signalling via MyD88 during S. typhimurium infection has been associated with B-cell production of IL-10 and mice with B cell-specific MyD88 deficiency were found to be more resistant to infection.290

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Regulatory T Cells Th1 responses that are required to control intracellular bacteria ultimately contribute to exacerbated tissue pathology requiring host countermeasures. This is achieved largely by production of IL-10 and TGF-β, which limit inflammation and control IFNγ production. Although other cells such as macrophages and DCs can produce these cytokines as well, regulatory T (Treg) cells are the main producers of these cytokines. Natural Treg cells are responsive to IL-2 due to constitutive CD25 expression and are characterized by expression of the transcription factor FOXP3.291 The inducible Treg are antigen-specific and hence can develop during infection to specifically deaccelerate ongoing immune responses and thus avoid excessive damage.292 Hence, expansion of Treg cells appears to be both antigen-dependent and -independent. Treg cells also selectively express TLRs and can be activated by TLR ligands. Therefore, Treg cells could act both during innate and adaptive immune responses. Although Treg act by controlling T-cell responses and immunopathology, they can also prevent the complete elimination of bacteria, and benefit intracellular bacteria by promoting the persistent chronic state of infection.

Memory T Cells Protective immunity against intracellular bacteria is believed to last for decades due to generation of immune memory. This notion remains a touchstone in efforts to develop effacious vaccines against intracellular bacteria, which aim at efficiently driving development of T- and B-cell memory.293 It is thought that during an ongoing immune response, most T cells and B cells become effector cells and eventually die of exhaustion during a primary immune response.294 A small proportion of these cells that receive stimulatory signals of intermediate strength change their phenotype and become long-lived memory T or B cells. The memory T cells respond to the homeostatic cytokines IL-7 and IL-15 via expression of the respective cytokine receptors.295 Central memory T cells and effector memory T cells are defined by differential surface molecules and functions.295 Effector memory T cells are not able to home to LNs and instead traffic to peripheral

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tissues where they secrete cytokines and express cytotoxic functions. Central memory T cells persist in LNs and are able to quickly propagate upon IL-2 stimulation due to IL-2 receptor expression. It is believed that memory T cells survive in the absence of MHC peptide recognition. Little is known about the induction and maintenance of long-lasting T cell immunity during chronic infections where antigen is everpresent.296 In experimental listeriosis of mice, induction of memory T cells is dependent on duration of infection.297 A clearer understanding of the requirements for memory T-cell development is essential if novel vaccines are to achieve longlasting T-cell immunity against intracellular bacteria.

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to combat the invader. Although direct toxicity of intracellular bacteria is generally low, their propensity to induce release of alarmins as a consequence of necrotic host cell death exacerbates inflammation.298 Inability to dispose of the intruders and to accomplish tissue healing can result in unresolved inflammation.299 As a consequence, host factors rather than microbial virulence factors drive pathology. Most of the factors involved in inflammation (ie, cytokines [TNF-α, IFNγ], ROIs/RNIs, and eicosanoids [prostaglandins, leukotrienes]) can act in both a pro- and anti-inflammatory direction, depending on the context. Thus, a fine-tuned balance of these factors is essential as recently demonstrated for infection with mycobacteria.300

BACTERIAL-TRIGGERED INFLAMMATION

Kinetics of Infection

Inflammation during infection with intracellular bacteria is initiated by sensing of bacterial components with PRRs. Immune cells are attracted to the site of infection in an attempt

The course of infection with intracellular bacteria can be conveniently separated into three stages (Fig. 40.5). At each stage, cytokines are produced that perform two functions.

FIG. 40.5. Kinetics of the Immune Response during Infection with Intracellular Bacteria. This figure depicts critical events in the development of immunity to intracellular bacterial infection over a temporal scale. Early innate responses (hours): Polymorphonuclear granulocytes (PNGs) phagocytose extracellular bacteria and kill them. Macrophages (MΦ) and dendritic cells (DCs) become infected. DCs secrete interleukin (IL)-12 and present antigen to naïve T cells. Natural killer T cells, nuocytes, and gammadelta (γδ) T cells respond to presented bacterial products, become activated and produce cytokines such as IL-4 and interferon (IFN)γ. The host cell epithelium also responds to infection by producing chemokines and cytokines. These molecules act in concert to trigger inflammation and help shape naïve T-cell differentiation. Intermediate innate/adaptive responses (days): Naïve T cells activated by antigen presentation and costimulation respond to a milieu of cytokines including IL-12, IL-6, IL-10, and transforming growth factor-beta (TGF-β). This results in programs of gene transcription causing T cells to embark on distinct pathways of differentiation. PNGs respond to inflammatory signals and produce IL-10. Natural T regulatory cells also produce IL-10 to dampen inflammation. Late adaptive responses (weeks): Cluster of differentiation (CD)4 T-cell differentiation gives rise to T-cell subsets that are able to produce different patterns of cytokines. These cytokines then act in concert to drive different MΦ activation states, such as classically activated macrophages (T helper 1 via IFNγ) and alternatively activated macrophages (T helper 2 via IL-4, IL-10, and IL-13) activation, control unregulated host immunity (inducible regulatory T cell via IL-10). T helper 17 drive inflammation, potentiate T helper 1 and mobilizes PNGs. CD8 T-cell activation and expansion result in cells able to directly lyse infected cells. These combined effects are critical in defining whether infection is completely cleared or remains persistent. TNFα, tumor necrosis factor-alpha.

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First, they execute effector functions directed at reducing the microbial burden, and second, they express regulatory functions that influence the subsequent course of infection. The early stage is initiated within minutes after microbial entry and dominated by cells of the innate immune system, in particular PNGs and MPs, which are attracted to the site of bacterial replication by chemokines and proinflammatory cytokines. Phagocytosis and intracellular killing of bacterial pathogens by PNGs and MPs probably represents the predominant effector function at this early stage. At the same time, DCs mature and migrate to dLNs, where they produce proinflammatory and immunoregulatory cytokines, notably IL-12 for Th1 priming and IL-6 and IL-23 for Th17 cell stimulation, 301–303 and thus influence the subsequent stages by promoting induction of the protective acquired immune response. In addition, during early infection, NK cells, NKT cells, and γδ T cells can produce IFNγ and IL-17. Because NKT cells express a lowvariant TCR, they recognize a limited set of antigens and hence lack the diversity of conventional T cells governing the late stage. Moreover, cells operative at the intermediate stage can be activated via nonclonally distributed receptors such as TLRs.304–306 The intermediate stage links the early (innate) with the late (acquired) immune response. This stage is essential for priming and polarization of conventional T cells. APCs, depending on the context and pathogen, tailor CD4 T-cell responses. Moreover, innate cells harboring the pathogen are activated by cytokines in an autocrine manner or by cytokines produced by innate lymphocytes, nuocytes, and tissue-resident cells. At the late stage, conventional αβ T cells with unique specificity become operative, which mobilize and sustain host defense that results in effective control and ideally sterile eradication of the pathogen. These include both MHC-I/peptide and group I CD1/lipid-specific αβ T cells. Subsequently, reparatory processes are initiated resulting in tissue repair and remodeling and return to homeostasis. In this last stage, the trophic functions of MPs surmount their antimicrobial capacities.185,307 The length and importance of each stage are markedly influenced by the type of intracellular pathogen. In experimental listeriosis of mice, the complete sequence of the host response lasts for less than 2 weeks, whereas in human TB it may endure for decades. The early stage is particularly important for control of L. monocytogenes organisms that divide rapidly and, at the same time, are highly susceptible to intracellular killing. The more robust and slowly dividing M. tuberculosis organisms are less vulnerable to this early stage of response. The relevance of the intermediate stage to microbial control is significantly influenced by the strength of the innate and the acquired immune response. The broader the window between these two stages, the more important the intermediate stage becomes. Upon secondary infection, the conventional T cells are activated more rapidly from the pool of memory T cells. The early and intermediate stages become largely dispensable because invading pathogens are rapidly confronted with the late-stage immune response. Suffice it to say that this is the major principle of vaccination. Deciphering the

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innate immune factors activated promptly after infection or vaccination, which determine the later ensuing protective immune response, has become a highly active topic of research, termed systems immunology or systems vaccinology.293 Some bacteria persist in the host lifelong. This is frequently the case for M. tuberculosis, which persists during latent M. tuberculosis infection without overt clinical signs. Control of latent TB, however, is T cell-dependent and hence, these T cells need to be fine-tuned to avoid deviation from protective immunity by confounding coinfections, for example, with helminths.308–310 Protective immunity, however, breaks down after coinfection with human immunodeficiency virus frequently causing active TB within months after coinfection.246

Cytokines and Chemokines Cytokines are central to resistance against intracellular bacteria. At all stages, cytokines are produced that perform regulatory and/or effector functions. Although cytokines are essential for control of infection, they can also cause harm to the host. To avoid such harmful consequences, downregulation of the immune response is required at later stages of infection. Neutralization of cytokines with specific antibodies and application of knockout mice lacking defined cytokines or cytokine receptor genes have provided deep insights into the role of single cytokines. The highly intertwined steps of the anti-infectious host response, which are controlled by cytokines, are listed in the following. • • • •

Leukocyte recruitment to the site of bacterial deposition Formation of granulomatous lesions Activation of antibacterial functions in MPs Induction and maintenance of a protective T-cell response • Downregulation of the antibacterial host response to avoid harmful sequelae of exaggerated immunity

Inflammation is usually a life-preserving process that needs to be tightly controlled. Certain steps are essential for recruitment of leukocytes, including PNGs and MPs, which phagocytose and—following activation—destroy the microbe. The essentiality of these processes is underlined by the high risks of lethal bacterial infection in humans with genetic defects affecting leukocyte motility/adhesion.311 Accumulation of leukocytes at sites of microbial assault is guided by cytokine/chemokine gradients, and the process has been coined leukocyte recruitment. Tissue influx of leukocytes is a consequence of their extravasation and is mediated by close interactions between circulating leukocytes and the blood vessel endothelia, in close proximity to the area of infection.

Leukocyte Recruitment Influx of inflammatory phagocytes occurs prior to the appearance of specific T-lymphocytes, and accordingly the relevant cytokines are primarily produced by MPs, as well as by epithelial and endothelial cells in response

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to microbial invasion. Early produced cytokines with effector functions include the proinflammatory cytokines, TNF- α , IL-1, IL-6, as well as chemokines.312–314 Four subgroups of chemokines can be distinguished on the basis of a conserved cysteine motif: the CC chemokines with two unseparated terminal cysteine residues, the CXC chemokines with nonconserved amino acids separating the two terminal cysteine residues, the CX3C chemokines with several amino acids separating the cysteine residues, and the C chemokine with only one terminal cysteine (C chemokine). Grouping of the large chemokine family can be further extended on the basis of different chemokine receptors.315 The CC chemokines preferentially act on MPs, whereas PNGs are primarily activated by CXC chemokines. Other leukocytes, including lymphocytes, eosinophils, and basophils, may also be stimulated by these chemokines. The C and CX3C chemokines primarily recruit NK cells and lymphocytes. Chemokines as a group play an important role in early mobilization of host defense. Knockout mice lacking the receptor for the CC chemokine MCP-1 are more susceptible to listeriosis than controls. 316 Knockout mice deficient in CC chemokine receptor 2 (CCR2) succumb to TB. In these mutants, the recruitment of macrophages, DCs, and T cells to the lung is impaired.317 On the other hand, MCP1-deficient knockout mice control M. tuberculosis infection similarly to wild-type animals. MCP-1 acts via the CCR2 receptor. It therefore appears that these chemokines are essential for antibacterial protection but are mutually compensatory.318 Experiments in other systems have revealed a central role of chemokines in early inflammation, in particular PNGs and monocyte extravasation. Chemokines, particularly those able to bind CCR2 receptor, are essential for bone marrow emigration of MPs and are therefore essential for infection with intracellular pathogens, as demonstrated for listeriae.319 In addition, some chemokines activate professional phagocytes and in this way probably promote early reduction of bacterial load. Abundant levels of CC chemokines are on the other hand deleterious, as mice lacking a chemokine decoy receptor (D6) have been shown to be impaired in controlling tissue inflammation during TB.320 The proinflammatory cytokines, IL-1, IL-6, TNF- α , and migration inhibitory factor are also involved in the early accumulation of inflammatory phagocytes at the site of bacterial growth. The essential role of IL-6 and TNF- α in antibacterial immunity is demonstrated by the exacerbated susceptibility to listeriosis and TB of knockout mice with a deficient IL-6 or TNF-type 1 receptor gene.321 Similarly knockout mice deficient in migration inhibitory factor suffer from exacerbated S. enterica infection.322 The proinflammatory cytokines, when produced in high amounts, cause acute-phase responses by inducing release of various plasma proteins from hepatocytes. They also serve as endogenous pyrogens that stimulate fever, and TNF- α is responsible for cachexia, the characteristic feature of wasting in infections with many intracellular bacteria, notably TB. Clinical trials showing that detrimental effects of excessive TNF- α production in patients with TB and leprosy can be ameliorated by treatment with

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thalidomide emphasize the double-sided role of TNF- α in chronic infections.323,324

Leukocyte Extravasation In the early phase of inflammation, extravasation of (and invasion by) PNGs and, subsequently, blood monocytes is induced by proinflammatory signals mediated by bacterial components (N-f-met-containing peptides, such as f-MetLeu-Phe or ligands for PRRs), complement components (C5a), arachidonic acid metabolites (PGE2), and cytokines. Infected MPs produce numerous proinflammatory cytokines, as well as various chemokines that stimulate local endothelial cells and blood phagocytes. The inflamed endothelium around the primary lesion expresses elevated levels of adhesion molecules, thus promoting extravasation of inflammatory phagocytes.325 Extravasation is mediated by interactions between leukocytes and endothelial cells by means of adhesion molecules. These include selectins, integrins, and members of the Ig superfamily. The L-selectins are found on leukocytes, whereas the Pand E-selectins are expressed by endothelial cells. Selectins bind to carbohydrate ligands on the corresponding cell type. The integrins are expressed on many cell types, including leukocytes and endothelial cells. Contact between leukocytes and endothelial cells is initiated when the blood vessel is suddenly broadened in diameter at inflammatory foci.326 This fast process is controlled by histamines, eicosanoids, and tryptases derived from tissue mast cells and recruited PNGs. Release of platelet activation factor following PRR stimulation in MPs and the subsequent coagulation cascade augment blood vessel permeability changes as well. Activated endothelial cells and leukocytes upregulate surface expression of adhesion molecules and thus promote leukocyte binding to the endothelium. This sets into motion the cascade of adhesion events. Selectin-mediated interactions result in leukocyte tethering and rolling. Subsequently, integrin interactions with Ig superfamily molecules cause tight leukocyte adhesion to endothelial cells. Once leukocytes firmly adhere to the endothelium, transmigration to the inflammatory focus occurs. Upregulation of P- and E-selectin expression primarily promotes PNG extravasation. In contrast, the L-selectins are constitutively expressed on virtually all leukocytes. Activated and memory T cells, as well as inflammatory phagocytes, however, express higher levels of integrins such as lymphocyte function-associated antigen-1 and very late antigen-4 and activated endothelial cells show elevated expression of Ig superfamily molecules. Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 upregulation is primarily important for monocyte and T-cell transmigration to inflammatory foci. Chemokine receptors are decisive factors for selective T-cell migration.314,327 Differential expression of chemokine receptors on human Th1 and Th2 cells, effector versus memory T cells, as well as central memory T cells versus effector memory T cells, direct preferential migration of selected T cells to the site of inflammation.328,329 Chemokines can bind to glycosaminoglycans bound to the endothelial surface without loss of biologic activity. Interactions between

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chemokine receptors on lymphocytes and chemokines bound to endothelial cells further promote selective T-cell migration. The lack of CCR7 (receptor type 7 for CC chemokines) combined with the low expression of CD62L and the high expression of lymphocyte function-associated antigen and of α4β1 integrin seems characteristic for effector memory T cells of Th1 type that migrate to sites of inflammation. In summary, tethering and rolling, which is then succeeded by tight adhesion and subsequent extravasation of leukocytes, results in leukocyte accumulation at the site of microbial colonization. This forms the basis for antimicrobial defense at site of microbial residence.

The Inflammasome IL-1β is a key inflammatory cytokine in host defense,330 and the generation of biologically active IL-1β is controlled by the inflammasome. Transcriptional regulation of this cytokine is primarily induced by PRR agonists and distinct cytokines, while cleavage of the proform is executed in the inflammasome. The inflammasome is a multiprotein cytosolic platform consisting of an NLR or PYHIN member, the ASC protein, and the procaspase-1.331 Depending on the NLR/ PYHIN cognate that is recruited to the cytosolic complex, four major inflammasomes have been described: NLRP1, NLRP3, NLRC4, and the AIM2 inflammasome. These inflammasomes detect intracellular cues from microbes, which provide the basis for optimal responses against infections with intracellular bacteria. Certain microbes induce activation of single or multiple inflammasomes.332–334 Thus, L. monocytogenes activates NLRP3 by virtue of its virulence factor LLO. Bacterial DNA is recognized through the AIM2 inflammasome and bacteria may also interfere with NLRC4 inflammasome assembly. AIM2 is relevant for recognition of other cytosolic bacteria, particularly for francisellae.70,75 Mycobacteria stimulate the NLRP3 inflammasome in macrophages, although evidence for inhibitory activity of tubercle bacilli has also been reported.334 Salmonellae induce activation of NLRC4 inflammasome via T3SS and flagella, and also trigger the NLRP3 inflammasome.335 Thus, most pathogens are sensed by multiple inflammasomes, suggesting that during coevolution, diverse pathways converge to ensure release of bioactive IL-1β upon infection. In addition, inflammasomes sense danger signals released by dying cells.332,334 Accordingly, inflammasome activation by intracellular microbes, which induce extensive tissue destruction, represents a double-edged sword: the benefit of IL-1β in the early phase of infection bears risk for the host in the case of prolonged release during chronic infection.

Interferons Both classes of interferons, IFN-I (comprising multiple members, with IFNα and IFNβ being of highest relevance) and IFN-II (IFNγ), are closely associated with host defense. IFN-I and IFN-II show partition in defence against viral and bacterial infections, respectively. However, this classification is oversimplified, and it is becoming increasingly clear that both IFN classes participate in infections with intracellular

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bacteria. IFN-II can be viewed as primarily beneficial, whereas type I IFN often seem to be detrimental. Activation of antibacterial macrophage properties by IFNγ represents a central step in acquired resistance against intracellular bacteria. IFNγ-activated macrophages rapidly kill susceptible intracellular bacteria, such as L. monocytogenes. Although the question as to whether IFNγ-stimulated MPs actually kill M. tuberculosis remains a matter of controversy, it is certain that they markedly inhibit growth of this pathogen. Consistent with such a central role of IFNγ in antibacterial immunity, IFNγ- or IFNγR-deficient knockout mice rapidly succumb to infections with L. monocytogenes, M. tuberculosis, S. enterica, and other intracellular pathogens.336–338 Support for the central role of IFNγ in control of intracellular bacterial infections in humans stems from the identification of hereditary IFNγR deficiency in children who suffer from frequent infections with various intracellular bacteria or even from M. bovis BCG vaccination.339,340 Conversely, IFNγ treatment in adjunct to chemotherapy has been used successfully in the treatment of leprosy, TB, and atypical mycobacteriosis.341 IFN-I cognates on the contrary are deleterious during infection with listeriae,342–344 mycobacteria,345–347 chlamydiae,348 and francisellae.349 These notions emerged from studies with mice lacking the receptor for the cytokine (IFNAR knockout mice), revealing impact of IFN-I on both innate and adaptive immune responses. Recently, crosstalk between type I and II IFN was described during infection with L. monocytogenes.350 The role of IFN-I during infections with other intracellular pathogens as well as detailed mechanistic explanations as to how these cytokines negatively impact on disease control awaits clarification.

Macrophage Heterogeneity and Polarization The myelomonocytic pathway, which comprises mature and immature cells, is characterized by a tremendous diversity and plasticity.351 The most prominent member of this pathway, the macrophage, performs a dual role in infections with intracellular bacteria. It may harbor, and at the same time dispose of, the intruder. These binary effects are correlated with the functional plasticity of the MP. From sensing of pathogens and initiation of inflammation to the resolution of infection and tissue repair/remodelling, MPs play diverse functions of critical importance. Similar to the dichotomy of Th1/Th2 cells and closely related to the cytokines produced by these lymphocyte subsets, macrophages may develop into classically activated macrophages (CAMs) or alternatively activated macrophages (AAMs).352 These two forms should be viewed as extreme poles of a continuum, with intermediate forms occurring in between. In stark contrast to T cells, chromatin reorganization in activated macrophages is not fi xed. As a corollary, macrophage activation mirrors functionality of these cells in a particular spatiotemporal context. More recently, epigenetic modifications (Jmjd3-Irf4 axis) were associated with preferential AAM development.353,354 CAMs or M1 macrophages are polarized by IFNγ or TLR agonists, such as LPS. Thus, Th1 cells and NK/NKT cells

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promote CAM genesis. The polarized CAMs secrete copious amounts of inflammatory cytokines (TNF-α, IL-12/23) and antimicrobial molecules (RNIs, ROIs), modulate iron homeostasis by repressing ferroportin and inducing ferritin, and upregulate costimulatory and MHC-II molecules on their cell surface. Production of CXCL9 and CXCL10 by CAMs boosts Th1-cell recruitment. Concomitantly, the beneficial antimicrobial effects may cause collateral tissue damage.355 AAM or M2 macrophages are polarized by the canonical cytokines IL-4 and IL-13 as well as by the more recently described mediators IL-21,356 IL-33,357 and IL-25.358 Accordingly, Th2 lymphocytes and nuocytes358–360 may induce AAM polarization. AAMs seem to exclusively derive from resident tissue macrophages.361 Functionally, AAMs are characterized by increased endocytic activity, release of IL-10, upregulation of IL-1RA and IL-1R2, and secretion of chemokines (CCL17, CCL22, CCL24) involved in recruitment of Th2 cells, Treg cells, eosinophils, and basophils. AAMs upregulate ferroportin and express folate receptors. A hallmark of these macrophages is the expression of arginase-1, which not only enables AAMs to perform tissue remodeling, but also induces expression of E-cadherin,362 involved in homotypic fusion. IL-4 and IL-13 induce homokaryon formation, a cell pathologic event particularly relevant for infections with intracellular bacteria. The metabolic and secretory properties of AAMs favor trophic functions and tissue tolerance.363 The precise role of AAMs and their cytokines in giant cell formation during chronic bacterial infection awaits further clarification. Although CAMs and AAMs are phenotypically and functionally different, both undergo a vivid crosstalk with adaptive lymphocytes (αβ T cells) and innate lymphocytes (γδ, NK, and NKT cells). The tremendous plasticity of polarized macrophages has been elucidated in malignancy,351,364 and should similarly hold true in bacterial infections. During L. monocytogenes infection, CAMs (Gr1low patrolling monocytes) remodel to AAMs.365 F. tularensis induces similar transition in macrophage polarization, to the advantage of the bacterium,366 and M. tuberculosis seems to hijack this polarization pattern toward AAMs.367,368 Blood cell gene transcripts from patients with TB show a combined IFN-I and IFN-II response accompanied by classical CAM gene expression.369,370 Patients suffering from typhoid fever present enrichment for genes encoding IFNγ-mediated immune responses. However, patients in which a CAM signature has been replaced by an AAM signature and have maintained it are prone to becoming carriers or to manifesting disease relapse.371 A clear scenario related to CAM/AAM balance has been reported for leprosy.372 Remodeling of recruited macrophages from alternative to classical phenotype occurs in patients who have converted from a lepromatous to a tuberculoid form of this disease.

GRANULOMA FORMATION: A PATHOLOGIC HALLMARK OF INTRACELLULAR BACTERIAL INFECTION A characteristic feature of many infections caused by intracellular bacteria is the need for tissue remodeling by the host at the site of infection when the ensuing inflammatory

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response is not successfully resolved. This process leads to formation of structures called granulomas373 (Fig. 40.6). The granuloma is primarily composed of T-lymphocytes and MPs, some of which contain indigestible antigen. In addition, granulomas also contain B-lymphocytes and DCs. MPs in granulomas demonstrate a degree of morphologic plasticity. Major macrophage phenotypes seen in granulomas include epitheloid-like and multinucleated giant cells. Macrophage plasticity may also be reflected in presence of both CAMs and AAMs, although the contribution of different states of macrophage maturation and activation is unclear at present. Granulomatous lesions are generally initiated by innate inflammatory signals mediated by microbial products, chemokines, and proinflammatory cytokines that are produced by endothelial cells and MPs at the site of infection. The best-studied granulomatous disease is TB where development of granulomas in the lung eventually leads to transmission of bacteria via cough. During TB, the granuloma is encapsulated by a fibrotic wall and its center becomes necrotic.374 The combined effects of prolonged macrophage activation, persistence of intracellular bacteria, and tissue hypoxia likely lead to enhanced cell death in the center of granulomas resulting in the formation of caseum. This caseum may favor the local replication of facultative intracellular bacteria in the cellular detritus, as well as microbial dissemination to distant tissue sites and to the environment to transmit infection. T cells are the critical driving forces of granuloma formation and maintenance in most granulomatous diseases, and T cell production of IFNγ and TNF-α is crucial. Experiments utilizing the respective knockout mice emphasize a role of IFNγ, TNF-α , and lymphotoxin-α3 in granuloma formation and maintenance during TB.337,375,376 Hereditary IFNγR deficiency has been described in humans, and these immunodeficient patients severely suffer and ultimately die of infections with intracellular bacteria.339,340 That this high susceptibility was accompanied by impaired granuloma formation is suggestive of the central role of granuloma formation in control of bacterial growth. The critical role of TNF-α in the containment of M. tuberculosis in humans has been impressively demonstrated by the increased risk of the reactivation of TB in patients with rheumatoid arthritis undergoing treatment with anti-TNF-α monoclonal antibody.377 Noncontagious diseases also characterized by granuloma formation include berylliosis caused by the undegradable irritant beryllium378 and sarcoidosis of unclear etiology.379

Early Immunologic Events in Granuloma Development The laboratory mouse model provides a plethora of genetic and immunologic tools to dissect the granulomatous response to infection. Although the classically used mouse model of TB fails to fully reproduce human-like granulomas, it represents a useful model for the generalized early stages of granuloma development during chronic infection. Orchestration of granuloma formation requires induction of M. tuberculosis –specific T-cell responses. In this section, we highlight recent findings using the murine model of TB to

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FIG. 40.6. Stages of Granuloma Development during Intracellular Infection. This figure describes a generalized scheme of granuloma development during intracellular infection, using tuberculosis (TB) as a paradigm. The upper panel depicts formation of a solid granuloma from bloodborne macrophages (MΦ), T cells, B cells, and dendritic cells (DCs) in response to infection. The center of the solid granuloma is composed of T cells as well as infected and uninfected MΦ which are, in turn, orchestrated by T-cell cytokines. Such signaling leads to classically activated macrophages and alternatively activated macrophages within granulomas. Eventually, the center of the granuloma shows extensive necrosis of MΦ to produce a necrotic granuloma. The necrotic granuloma may spontaneously heal or become sterilized by chemotherapy. Complete inactivation and scarring then ensues or in the event of incomplete clearance of persistent bacteria, reactivation of disease may occur later. In the case of TB, the necrotic granuloma may also further develop to produce a large central caseous mass that eventually liquefies to give rise to a caseous granuloma. Absorption of bronchi by this structure allows egression of bacteria into the airway mediating transmission. IFNγ, interferon gamma; IL, interleukin; TNF-α, tumor necrosis factor-alpha.

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give insight into events governing how granulomas develop during the early stages of infection. These responses can be enhanced by early production of the cytokine IL-17 by activated γδ T cells.283 IL-17 enhances influx of Th1 cells producing IFNγ and TNF-α into the infected lung that are in turn able to control bacterial replication.380 Excessive IL-17 may also enhance host pathology due to enhanced Th1 responses and require counter measures of control.381 Such control could be provided by IL-10 as Th1 cell influx into the lung during murine TB is enhanced in its absence.382 Furthermore, enhanced IL-17 production in the lung of mice after repeated application of M. bovis BCG during TB results in exacerbated disease, demonstrating the need to regulate inflammation to maintain a protective immune balance.383 In addition, IL-12 signalling of DCs represents a key event initiating Th1 responses, and this IL-12 signaling shows multifunctionality during the early granulomatous response to infection. Signaling of immature DCs via engagement of IL-12 receptor β1 chain by the IL-12p40 homodimer, IL-12p80, mediates DC migration to dLNs, a prerequisite for driving rapid T-cell responses in the lung.384 DCs can also produce an IL-12 receptor splice variant that not only drives naïve T-cell proliferation in lymph nodes but also enhances migratory activity of T cells.385 Chemokines, which play multiple roles in early immune responses, are also involved in granuloma formation. CCR7 is required for murine granuloma formation386 ; CCL19 and CCL21 participate in development of the Th1 responses while another homeostatic chemokine, CXCL13, is involved in the spatial construction of granulomatous lesions.387 CCL19 and CCL21 are ligands of CCR7 on DCs and also signal their trafficking to dLNs to prime T-cell responses.254 Therefore, homeostatic chemokine function demonstrates an intimate relationship between the need to drive T-cell responses in dLNs and the orchestration of granuloma formation in the lung during early stages of TB. During later stages of this disease, when granuloma formation has been fully established, T-cell activation can occur in the lung via ectopic or tertiary lymph node structures, such as inducible bronchus-associated lymphoid tissue.388 This ability to prime T cells directly in the lung may become more relevant in later stages of human disease when cavity formation and extracellular growth of M. tuberculosis results in the need to provide protective immunity in the face of increased bacterial loads in the lung.388,389 In addition, IL-17 produced by T cells enhances CXCL13 expression, which is required for inducible bronchus-associated lymphoid tissue formation, indicating an additional role for this cytokine in protective immunity.388,390

Contribution of Granuloma to Disease In addition to its central role in TB, granuloma formation and development represents a central feature of disease resulting from intracellular infection with brucellae.391 Brucellosis is a multiorgan disease in which granulomas contain largely epithelioid macrophages occurring in lymphatics, brain, lung, and bone. Hepatic granulomas can develop central necrosis. The structural similarity of these granulomas to those seen in other infections makes a definitive diagnosis of brucellosis complicated.392

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Melioidosis represents another systemic disease with an important granulomatous component in bone, brain, and lung.393 The disease in humans is caused by B. pseudomallei. Granulomas often show layers of epithelioid macrophages and presence of giant cells. These granulomas are often unable to contain bacteria because systemic spread of bacteria and septicemia can occur. Lymphogranuloma venereum, a sexually transmitted disease where granuloma formation is central, is caused by the L1, L2, and L3 serotypes of C. trachomatis. Trachoma, which results from a granulomatous response of the upper eyelid due to C. trachomatis infection, is a leading cause of infectious blindness worldwide.20 In human infection with S. enterica serovars, Typhi and Paratyphi, bacteria are disseminated within MPs to the liver, spleen, and lymph nodes where granuloma are formed.394 The definitive role of granuloma formation in development of active TB has been difficult to establish. A century ago, Anton Ghon defined latent subclinical TB in humans as the Ghon complex, which is composed of single subpleural caseous granulomas accompanied by caseation in the corresponding dLNs.395 Animal models currently in use are biased toward accelerated disease, where granuloma development consistently accompanies pathology, making understanding the role of the granuloma in protection difficult to discern. Key to initial control of M. tuberculosis very early in infection is how the infected cell dies. Should the cell die by apoptosis, bacterial growth is primarily prevented. Should the cell die to necrosis, where the host cell membrane is breached, M. tuberculosis can proliferate and disseminate.396,397 Central necrosis and caseation in granulomas could in some way reflect the balance between these mechanisms of cell death. Infected macrophages also employ antibacterial mechanisms to control intracellular bacterial growth, such as autophagy126,142 and production of antimicrobial peptides.398,399 IFNγ enhances these mechanisms, but intracellular bacteria are often incompletely eradicated requiring consecutive waves of specific T cells and monocytes to arrive in the lung or dLNs, which then arrange into the classic stratified granuloma structure.374,400,401 The murine model of mycobacterial disease has addressed the contribution of T-cell immunity to granuloma development using intravital imaging. These studies have shown that antigen presentation to T cells within granulomas leads to production of TNF-α , which is critical to maintenance of granuloma structural integrity.402 Furthermore, the T-cell compartment within granulomas was shown to be highly dynamic with T cells entering and exiting lesions. T-cell effector function in this model could be potentiated by exogenously introduced antigen, and this strategy might be explored using therapeutic strategies to augment the protective properties of granulomas.403 The understanding of granuloma development has benefited remarkably from use of zebrafish infected with Mycobacterium marinum to simplify study of the early events of granuloma formation in vivo. In particular, intravital imaging of zebra fish embryos to model early events in granuloma formation in the absence of adaptive T-cell responses showed that infected macrophages traffic readily

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between multiple nascent granulomas.404 This suggests that granuloma formation before the onset of adaptive immunity may in fact assist stable establishment of infection at an early stage. Later, T cells may be required to imprint on the granuloma the ability to contain mycobacterial growth. The zebrafish has been exploited for forward genetic screening revealing a critical function of Ita4h, whose gene product catalyses leukotriene B4 synthesis. Leukotrienes are eicosanoids derived from essential fatty acid metabolism that demonstrate chemotactic and anti-inflammatory activity. Increased leukotriene B4 synthesis was found to suppress TNF-α production, which in turn abrogated protection in nascent granulomas.300 This finding highlights the utility of using diverse models to address fundamental issues in granuloma biology. Other animal models of TB, namely nonhuman primates (NHPs), rabbits, and guinea pigs show some ability to model granuloma formation. In contrast to the murine model, granulomas formed in rabbits and guinea pigs demonstrate central caseation and hypoxia.405,406 The NHP represents the closest model to human granuloma formation. Infection of NHPs results in a portion of animals with latent M. tuberculosis infection. Unlike other animal models of TB, disease in NHPs transitions through formation of a Ghon complex, suggesting that this initial event may represent the very early response in all cases.407 The lymphatic lesion then undergoes necrosis and caseation as early as 3 to 4 weeks postinfection. These data and those seen in humans suggest that the Ghon complex indeed represents the site at which disease is either contained or progresses. Data from NHPs indicate that granuloma cellular composition and T-cell function can vary greatly in the same host and even in the same organ, for example, the lung. Additional studies in NHPs have established that early granuloma formation is accompanied by potent inflammatory responses, which are characterized by transcriptional networks controlled by the cytokines IFNγ and TNF-α as well as the intracellular JAK and STAT signaling pathways, while later granulomas show transcriptional networks reflective of tight control of inflammation and chemokine production.408 This inhibitory property of the late-stage granuloma could reflect the necessity to limit host tissue damage resulting from a nonresolved inflammatory response.

Cellular Mechanisms Active within Granulomas In terms of understanding of cellular mechanisms operative in granulomas and how these impact granuloma biology, TB is the most widely studied granulomatous disease. Macrophages within granulomas demonstrate remarkable morphologic plasticity. Different macrophage morphologies in granulomas include epithelioid and foamy cells. In addition, macrophages can fuse to produce multinucleated giant cells.374 On the tissue scale, one of the most characteristic gross morphologic features of granulomas in TB is their propensity to form a region of central necrosis, which softens in end stages to become caseous or cheese-like. This white mass results from extensive MP cell death.409 Central necrosis appears at the onset of vigorous T-cell immunity,

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suggesting that it could represent a sacrifice of tissue to allow efficient killing of intracellular bacteria. At this stage, granulomas can become inert and eventually calcify. However, if intracellular bacteria continue to grow to up to hundreds of billions of microorganisms, granulomas in the lung become enlarged and can erupt into a bronchus with the central caseous mass liquefying. Coughing up the liquefied caseous mass containing bacteria allows transmission of TB. The molecular and cellular events that define these processes enable understanding of how TB is transmitted and offer important targets for therapy. In a model of granuloma caseation in the lung after dermal infection of knockout mice unable to produce RNIs, central granuloma caseation is accompanied by a local increase in serine protease activity. The serine proteases cathepsin G and neutrophil elastase are both active at neutral pH. Although both serine proteases show antibacterial activity,398,410 cathepsin G enhances necrosis of IFNγ-activated macrophages infected with M. tuberculosis.410 Therefore, deployment of serine protease activity in the lung may represent a double-edged sword, reflecting at a molecular level the need to balance mycobacterial killing with macrophage cell death in the granuloma. Recent studies suggest that products of the sst1 genetic locus in mice prevent development of necrotic lung lesions during murine TB. A protein product encoded at this locus termed intracellular pathogen resistance 1 shows ability to direct infected macrophages to undergo apoptosis rather than necrosis.411 For disease transmission to occur, the lung extracellular matrix must be remodeled. Fibrillar collagens, highly resistant to enzymatic degradation, lend the lung extracellular matrix its extraordinarily tensile strength. In matrix remodelling, matrix metalloproteinases (MMPs), which cleave collagen at neutral pH, appear to be critical players. Patients with TB demonstrate increased levels of MMP-1 activity in sputum, and a transgenic mouse expressing human MMP-1 suffered increased lung tissue destruction during TB, suggesting MMP-1 is a key proponent of lung remodeling.412 Tissue containing caseous granulomas shows an increase in host cell lipid metabolism.413 Foamy macrophages, containing multiple lipid droplets, are also found in regions surrounding central necrosis in granulomas.413 In addition, human macrophages infected in vitro with M. tuberculosis demonstrate upregulated lipid metabolism.414 M. tuberculosis in sputum from patients with TB are contained in lipid droplets and show reduced antibiotic susceptibility.415 It is therefore possible that persistence of mycobacteria in foamy macrophages my represent a survival strategy in the face of drug therapy.

GENETIC CONTROL OF RESISTANCE AGAINST INTRACELLULAR BACTERIA Resistance against intracellular bacteria is genetically controlled, and inherited factors are of particular importance in chronic infections with broad clinical spectrum, such as TB and leprosy. Although the impact of host genetic mechanisms on the outcome of infectious disease has been recognized for a long time, our understanding of the underlying

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factors remains fragmented.416,417 First, resistance to infection is highly polygenic. Second, a marked heterogeneity exists within populations. Third, variability in the genome of the pathogen, as well as environmental factors such as the availability of nutrients, further affect the outcome of the host–pathogen relationship. However, technologic advances in recent years have markedly facilitated the progress of immunogenetic studies. Single nucleotide polymorphism arrays and DNA sequencing are frequently applied in such investigations and genome-wide association studies have allowed identification of distinct loci associated with susceptibility to infection.418–420 The significance of genetic factors is perhaps most dramatically illustrated by the Lübeck disaster in 1927, when 251 babies were accidentally vaccinated with viable M. tuberculosis instead of BCG. At the end of the 6-year observation period, six children (2%) still suffered from TB, 129 (51%) had become ill but recovered, 77 (31%) had died, and in 39 children (16%) clinical signs of TB had never developed.421 The marked influence of ethnic differences on the prevalence of TB further supports the role of genetic factors.422 In the 1940s, Lurie studied native resistance to TB in rabbits, and by selective inbreeding he succeeded in establishing strains of rabbits that differed remarkably in their susceptibility to infection with M. tuberculosis.219 Similarly, congenic mouse strains that differ in their susceptibility to experimental infection with several intracellular bacteria have been developed.423 At least three levels of the host–pathogen relationship serve as potential targets for genetic control, which are briefly described in the following. 1. Genetic factors decide whether infection becomes abortive or establishes itself in a stable form. Convincing evidence for genetic control mechanisms at this level does not exist. 2. Genetic factors control transition from infection to disease. This control step distinguishes susceptible from resistant individuals. Such inherited influences are well proven in mice and begin to unfold in the human population. 3. Severity and/or form of disease are controlled by genetic factors.

Primary Immunodeficiencies Numerous single-gene (Mendelian) disorders that perturb immune functions (ie, currently > 300 primary immunodeficiencies) have been reported.424 These monogenic diseases are rare, arise from major functional aberrations at single genes, and confer predisposition to a certain type of infection. The most thoroughly characterized of these syndromes in context of infection with intracellular pathogens is the Mendelian susceptibility to mycobacterial diseases.423 The IFNγR1 deficiency was described first.339,425–427 Subsequently, deficiencies in other Th1-associated molecules, namely IFNγR2,428,429 STAT1,430,431 IL-12B,432 and IL-12B1433 were reported. More recently, IRF8 mutations, which affect development of monocytes and DCs, have been associated with susceptibility to mycobacterial diseases.434 Patients with

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Mendelian susceptibility to mycobacterial diseases also develop infections with typhoidal and especially nontyphoidal salmonellae with high frequencies.435

Multigenic Predispositions/Major Genes Linkage studies, and more recently genome-wide association studies, suggest polygenic characteristics of most infectious diseases. Studies in the mouse system have revealed a single dominant autosomal gene on chromosome 1, which is responsible for resistance against M. bovis BCG, M. lepraemurium, M. avium/M. intracellulare, S. enterica, and the protozoan pathogen Leishmania donovani. In contrast, murine resistance against other intracellular bacteria, most remarkably M. tuberculosis, is apparently not controlled by this gene.436 The responsible gene has been named Nramp1 for natural resistance-associated macrophage protein.437 The role of Nramp1 in controlling infectious diseases in humans remains controversial.438,439 Other mouse gene loci affecting susceptibility to mycobacterial disease are Icbp,440 Trl-1,441 Trl-4,442 Tbs1,443,444 and Ipr1.411,445 Certain loci impact on resistance against multiple intracellular bacteria. Ipr1 affects susceptibility to listeriae,411 whereas Icbp affects responses against salmonellae.446 The contribution of human homologues of the previously mentioned loci to antibacterial resistance needs further in-depth investigations. Genomewide association studies have described susceptibility loci for TB on chromosome 18q11.2447 and chromosome 8q12.448 Susceptibility to leprosy is associated with multiple innate immune-relevant genes.449,450 Legionnaires’ disease seems to be differently associated with polymorphisms in TLR-4 and TLR-5.451–453 Single nucleotide polymorphism arrays, to analyze the association between clinical TB and certain PRRs (TLR-1, 2, 4, 6, 8, 9; DC-SIGN; NOD2; mannan-binding lectin) and adaptor molecules (TIR domain-containing adaptor protein), came to divergent results indicating a need for further studies.423,454 Polygenic predispositions and immune defects in innate elements are currently being investigated and seem to bear an unanticipated role in resistance against infectious diseases.455

Major Histocompatibility Complex Control of Severity and Form of Disease Segregation analyses in various human populations also indicate linkage of HLA types with severity of TB and leprosy. Strong evidence exists suggesting an influence of HLA on the development toward the tuberculoid or the lepromatous pole of leprosy. Although some linkage with MHC-I molecules has been observed in certain populations, MHC-II control appears to be more important.456 Originally, it was found that HLA-DR2 subtypes are linked with increased incidences of lepromatous leprosy and that HLA-DR3 represents a linkage marker for tuberculoid leprosy. Recent population-based association studies, however, have provided evidence for an association between distinct HLA-DR2 alleles and susceptibility to tuberculoid leprosy. In TB, evidence for association of HLA-DR2 subtypes with pulmonary TB has been found. With more data from various population groups

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becoming available, it is increasingly clear that HLA-DR associations with distinct disease forms differ among population groups, thus making it impossible to extrapolate from one population to another. These discrepancies underline the polygenic nature of resistance to infectious diseases.

CONCLUSION AND OUTLOOK It is hoped that the reader of this chapter has not only become familiar with the principal mechanisms underlying immunity against intracellular bacteria, but also realizes the great complexity at the interface between prokaryotic and mammalian eukaryotic systems. Understanding intracellular bacterial infections requires knowledge not only of immunology, but also of molecular biology of the infectious agent and biology of the target cell. In vitro analyses can only provide incomplete answers to the questions relevant to antibacterial immunity and must be complemented by in vivo experiments. Despite the high degree of complexity, such interdisciplinary research efforts certainly provide rewards. First, understanding the performance of the immune system in bacterial infections can provide clues to questions pertinent to basic immunology. Knowledge of the rules underlying the extraordinary plasticity and adaptability of the immune system required for coping with transmutable “viable antigens” that developed during millennia of coexistence will provide deeper insights into the immunoregulation and evolution of the immune system. Second, applied questions will benefit equally well from these approaches. With the increasing inadequacy of chemotherapy in the control of bacterial infections, the need for adjunctive immune

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measures is gaining importance. Rational strategies toward vaccination and immunotherapy will benefit from the deeper understanding of the immune mechanisms operative in intracellular bacterial infections. With the elucidation of the genomes of major intracellular pathogens, as well as of the human and murine genomes, this type of interdisciplinary research has, in fact, entered a new phase and novel next-generation deep-sequencing technologies promise more rapid and less costly progress. Global analyses of the transcriptome and proteome down to the single-cell level will undoubtedly provide a comprehensive view of this dynamic interplay in the near future. The reader may find it ironic that the spirit of these investigations remains the same as it was at the early beginnings of immunology, which started as an approach to the intervention of bacterial infections.

ACKNOWLEDGMENTS The authors acknowledge financial support from the German Ministry of Science and Technology (Bundesministerium für Bildung und Forschung; BMBF grant nos. 01KA1010, 01KI1007C and 01KI0781, 01GS0814, 0313801K), The Bill & Melinda Gates foundation (BMGF GC6-74 37772), the European Union Framework Programme 7 (EU FP7 NEWTBVAC, PHAGOSYS, SysteMTB, TRANSVAC), the European and Developing Countries Clinical Trials Partnership (EDCTP projects AE-TBC and TB-TEA) and the National Institutes of Health (NIH grant no. 745090-HHSN272200800059C). We are grateful to M.L. Grossman for help preparing the chapter and to D. Schad for preparing graphics.

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41

Immunity to Extracellular Bacteria Moon H. Nahm • Jannet Katz

INTRODUCTION Human interactions with bacteria are complex, with a consortial relationship having been developed between humans and microbes. Each human being is composed of 1012 human cells and is inhabited by 1014 bacteria composed of innumerable species.1 More than 500 distinct bacterial species are estimated to reside in the human oropharynx alone.2 Recent studies of gut microbiota have begun to reveal both the extensive diversity of microbes in the gut as well as their complex relationship with the host.3 Relatively few of these bacteria are harmful to us in any way. We know little about the innate or acquired immune mechanisms that maintain this equilibrium. In large part, many diseases caused by bacteria are mistakes in which this consortial relationship breaks down and the lines that define the relationship are crossed. The innate and adaptive responses to these transgressions can in themselves lead to dire consequences. In Lives of the Cell, Lewis Thomas points out: The microorganisms that seem to have it in for us in the worst way—the ones that really appear to wish us ill—turn out on close examination to be rather more like bystanders, strays, strangers in from the cold. They will invade and replicate if they get the chance, and some of them will get into our deepest tissues and set forth in the blood, but it is our response to their presence that makes the disease. Our arsenals for fighting off bacteria are so powerful, and involve so many different defense mechanisms, that we are in more danger from them than from the invaders. We live in the midst of explosive devices; we are mined.4 While certain bacterial species are classified as pathogens, they can live in harmony on our surfaces for long periods and never cause disease. The bacteria that can cause human disease are quite diverse. Based on the pathogenesis of infection and the resulting immune response, these bacteria can be categorized into two general types: those causing intracellular infections and those causing extracellular infections. Most bacteria causing intracellular infections avoid being killed after phagocytosis by either interfering with phagosome-lysosome fusion or by escaping from the phagosome and into the cytoplasm. Cellular immunity is critical against bacteria that reside mainly within an intracellular milieu. In contrast, the bacteria causing extracellular infections survive in the host by avoiding engulfment by professional phagocytic cells such as neutrophils and macrophages. They do this by presenting a surface that

minimizes the opsonic and lytic effects of antibody, complement, and other opsonins. Although extracellular bacteria have the ability to enter and pass through cells as a means of moving from one in vivo environment to another, they are readily killed once captured by phagocytes. Accordingly, the host defense against extracellular bacteria is critically dependent on humoral immunity: complement and the production of specific antibody. Table 41.1 lists many of the important bacteria that can cause extracellular infections in humans, together with the diseases they cause and some of their major virulence factors. In this chapter, we describe the surface structures of many of these bacteria and provide examples of how they are able to infect their hosts and cause disease. We also describe the salient aspects of the innate immunity and antigen-induced immunity important in the host’s defense against these bacteria.

SURFACE STRUCTURE OF GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA Differences among bacteria contribute to their specific adaptation to either a particular host species or to microenvironments within their host. In general, we currently have only a limited understanding of the limitations in nutrients and the specificity of enzymes and bacterial adhesins, or host receptors, that account for these highly specific microbial tropisms. The diversity of bacterial structures encountered by the host offers a major challenge to immune detection of potential pathogens. There are no structural features that reliably differentiate pathogens from nonpathogens. Moreover, many important extracellular pathogens exist mainly as commensals, only causing damage when the balance between host and microbe is perturbed (opportunistic pathogens). Thus, their diversity requires that initial recognition through innate immunity be focused on the more conserved structural features or “molecular patterns” of bacteria. These molecular patterns are, in general, structures such as peptidoglycan, lipoteichoic acid (LTA), and lipopolysaccharide (LPS) that are also essential for bacterial viability and are, thus, unlikely to be modified so as to evade innate immunity. A further consideration is that these structural differences between types of bacteria may dictate differences in host responses and contribute to distinct patterns of disease. That said, different types of bacteria may also give rise to remarkably similar host responses. The syndrome of sepsis, for instance, looks very similar regardless whether it is caused by a gram-positive or gram-negative organism, although there may be little overlap in the specific bacterial mediators involved.

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Extracellular Bacteria Commonly Associated With Diseases

Species

Disease

Important Virulence Structures/ Molecules

Special Adaptations Critical to Host Infection

Neisseria gonorrhoeae

Urethritis, cervicitis, salpingitis, endometritis, prostatitis, arthritis, proctitis, pharyngitis Bacteremia, meningitis, septic arthritis

Lipopolysaccharide, fimbria, peptidoglycan, Opa protein adhesin, IgA1 protease Capsule, lipopolysaccharide, fimbria, IgA1 protease

Haemophilus influenzae

Otitis media, bronchitis, pneumonia, sepsis, meningitis (encapsulated strains)

Bordetella pertussis

Whooping cough in children, chronic cough syndrome in adults Infections in compromised hosts, pneumonia, sepsis

Lipopolysaccharide with phosphorylcholine, fimbria, high-molecular-weight adhesions, IgA1 protease Pertussis toxin, pertactin, filamentous hemagglutinin, fimbria

Phase and antigenic variation, molecular mimicry of human antigens Phase and antigenic variation, molecular mimicry of human antigens, asymptomatic carriage common Phase and antigenic variation, molecular mimicry of human antigens

Neisseria meningitidis

Pseudomonas aeruginosa

Escherichia coli

Lipopolysaccharide, proteases, lipases, lecithinases, exotoxin A, elastase, flagella

Urinary tract infection, sepsis, traveler’s diarrhea, dysentery, neonatal meningitis, hemolytic-uremic syndrome Diarrhea

Cholera toxin, fimbria

Helicobacter pylori

Peptic ulcer disease

Urease, flagella, CagA

Streptococcus pneumoniae

Pneumonia, otitis media, meningitis, sinusitis

Streptococcus pyogenes (group A Streptococcus)

Acute pharyngitis, scarlet fever, necrotizing fasciitis, streptococcal toxic shock syndrome, rheumatic fever, and glomerulonephritis Bacteremia, neonatal pneumonia and meningitis

Capsule, PspA and C, pneumolysin, neuraminidase, hyaluronidase, teichoic acids with phosphorylcholine, IgA1 protease Hyaluronic acid capsule, M-protein, streptococcal pyrogenic exotoxins, streptolysin O, streptolysin S, NAD-glycohydrolase, C5a peptidase Capsule, beta hemolysin, hyaluronidase, C5a peptidase

Vibrio cholerae

Streptococcus agalactiae (Group B Streptococcus) Staphylococcus aureus

Capsular polysaccharide, lipopolysaccharide, fimbria, toxins, siderophores

Coordinate regulation of multiple virulence factors upon exposure to the host environment Relatively large genomic size (approximately six megabases) allows considerable adaptability to changes in environmental conditions, biofilms Antigenic heterogeneity of capsule and lipopolysaccharide

Bacterial dispersal via cholera toxin, which induces copious watery diarrhea Ability to survive at low pH provides a niche lacking bacterial competition or efficient immune surveillance Asymptomatic colonization, genetic transformation permitting continual generation of new genotypes Molecular mimicry of human antigens, high diversity of M-proteins

Asymptomatic colonization, acquisition by infants during parturition Resistant to dehydration, asymptomatic colonization

Impetigo, folliculitis, boils, cellulitis, wound infections, toxic shock, osteomyelitis, endocarditis, bacteremia, pneumonia, food poisoning Cutaneous infection, inhalation anthrax Diphtheria (pharyngitis/tonsillitis)

Tissue-degrading enzymes, alpha toxin and other membranedamaging toxins, epidermolytic toxins, enterotoxins, capsule, protein A, TSST1, pigment Capsule, lethal and edema factors

Clostridium tetani

Tetanus (spastic paralysis)

Clostridium perfringens

Gas gangrene, anaerobic cellulitis, endometritis, food poisoning Flaccid paralysis: cutaneous, infant, and ingestion forms

Tetanus toxin (blocks inhibitory neurotransmitters) More than 10 exotoxins

Opportunistic infection by a spore-forming soil organism Toxin gene contained in temperate phage and expression regulated by iron concentration Opportunistic infection by a spore-forming soil anaerobe Opportunistic infection of wounds

Botulism toxin blocks acetylcholine release at synapses

Opportunistic infection by a spore-forming anaerobe

Bacillus anthracis Corynebacterium diphtheria

Clostridium botulinum

Diphtheria toxin

CagA, cytotoxin-associated gene A, Ig, immunoglobulin; Psp, pneumococcal surface protein; TSST1, toxic shock syndrome toxin 1.

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Most extracellular, as well as intracellular, pathogenic bacteria can be divided into two major groups (gram-negative and gram-positive) based on their response to staining with Gram stain. To illustrate the surface of the bacteria in the two groups, the surface structures of Streptococcus pneumoniae (Panel A) and Neisseria meningitidis (Panel B) are shown in Figure 41.1. Three layers are commonly recognized: cytoplasmic membrane, cell wall, and outer layer. Although these layers are described in detail in the following, it is important to note that these definitions are operational and that, in reality, the layers are not entirely distinct. For instance, molecules anchored in the cytoplasmic membrane or cell wall may extend into or through other layers.

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It is also important to note that the capsule, O antigens, and cell wall are not contiguous shields; rather they are permeable enough to allow through secreted products and nutrients as well as some immunologic factors (eg, antibodies and complement). All bacteria have a cytoplasmic membrane, a non–sterolcontaining phospholipid bilayer. This membrane is an osmotic barrier and also forms a barrier for most molecules. The cytoplasmic membrane has various proteins, many of which function in transport. Some of these proteins, referred to as lipoproteins (eg, pneumococcal surface adhesion A, which is a manganese permease in S. pneumoniae), are noncovalently anchored to the membrane through

A

B

FIG. 41.1. Schematic representation of the surfaces of Streptococcus pneumoniae (A) and Neisseria meningitidis (B) as examples of gram-positive and gram-negative bacteria, respectively. The cell wall polysaccharide of S. pneumoniae is often called C-polysaccharide. The inset in B shows lipopolysaccharide anchored to the outer leaflet of the outer membrane.

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lipid modifications and are especially common among some bacteria (eg, Borrelia burgdorferi and Mycobacterium tuberculosis). Proteins not exposed on the surface generally display a greater degree of structural and functional conservation compared with those exposed to the selective pressure of host immunity. A cell wall is found in all of the pathogenic bacteria of both groups, with the exception of mollicutes (which include the genus Mycoplasma). The cell wall surrounds the cytoplasmic membrane and is made of peptidoglycan, which is a polymer of alternating sugars N-acetylglucosamine and N-acetylmuramic acid, the latter being connected to a stem peptide. The stem peptides include four to five Dand L-amino acids that are extensively cross-linked by bridges that provide rigidity to the cell wall and protect it from environmental extremes (especially differences in osmolarity). These cell wall peptides include atypical amino acids such as diamino pimelic acids, which are the anchoring site for Braun lipoprotein of gram- negative bacteria and are found in most gram-negative bacteria but in few gram-positive bacterial species. Peptidoglycan polymerization is carried out by enzymes, many of which are the target of β -lactam antibiotics and are referred to as penicillinbinding proteins. Compared with gram-negative bacteria, gram-positive organisms may have different stem peptides and cross-linking as well as a thicker (20 to 30 nm compared with 2 to 4 nm) cell wall layer that can retain the Gram stain better. The thick cell wall of the gram-positive bacteria may be responsible for their greater resistance to complementmediated lysis. Other features of the cell wall such as the O-acetylation of N-acetylmuramic acid or the deacetylation of N-acetylglucosamine5,6 found in some species mediate resistance to lysozyme, an enzyme that lyses bacteria by cleaving the peptidoglycan backbone.6 In addition to peptidoglycan, many gram-positive bacteria have polysaccharide (PS) associated with their cell walls, with this cell wall PS often extending into the outer layer. The structure of the cell wall PS of gram-positive bacteria varies between species but is relatively invariant within a species. Differences in the antigenicity of the cell wall PS have been used to distinguish species (eg, separate streptococci into groups A, B, C, etc.).7,8 Cell wall PS often has phosphate group(s) in repeating units of glycerol or ribitol in a structure known as teichoic acid. Teichoic acid may also be linked to lipid molecules and is then called LTA, which is anchored to the cytoplasmic membrane and extends out through the cell wall.9 In pneumococci, the overall PS structures of LTA and cell wall teichoic acid (also referred to as C-polysaccharide) are very similar, with the difference being their mode of attachment to the bacterial surface.10,11 Another major difference between the surface structures of gram-negative and gram-positive bacteria is the presence of an outer membrane on gram-negative bacteria. The outer membrane contains many proteins, including channel-forming porins. The outer membrane is an asymmetrical lipid bilayer. The inner leaflet is comprised primarily of phospholipids while the outer leaflet contains lipid A, the hydrophobic component of LPS. LPS, also called endotoxin, is an amphipathic glycolipid with four distinct regions: lipid

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A, the inner core, the outer core, and, in some species, the O antigen. Lipid A is composed of a dihexosamine backbone to which between five and seven saturated (12- to 16-carbon) fatty acids are attached through amide and ester linkages. Lipid A is the principal “toxin” associated with most gramnegative bacteria, although it is now clear that lipid A is not a true toxin. Rather, its ability to induce cytokines accounts for its potentially detrimental effects. The carbohydrate portion of the LPS, which makes a minimal contribution to its endotoxin activity, is attached to lipid A through a molecule unique to gram-negative bacteria called ketodeoxyoctanoate. Together with heptose moieties, this molecule forms the inner core of the LPS. The outer core is composed of 7 to 10 monosaccharide units whose arrangement is relatively conserved among gram-negative species.12 In many gram-negative bacteria, the outer core of LPS is connected to a repeating series of carbohydrates called the O antigen. The O antigen forms a hydrophilic shield around the bacterium that provides a barrier to complement deposition on the bacterial cell surface. The O antigen is variable in length, is antigenically diverse, and confers serotypic specificity. The O antigens of Escherichia coli, Klebsiella, and Salmonella have as many as 30 repeating units composed of four to six sugars each.12 Members of the genera Neisseria and Haemophilus, on the other hand, lack LPS with O antigens but instead have lipooligosaccharides, which have short oligosaccharides that do not exceed 7000 daltons. For many pathogenic extracellular bacteria, PS components dominate the outer layer. In addition to the PS on LPS (gram-negative) and teichoic acid (gram-positive), there is often another thick layer of carbohydrate referred to as “capsule” that may account for more than half of the bacterial mass. An exception to this general rule is Bacillus anthracis, whose capsule is made of poly-D-glutamic acid rather than a polysaccharide.13 S. pneumoniae has capsular PS that is covalently attached to the cell wall in most (but not all) serotypes.14 In contrast, the capsule PS is anchored to the outer membrane by acyl chains in N. meningitidis15 and Haemophilus influenzae type b.16 Capsular PSs may be highly diverse both within and between species. In the case of S. pneumoniae, each strain expresses a single type of capsular PS, with members of this species being capable of synthesizing more than 90 structurally distinct types.17,18 This diversity limits immune recognition until antibody is generated to the capsular PS of the infecting strain (antigenic variation). The outer layer is well developed in bacteria that cause extracellular infections and has many features that help the bacteria circumvent the host immune system. First, the outer layer has properties that reduce the attachment of extracellular bacteria to eukaryotic surfaces, including those of phagocytes. Generally, the PS capsules render the bacteria hydrophilic and negatively charged like eukaryotic cell surfaces, which are rich in sialic acid. By enhancing the degradation of C3b, the negatively charged surface makes the bacteria partly resistant to the deposition of complement by the alternative pathway.19 Second, in some cases, elicitation of antibody is minimized because the capsular PS or

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LPS mimics host antigens, as is more fully described in the section on Antigen-Specific Host Defense Response of this chapter. Third, by physically masking most of the other bacterial surface components, the outer layer minimizes the number of exposed epitopes that can be recognized by the antibody and complement. Although the capsule is porous to antibodies and complement, the binding of antibodies and fi xing of complement beneath the capsule surface are relatively ineffective in promoting opsonophagocytosis and clearance.20

BACTERIAL VIRULENCE FACTORS Extracellular bacteria often elaborate molecules called “virulence factors” that are useful to their survival and proliferation in the host. For example, the shielding function of the outer layer is further augmented by the presence of surface proteins that can interfere with host clearance mechanisms. Proteins inhibiting effective complement deposition include M-protein in Streptococcus pyogenes,21 pneumococcal surface protein A (PspA), and pneumococcal surface protein C, which is alternatively called choline binding protein A or the C3-binding protein in S. pneumonia.22,23 An example of a protein that interferes with antibody is protein A, which is expressed on the surface of Staphylococcus aureus and binds immunoglobulin (Ig) in a manner that precludes recognition of its target antigen.24 In addition, many successful mucosal pathogens, including members of the Neisseria, Haemophilus, and Streptococcus genera, express proteases with specificity for the hinge region of human IgA1.25 These IgA1 proteases remove the Fcα component required to promote the inflammatory process, leaving the organisms’ antigens obscured by the binding of inert Fabα fragments. By inhibiting the deposition of complement or antibody, many of these proteins act to diminish phagocytosis. The best-known virulence factors are toxins, which interrupt specific host functions. These proteinaceous molecules (also referred to as exotoxins to differentiate them from endotoxin) can be grouped on the basis of their molecular structure and their mechanism of action.26 The largest group are called A-B toxins, which are comprised of two subunits, each with a different function. The A subunit has enzymatic activity, and the B subunit targets the A subunit to the host cells. This group includes diphtheria toxin, cholera toxin, pertussis toxin, and two anthrax toxins (lethal factor and edema factor). For instance, the lethal factor of B. anthracis behaves as the A subunit and requires a B subunit protein named “protective antigen” to enter into target cells. In some cases, the toxin alone is sufficient to account for the detrimental symptoms of its respective infection. Cholera toxin causes ADP ribosylation of G proteins, which stimulates adenylate cyclase and increases cyclic adenosine monophosphate in cells lining the gut. This results in the secretion of electrolytes and is responsible for a severe diarrhea, which promotes transmission of Vibrio cholerae but often also causes dehydration that, if not treated, may be fatal. Uptake of botulism toxin by nerve endings leads to retrograde transport that interrupts synaptic transmission and causes a flaccid paralysis.27 Staphylococcal enterotoxin

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A (a toxin that acts from the gut lumen), which is one of five membrane-damaging toxins produced by staphylococci, is the primary cause of staphylococcal food poisoning and plays a major role in invasive infections.28 Some strains of E. coli produce a protein synthesis–inhibiting verotoxin, which may damage the microvasculature of the kidney and cause hemolytic uremic syndrome.29 Another group of proteins secreted by S. aureus and S. pyogenes have toxin-like effects but lack any enzymatic activity. These “superantigens” cause a nonclonal stimulation of T cells by bridging major histocompatibility complex (MHC) class II molecules (outside the antigen-binding site) on antigen-presenting cells with the Vβ region of the T-cell receptor on T cells. The ensuing massive release of cytokines by localized release of a superantigen, such as the toxic shock syndrome toxin 1 expressed by some strains of S. aureus, results in systemic symptoms that are collectively known as “toxic shock syndrome.”30 Another class of virulence factors neutralizes host defenses. As stated previously, many virulence factors interfere with complement deposition on bacteria. S. pyogenes and group B streptococci produce a C5a peptidase that inhibits the chemotaxic effects (recruitment of host phagocytes to the sites of infections) of C5a, a product of complement activation.31 Pneumolysin produced by S. pneumoniae is a member of a large class of cholesterol-dependent cytotoxins that oligomerize to form large pores, which interfere with a number of host cell functions or induce cell death when present in higher concentrations.32 Pneumolysin also depletes complement at a distance from the pneumococci and interferes with both the function of phagocytes and the development of protective immunity.33 Helicobacter pylori produces urease, which can generate ammonia that can neutralize acid in the stomach and thereby promotes the survival or the organism. S. aureus produces a pigment that makes the bacterium more resistant to oxidative stress and killing by neutrophils.34 While evasion of professional phagocytes is critical for extracellular pathogens, the ability to attach to other cell types, including both mucosal and nonmucosal surfaces, is important for their persistence. Many bacterial surface proteins have an adhesive function that confers a high affi nity for binding to specific host cell receptors. Nasopharyngeal carriage of pneumococci is mediated largely by adherence to the host molecules N-acetylglucosamine β1→3 galactose or N-acetylglucosamine β1→4 galactose.35 Bacteria often mimic host ligands in order to coopt host receptors for their own purposes. Many pathogens of the airway express phosphorylcholine (phosphocholine [PC]) on their surfaces.36 This molecule, which is otherwise unusual in bacteria, is found on platelet-activating factor (PAF) and allows bacterial binding to its receptor (rPAF).37 To facilitate their attachment to host cells, many bacteria use pili (fi mbriae), long fi lamentous structures extending from the organism. The Pap pilus of E. coli binds the galactose α1→4 galactose unit of cell surface globoside in urethral epithelial cells.38 The V. cholerae pilus allows that bacterium to attach to the enterocyte for more efficient toxin delivery.39,40 Bordetella pertussis has three adherence factors—a fi lamentous hemagglutinin,

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pertactin, and a pilus—that allow it to attach to ciliated respiratory epithelial cells in the trachea and bronchi and thus resist the cleansing action of mucus flow.41,42 Another group of virulence factors helps bacteria acquire essential nutrients. Motile bacteria (eg, Pseudomonas aeruginosa) express flagellin, a complex motor apparatus that allows the bacterial cell to transit along a concentration gradient of nutrients.43 While in some cases the host and microbe provide nutrients for one another, for other nutrients there is fierce competition. Mucosal fluid and blood are low in free ferric iron due to the presence of iron-binding proteins such as lactoferrin and transferrin. To successfully compete with the host for this vital metabolite, N. meningitidis, Neisseria gonorrhoeae, and H. influenzae have complex surface transport systems that can obtain iron from human transferrin, lactoferrin, and hemoglobin.44 Other bacteria such as E. coli and salmonella use a different mechanism to acquire iron; they secrete small, high-affinity iron chelators, called siderophores, that remove iron from human proteins in the environment surrounding the bacteria. The iron-siderophore complex is then taken up by the bacterium, which then degrades the siderophore so that the iron can be freed for its use.45 Production of virulence factors is often highly regulated by bacteria in order to adapt to different environments, such as the natural environment outside of a host, the mucosa of a host, or more invasive sites within a host. For staphylococci, it has been shown that the amount of capsule is regulated in response to environmental stimuli.46 One of the best studied of such regulatory systems is the BvgAS, a two-component regulatory system in B. pertussis.47 This system, which regulates the expression of adhesins, toxins, and other virulence factors, is controlled by external signals including Mg 2 +, temperature, and nicotinic acid. Two proteins are involved in this regulatory system: BvgS and BvgA. BvgS, the sensor, is a kinase and is able to autophosphorylate itself in response to the environmental signal. BvgA, the response regulator, is in turn phosphorylated by BvgS. Phosphorylated BvgA is able to activate transcription of virulence genes through a change in its interaction with a 70-bp consensus sequence repeated in bvg-regulated promoters.47 Analogous two-component regulatory systems in other pathogens are frequently used to regulate the expression of genes associated with virulence.48 Another strategy used by extracellular pathogens depends on selection among a heterogeneous population for those members with permissive characteristics. This heterogeneity in a population is commonly generated through genomic rearrangements, such as recombinational events or slipstranded mispairing in highly repetitive deoxyribonucleic acid (DNA) sequences.49 This latter mechanism allows for reversible on-off switching (phase variation) and is especially prevalent in genes encoding cell surface structures subject to immune pressure. For instance, the capsular PS on N. meningitidis is needed to protect the organism during invasive infection but inhibits adherence on the mucosal surface where complement is less abundant. Phase variation of a gene required for capsule synthesis allows for selection of organisms without capsule (phase-off).50 This change facilitates the bacterial adhesion to the epithelial cells, perhaps by exposing the bacterial adhesins. Alternatively, by decreasing capsule

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production, the bacteria become less hydrophilic and less negatively charged. This change facilitates their entry into the epithelial cells and their subsequent invasion into deeper tissues. Upon the emergence of the bacteria from the epithelial cells into the submucosa, capsule synthesis is restored (phase-on) because of the selective pressure of complementmediated clearance and the requirement for the capsule to survive where the concentration of complement is higher. The flexibility to express different surface properties helps bacteria successfully evade the host immune system and survive in many niches within the host. Bacteria-to-bacteria signaling is another important mechanism for the control of virulence factors. This phenomenon, called “quorum sensing,”51 has been shown to be operative in a large number of gram-negative and grampositive species. The signal transmitted between the bacteria can be an acylated lipid (eg, homoserine lactone) in gramnegative bacteria (eg, vibrios) or a peptide in gram-positive bacteria (eg, S. aureus). Quorum sensing has been shown to be important in biofi lm formation in a number of bacterial species, in the expression of “competence” for the uptake and incorporation of exogenous DNA (transformation), and in the regulation of a number of virulence factors.52 Biofi lms are communities of one or multiple bacterial species that adhere both to each other and to a target surface. Bacteria in biofims are particularly resistant to many host clearance mechanisms and to antibiotics that are effective against free-living (planktonic) bacteria. Biofilms, therefore, are often a contributing factor in more chronic bacterial infections such as those involving foreign bodies or chronic otitis media.53 An important characteristic of virulence factors is their structural polymorphism. For instance, there are at least 100 different serologic types of M proteins of S. pyogenes.54 Similarly, pneumococci have more than 90 serologically distinct capsular PSs.17,18 The polymorphism in the structure of many virulence factors allows the bacteria making them to avoid antigen-specific host immunity. For example, antibodies to the immunodominant region on one serotype of M protein do not cross-react with M proteins of other serotypes and thus do not provide protection against strains expressing other serotypes. Similarly, newly acquired pneumococci can escape recognition by anticapsular antibodies produced in response to previous pneumococcal infections with other serotypes. The polymorphism in virulence factors is achieved by various genetic mechanisms. Variation in M proteins is the result of sequence differences in the N-terminal (but not C-terminal) half of M proteins.55 S. pneumoniae has the genes for synthesizing capsular PS as a “genetic cassette” that can be exchanged among different strains56 and may result in the shift in the serotype distribution following the use of vaccines designed to elicit serotype-specific protection.57,58 Neisseria has genetic machinery for rapid gene rearrangement59 through gene conversion using the multiple “silent” pili genes with different sequences. This process, similar to gene rearrangements that generate specific Ig, permits an individual bacterium to quickly produce progeny expressing pili with different characteristics. The number of potential

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pilus-antigen variants within the progeny of a single organism is estimated to be greater than 100,000.60 In addition, Neisseria contain large numbers of genes with tandem repeats that undergo phase variation through slip-stranded mispairing of these sequences. Based on predictions from whole genome sequencing, through this mechanism alone the organism may be able to generate more than 265 different phenotypic variants.61 Sequencing of the entire genomes of bacteria has shown that the genes for virulence factors have generally originated from other organisms and exist as a part of large blocks of DNA containing multiple genes. These DNA blocks are called “pathogenicity islands” (PAIs). For instance, strains of “enteropathogenic” E. coli contain a PAI encompassing about 41 genes encoding a surface ligand required for intimate association of the bacterial and host cells and for a bacterial secretion apparatus.62 This system (type IV secretion system) allows for delivery of the receptor for its own adhesin, encoded on the same PAI, into host cells. Elaborate secretion mechanisms (types III and IV) and pore-forming toxins are now known to be mechanisms whereby extracellular organisms gain access to the host cell cytoplasm to modify its activity to suit their needs. Another example is H. pylori, which injects cytotoxin-associated gene A (CagA) molecules directly into host cells using a syringe-like type IV secretory apparatus. CagA is then phosphorylated by the host cells, and the phosphorylated CagA alters host cell function, with the H. pylori strains producing the CagA molecules being more likely to cause ulcers.63 In the case of S. pyogenes, its pore-forming toxin, streptolysin O, allows for translocation of an enzyme (NAD-glycohydrolase) that is capable of producing the potent cytoplasmic second messenger, cyclic ADP-ribose.64

BACTERIAL INVASION OF THE HOST Both keratinized skin and mucosal surfaces have inherent nonimmune defense mechanisms that modulate bacterial growth and minimize the risk of invasion. Healthy human skin is an effective physical barrier to infection by most human extracellular and intracellular pathogens. The keratinization of fully differentiated skin epithelium results in a relatively impermeable surface. In addition, lysozymes, toxic lipids, and hydrogen ions secreted by cutaneous glands offer bacteriostatic protection for cutaneous pores and hair follicles. Occasionally, this defense can be breached by extracellular bacteria such as S. pyogenes or S. aureus, causing cellulitis and abscess. More commonly, bacterial invasion through intact skin requires physical damage, such as abrasions, burns, or other trauma. For instance, cutaneous anthrax develops when B. anthracis enters the body through a break in the skin. Staphylococcus epidermidis, a member of the commensal skin flora, can infect indwelling catheters by invading through the puncture site in the skin and may lead to bacteremia or colonization of prosthetic devices, including artificial heart valves and shunts. A major factor allowing these bacteria to cause disease is their ability to form a biofi lm, which facilitates their adhesion, is antiphagocytic, and acts as a barrier to antibiotic penetration.65

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Unlike the skin, the mucosal epithelium is not keratinized. Instead, mucosal areas, such as the gastrointestinal tract, nasopharynx, upper airway, and vagina, are moist and nutritionally rich. Thus, it is not surprising that mucosal areas contain a large number of bacteria. In oral secretions and gastrointestinal products, 108 and 1011 bacteria/mL may be found, respectively. To ensure their survival in the mucosal environment, extracellular bacteria elaborate many virulence factors required for the acquisition of essential nutrients or for their adherence to the host cells. In some cases, bacteria may subvert the host inflammatory response. Salmonella species can block the activation of NF-κ B and the subsequent activation of the inflammatory response. They achieve this by preventing the degradation of Iκ B, which is essential for the translocation of NF-κ B from the cytoplasm to the nucleus.66 In some cases, pathogens locate in a less well-protected microenvironment within the mucosal areas. H. pylori survives in the very acidic stomach by burying itself in the mucus, which protects it from direct exposure to the acid and from phagocytes. There are also few other species for H. pylori to compete with in this more hostile environment. Mucosal sites play host to an especially diverse array of bacterial species. Most of the bacteria species found at mucosal sites are harmless. In addition, polymerase chain reaction analysis of 16S ribosomal ribonucleic acid sequences suggests the presence of many additional unidentified (and so far unculturable) bacterial species on the mucosal surface.67 Many potentially pathogenic bacterial strains are also often found in the mucosal areas of healthy individuals without causing symptoms. S. pneumoniae, N. meningitidis, H. influenzae, and S. aureus are examples of pathogenic extracellular bacteria that are frequently carried in the nasopharynx of healthy individuals. The carriage rate of pathogenic bacteria can be relatively high; for example, 50% to 60% of healthy young children may carry S. pneumoniae in their throats.68 Maintenance of species diversity in mucosa is dynamic. In some situations, collaboration among bacteria is essential for their successful colonization, as seen among the complex hierarchical communities adhering to tooth surfaces. In other situations, bacterial species compete and regulate diversity among themselves.36 Many bacterial species produce molecules that suppress the growth of other bacterial species. These molecules may include bacteriocins, small molecules that target members of the same or different species that do not express the same bacteriocins.69 Some species can take advantage of host responses to which they are resistant to outcompete another member of the same niche that is less resistant.36 The host may also control the diversity of colonizing bacteria by modifying the pH or other environmental conditions in the mucosal area. Interference with these homeostatic mechanisms, as occurs with antibiotic therapy, may alter the flora and predispose the host to disease. As noted previously, stomach acid is an effective barrier to reaching the nutrient-rich environment of the gut. When stomach acid is pharmacologically reduced, the inoculum of organisms like V. cholerae required to infect the intestines is greatly diminished.

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Several explanations have been advanced to explain why many pathogens that colonize mucosal sites do not cause disease. One explanation is that the maintenance of diverse bacterial population is responsible for the prevention of the disease. For instance, the destruction of the normal gastrointestinal bacterial flora with some antibiotics can be associated with the selective expansion of Clostridium difficile and the development of pseudomembranous colitis.70 Another explanation may be that the pathogenic bacteria carried in healthy persons are different from those isolated in disease settings. For instance, during nonepidemic periods, approximately 5% to 10% of the population carries N. meningitidis, which are mostly nonencapsulated.71 During epidemics, 30% to 60% of the population may carry meningococci, which are mostly encapsulated and the majority of which are of the same capsular type as the case strain causing the epidemic.72 A third explanation is that the pathogenic bacteria are effectively confined to the mucosal surface where they do not cause damage or induce inflammation. Group B streptococci are carried asymptomatically in the lower intestine and the female genital tract. In the same host, in the setting of parturition, group B streptococci may access the bloodstream and cause septic infection. A fourth explanation is centered on the differences among hosts. Group B streptococci that colonize the mother may cause lifethreatening infection when the same strain is passed to the neonate at or before birth.73 Although pathogenic extracellular bacteria can exist asymptomatically in the mucosa, they can passively enter into less-well-defended sites and cause focal infections. For instance, E. coli, normally present in the gut, may enter the normally sterile urogenital tract and cause urinary tract infections. S. pneumoniae and H. influenzae are often carried in the nasopharyngeal space, but they can invade nearby normally sterile cavities (eg, lungs, sinuses, and the middle ear) and cause focal infections. Aspiration of bacteria from the nasopharynx into the lungs most likely occurs frequently with no ill effects; however, aspiration may lead to an infection when there is damage to the epithelial surface, particularly when the protective effects of mucociliary activity are lost, as often occurs in a smoker or during recent viral infection (respiratory syncytial virus or influenza).7 Some bacteria produce enzymes such as hyaluronidase,74 which may aid in their passage through tissue barriers. Bacteria can actively invade deeper tissues by multiple pathways. They can enter through specialized cells. Shigella, for example, can breach the gut mucosa by transcytosing through the M cells in the gut.75 Alternatively, extracellular bacteria can breach a cellular barrier (epithelium or endothelium) by going through (transcytosis) or between (paracellular pathway) the cells.75 Porphyromonas gingivalis, an organism associated with adult periodontitis, may breach the epithelial layer by the paracellular pathway through the production of enzymes useful in digesting the tight junction.76 Two different mechanisms of transcytosis have been described for pneumococci. In one, pneumococci may cross the bronchial epithelial cells by binding the polymeric Ig receptor of the epithelial cells and traveling

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in a retrograde manner by the IgA secretory pathway.77 In the other, pneumococci may use PC to bind to rPAF, which is abundant on activated endothelial cells, epithelial cells, or pneumocytes.35,78 In many cases, bacterial adhesion triggers changes in the host cell function, and these changes can assist transcytosis. For instance, nontypeable H. influenzae with LPS glycoform-containing PC can bind to rPAF on endothelial cells and initiate signaling through this receptor.79

ANTIGEN-NONSPECIFIC HOST DEFENSE RESPONSE To protect from infections caused by highly adaptable bacteria, the host employs a multilayered defense. This includes the mechanical barriers and iron sequestration described previously as well as phagocytes, complement fi xation, lysozyme, and (cytokine-induced) local inflammation. In addition, the host is protected with antigen-specific antibody (see section on Antigen-Specific Host Defense Response) and T-cell– mediated cellular immunity. Antigen-specific immunity, although exquisitely protective, takes several days to weeks to develop following exposure to a pathogen. As many extracellular pathogens are capable of causing overwhelming infection in periods of hours to days, other more rapidly acting forms of protection are needed. Consequently, the primary defense against bacteria during the early phase of infection remains antigen-nonspecific host immunity. The importance and significance of nonspecific immunity is readily demonstrated by the relative ease with which colonies of SCID mice, which lack antigen-specific immunity, can be maintained.80 This section describes several antigennonspecific host defense mechanisms, but see the chapter on innate immunity for additional information.

Mucosal Defense Although mucosal areas are rich with nutrients for bacteria, uncontrolled local proliferation of bacteria is held in check by mechanical cleansing actions and the lack of available iron. In the gastrointestinal tract, normal peristaltic motility, the secretion of mucus, and the detergent action of bile limit the number of bacteria. The normally sterile lower respiratory tract is protected by the movement of mucus by cilia lining the airway, which continually remove aspirated bacteria. Normal epithelial and tissue architecture are essential for drainage and expulsion of bacteria, and disruption of this mechanism by smoking, viral infections (eg, influenza), or bacterial infection (eg, pertussis) makes the host markedly susceptible to infection by bacteria that otherwise exist only as commensals of the upper airway. The increased frequency of lower respiratory tract infections in the elderly is due, in large part, to the loss of function of the mucociliary elevator and the increased aspiration from the upper respiratory tract of secretions containing bacteria.81,82 In addition to the removal of bacteria by mucus flow, mucosal fluid contains many antibacterial products such as lactoferrin, lactoperoxidase, mucin, lysozyme, and defensins.83 Lactoferrin—found in various body fluids such as milk, saliva, and tears—binds iron and lowers the level

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of available iron (especially in areas with a low pH).84 Mucin traps microbes and facilitates their removal.85 IgA antibodies in mucosal fluid may inhibit colonization by interfering with microbial adherence or by inactivating toxins.86 In addition, the polymeric structure of secretory IgA promotes agglutination, which in turn facilitates removal by mucus. Lysozyme reduces the bacterial load by cleaving the 1→4 linkage between N-acetylmuramic acid and N-acetylglucosamine of peptidoglycan. A number of antimicrobial peptides, including defensins, disrupt bacterial membranes.87 In the intestine, Paneth cells at the base of intestinal crypts produce defensins that are important in defense against intestinal infections.88 In the lungs, collectin-like surfactant proteins, such as SP-A and SP-D, may be important in host defense by opsonizing bacteria for alveolar macrophages.89 SP-A–deficient mice, for example, are more susceptible to group B streptococcal infection of the lungs.90 Epithelial cells that interface with the microbial world must exist in a quiescent state in response to colonizing organisms. If not, chronic inflammation may result in a disease (eg, chronic inflammatory bowel disease). When the epithelial barrier is breached, these cells are able to elaborate cytokines and chemokine as an early trigger to the inflammatory response.

Local Response to Bacterial Invasion (Acute Inflammation) Upon entry into the host, many bacteria initiate local inflammatory processes by providing various inflammatory products such as peptidoglycan, LPS, LTA, exotoxins, lipoproteins, and glycolipids.91 Antibiotics used for treatment may destroy bacteria and consequently release additional inflammatory products. These molecules are called pathogen-associated molecular patterns and trigger responses through their interaction with pathogen pattern recognition receptors (PRR), which often reside on the host cell membrane or in the cytoplasm. The best-known PRRs residing on the cell membrane are the toll-like receptors (TLRs), which have a transmembrane region that separates the cytoplasmic signaling domain from the extracellular ligand binding domain. TLRs are generally most abundant on inflammatory cells but are present in lower levels in the epithelial barrier, which is continuously exposed to microbial products.92 TLR2 detects lipoproteins and lipoteichoic acid and requires a binding partner (TLR1 or 6) to transmit signals leading to cytokine production.93 Mice lacking the TLR2 gene are more susceptible to mucosal and systemic infection with staphylococci and streptococci.94,95 LPS binds to TLR4 with the help of MD2, cluster of differentiation (CD)14, and lipid-binding proteins. Mice without functional TLR4 are unresponsive to LPS. Bacterial flagellin signals through TLR5.96 Bacterial DNA, rich in unmethylated CpG motif, is a potent inducer of inflammation through its binding to TLR9. TLR9 receptors are found inside the cells as they occur in endocytic vesicle membranes and react to phagocytosed bacteria. TLR activation increases expression of inflammatory cytokines (eg, tumor necrosis factor [TNF]-α) through increased transcription of their

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genes. The protective effect of TLR can be seen early in an infection. For instance, TLR9 can protect against pneumonia caused by S. pneumoniae even before circulating inflammatory cells enter into the lungs.97 Bacterial invasion is also detected by various intracellular PRRs. The best-known PRR family may be the nucleotide oligomerization domain (NOD)-like receptors (NLRs). At least 23 NLR family members have been identified in humans, with each member having a central nucleotide-binding domain (NACHT domain), an N-terminal effector domain, and a C-terminal receptor domain with leucine-rich repeats. Ligands may bind the leucine-rich repeat domain and initiate oligomerization of the NATCH domain to form a signaling platform that allows the binding of adaptor and effector molecules, ultimately leading to activation of caspase 1 and then interleukin (IL)-1β.98–100 This molecular assembly is named an “inflammasome,”101 with the best-known infammasome being Nlrp3 (also known as NALP3 or cyropyrin), which can sense various bacterial products as well as alum, silica, and urate crystals.101 Another inflammasome is Nlrc4 (also known as CARD12 or IPAF), which can detect flagellin. Flagellin, however, is also detected by TLR5.102 Other well-known NLRs are NOD1 and NOD2. NOD1 is expressed in various cell types, but NOD2 is expressed primarily among the epithelial cells of the lungs and intestine, macrophages, and dendritic cells (DCs).103–106 NOD1 recognizes peptidoglycan fragments containing meso-diaminopimelic acid primarily from gram-negative bacteria,107 but NOD2 recognizes muramyl dipeptide, the minimal motif of peptidoglycan that is shared by both gram-positive and gram-negative bacteria.108 Interestingly, the lack of signaling by muramyl dipeptide results from the NOD2 gene mutation involved in Crohn disease.98,108,109 During the initial phase of infl ammation following a bacterial invasion, many cell types residing in the mucosa or skin (eg, keratinocytes) may produce molecules important in controlling infections. Several studies revealed that mast cells are one of the important resident host cells involved in the innate immune response. Mast cells, classically known for their stores of histamine and serotonin,110 are abundant along the bronchial tree and the epidermis of the skin. They are now known to both contain preformed TNF- α as well as be a major source of various cytokines. Mast cells account for 90% of IL-4– and IL-6–producing cells in the nasal cavity.111 Upon exposure to various bacterial products (eg, LPS), mast cells release these cytokines, which are essential for the recruitment of neutrophils to the site of infl ammation. The absence of mast cells can increase the susceptibility of animals to bacterial infections in the peritoneum or the lungs; their absence can be partially compensated for by administration of TNF- α .112 As the inflammatory process persists, additional cell types come to the site of inflammation. In the case of experimental pneumococcal pneumonia, neutrophils come to the lungs in 12 to 24 hours, followed by the appearance of monocytes and macrophages in 48 hours.113 Similar phagocyte entry sequence was observed for nasopharyngeal

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colonization by S. pneumoniae.114 Few lymphocytes are observed in the lungs during this time period. Upon their arrival at the site of infection, neutrophils and macrophages, which can rapidly phagocytize and kill the bacteria, become activated by the bacterial products (eg, LPS) and chemokines (eg, IL-8). Phagocytosis can occur when phagocytic cells recognize certain native molecular structures of the bacteria such as lectins, PS, and peptides (RGD sequence),115 or aided by their CR3 and Fc receptors, recognize the host opsonins on the bacterial surface. Inflammatory processes trigger the cascade of chemokine and cytokine release at the site of inflammation. Sequential appearance of chemokines has been noted in the pneumonia model.113 The peak levels of chemokines macrophage inflammatory protein (MIP)-2 and KC are achieved in the lungs less than 6 hours after infection. The peak levels of MIP-1a and MCP-1 are observed in 12 to 24 hours. Neutralizing MIP-1a and MCP-1 along with RANTES reduces macrophage recruitment. The cytokines produced during acute inflammation can be divided into two groups: proinflammatory cytokines (eg, IL-1 and TNF-α) and anti-inflammatory cytokines (eg, IL-4). The molecules produced during inflammation can induce the expression of ELAM, ICAM, and VCAM on endothelial cells and of selectins and integrins on leukocytes, thereby modifying the properties of the cells (cell adhesion, vascular permeability, etc.) at the site of inflammation. Inflammation also draws phagocytes to the site of pathogen invasion, where the phagocytes generally efficiently recognize, ingest, and kill the extracellular pathogens. Among many surface receptors, CR3 may be the most relevant to recognizing extracellular bacteria. Indeed, persons with CR3 deficiency (leukocyte adhesion deficiency type 1) have leukocytes ineffective in phagocytic killing.116 The phagocytic killing occurs rapidly (generally within 15 minutes) when phagosomes fuse with lysosomes and the ingested bacteria are exposed to lysosomal enzymes, although some extracellular bacteria can survive in phagocytes for a significant period.117 In addition to this classic killing mechanism, a new mechanism called “autophagy” has been described. This process is characterized by the engulfment of portions of the cytosol into a characteristic double-membrane vacuole called an “autophagosome.”118,119 After maturation, autophagosomes fuse with lysosomes followed by degradation of the sequestered structures and recycling of the degraded products.120 While the autophage is important in controlling infections by intracellular pathogens (eg, virus or intracellular bacteria), it may also be involved in defense against extracellular pathogens. S. pyogenes may be killed by autophagy.121,122 However, S. aureus may also exploit autophages to its advantage as S. aureus activates an autophagic response to enter the double-membraned autophagosomes but prevents autophagosome maturation.123,124 Another phagocyte response involves neutrophil extracellular traps (NETs).125 In this situation, when phagocytes encounter pathogens, the phagocytes decondense the phagocytes’ DNA and release their DNA as well as their cellular contents. This decondensed DNA forms a net with

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cellular debris with the NET being able to capture and destroy many bacteria. However, some pathogens such as S. pneumoniae can escape the NET by destroying it with endonuclease.126

Systemic Response to Bacterial Invasion In response to inflammatory bacterial products, cytokines such as IL-1, TNF-a, and IL-6 are released into the systemic circulation and trigger many systemic changes, such as fever and accumulation of leukocytes at the sites of infection. The cytokines also trigger an acute-phase response by the liver.127,128 The acute-phase response occurs when hepatocytes, in response to the cytokines, activate transcription factors such as NF-κ B and STAT3,127,128 and increase production and secretion of a variety of molecules that are termed acute-phase reactants, such as coagulation factors, serum amyloid A protein, C-reactive protein (CRP), TREM-1,129 and collectins.130 CRP was named for its ability to bind to pneumococcal C-polysaccharide. Its serum level begins to rise 2 to 3 hours after infection and increases more than 1000-fold within 2 to 3 days after infection.131 CRP recognizes pathogens by binding to PC, which is expressed on many respiratory tract pathogens, and activates complement on the bacteria or functions like anti-PC antibody by engaging FcR. Indeed, CRP can kill PC-expressing H. influenzae in vitro in the presence of complement. In addition, while many pathogens use PC to bind to the host cells via rPAF, CRP also blocks bacterial adhesion that involves this receptor.132 Transgenic mice expressing human CRP are more resistant to systemic pneumococcal infection.133 The liver produces mannan-binding lectin (MBL), which is a member of the collectin family. The structural hallmark of these family members is a cystine-rich N terminus, a collagen-like domain, and a C-type lectin domain. These molecular features allow MBL to assemble into a C1q-like structure. Although it is an acute-phase protein, MBL levels change only two- and threefold during the course of an infection.134 However, the population distribution of MBL levels ranges between 100- and 1000-fold,135,136 with about 5% of Europeans being MBL deficient (< 100 ng/mL).137 MBL uses the lectin domain to recognize the target carbohydrate on a microbe, activates MBL-associated protease-2, and initiates the lectin pathway of the complement system. MBL is the innate opsonin for baker’s yeasts, and MBL-deficient individuals may have an increased incidence of infection.138 MBL significantly reduces bacteremia from N. meningitides.139 Although this relationship is controversial,140,141 MBL-deficient persons may be more susceptible to infections by S. pneumoniae with a low invasion index.142 Interestingly, transplant patients who received the liver from MBL-deficient donors are more susceptible to infections.143 The collectin family includes SP-A and SP-D, which may play important roles in lung immunity.144,145 The liver produces another group of innate opsonins, called ficolins. Humans produce three ficolins: L- and H-ficolins are from the liver,146 but M-ficolin is in the secretory granules of neutrophils, monocytes, and type II alveolar

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epithelial cells.147 Ficolins have a fibrinogen-like domain at the C terminus instead of a C-type lectin domain, but they assume an overall structure similar to those of MBL and C1q. Ficolins also trigger the lectin pathway of the complement system by activating MBL-associated protease-2.148,149 L-ficolin can bind many structures with acetyl groups150 but many bacterial surface molecules that appear to be the natural ficolin ligands contain carbohydrate moieties. Thus, ficolins display lectin-like functional properties. Once extracellular bacteria enter the systemic circulation, these opsonins together with antibodies opsonize bacteria for rapid removal by the spleen or the liver.20,151 Persons lacking splenic function (due to sickle cell disease or splenectomy) are at an increased risk of overwhelming sepsis from encapsulated pathogens (eg, S. pneumoniae).133 Clearance of bacteria from the blood by these organs is facilitated because phagocytes are abundant and blood circulates slowly in these organs. S. pneumoniae serotype 27 produce capsular PS-containing PCs. CRP can bind and opsonize serotype 27 pneumococci for phagocytes,152 which may be why serotype 27 pneumococci are nonpathogenic.153 The liver is also the major source of transferrin, which increases iron storage by tissues and lowers the serum concentration of iron. Iron at the site of inflammation may be reduced by neutrophil-secreted lactoferrin. The reduction in the amount of iron available to bacteria can be a significant defensive measure.154 Moreover, even a moderate reduction in iron intake155 and the use of an iron chelator have both been shown to be beneficial against infections by extracellular bacteria. In contrast, an excess of iron may predispose an individual to infections.156

ANTIGEN-SPECIFIC HOST DEFENSE RESPONSE Accompanying antigen-nonspecific responses, the host also mounts an adaptive, antigen-specific immune response. For protective responses to extracellular bacteria, B-cell– mediated (but not T-cell–mediated) immune responses are critical, as shown by clinical observations of patients with Bruton agammaglobulinemia. These patients, who have relatively normal T-cell function but lack B cells, suffer primarily from infections caused by extracellular bacteria, infections that can be successfully treated with the passive administration of pooled gammaglobulin.157 Consequently, protective B-cell responses are described in detail subsequently.

Responses of the Host (B Cell) Immune System to Bacteria Following an asymptomatic exposure to extracellular bacteria or to an infection, the host develops antibodies to many different bacterial antigens. For instance, the level of antibodies to various pneumococcal antigens increases in young children as they age, even if they never have clinical infections.158 However, the antibody levels remain low in those young children without evidence of asymptomatic nasopharyngeal carriage of pneumococci. This finding suggests that asymptomatic carriage of pneumococci is sufficient to raise antibody levels.158

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When an infection occurs, it presents the host with a large load of free antigens released from bacteria such as capsular PSs and proteins (eg, toxins). Bacterial proteins induce strong immune responses in a conventional T-cell– dependent manner; indeed, the antibody response induced by bacterial proteins has been used to diagnose infections.159 In addition, the released PSs that are readily detectable in the urine of many patients160 are used to diagnose pneumococcal pneumonia.161 The released PSs may neutralize the anti-PS antibodies in the host. For example, vaccination with hemophilus vaccines may neutralize preexisting antiPS antibody and briefly increase disease susceptibility immediately after the vaccination.162 In contrast to proteins, bacterial PSs generally elicit antibody responses with minimal help from T cells.163 As bacterial PSs usually have many repeating units and multiple epitopes, they can efficiently cross-link B-cell receptors and stimulate B cells. The PSs primarily stimulate two subsets of B cells: B1 B cells164 and marginal zone (MZ) B cells.165,166 These two types of B cells together with follicular B cells are the three recognized subsets of mature B cells with preferential anatomic locations. B1 B cells are associated with the peritoneum, MZ B cells are found in the splenic marginal zone, and follicular B cells are in splenic follicles. In mice, the subsets can be distinguished by their surface phenotypes. Follicular B cells are IgMlo, IgDhi, CD23 +, CD21int, and CD1d low, whereas MZ B cells are IgMhi, IgDlo, CD23low, CD21hi, and CD1dhi.167 B1 B cells express CD11b and B220, with CD5 expression being used to divide them into B1a (CD5 +) and B1b (CD5–) subsets.164 Furthermore, these subsets have distinct developmental requirements. MZ B cells require a proline-rich tyrosine kinase (Pyk-2),165 Aiolos, and Notch2.167 B1 B-cell deficiency was noted in mice without the regulatory B1 subunit of calcineurin.168 B1a B cells are absent in CD19-deficient mice,169 and the development of B1 and follicular B cells requires BTK.170 Several observations support the contention that the antibody response to PS antigens is largely independent of T cells. Athymic mice can produce antibodies to PS antigens. PS antigens do not bind class II molecules as protein antigens do171 and may actually interfere with the presentation of protein antigens.172 In addition, they do not usually induce the formation of germinal centers,173 they elicit poor immune memory,174 and they easily tolerize B cells.175 Nonetheless, there have been past reports of T-cell involvement in the antibody response to PS antigens,176 and recent studies suggest that CD40 is involved in this response.177 Because the PS antigens used for the studies may have had contaminants that affected their immune properties178 and as zwitter ionic PSs may behave differently from other PSs,179 additional studies with pure PSs would be needed to define the role of non–B cells in the antibody response to PS antigens. PS antigens commonly elicit oligoclonal antibodies, which utilize a restricted number of V region genes180,181 even among genetically unrelated humans.182 In addition, the antibodies to PS exhibit few somatic mutations182,183 and generally have a low affinity to the antigen.184 However, because the capsular PS and LPS O antigens present repeating epitopes, even low-affinity antibodies can bind with enough

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avidity to fi x complement and cause opsonization and/or bacteriolysis. Capsular PSs have been used as vaccines because antibodies specific for them are protective. Young children, however, do not produce antibodies to most PS antigens until they are several years old,185 and they are particularly susceptible to infections by encapsulated bacteria during their first few years of life.186 However, young children readily produce antibodies to PS when it is conjugated to a protein carrier. The clinical use of “conjugate” vaccines to induce antibodies to H. influenzae type b (Hib)-PS in young children has virtually eliminated Hib meningitis as well as oropharyngeal colonization by Hib.187 Similar “conjugate” vaccine approaches have been used to produce 4-valent meningococcal and 7-, 10-, and 13-valent pneumococcal conjugate vaccines.188–190 The pneumococcal conjugate vaccine has been used for young children since 2000 and has markedly reduced the incidence of invasive pneumococcal infections in both young children as well as old adults.191 The immunobiology of conjugate vaccines can differ based on the protein carriers used. Among hemophilus conjugate vaccines, Hib-PS conjugated to the meningococcal outer membrane protein complex can elicit antibody responses after only one immunization,192 presumably because, unlike other protein molecules used as carriers, outer membrane protein complex stimulates TLR2.193 Not all bacterial antigens are presented to the host as free molecules, and some antigens remain associated with the bacteria. When bacteria enter the blood circulation, they preferentially localize at the marginal zone of the spleen. The marginal zone has features useful in capturing particles in the blood: the zone is where the terminal arteriole ends and empties into sinuses and has several characteristic macrophages and DCs.167 Following the localization of bacteria at the marginal zone of the spleen, even without T cells, within 2 to 3 days MZ B cells can be activated and become plasma cells secreting antibodies to bacterial PSs.166 MZ B cells have additional unique characteristics. They are rapidly stimulated by LPS,194 and their maturation may be facilitated by other bacterial molecules as well. MZ B cells may facilitate the activation of follicular B cells as they can capture IgM immune complex and transport it to follicular DCs.195,196 Although MZ B cells can mature and differentiate independently of T cells,166 other cells, including T cells, may help their maturation and antibody responses to various bacterial antigens. MZ B cells can present bacterial (protein) antigens to naïve T cells.195 In addition to protein antigens, antibody responses to bacterial PSs require cofactors such as B7-2197 and CD40,197,198 and can be reduced with the simultaneous injection of anti-CD4 and anti-CD8 antibodies.199,200 Indeed, studies have shown antibody responses to PSs attached to bacteria to be T-cell–dependent.201 MZ B-cell response may depend on DCs as well. Upon taking up dead bacteria, CD11clow CD11bhigh DCs in the blood locate to the spleen and may provide transmembrane activator and CAML interactor ligand(s) helpful in MZ B-cell survival.202 Adoptive transfer of live DCs after an in vitro exposure to dead pneumococci can transfer

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antibody responses to pneumococcal proteins and PS antigens.203 Natural killer T cells may be involved in MZ B-cell maturation as MZ B cells express CD1 and may activate natural killer T cells.195 Preimmune animals have antibodies that cross-react with many structurally unrelated antigens. These antibodies are often labeled as “natural antibodies.” The majority of these antibodies is of the IgM isotype and frequently bind autologous antigens. Anti-PC antibodies may be an example of natural antibodies. Recent studies suggest that these natural antibodies are important in the early phase of bacterial and viral infections.204 For instance, anti-PC antibodies react with a PC epitope found on S. pneumoniae, H. influenzae, and Wuchereria bancrofti (a tissue nematode).205–207 Anti-PC antibodies can reduce the susceptibility of mice to pneumococcal infections.206 A recent study suggests that the natural antibodies are from B1a B cells. CD19-deficient mice lack B1a B cells and have a reduced number of MZ B cells. These mice also lack anti-PC antibodies and are susceptible to pneumococcal infections.169 In contrast, another mouse strain that exhibits both a reduced number of MZ B cells and a deficiency of B1b cells can produce anti-PC antibodies and is as resistant to pneumococcal infections as are normal mice.169 In addition to natural antibodies, animals often have preexisting antibodies to a PS that cross-react with structurally similar PSs, 208–210 probably because many PS molecules have very similar structures. Sometimes it is difficult to distinguish usual “anti-PS antibodies” from “natural antibodies.” Cross-reactions may play an important role in protecting the host against its fi rst exposure to a bacterial species. For instance, human adults carry detectable amounts of antibodies to the Hib-PS—even in the absence of vaccination—and are thus relatively resistant to H. influenzae infections.186 While some of the antibodies may be the result of immunization by subclinical infections, the majority of human preimmune (but not postimmune) anti–Hib-PS antibodies cross-react with E. coli K100, the PS capsule of which is an isopolymer of Hib-PS.211 Experimental colonization of rats with E. coli K100 can protect them against Hib.212 About 1% of human IgG binds a carbohydrate epitope (galactose [1→3] galactose), 213 and this antibody can kill Trypanosoma and Leishmania in vitro.214 Cross-reactive antibodies binding the LPS core components are thought to be responsible for the protection from bacteremic dissemination of gonococci in nonimmune patients, 215 although they cannot prevent infection of the genital tract.215 Normal gut flora may be the antigenic stimulus for many of the cross-reactive anti-PS antibodies. About 1% of the human population carries E. coli K100 in their gut at any moment.216 Antibodies to (galactose [1→3] galactose) bind many species of bacteria isolated from normal stool specimens.213 The gut flora may have additional interesting impacts on the immune system. For example, in some transgenic mice, inflammatory bowel diseases develop in the presence of normal intestinal flora but not in the absence of gut flora. In addition, in some animals, such as chickens and rabbits, microbial colonization of the gut

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appears to be necessary for the normal development of antibody V region repertoires.217 Bacteria should therefore be considered as active participants in shaping the host immune system.

Protective Mechanisms of Antibodies Antibodies to virulence factors may act by neutralizing the function of those factors. Anti-toxin antibodies can protect a host by blocking the action of the toxins (eg, blocking the binding of the toxins to the host cell receptors) or by increasing the removal rate of the toxins. Antibodies to superantigens or tetanus toxin can inactivate them and thereby provide protection to the host. Antibodies to an E. coli adhesin can prevent experimental infections by E. coli.218 Antibodies to M protein neutralize its ability to interfere with complement and provide protection against S. pyogenes infections. Antibodies to LPS,219,220 and perhaps to LTA,221 can be protective. Antibodies to PspA, pneumolysin, autolysin, or pneumococcal surface protein C can protect animals from fatal pneumococcal sepsis. Although these antigens are being investigated as potential replacements for the expensive pneumococcal conjugate vaccines, the protective mechanisms that they employ are still unclear. The most recent hypothesis suggests that antibodies to PspA may inhibit its decomplementation properties and the antibodies may increase the complement fi xation on pneumococci.33,222 Antibodies to IgA1 protease or iron-transport systems223,224 can also protect against bacterial infections, most likely by neutralizing the normal functions of the target antigens. Finally, in the presence of antibodies and complement, the ability of the liver to remove bacteria increases significantly.20 Thus, another protection mechanism provided by antibodies may be to facilitate the in vivo removal of bacteria from circulation by enhancing the ability of the reticuloendothelial system to clear bacteria. Antibodies to capsule PS can provide protection by fi xing complement on the surface of bacteria and by inducing bacteriolysis or opsonization. The bacteriolysis pathway can provide significant in vivo protection against gramnegative bacteria, as illustrated by the susceptibility to N. meningitidis infections of persons with deficiencies in C5–9 components.225 In contrast, antibodies and complement do not lyse gram-positive bacteria but opsonize them for phagocytic killing, as explained in the following.226 Host phagocytes cannot readily recognize and kill the intact encapsulated gram-positive bacteria. However, once bacteria are coated with antibodies and complement, the host phagocytes can readily recognize the bacteria via various recognition receptors and engulf them for intracellular killing. The Fc receptor (CD16b) and the complement receptor (CR3) are some of the important recognition receptors. CR3, an integrin molecule, is a heterodimer of CD11b and CD18. Protection mediated by this antibody/complementmediated opsonization is probably important in vivo, as both complement deficiency and agammaglobulinemia predispose individuals to infections by many different extracellular bacteria.157,225 To be effective for opsonization, the epitope of the surface antigens must be exposed on the

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surface of the bacteria. Effective antibodies to the porins of N. meningitidis recognize the surface loop of the molecule.227 In most pneumococci, C-polysaccharide (C-PS) is mostly buried underneath the PS capsule. Although antibodies to the C-PS can fi x complement,20 anti–C-PS antibodies were ineffective in protecting mice against most S. pneumoniae unlike antibodies to capsular PS, which are protective.228 However, a recent study found that purified human anti–C-PS antibodies can opsonize pneumococci229 ; thus, additional studies are needed. Because antibody-mediated opsonization and bacteriolysis are dependent upon the complement-fi xing properties of the Fc region, the relative efficacies of antibodies of different Ig isotypes have been compared. IgM antibodies are produced early in the course of infections and should be important in the early phase of infections because they fi x complement very efficiently and can opsonize bacteria. Selective deficiency of IgM antibodies was found to increase susceptibility to bacterial infections.230 Studies found that specific IgM antibodies agglutinate erythrocytes, fi x complement, and lyse erythrocytes more readily than IgG antibodies,231 and IgM antibodies are more effective in complement-mediated bacteriolysis232 ; however, IgG antibodies are more effective than IgM antibodies in preventing pneumococci infections of mice233 or in opsonizing Hib in vitro.234 Moreover, antibodies of some IgG subclasses have been reported to be more protective against specific viral235 and fungal236 infections than antibodies of other subclasses. These results suggest that optimal opsonization requires not only complement receptors but also Fc receptors for IgG. In the absence of inflammation, IgM antibodies are confined to the intravascular space, whereas IgG antibodies can enter the extracellular space. However, inflammation can make the vessels at the infection site permeable, at which point, antibodies of all isotypes may enter the infection site. Compared with IgM antibodies, IgG antibodies may be especially efficient at neutralizing toxins because they have a longer half-life, generally have a higher affinity, and are already present in extravascular spaces prior to infection.237 IgG subclasses differ in their ability to fi x complement and to bind Fc receptors.238,239 It has also been reported that IgG1 mouse monoclonal antibody is protective against Cryptococcus neoformans but that IgG3 mouse antibody is not.236 Consequently, the fact that antibodies to bacterial PS are found to be largely restricted to a single IgG subclass (IgG2 in humans and IgG3 in mice) has led to many studies of the differences in the protective properties of anti-PS antibodies of different isotypes. Mouse IgG3 antibodies (but not antibodies of other IgG subclasses) can associate with each other through their Fc regions.240 This feature may make the IgG3 antibodies with a low affinity to PS more effective in binding the antigen than antibodies of other isotypes of the same affinity. Although these observations provide a theoretical advantage for mouse IgG3 antibodies, this same aggregation phenomenon has not been observed for human IgG2 antibodies even though some human IgG2 can form covalently joined dimers.241 The full significance of IgG3 aggregation is not clear, however, as anti-PS antibodies of the IgG3 isotype have not been observed to be any more efficacious

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against pneumococcal infections than antibodies of other isotypes.242 IgG2 antibody levels can be significant, however. People expressing the Gm23 + IgG2 allele have higher IgG2 antibody levels than people with the Gm23– allele. Among C2-deficient persons, Gm23– persons are more susceptible to bacterial infections than Gm23 + individuals.243 Moreover, in contrast to expectations, many studies found that human IgG1 antibodies are slightly more effective at opsonization and bacteriolysis than are human IgG2 antibodies.239,244 Neither of these isotypes appears to be essential, however, as individuals lacking IgG1 and IgG2 subclass genes are healthy.245 Furthermore, human IgG2 antibodies bind less strongly to CD16, CD32, and CD64 than do IgG1 or IgG3 antibodies246 and may not be effective for neutrophil opsonization in individuals homozygous for a specific CD32 allele.244 These observations, taken together, strongly suggest that the human IgG2 (or mouse IgG3) subclass may not provide any unique advantage in defense against bacteria. IgA is highly heterogeneous in structure: it can exist as a monomer, a polymer, or in secretory forms. In addition, its function is still unclear. Although it has been reported that IgA can opsonize,247 fi x complement,248 and facilitate the lysis of N. meningitides,249 other studies have found that IgA does not fi x complement in vitro250 and may even inhibit IgG-mediated complement-dependent killing.251 The ability of IgA to fi x complement may also depend upon its denaturation or its glycosylation status.252,253 Nevertheless, other studies indicate that IgA antibodies may fi x complement by the MBL pathway254 and that human IgA antibodies against pneumococcal capsular PS can opsonize pneumococci for killing by neutrophils.255 Bacteria that commonly colonize or infect mucosal areas often produce IgA1 protease, and IgA antibody has been found to provide protection in at least some of these cases.256 These findings suggest that IgA may play an important role as a part of the complex mucosal immune defense. For example, IgA antibodies may be important in reducing nasopharyngeal colonization by bacteria inasmuch as the mice deficient in IgA or polymeric Ig receptor can carry pneumococci in the nasopharynx even after an immunization against pneumococci.257 IgA may function by aggregating the bacteria and facilitating their expulsion from mucosal areas. IgA may also block the invasion of bacteria through mucosal epithelial cells, as endocytosed IgA has been found to block the transport of virus through epithelial cells.258 However, IgA-deficient persons or mice are relatively healthy, and IgA-deficient mice can elicit normal protective immunity to experimental infections with influenza virus. IgM antibodies may function as secretory antibodies in IgAdeficient individuals.259

T-Cell Immune Responses to Extracellular Bacteria Although immune responses to toxins from extracellular bacteria are T-cell–dependent, antitoxin antibodies mediate protection; therefore, the protective immunity against extracellular bacteria is clearly centered on the B-cell responses. However, recent studies suggest additional roles for T cells in responses to extracellular bacteria and their products.

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PS associated with lipid can stimulate T cells in association with CD1 molecules.260 Also, studies of abscess formation in response to Bacteroides fragilis infections led to a discovery that zwitterionic PS can be taken up by antigen-presenting cells, which can process the PS via a nitric oxide– dependent mechanism and present it in association with MHC class II molecules179 to stimulate CD4 + T cells to produce IL-17.261 IL-17–producing CD4 + T cells are now named “Th17 cells” and are found to be distinct from Th1 and Th2 cells. Th17 cells produce unique set of cytokines, such as IL-17 (IL-17A), IL-17F, IL-21, and IL-22, and require a distinct cytokine milieu (IL-1, IL-6, and TGF-β) for their development.262–265 Th17 cells may be involved in abscess formation by B. fragilis infections because Th17 cells are found in these abscess.266 Several studies found that IL-17 and Th17 cells are important in the nasopharyngeal carriage of S. pneumoniae in mice.114,267 A recent study showed that Th17 cells may recruit monocytes and macrophages to the nasopharynx where the monocytes/macrophages may actually remove pneumococci. Clinical examples clearly demonstrate the importance of IL-17 on some extracellular bacterial infections in humans. Patients with autosomal dominant hyper-IgE syndrome are deficient in Th17 cells and are very susceptible to infections by fungus and S. aureus.268 Patients with mutations in IL-17F or IL-17 receptor A (IL-17RA) are susceptible to fungal and staphylococcal infections.269 In view of these new findings, some researchers are investigating the possibility of using antigens stimulating Th17 cells as vaccines against S. pneumoniae.

DELETERIOUS HOST RESPONSES Inflammatory responses by the host inevitably cause some tissue damage. In some bacterial infections such as pneumonia and meningitis, this damage plays a significant role in disease pathology and symptoms. For instance, animal models of meningitis have shown that inflammation associated with bacterial products (primarily bacterial cell walls) is the primary cause of neurologic damage. Treatment of animals with antibiotics alone can eradicate the bacteria, but it does not prevent neurologic damage. In contrast, when inflammation was controlled by steroids administered along with the antibiotics, neurologic damage was considerably reduced.270

Antigen-Nonspecific Deleterious Response Uncontrolled inflammation at the systemic level can produce septic shock, which can be triggered by several factors, including exotoxins (eg, staphylococcal enterotoxin B) of gram-positive bacteria, the combination of LTA and peptidoglycan from gram-positive bacteria,271 or LPS from gram-negative bacteria. The staphylococcal enterotoxin B superantigen binds the host’s class II molecules of the MHC region and can stimulate large numbers of helper T cells to release cytokines. Septic shock can also be initiated when LPS from gram-negative bacteria binds CD14 and a TLR and stimulates macrophages or monocytes to secrete inflammatory cytokines. In addition to resulting in the release of cytokines, the stimulation of host cells by bacterial

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products leads to the release of other mediators of inflammation, such as arachidonic acid metabolites, activation of the complement cascade, and activation of the coagulation cascade. Excess release of the mediators leads to the failure of the vascular system and, finally, the failure of multiple organ systems. Studies using transgenic mice with defective genes have identified several molecules critical in developing septic shock, such as TNF- α , one of its receptors TNFRI, caspase 1, and ICAM-1.272 This approach also showed that CD14 and TLR4 are critical for LPS-induced septic shock and that CD28, a T-cell costimulation molecule, is necessary for superantigen-induced septic shock.272 Anthrax infections provide another example of uncontrolled host responses. “Lethal factor” binds the “protective antigen” immobilized on the macrophages and then stimulates the cells to secrete cytokines and reactive oxygen intermediates. These macrophage products are thought to kill the host, as the host dies even when the proliferation of the bacteria is controlled. When macrophage cells are removed from animals, the animals are resistant to anthrax toxins.273 This suggests that the macrophage response to the toxins is actually responsible for the death of the host. Although inflammation is a significant cause of morbidity and mortality, it must also be regarded as the host’s primary protection against bacterial infections. Evidence for this hypothesis comes from studies with TLR4-deficient mice, which, although nonreactive to LPS and completely resistant to LPS shock, are more susceptible to infection with gram-negative bacteria than are normal mice.274,275 Perhaps LPS is “toxic” because the host has evolved to use this common bacterial component as a trigger for host responses.

Autoimmune Disorders Autoimmune diseases are characterized by an overactive host immune response toward self, the host’s own cells and tissues. Various factors are involved in autoimmune diseases, including genetic predisposition and environmental triggers. The pathogenesis of autoimmune diseases has at its core the development of autoreactive effector lymphocytes, and these can involve, among others, T-cell bypass and molecular mimicry. T-cell bypass is based on the notion that activated T helper cells provide the necessary factors to activated B cells for the production of antibodies. Some microorganisms can provide the bypass with superantigens, which can bind to many T cells expressing certain types of Vβ regions and stimulate them to create a nonspecific polyclonal T-cell activation.276,277 The best-studied examples of superantigens are the exotoxins secreted by S. aureus and S. pyogenes.278–280 Molecular mimicry occurs when a bacterial antigen shares structural similarities with a host antigen, and hence, antibodies produced against the bacterial antigen could also bind to the host antigen, thereby casuing an autoimmune disease. For instance, the LPS of many strains of N. meningitidis, N. gonorrhoeae, H. influenzae, and Haemophilus ducreyi expresses the epitope of blood group antigen pK.281 The PS capsule of N. meningitidis group B mimics epitopes expressed in the central nervous system,282 such as the N-acetylneuramic acid epitope in the embryonic N-CAM.283

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While infection by these pathogens may cause autoimmune diseases, epidemiologic studies have clearly associated Campylobacter jejuni infections, a leading cause of acute gastroenteritis, with development of antiganglioside antibodies and autoimmune diseases: Guillain-Barre syndrome284 or its variant Miller-Fisher syndrome. Patients with Miller-Fisher syndrome have ophthalmoplegia and generally have autoantibodies to GQ1b, but Guillain-Barre syndrome is associated with autoantibodies to GM1 or GD1a.285 Production of the two different types of autoantibodies has been associated with an allelism of sialyl transferase II of C. jejuni. One allele produces lipooligosaccharide-mimicking GM1 and GD1a, and infection by C. jejuni with this allele has been associated with Guillain-Barre syndrome. On the other hand, the other allele produces lipooligosaccharide-mimicking GQ1b, with its infection being associated with Miller-Fisher syndrome. Perhaps the most classical example of infection-associated autoimmunity may be rheumatic fever and acute glomerulonephritis associated with S. pyogenes infections. Studies found that S. pyogenes can be divided into two classes with a monoclonal antibody to M protein286 and that rheumatic fever develops only after infections with class I strains of S. pyogenes.286 Class I and class II strains of S. pyogenes can also be readily distinguished by the linkage relationship of the M protein genes with the genes encoding related surface proteins.286,287 M proteins from some class I S. pyogenes express epitopes highly cross-reactive with epitopes of cardiac myosin, tropomyosin, vimentin, laminin, and keratin.288–290 An antibody molecule may bind to all of these protein molecules because a major portion of these proteins is coiled–coil α –helix.290 The polyreactive antibodies to M protein may directly damage myocardial and endothelial cells.291 In addition to antibodies, CD4 + and CD8 + T cells are found at rheumatic heart valves,292 and the T cells proliferate to M protein peptides and heart proteins.293 These observations suggest that the T cells with crossreactivity between M protein and myosin may be involved in the pathogenesis of rheumatic fever as well.

CONCLUSION Because extracellular bacteria can grow rapidly and produce toxins, some are potent pathogens. To combat these bacteria, higher organisms primarily depend on two arms of the immune system: innate immunity and adaptive immunity centered on antibody molecules. The two arms of the immune system are comprised of multiple layers of protection. In the early stage of an infection, innate immunity involving pattern recognition receptors, complement, phagocytes, and natural antibodies cross-reacting with many antigens are important in host defense. During the late stage of an infection, pathogen-specific antibodies appear. These antibodies generally mediate the ultimate protection against extracellular bacteria by triggering the protective effects of complement and phagocytes. Nevertheless, innate and adaptive immune responses may cause damage instead of protection. A better understanding of how our immune system protects against each pathogen will aid in the development of more effective preventive and therapeutic measures against these pathogens.

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36 42

Immunology of Human Immunodeficiency Virus Infection Douglas S. Kwon • Bruce D. Walker

INTRODUCTION In June 1981, the U.S. Centers for Disease Control and Prevention (CDC) reported five cases of Pneumocystis pneumonia (PCP) in otherwise healthy young gay men in Los Angeles, California,1 representing the first published evidence of what was to become the acquired immunodeficiency syndrome (AIDS) epidemic. At that time PCP was rare, known as an “opportunistic infection” that occurred in the setting of severe immune compromise resulting from cancer treatment. At the same time, there were also reports of an aggressive form of Kaposi sarcoma, a generally benign cancer, in the same demographic, namely young gay men.2 Like PCP, Kaposi sarcoma was infrequent and was typically seen in older men or those who were immunosuppressed following organ transplantation. It made sense in the setting of severe immune compromise to see PCP or Kaposi sarcoma, but what was alarming in those initial reports was that it was being seen in otherwise seemingly healthy people. At that time pentamidine, the main drug used to treat PCP, was only available through the CDC. Coincident with the first PCP cases reported in Los Angeles, there was a dramatic increase in requests for pentamidine from many other cities across the United States. A consistent and disturbing pattern was quickly noted—the drug was largely being requested for young men who had no known reason to be immune compromised and at risk for PCP. An emerging epidemic was thus recognized, and already from those first patients there were clues pointing to the central problem induced by what was ultimately recognized to be infection by a new human retrovirus: that human immunodeficiency virus (HIV) is an infection of the immune system. When an antibody test for HIV became available in 1985, the kinetics of the expanding epidemic first began to be realized through retrospective examination of stored serum samples. Results from analysis of samples from a hepatitis B vaccine trial that had coincidentally been initiated in 1980 in gay and bisexual men in San Francisco, California, were startling.3 By 1982, the cumulative incidence of infection in those at-risk men had risen to 42.6%. HIV has since gone on to infect over 60 million people worldwide and has caused over 30 million deaths.4 In some of the most affected areas of the world, HIV prevalence exceeds 50% in certain communities and age groups (Fig. 42.1).5 Thankfully, the development of effective HIV drug therapy has turned HIV infection into a treatable condition for those fortunate to have access to these medications,6 but viral integration into the host chromosome makes the need for treatment

lifelong.7 Regrettably, for each person who begins antiretroviral therapy (ART) in resource-limited settings, there are two who become newly infected.4 The ultimate resolution of the epidemic will almost certainly require an effective vaccine, but despite nearly a billion dollars invested annually in AIDS vaccine development, this has remained an elusive goal. Unlike other infections for which effective vaccines exist, for HIV there is a lack of natural clearance and subsequent durable protection.8 As natural protective immunity does not exist, an effective HIV vaccine will have to do better than nature does.

HUMAN IMMUNODEFICIENCY VIRUS ORIGIN, TRANSMISSION, AND DISEASE PROGRESSION Origin of Human Immunodeficiency Virus and Relationship to Other Retroviruses The isolation of a retrovirus in 1983 from a patient with AIDS in France led to wider sampling of at-risk persons and was paralleled by the discovery of other primate retroviruses. Sequencing of HIV revealed enormous variability among isolates, which were ultimately phylogenetically characterized as belonging to four distinct groups. Group M is the largest and accounts for over 98% of infections worldwide.9 Based on geographically targeted sequencing, it appears that the epicenter of the global group M epidemic was in Kinshasa in the Democratic Republic of the Congo,10 and that a cross-species transmission of simian immunodeficiency virus (SIV) from chimpanzees occurred in the early 1900s.11,12 In turn, chimpanzees had acquired SIV from cross-species transmission of two viruses in monkeys that recombined. The actual origin of the group M epidemic in humans appears to have been in a remote region of southeastern Cameroon, likely by exposure through butchering of a chimpanzee.13 Much less frequent are groups N, O, and P, the result of separate cross-species transmissions from chimpanzees (groups N and O) and gorillas (group P), which have remained localized to Cameroon and neighboring countries in west Africa.9,14,15 HIV is a typical retrovirus, morphologically characterized by a core containing the viral ribonucleic acid (RNA) and two copies of reverse transcriptase (RT), surrounded by an outer envelope that is heavily glycosylated. The errorprone RT leads to the rapid generation of new mutations, resulting in enormous genetic variation within group M viruses globally. This is largely due to lack of 3’ exonuclease proofreading activity of RT, which gives rise to the mutation of approximately 1 in every 10,000 nucleotides during viral genome transcription; in other words, once per replication

1016

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No data

600 named

TABLE

46.4

Class I antigens

Class II antigens

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Summary of Features of Major Histocompatibility Complex Single polymorphic chain Three domains: alpha 1, 2, and 3 MW: 45,000 Associated with beta 2 microglobulin A, B, and C loci in humans Expressed on all tissues and cells Two polymorphic chains: alpha and beta Each with two domains: alpha 1 and 2, beta 1 and 2 MW: 33,000 and 28,000 DP, DQ, and DR loci in humans Expressed on macrophages, dendritic cells, and B cells; vascular endothelium; activated human T cells

alleles at each of the HLA-A and B (class I) and >300 at the DRB1 (class II) locus.12 The high degree of polymorphism has important consequences for transplantation. Given that there are three class I loci (A, B, and C) and three to four class II loci (DQ, DP, DRB1, ± an expressed DRB 3, 4, or 5 locus present in some haplotypes) on each haplotype, the likelihood of achieving identity for MHC antigens in two unrelated humans is extremely small, though for individuals with two major conserved HLA haplotypes,13,14 the likelihood is increased. Tissue Distribution. The tissue distribution of the two types of MHC antigens differs. Class I antigens are constitutively expressed on all nucleated cells, but at low levels on some types of cells.15 Class II MHC antigens are more selective in their distribution.16 They are especially frequent on macrophages, dendritic cells (DCs), and B-lymphocytes. They are present on other lymphoid cells under some circumstances and on vascular endothelium. Their expression on some tissues of the body is regulated (eg, by interferons [IFNs]).17 One of the important distinctions between rodents and many larger species is the lack of constitutive expression of class II antigens on the vascular endothelium and other cell populations in rodents. In contrast, pigs, monkeys, and humans express class II antigens on these tissues.18–20 Physiologic Function of Major Histocompatibility Complex Antigens. MHC antigens are called “histocompatibility” antigens because of their powerful role in causing graft rejection; however, they did not evolve in nature to prevent tissue grafting. While the name serves to emphasize the historical importance of transplantation in the discovery of the MHC, the essential role of MHC antigens is now understood to involve the presentation of peptides of foreign antigens to responding T cells (see Chapter 22). The Importance of Major Histocompatibility Complex Antigens in Alloreactivity. MHC antigens are exceptionally important in stimulating T- and B-cell alloresponses. This section will

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focus mainly on T-cell responses to MHC, as alloantibody responses are discussed later in the chapter. Vigorous Graft R ejection. Mouse skin grafts differing only in their MHC antigens are typically rejected in 8 to 10 days, whereas MHC-matched grafts may be rejected more slowly, depending on the number of minor histocompatibility antigen (MiHA) differences. In pigs, primarily vascularized organs, such as the kidney, may survive indefinitely in some cases, even without immunosuppression, if all of their MHC antigens are matched, whereas MHC-mismatched kidneys are always rejected within 2 weeks.18 Strong In Vitro A lloresponses. Allogeneic MHC antigens stimulate an extraordinarily strong T-cell response in vitro, whereas responses to non-MHC antigens generally require in vivo priming. For the most part, cluster of differentiation (CD)4 cells recognize class II alloantigens and CD8 cells recognize class I alloantigens. However, this strong bias is more stringent for CD4 cells than CD8 cells. The standard in vitro assay of T helper function is the mixed lymphocyte response, which measures proliferation of T cells after allogeneic stimulation. Limiting dilution assays can be used to quantify alloreactive proliferating or cytokine-producing cells. Such analyses have led to frequency estimates of approximately 1% to 7% of T cells responding to a particular allogeneic donor,21–24 whereas naïve T cells reactive with an exogenous peptide generally represent only approximately one in tens to hundreds of thousands of the same T-cell pool.25–27 Strong primary direct alloresponses of CD8 + T cells can be measured in vitro, either in standard cell-mediated lympholysis assays or in limiting dilution assays measuring cytotoxic T-lymphocyte (CTL) precursor frequencies. CTLs against MHC alloantigens can easily be generated from naive T cells following stimulation in vitro, whereas generation of CTLs to MiHAs generally requires that the T cells first be primed in vivo. Direct (without in vitro stimulation) cytotoxic activity,28 increased alloreactive precursor frequencies, and modified CTL assays29 have been used to demonstrate in vivo activation by alloantigens. ELISpot and flow cytometric

carboxyfluorescein diacetate succinimidyl ester dye dilution assays and intracellular cytokine staining have enhanced the ability to detect alloreactive T cells.24,30 Direct Recognition of Allogeneic Major Histocompatibility Complex Antigens. The extraordinary strength of alloreactivity largely reflects the ability of T cells to recognize allogeneic MHC antigens presented on donor APC, referred to as “direct” allorecognition. Three different but not mutually exclusive hypotheses have been proposed to explain the high frequency of alloreactive T cells. Genetic Bias. Because the thymus only positively selects T cells with some MHC reactivity, a T-cell receptor (TCR) gene pool with intrinsic affinity for MHC molecules would allow for more efficient thymic selection. TCRs indeed have intrinsic affinity for MHC molecules.31–40 Intrinsic allogeneic MHC reactivity is thereby prominent within a T-cell repertoire that has been negatively selected only by “self” MHC-peptide complexes. The “Determinant Density” Hypothesis. As illustrated in Figure 46.6A, the density of specific peptide determinants presented by an APC would be quite low (as most MHC antigens present other peptides), whereas the density of a peptide-independent allogeneic MHC determinant on allogeneic APCs would be very high (as every MHC antigen would include the foreign determinant). These abundant allogeneic MHC determinants would activate many crossreactive T cells with relatively low affi nities. This hypothesis requires that allogeneic MHC molecules can be recognized at least partly independently of the peptides they present. While peptide-independent and peptide-“promiscuous” alloreactive T cell clones have been described,41–50 potential artefacts of in vitro culture and assay systems might have biased these results. Notably, human alloreactive T cells expanded in vivo in a graft-versus-host response were strongly dominated by peptide-specific clones,51 and recent studies demonstrated that the requirement for peptide recognition limits TCR alloreactivity from being extended more broadly by their inherent MHC-binding capacity.52

Y6

Y4

Y5

Y7

Y5

Y3 Donor APC

Y4

Self APC

Donor APC

Y1

Y3 X Donor APC with donor MHC antigens ( ), all of which are foreign.

AA

Self APC with self MHC molecules ( ). The rare self MHC molecule presents a peptide (X) of an environmental pathogen.

Y8

Y2 Donor MHC antigens ( ). Each presents different “self peptides,” generating different foreign determinants.

Self APC

Y6 Y7

Y2 X

Self MHC molecules ( ) also present self peptides (Y1...N), but these are all self determinants.

B

FIG. 46.6. A: Determinant density hypothesis. B: Determinant frequency hypothesis. APC, antigen-pressenting cell; MHC, major histocompatibility complex.

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The “Determinant Frequency” Hypothesis. The third explanation for alloreactivity is based on the idea that allospecificities include the specific peptides presented by allogeneic MHC molecules,53 for which there is strong evidence. Positive selection, which requires lower TCR affinity for self-MHC/peptide complexes than that involved in negative selection,38 enriches for TCRs capable of seeing modified self-MHC antigens, which may cross-react on allogeneic MHC molecules. The repertoire of T cells positively selected with low affinity for self-MHC molecules plus peptides of self-proteins (say X1, 2 . . . n), may cross-react with allogeneic MHC antigens presenting peptides of polymorphic or nonpolymorphic allogeneic proteins (eg, “Allo + X1, Allo + X 2, ...Allo + X n”) (see Fig. 46.6B). Nonpolymporphic peptides presented by allogeneic MHC molecules would be seen by different TCRs than those recognizing the same peptide with self-MHC, as both peptide and MHC alpha helix residues contribute to the surface that is recognized by a TCR.54–57 Thus, the set of determinants represented by “Self + X1 . . . n” would differ from that represented by “Allo + X1 . . . n.” T cells strongly responsive to self-peptides on selfAPCs (Self + X1, Self + X 2, etc.) are eliminated by negative selection, which would not affect the response to the many peptides on allogeneic APCs (Allo + X1, Allo + X 2, etc.). Consistent with this hypothesis, many alloreactive T cells have been shown to be peptide-specific or at least partially peptide-selective.41,42,48,58–63 Cardiac allografting studies using DM−/− mice, which lack the capacity to replace invariant chain-derived CLIP peptide with a more diverse array of peptides, provide strong in vivo evidence for the importance of peptides in direct allorecognition.64 Overall, the available information supports the inherent MHC binding capacity of TCRs, combined with the determinant frequency notion, to explain the high frequency of T cells recognizing alloantigens directly.

Minor Histocompatibility Antigens While initially defined by their ability to cause rapid graft rejection, MHC antigens are defined in part by the location of the genes encoding them and in part by the wellcharacterized structure of both class I and class II antigens (see Chapter 21). MiHAs, on the other hand, are those capable of eliciting a T-cell immune response, but which lack the structural characteristics of MHC products.65 Rather than being allelic cell surface proteins, MiHAs are donor-specific peptides presented by MHC molecules that are shared by donor and recipient.66–73 As individuals are tolerant to the peptides derived from their own proteins, they only respond to the peptides of another individual’s proteins that have allelic variation. Unlike MHC antigens, MiHAs do not readily stimulate primary in vitro cell-mediated responses in mixed lymphocyte response and cell-mediated lympholysis assays, reflecting the low frequency of T cells recognizing them in the unprimed T-cell repertoire. It has been estimated that there may be as many as 720 minor histocompatibility loci in mice,74 some of which are autosomal and others of which are encoded on the Y chromosome. MiHAs can be expressed ubiquitously or

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in a tissue-selective manner.75 Many proteins producing MiHAs have been identified,71–73,76–89 some of which are intracellular proteins such as nuclear transcription factors and myosin, while others, like CD31 and CD19, are polymorphic cell surface glycoproteins.90–92 Some MiHAs are diallelic peptides, both of which can be represented by a particular MHC molecule,76,78 resulting in bidirectional recognition (eg, the murine H13 locus78). Alternatively, allelic variation in MHC-binding capacity of a peptide can result in one allele being presented and the other not (eg, the human HA-1 minor antigen, in which only one of two allelic peptides binds effectively to HLA-A276). Minor antigenic determinants can also result from the failure of one allele to be processed to a peptide. An example is HA-8, for which only one allele is effectively transported by the TAP complex, resulting in a null allele despite the presence of the MHC-binding peptide sequence in the molecule.79 Both helper determinants, recognized by CD4 + cells, and cytotoxic determinants, recognized by CD8 + cells, are required for effective cytotoxic T-cell responses to MiHAs.93,94 When multiple MiHA disparities exist, a phenomenon known as “immunodominance” may occur.95–100 Removal of the immunodominant recognition can reveal strong responses to antigens that evoked weak or no responses before. This phenomenon may be due to competition between peptides for presentation by MHC molecules,74 as well as differing durations of antigen presentation and TCR avidities.101 An exceptional peptide, H60, is derived from an NKG2Dbinding protein and produces responses that are comparable in potency to those elicited by MHC alloantigens, apparently due to the existence of a very high frequency of TCR in the naïve repertoire recognizing this peptide,102 which may be a useful target for graft-versus-leukemia (GVL) effects in HCT.103 However, immunodominance of CTL responses measured in vitro does not necessarily reflect the immunodominance of the same antigens in vivo.74,104 possibly reflecting the importance of tissue distribution of minor antigen expression or of helper T-cell responses.84 The H-Y antigens are encoded on the Y chromosome and therefore expressed only by males.81,82,105–108 They are of biologic significance, as they cause rejection of male skin grafts in syngeneic female mice and, in humans, female-to-male HCT is associated with increased GVHD rates compared to other combinations in the HLA-identical–related donor setting. Human MiHAs have been identified as determinants recognized by CTLs, mainly in the setting of HLA-identical sibling donor HCT, in association with GVHD and marrow graft rejection.77,79,84,85,109,110 Certain MiHA incompatibilities (eg, HA-1) may predispose to GVHD.77,111 Immunodominance of CTL responses to particular H-Y and HA determinants as well as expansion of minor antigen-specific CTLs detected with tetrameric complexes of HLA molecules and minor antigenic peptides85,110,112 have been associated with GVHD and marrow graft rejection.113 Most MiHAs identified to date are determinants recognized by CTL, reflecting the relative ease with which CTL assays can be used to measure peptide-specific responses. Newer techniques have allowed the recent identification of minor antigenic peptide epitopes recognized by CD4 + T cells.114–116

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It is difficult to detect humoral responses to MiHAs, presumably because individual peptide/MHC complexes are too low in abundance on the cell surface to either stimulate an antibody response or be detected on the cell surface with antibodies. An exception is the recent discovery of antibodies against H-Y (Y chromosome–encoded) antigens in association with chronic GVHD (cGVHD).

Other Antigens of Potential Importance in Transplantation Superantigens Superantigens include products of endogenous retroviruses in mice, such as mammary tumor viruses, and bacterial products such as stapholococcal enterotoxin B. Like MHC antigens, superantigens can stimulate primary in vitro T-cell proliferative responses and activate a high proportion of the T-cell repertoire. However, these antigens are not presented as peptides in the binding groove of MHC molecules, but instead bind to distinct regions of class II MHC molecules, and engage nonvariable portions of Vβ components of the TCR, rather than the hypervariable regions that recognize peptides. Endogenous superantigens are not classical transplantation antigens, perhaps because of their restricted tissue expression patterns.117–121 However, they may contribute to GVHD in mice.122

Tissue-Specific Antigens Some peptides are derived from proteins with limited tissue distribution,123–125 which may not include hematopoietic cells used in traditional assays of alloreactivity. One implication is that transplantation tolerance induced by one set of donor cells might not always induce complete tolerance to donor cells of a different sort. For example, skin-specific antigens may be targets of skin graft rejection despite the presence of stable hematopoietic chimerism induced by HCT.126 Tissue-specific proteins lacking allelic variation may still serve as alloantigens because the determinant formed by a given peptide with an allogeneic MHC molecule would be different from that formed by the same peptide with a recipient MHC molecule. Several human MiHAs may be expressed only on hematopoietic cells,75,127 and these may provide an opportunity to use graft-versus-host–alloreactive donor T cells (that recognize such antigens) to achieve GVL effects without GVHD (largely a disease affecting epithelial tissues). However, disparities for some of these minor antigens (eg, HA-1) have in fact been associated with an increased incidence of GVHD.77,111 Endothelial Glycoproteins Blood Group Antigens. Blood group antigens are the products of glycosylation enzymes that are not the same in all individuals. They are expressed on erythrocytes and other cells and, importantly, on vascular endothelium where they may serve as the targets for “natural” antibody-mediated attack on blood vessels of organ grafts. Blood group A and B individuals each express their respective antigen, but O individuals have neither. “Natural“ antibodies against blood group antigens an individual lacks probably arise due to

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cross-reactions with common carbohydrate determinants of environmental microorganisms. Type O individuals have antibodies to the antigens of A and B donors, whereas A and B individuals only have antibodies reactive with antigens from each other, and AB individuals have antibodies to neither. Therefore, O recipients can only receive transfusions from O donors, A and B recipients can receive transfusions from O donors or from individuals sharing their blood type, and AB recipients can receive blood from donors of any blood type. The same rules apply to the transplantation of most organs.128 Recently, advances have been made in the ability to successfully transplant kidneys across ABO barriers by adsorbing antibody from the plasma and depleting B cells.129–131 Other non-ABO blood group antigens on erythrocytes are irrelevant to organ transplantation because they are not expressed on vascular endothelium. Blood group antigens are of lesser importance, but nevertheless significant, in HCT. ABO incompatibility in the hostversus-graft direction (“major” ABO mismatch) can lead to prolonged red cell aplasia following HCT; incompatibility in the graft-versus-host direction (“minor” mismatch) can result in initial hemolytic anemia, but this complication can be avoided by washing the donor HCT preparation to rid it of plasma.132,133 Species-Specific Carbohydrate Determinants. Closely analogous to the blood group antigens are the carbohydrate determinants expressed on vascular endothelium that show species selectivity. For example, pigs, which are of interest as an organ source for xenotransplantation, have a glycosyltransferase enzyme not expressed by humans that glycosylates β-galactosyl N-acetyl glucosamine to form a Galα1-3Galβ14GlcNAc (αGal) determinant. This enzyme is present in most species but underwent a loss of function mutation in our nonhuman primate ancestors. In humans, a fucosyltransferase generates instead the H-substance from the same substrate, leading to blood group O. Preformed or “natural” antibodies are present in human serum that react to the nonself-pig determinant. Like the blood group antibodies, these natural antibodies probably arise from cross-reactions with environmental microorganisms,134,135 and they also cause hyperacute rejection (HAR) of most primarily vascularized xenogeneic transplants.

“Missing Self” and Natural Killer Cell Recognition In apparent violation of the laws of transplantation described previously, (AxB) F1 mice are capable of rejecting bone marrow from parental donors, a phenomenon termed hybrid resistance. This phenomenon, as well as rapid rejection of fully allogeneic marrow, is mediated by natural killer (NK) cells.136 NK cells are large granular lymphocytes that lack TCRs and that have the ability to mediate cytolysis against certain tumor targets and hematopoietic cells. NK cells also produce a number of proinflammatory, hematopoietic, and even anti-inflammatory cytokines, and may be divided into subsets on the basis of their cytokine production pattern.137 The originally puzzling specificity of NK cell–mediated marrow rejection is due to the expression by NK cells of inhibitory receptors that recognize specific groups of class I

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CHAPTER 46 AxB F1 mouse

Rejects “B” marrow

Inhibitory receptor for “A” class I

Rejects “A” marrow Rejects neither “A” nor “B” marrow

Inhibitory receptor for “B” class I

FIG. 46.7. An Explanation for Hybrid Resistance. Each filled circle represents a subset of natural killer cells.

MHC alleles on target cells and prevent cytolysis by the NK cell. These class I receptors are type II C lectins (Ly49 family) or dimers of CD94 with NKG2 lectins in the mouse, and are either immunoglobulin family members (KIR) or CD94/NKG2 in humans. Recognition by an inhibitory receptor of a class I ligand results in intracellular transmission of an inhibitory signal via an immune receptor tyrosinebased inhibitory motif that interacts with a tyrosine phosphatase and counteracts activating signals transmitted from other cell surface molecules. Recognition of “self” class I inhibitory ligands prevents NK cells from killing normal autologous cells.138,139 Other molecules expressed by infected or stressed cells activate NK cells, counterbalancing these inhibitory signals (see Chapter 17). Inhibitory receptors are clonally distributed on NK cells, each of which may express one or more different inhibitory receptor. For NK cells to be functional yet tolerant of “self,” they must express at least one inhibitory receptor for a “self” class I MHC molecule.140,141 Thus, as is illustrated in Figure 46.7, an AxB F1 recipient will have subsets of NK cells with inhibitory receptors that recognize MHC of either the A parent, the B parent, or both. The absence of “B” class I molecules on, for example, AA parental hematopoietic

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cells, permits subsets of (AxB)F1 NK cells that have inhibitory receptors only for class I molecules from the B parent to destroy AA cells. Thus, hybrid resistance and NK cell– mediated resistance to fully allogeneic marrow grafts can be explained on the basis of “missing self.”142 The roles of NK cells in transplantation are discussed in later sections.

MECHANISMS OF REJECTION At least four distinct mechanisms that can cause graft rejection have been defined. According to the timeframe in which they tend to occur in clinical practice, they are, namely, HAR, accelerated rejection, acute rejection, and chronic rejection.

Rejection Caused by Preformed Antibodies (Hyperacute Rejection) HAR occurs within minutes to hours after blood flow is established to a transplanted vascularized organ.143–145 The phenomenon is visible and dramatic: the organ turns blue and its function ceases. Microscopically, there is extensive evidence of vascular thrombosis and hemorrhage. The important components involved in the mechanism of HAR include 1) donor endothelial antigens, 2) preformed antibodies that can bind these antigens, 3) complement, and 4) coagulation cascades, which are activated by the binding of preformed antibodies to the vascular endothelium. The interaction of these components leading to hyperacute rejection is diagrammed in Figure 46.8. The role of complement in HAR is inferred both from the accumulation of various complement components in the grafts and from the fact that complement depletion leads to prolonged survival of xenografts.146 Complement activation leads to production of active protein fragments and complexes of complement components, which cause tissue injury either directly or by recruiting effector cells that mediate destruction of the graft. In allogeneic combinations, this is initiated by antibody-mediated activation of complement through the classical pathway, whereas in xenogeneic combinations, the alternative pathway may also be involved.147 In both cases, the membrane attack complex, produced by the ordered interaction of several complement components, initiates the destructive pathway.

Antibody Complement activation Antigen

Endothelium

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Complement regulatory proteins

Endothelial activation Type 1

DAF MCP CD59 sCr1

FIG. 46.8. Schematic Representation of Hyperacute Rejection.

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Complement activation is controlled by several regulatory molecules, including complement receptor 1, decay accelerating factor (DAF; CD55), membrane cofactor protein (CD46), and CD59, which act at different stages along the cascade (see Chapter 36). Many of these molecules are produced by the vascular endothelial cells. Because these regulatory proteins prevent unwanted complement activation in the face of low levels of perturbation to the system, the titer and avidity of the preformed antibodies must be high enough to activate despite these downregulating molecules. Thus, preformed antibodies directed at MHC antigens almost always accomplish this activation, whereas the lower-affinity blood group antibodies lead to hyperacute rejection in only about 25% of kidneys. One of the reasons that hyperacute rejection is such an important feature in xenogeneic transplantation is that the complement regulatory proteins produced by the donor vascular endothelium of one species often do not function effectively with complement molecules derived from a different species. Because of this incompatibility, lower levels of an initial triggering signal lead to explosive complement activation. Although the membrane attack complex is often thought of as a lytic molecule, its effect on the donor vascular endothelium, even before cell lysis, is to cause endothelial activation. This occurs rapidly, before there is time for new gene transcription or protein synthesis, and has been referred to as type I endothelial activation. The two principal manifestations of this activation are cell retraction, leading to gaps between endothelial cells, and initiation of coagulation pathways due to the loss of antithrombotic molecules from the endothelium.148 Thus, type I endothelial activation is responsible for the two principal pathologic findings in hyperacute rejection: extravascular hemorrhage and edema, and intravascular thrombosis. There are no known treatments that can stop the process of hyperacute rejection once it has started and, thus, it is essential to avoid the circumstances that initiate it. Experimentally, this can be accomplished for relatively short periods of time by administration of complement inhibitors, such as cobra venom factor, which depletes complement. In clinical practice, this is accomplished by avoiding transplantation in the face of preformed antibodies, both by avoiding blood-group antigen disparities and by testing recipients before transplantation (ie, a “cross-match”) to determine whether they have performed antidonor antibodies. Not all organs and tissues are equally susceptible to hyperacute rejection. Most primarily vascularized organs, such as kidneys and hearts, are very susceptible, but the liver can often survive without hyperacute rejection despite preexisting antidonor antibodies.149 It is not clear whether this unusual feature of the liver reflects the large surface area of its vascular endothelium or an intrinsic property of liver endothelial cells. Nonetheless, hyperacute rejection of the liver has occurred in some cases, especially involving xenogeneic transplantation, indicating that its resistance to hyperacute rejection is not absolute. Skin grafts are relatively resistant to hyperacute rejection but high levels of antibody can cause a “white graft” (ie, a failure of blood vessels to communicate with those of the recipient)150 Pancreatic islets are likewise resistant to this

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form of rejection.151 Free cellular transplants, such as bone marrow cells or hepatocytes, that express some of the antigens recognized by preformed antibodies, are cleared quickly from the circulation by the reticular endothelial system, leading to resistance to engraftment.152 In the case of HCT, this resistance can be overcome by transplanting larger numbers of cells.152–154 Additionally, antibody-independent complement activation has been shown to be a significant factor diminishing the engraftment of porcine bone marrow in mice.155

Acute Humoral Rejection (Accelerated Rejection) A second mechanism of rejection, also caused by antibodies, occurs as a result of antibodies that are induced very rapidly after a transplant is performed. This type of rejection has been called “acute humoral” or “accelerated” rejection because it typically occurs within the first 5 days after transplant. The process is characterized by fibrinoid necrosis of donor arterioles with intravascular thrombosis.156 Accelerated rejection is rare in allogeneic combinations because it requires that an antibody response occur before the T-cell response that is typically responsible for acute rejection episodes (see following discussion). Indeed, most allogeneic B-cell responses are T-cell–dependent. The best examples of accelerated rejection are probably those observed in vascularized organ transplants between closely related, concordant xenogeneic species and between discordant species following adsorption of anti-Gal antibodies. In these cases, the levels of preformed antibodies are not sufficient to cause hyperacute rejection, but antidonor antibodies appear rapidly (within 3 to 4 days) in association with the onset of rejection. The pathology of acute humoral rejection reveals a paucity of lymphocytes infi ltrating the donor graft, antibody binding to donor vascular endothelium, and fibrinoid necrosis of the donor vessels. Vigorous anti–T-cell immunosuppression has little effect on acute humoral rejection, whereas immunosuppression with reagents that affect B-cell responses, such as cyclophosphamide, delays its onset until more typical T-cell–mediated rejection occurs.157 As in hyperacute rejection, the process of acute humoral rejection is usually initiated by antibody binding to antigens on the donor vascular endothelium. In this case, however, the subsequent endothelial changes occur more slowly, allowing time for gene transcription and new protein synthesis. This later form of activation has been called type II endothelial activation.147,158 Many of its features appear to be mediated by the transcription factor NF-κ B, which generates many of the responses associated with inflammation, including the secretion of inflammatory cytokines such as interleukin (IL)-1 and IL-8, and the expression of adhesion molecules such as E-selectin and intercellular adhesion molecule (ICAM)-1.159 In addition, type II endothelial activation causes the loss of thrombomodulin and other prothrombotic changes.160 Thus, the events following type II endothelial activation are associated with the pathologic changes that occur with “accelerated” rejection, including the tendency toward intravascular thrombosis and the inflammatory destruction of donor vessels that occurs in the absence of infi ltrating lymphocytes.

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CHAPTER 46

Just as there are regulatory processes for complement activation, there are regulatory molecules that counter the tendency toward intravascular coagulation and the process of type II endothelial activation (eg, tissue factor protein inhibitor [expressed by vascular endothelium, which inhibits factor Xa of the clotting cascade] and a number of other protective molecules, including Bcl-x L, Bcl-2, and A20).147,161 Although these are often thought of as antiapoptotic molecules, they also tend to inhibit activation mediated by NF-κ B. Like the regulatory molecules of complement, some of these regulators may not function across species differences, leading to disorder regulation of the coagulation system.162 Although vigorous early antibody responses generate type II endothelial activation and accelerated rejection, later antibody responses often fail to do so. The process that enables transplanted organs to survive in the face of circulating antibodies that can bind endothelial antigens has been called “accommodation.”147,163 This phenomenon has been observed in some allogeneic and xenogeneic combinations with preformed antibodies, but has so far been disappointingly ineffective in discordant xenotransplants. Resistance to type II endothelial activation has been achieved in vitro by pretreatment with low levels of antiendothelial antibodies that are insufficient to trigger activation.164 The achievement of accommodation is associated with increased expression of the antiapoptotic genes described previously and with changes in the isotype of the recipient’s antibody responses.158,165 An important difference between HAR and acute humoral rejection is that there is no known therapy to stop graft destruction by HAR, whereas acute humoral rejection can sometimes be reversed by desensitization.

Rejection Caused by T Cells (Acute Rejection and Graft-versus-Host Disease) “Acute cellular rejection,” which is characterized by a mononuclear cell infi ltrate in the graft, is the most common type of organ allograft rejection. Acute rejection is most common during the first 3 months after transplant, but may occur at any time, especially if immunosuppressive medication is withdrawn. Acute rejection is T cell–dependent, and its treatment, which is usually successful, includes increased doses of standard immunosuppressive drugs or antilymphocyte antibodies. The use of newer immunosuppressive drugs and anti– T-cell antibodies has markedly reduced acute rejection rates. For example, the vast majority of kidney transplant recipients never experience an episode of acute rejection. It is now quite rare to lose a transplanted organ to cellmediated rejection during the fi rst year after transplantation. However, the use of these highly effective immunosuppressive treatments is associated with significant morbidity. Experimental models for acute rejection include nonprimarily vascularized skin grafts, heart graft fragments, artificial “sponge” allografts, or islet transplants in rodents, which may not accurately reflect the processes of rejection

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for primarily vascularized organs. While there are models of heart, kidney, liver, and other types of primarily vascularized organ transplants in rodents, these types of transplants are more tolerogenic and hence more easily accepted than similar transplants in large animals and humans. Studies of primarily vascularized organ transplants in large animals, such as monkeys or pigs, have obvious clinical relevance, but are expensive and require many special resources. Acute GVHD is the counterpart of cellular rejection that involves graft-versus-host alloreactivity, usually in the context of HCT, but also sometimes with organ transplants that carry significant amounts of donor lymphoid tissue (eg, liver). Like acute rejection, acute GVHD is T cell– dependent. T-cell depletion of the donor hematopoietic cell graft prevents GVHD but is associated with increased rates of graft rejection and relapse of malignant diseases. The concepts of “direct” and “indirect” allorecognition introduced previously must be considered at both the sensitization and effector phases of an immune response. The definition of “indirect” recognition used in this chapter is based on which set of APCs (donor versus recipient) is presenting donor antigen. “Cross-priming” is a term specifically denoting sensitization of CD8 T cells through the indirect pathway. As shown in Figure 46.9, there are three major T-cell pathways to consider in relation to graft rejection. These include 1) direct recognition of donor alloantigens by CD4 + T cells, which generate effector CD4 cells and provide help for the generation of effector CD8 cells; 2) direct activation of CD8 T cells by donor APCs; and 3) CD4 + T-cell activation by recipient APCs presenting re-processed donor antigens (the indirect pathway of sensitization). This pathway is important in providing help for immunoglobulin production by B cells. A role in rejection for cross-primed CD8 + T cells is not included in Figure 46.9, because this pathway has not been shown to play a role in allograft rejection except under certain circumstances, as described in the following. The multiplicity of T-cell sensitization and effector pathways involved in graft rejection and GVHD is demonstrated by the frequent observation of rejection or GVHD when only CD4 + or CD8 + T cells are depleted.166–168 As a result of the high precursor frequency of T cells that respond to allogeneic MHC antigens directly, populations of T cells that ordinarily have minimal significance become functionally important.

Sensitization and Cell Trafficking during Rejection and Graft versus Host Disease Transplanted tissue contains passenger leukocytes of donor origin that have the characteristics of immature DCs.169 In response to the inflammatory signals that are triggered by retrieval and transplantation, both within the tissue itself as well as in the recipient,170 the donor-derived passenger leukocytes rapidly leave the graft and migrate to the secondary lymphoid tissues of the recipient.171 Secondary lymphoid tissues comprise the spleen, lymph nodes, and gut- or mucosal-associated lymphoid tissue, and depending on the location of the graft, the passenger leukocytes will migrate to the tissue that drains the graft site where they encounter naïve T cells. After transplantation, both in situ within the tissue and during migration, the passenger leukocytes

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A

B7, IL-12, etc.

Donor APC

APC Class II MHC

activation by Th (CD40L, CD4+ Th

Help

CD8+ pCTL

Donor APC Class I MHC CD4+

Class I MHC

IFN-g, etc.)

Graft Target Cell Class I MHC

Th

CD8+ CTL

Recipient Class II MHC plus donor peptide

CD4-independent CD8+ cell

X Recipient APC

FIG. 46.9. Model of T-Cell–Mediated Rejection. A: Interactions between cluster of differentiation (CD)4+ Th, donor antigen-presenting complex, and CD8+ cytotoxic T lymphocyte. B: Additional pathways of T-cell sensitization that can lead to rejection. MHC, major histocompatibility complex.

acquire the phenotypic and functional characteristics of mature DCs, expressing high levels of MHC class I and II molecules as well as other cell surface costimulatory molecules necessary to fully activate naïve CD4 + and CD8 + T cells.172 Once in the secondary lymphoid tissues they act as professional APCs, presenting antigens expressed in the transplanted tissue to recipient T cells via the direct pathway of allorecognition.173 Naïve T cells recirculate around the body and are constantly moving through the secondary lymphoid tissues sampling the APC, both host- and (after transplantation) donor-derived, for antigen.174 If a naïve T cell with a TCR that can recognize a donor MHC molecule encounters the donor-derived passenger leukocyte in the draining lymphoid tissue as it recirculates, it will stop, interact, and differentiate into an antigen-experienced effector T cell. In support of the secondary lymphoid tissue being the primary site for sensitization of naive T cells and initiation of rejection after solid organ transplantation, Lakkis and colleagues175 showed that cardiac allografts were not rejected in splenectomised aly/aly mice that lack secondary lymphoid tissue as a result of a mutation in the gene encoding NF-kB–inducing kinase176 and suggested that in this situation permanent graft acceptance was due to immunologic ignorance. Other studies supporting the concept that secondary lymphoid tissues draining the graft are the key site for initiation of the immune response have followed the fate of T cells of a known specificity for donor antigen as they respond.177 Similarly, after bone marrow transplantation (BMT) the initiation of GVHD also takes place in the secondary lymphoid tissue, with evidence for the initial proliferation of donor CD4 + T cells followed by CD8 + T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin.178–180 Visualization of T cells responding during the initiation of GVHD showed that while Peyer patches are involved, other secondary lymphoid tissues contribute to the activation of T cells that can home to the gut; mesenteric lymph nodes and spleen are also sites where gut homing T cells were activated.179

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In solid organ transplantation, exclusive initiation of rejection in the secondary lymphoid tissues conflicts with the earlier hypothesis that rejection was initiated within the graft itself by donor endothelial cells lining the vessels that could activate T cells directly as they passed through the graft.181,182 Since these early papers, there have been a number of studies that support this hypothesis. For example, human endothelial cells have been shown to activate naïve T cells in vitro.183 In the mouse, APCs that are not of hematopoietic origin have been shown to activate CD8 + T cells in vitro and in vivo,184 thus supporting the concept that T cells may be activated in the graft rather than in the secondary lymphoid tissue. Moreover, splenectomized lymphotoxin α and lymphotoxin β knockout mice that also lack secondary lymphoid tissues were found to reject cardiac allografts, albeit at a slower than normal tempo.185 Each of these models is subtly different immunologically, and therefore different components of the immune response to an allograft may be differentially affected by the presence or absence of secondary lymphoid tissues. Clearly, in the absence of secondary lymphoid tissue, the initiation of the rejection response by naïve T cells is less aggressive. While antigen presentation via the direct pathway plays a dominant role in initiating the response to a transplant, a finite number of donor-derived passenger leukocytes is transferred within a transplanted organ. Thus the role of the direct pathway initiated by passenger leukocytes may diminish with time as eventually only “nonprofessional” APCs, including endothelial cells, remain to stimulate direct pathway T cells. Thus the role of endothelial cells within the graft may assume a greater significance with time after transplantation both for the initiation of the response and as a target for direct pathway effector cells. While activation of naïve T cells may occur predominantly in the secondary lymphoid tissues after transplantation, activation of memory T cells in presensitized recipients is quite different. Unlike naïve T cells, memory T cells can migrate to nonlymphoid tissues in the periphery186 and can trigger rejection through pathways that are independent of secondary lymphoid tissues.187

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CHAPTER 46

Thus, in humans, where there are likely to be both naïve and memory T cells that can recognize or cross-react with donor MHC molecules, rejection may be initiated both within the secondary lymphoid tissue as well as within the allograft by naïve and memory T cells, respectively. At the same time that donor-derived passenger leukocytes are leaving the graft, recipient leukocytes including APCs are attracted to the graft by the inflammatory mediators and chemokines released in the vicinity of the transplanted tissue. As these cells traffic through the graft, they phagocytose debris arising from tissue damage at the time of transplantation before migrating to the draining lymphoid tissue. The ingested antigens are processed and presented on recipient MHC molecules to T cells in the recipient lymphoid tissue.188 In addition, soluble antigens released from the graft will also be transported in the blood to the draining lymphoid tissue, where they will be taken up and presented by resident APCs. Common antigenic peptides presented by the indirect pathway are the hypervariable peptide binding regions of MHC molecules.189 Indirect pathway responses undoubtedly contribute to acute rejection, although the tempo of rejection may be slower due to the lower frequency of T cells that can respond. However, unlike direct pathway allorecognition, the indirect pathway is available for antigen presentation for as long as the graft remains in situ, and therefore becomes the dominant mode of allorecognition long term. A third pathway of allorecognition has been described more recently, the so-called semidirect pathway that involves the capture of donor MHC-peptide complexes by host APCs. The exchange of fragments of cell membrane between cells that interact with one another is a well described phenomenon in cell biology. In the context of the immune response to an allograft, the transfer of membrane fragments from allogeneic cells expressing donor MHC molecules can result in the presentation of intact donor MHC molecules by recipient APCs to T cells. The significance of the semidirect pathway is still under investigation.190 Traffic of naïve lymphocytes is usually restricted to recirculation between the blood and lymphatic systems. However, once they have been primed in the secondary lymphoid tissues, activated lymphocytes as well as other activated leukocytes must be able to migrate into the graft in order to destroy the transplanted tissue, a process known as leukocyte recruitment. The inflammatory processes at the site of transplantation generate chemotactic cytokines called chemokines, and upregulation of chemokine receptor expression by activated leukocytes enables them to migrate along the chemoattractant gradient to reach the graft.191 Inflammatory signals also affect blood vessels in the vicinity of the transplant, causing vasodilation and endothelial activation. Activated endothelial cells rapidly externalize preformed granules called Weibel-Palade bodies that contain the adhesion molecule P-selectin192 and rapidly upregulate expression of vascular cell adhesion molecule and CD62E (E-selectin). At the same time, chemokines released from the graft become tethered to the endothelium, and these alterations in endothelial surface markers advertise to passing leukocytes that an inflammatory process is occurring in the neighboring tissue.

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Leukocytes are usually conveyed within the fast laminar flow at the center of blood vessels, but once activated leukocytes reach postcapillary venules in proximity to the graft, they are able to leave this rapid flow and move toward the edge of the vessel. This occurs in response to the local chemokine gradient and is assisted by the slower blood flow in the vasodilated blood vessels near the graft. Leukocyte extravasation is a multistep process. Initially, low-affinity interactions develop between endothelial P-selectin and sialyl-LewisX moieties that are present on the surface of activated leukocytes. These interactions continually form and break down, and the leukocyte “rolls” along the endothelial surface. If chemokines are present on the endothelial surface, conformational changes in leukocyte integrin molecules occur that allow them to bind other endothelial adhesion molecules such as ICAM-1. These higher-affinity interactions cause arrest of the leukocyte on the endothelial surface, allowing it to commence extravasation. Having entered the tissues, the activated leukocytes continue to migrate along chemokine gradients in order to invade the graft.

Antigen Recognition and T-Cell Help in Graft Rejection and Graft-versus-Host Disease Role of Direct Cluster of Differentiation 4 Allorecognition. Priming of naïve, directly alloreactive T cells requires professional APCs that leave the graft and enter the recipient’s lymphoid tissues. Direct CD4 T-cell sensitization by donor class II MHC antigens may both generate CD4 + effector cells and provide help for the activation, differentiation, and proliferation of cytotoxic CD8 + cells that directly recognize donor class I MHC antigens and destroy the graft (see Fig. 46.9). Depletion of donor APCs can markedly prolong graft survival,193–198 illustrating the importance of direct allorecognition in inducing rejection. The CD4 help for CD8 cells consists of both cytokine (eg, IL-2) production and “conditioning” of the APC, for example by interactions of CD40 on the APC with CD40L on the activated CD4 cell. These interactions upregulate APC expression of CD80 and CD86 costimulatory molecules and cytokines such as IL-12 and MHC, making the cell a more effective APC. Studies of antiviral immunity indicate that CD4 help is needed for development of full effector function,199 and for CD8 memory cell survival200 and function.201 Studies involving very limited (not clinically relevant) antigenic disparities between donors and recipients suggested that a “three-cell cluster” model involving interactions between helper T cells, effector T cells, and APCs was essential for rejection.202–205 However, studies involving more extensive, clinically relevant histoincompatibilities206,207 suggest that CD4 helper cells sensitized by antigen presented on recipient APCs can provide help for directly alloreactive CD8 + effector cells. It remains possible that a “three-cell cluster” is still essential for CD4 cells to provide help to CD8 cells mediating rejection, and that donor class I MHC/peptide complexes are transferred and picked up by recipient APCs. Recipient APCs with directly alloreactive CD8 T cells would thereby encounter their ligands on the same recipient APC that an indirectly alloreactive CD4 cell recognizes. Such transfer of class I/peptide antigens, resulting in this type of

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“semidirect” antigen presentation, requires consideration in transplant models.208–211 CD4 + T cells alone can cause graft rejection (without CD8 cells) in the setting of class II or multiple minor histoincompatibilities,198,212–215 indicating that CD4 T cells can mediate rejection effector functions. In BMT recipients, they can induce GVHD in the absence of CD8 cells in the setting of class II, full MHC, or multiple minor histoincompatibilities,216–219 and can reject class II and minor antigen– mismatched bone marrow.220,221 Role of Indirect Cluster of Differentiation 4 Cell–Mediated Allorecognition. Indirectly alloreactive CD4 T cells have roles in skin and solid organ graft rejection,210,222–232 including the provision of help for class-switched alloantibody responses.233 This help requires cognate interactions between recipient class II–restricted indirectly alloreactive CD4 cells and host B cells that recognize donor MHC molecules through their immunoglobulin receptors, process them, and present donor MHC peptides with their class II molecules. CD4 cells also contribute to rejection of bone marrow grafts differing only at class I MHC loci, possibly implicating indirect allorecognition.220,221 Rejection by CD4 cells of skin grafts lacking class II MHC shows the strength of the indirect pathway of rejection.206,222 Rejection of islet xenografts in mice may depend on indirectly xenoreactive CD4 + T cells.234 Sensitization of indirect CD4 responses to donor MHC–derived peptides has been demonstrated in patients undergoing graft rejection, and these may be correlated with poor outcomes.235–242 A major role for indirect allorecognition has been suggested in the setting of chronic rejection,230,243–246 in part because of its role in inducing antibody responses, which are implicated in chronic rejection.247–251 Moreover, the eventual replacement of donor APCs by recipient APCs implicates the latter in long-term graft recipients.196,241,252,253 Consistently, direct alloresponses tend to subside over time in transplant patients.254–256 Nevertheless, donor APC depletion or the lack of donor class II MHC can prevent rejection in some situations.193–198 An essential role for indirect allorecognition has not been demonstrated for acute rejection.21,253,257,258 Indirectly alloreactive CD4 + cells alone fail to reject skin grafts with minimal class I or minor histoincompatibilities,202,259,260 or to induce GVHD against class I MHC or minor histocompatibility barriers alone.216,217 With rodent primarily vascularized allografts, donor APC depletion may, by preventing the strong direct alloresponse, allow the inherent tolerogenicity of the organ to prevail. Role of Helper-Independent Cluster of Differentiation 8 + T Cells. CD8 T cells can readily reject skin and bone marrow allografts in the absence of CD4 cells,166,202,220,221,261,262 and alloreactive CD8 T-cell memory can be generated and maintained without CD4 cells.263 CD8 cells can also induce GVHD without CD4 T cells in the setting of full MHC, class I only, and minor antigen histoincompatibilities.264,265 Direct recognition of recipient MiHAs on recipient APCs is essential for the induction of CD8-dependent,

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CD4-independent GVHD in MHC-identical, lethally irradiated mice,266 but indirect267 or “semidirect”211 CD8 recognition of recipient antigens presented by donor APCs amplifies the process. Together, these studies show that CD4 help is not critical for CD8 cell–mediated rejection or GVHD. However, the requirement for CD4 help may increase in the absence of inflammatory stimuli, as indicated by marked differences in the need for CD4 help for CD8-cell activation and GVL effects in the presence and absence of inflammation.268,269 Grafts expressing only class I antigen disparities are usually rejected quite slowly, and CD4-independent rejection is relatively easily suppressed by cyclosporine.270–272 Many primarily vascularized grafts that express only a class I antigen disparity require CD4 + cells to initiate rejection, and, when it occurs, CD4-independent rejection by CD8 + cells is dependent on the number of donor APCs in a graft.166,222 CD4-indepenent CD8 + cells do not reject grafts expressing only a small number of minor antigen disparities and generate only weak helper responses even in the presence of multiple MiHA disparities. CD8 + helper cells also differ from CD4 + helper cells in being unable to provide help for other cell populations.273 CD8 + cells alone cannot reject skin grafts with only limited class II antigen disparities.202,218,259,274 Cross-Primed Cluster of Differentiation 8 Cells. Peptides of exogenous antigens were originally thought to be presented by MHC class II antigens, whereas those of endogenous cellular antigens are presented by MHC class I molecules.275,276 However, it is now clear that class I presentation of exogenous peptides (cross-presentation) is essential for many immune responses, including those against microbial and tumor antigens.277–281 Several pathways have now been delineated for cross-presentation by class I molecules.282–284 CD8 cell crosspriming was originally demonstrated in a transplantation model by Bevan70 when minor antigen–disparate grafts with MHC antigens of type A were placed on MHC (A × B) F1 recipients and CD8 + cells became sensitized to the minor antigens presented by both A and B types of class I MHC molecules. Activation of cross-primed CD8 cells is strongly dependent on CD4 help and IL-2.285 Cross-primed CD8 cells recognizing donor MiHAs and MHC-derived peptides are most likely to participate in rejection when there is sharing of class I alleles between the donor and recipient. Without such sharing, the self-class I/allogeneic peptide epitope cannot be presented by the parenchymal or endothelial cells of the graft.286 However, even without class I sharing, indirect CD8 + -cell sensitization can lead to skin allograft rejection, perhaps due to recognition of donor peptides presented by recipient endothelial cells on host-derived vessels that revascularize the graft.287,288 Cross-primed CD8 cells might also contribute to graft rejection via indirect effector mechanisms upon antigen recognition on host APCs in the graft or by producing inflammatory cytokines.289 Some of the rejection processes previously attributed to cross-primed CD8 cells may in fact be mediated by CD8 cells seeing intact donor MHC-peptide complexes on recipient APCs (“semidirect” presentation).

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Effector Mechanisms of Rejection and Graft-versus-Host Disease While cytotoxic T cells are important effectors of graft rejection and GVHD, additional mechanisms involve effector cells of the innate immune system and cytokines as final mediators of tissue destruction. The net result of this multiplicity of pathways is considerable redundancy of mechanisms of graft rejection and GVHD.

Another non-CTL graft rejection mechanism involves antibodies, which cause hyperacute rejection, acute humoral rejection, or chronic rejection through Fc receptor, complement-mediated, and other inflammatory pathways. B cells326,327 and antibodies327 contribute to cGVHD in animal models and are implicated in human cGVHD.328–330 B-cell depletion with rituximab has been reported to have efficacy against cGVHD.330–332

Cytotoxic Mechanisms of Graft Rejection and Graftversus-Host Disease. Rejecting organs contain proteins and messenger ribonucleic acid (RNA) encoding perforin, granzymes, and proteases associated with cell-mediated cytotoxicity.290–297 The presence in urine of RNA encoding perforin and granzyme B has been associated with renal allograft rejection in humans.298 Although the perforin/granzyme pathway is the major cytolytic pathway for CD8 T cells and CD4 cells tend to utilize the Fas/FasL pathway,299 both subsets are capable of both types of cytolytic activity,300,301 and the perforin pathway is available to both T-cell subsets mediating GVHD.302 All of these cytotoxic proteins play contributory roles, and no single protein has been found to be critical for solid organ graft rejection,303–306 GVHD,307–310 or bone marrow graft rejection311 in the presence of clinically relevant mismatches. Critical cytotoxic interactions have been identified in less relevant animal models involving Fas-dependent GVHD directed at isolated class II MHC disparities310 and perforin-dependent rejection of K b mutant class I–only mismatched heart allografts.303 Fas ligand promotes lymphoid hypoplasia312 and skin and liver GVHD,312 and both Fas ligand and TRAIL are required for GVHDrelated thymic destruction.313 While the perforin-granzyme pathway contributes to GVHD,310,312 the Fas pathway appears to be of greater overall importance. In contrast, the perforin/granzyme and TRAIL pathways predominate in antileukemic effects, especially of CD8 cells, and selective blockade of the Fas/FasL pathway may ameliorate CD8mediated GVHD without eliminating GVL effects.309,314–318

Cytokines as Mediators of Graft Rejection and Graftversus-Host Disease . Interactions between alloreactive CD4 helper cells producing cytokines of the “Th1” type and alloreactive cytotoxic CD8 + effector cells can mediate rejection and GVHD via direct cytotoxicity.333 The “indirect” mechanisms of graft rejection and GVHD are likely to include cytokines.298 “Th17” cells producing IL-17 and other proinflammatory cytokines promote rejection and GVHD.334–348 The generation of Th-17 cells is antagonized by Th-1 cells and promoted by IL-23, transforming growth factor (TGF)-β, and IL-6. A great redundancy of rejection pathways is suggested by studies detecting both Th1 (IL-2, IFNγ ) and Th2 (IL-4, IL-5, IL-10) cytokines in rejecting allografts.296,349–356 Th2 can also mediate GVHD and rejection. There appears to be strain-dependent tissue specificity to the type of GVHD induced by the various Th subsets.337,357,358 Thus, while the concept that Th2 cytokines are anti-inflammatory attracted interest in the transplantation field, 359–367 Th2 responses can clearly contribute to both graft rejection359,368–370 and GVHD.357,371–373 With a few special exceptions, 374–377 studies using various cytokine knockout mice as recipients have failed to reveal any single molecule that is essential for rejection378–384 or GVHD.371,385,386 In GVHD, cytokines such as tumor necrosis factor (TNF)- α and IFNγ play a role. Macrophages are activated by lipopolysaccharides from the damaged gut epithelium and by IFNγ to release TNF- α , nitric oxide, and other mediators of tissue injury. 387–391 In certain models, TNF-α had been shown to be critical for wasting disease and intestinal GVHD. 392,393 While the relative contribution of cytokine-dependent mechanisms versus direct cell–mediated cytotoxicity to GVHD is still a matter of debate, GVHD is induced by T cells incapable of both perforin-mediated and Fas-mediated cytotoxicity, even in recipients lacking TNF receptor 1–mediated signaling, 310,394,395 demonstrating the redundancy of GVHD effector mechanisms.

Non-Cytotoxic T-Lymphocyte Effector Mechanisms in Graft Rejection and Graft-versus-Host Disease. T cells can effect rejection of grafts whose parenchymal cells do not express the TCR ligand, indicating the existence of “indirect” effector mechanisms. Entire skin grafts can be rejected when only the APCs are foreign,319 indicating that nonselective destruction of grafted tissue can occur. Several studies have implicated indirect CD4 cell–mediated rejection of skin320,321 and cardiac322 allografts. Replacement of graft endothelium by the host was shown to be needed for rejection through this indirect effector mechanism.322 GVHD of the liver and intestine can be induced by donor T cells in MHCdeficient hosts receiving wild-type host DCs, suggesting that indirect effector mechanisms may also mediate tissue injury of GVHD.323,324 However, CD4-mediated GVHD against MiHAs is markedly attenuated when the target antigens are expressed only on hematopoietic cells.325 Thus, “indirect” effector mechanisms can destroy transplanted tissue or recipient tissue in the case of GVHD, but less efficiently than direct cytotoxic mechanisms.

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Graft-Infiltrating Cells. Many types of cells infi ltrate rejecting grafts, including CD4 + and CD8 + T cells, NK cells, and macrophages.396–404 While B cells may be less prominent,405 their presence has been associated with both acute and chronic rejection, and they are attracting increasing interest for their role not only as producers of antibody effectors of rejection, but also as APCs.406 B cells may be located in tertiary lymphoid organs found in chronically rejecting allografts.407 The number of invading T cells in a graft is not necessarily correlated with the speed of rejection.405 This finding has

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suggested that certain critical elements of the graft, such as its blood vessels, are the actual site of graft destruction and, indeed, endothelialitis is an important hallmark of clinically significant rejection activity.408 Repertoire analysis of graft-infiltrating T cells in acutely rejecting grafts reveals marked polyconality,22,409–412 but only the donor-reactive CTLs show evidence of having been activated in vivo.413 Oligoclonal dominance has been suggested in studies of tolerated rodent allografts412 and in long-term rejected human kidneys.414 T cells infi ltrating xenografts included a broad TCR repertoire.415–417 T cells mediating GVHD in the setting of multiple minor histoincompatibilities demonstrated a markedly skewed repertoire involving several different Vβ families.418,419 Clinical studies suggest that the anti-MiHA TCR repertoire is most often polyclonal.420 Role of Natural Killer Cells. Although the role of NK cells in marrow rejection is well established in mice, the amount of resistance mediated by NK cells to allogeneic hematopoietic stem cells is limited and can be readily overcome by increasing the dose of donor stem cells administered.421,422 Furthermore, a role for NK cells in resisting human allogeneic marrow engraftment has not been clearly demonstrated, although they might be expected to be important in recipients of reduced toxicity conditioning regimens. Indeed, patients with severe combined (T- and B-cell) immunodeficiency who have functional NK cells require cytotoxic conditioning to permit engraftment of haploidentical marrow, whereas those lacking NK cells do not.423 The ability of NK cells to be triggered by “missing self” may have utility in HCT.424 Donor-derived NK cells with graft-versus-host reactivity due to the lack of donor class I MHC inhibitory ligands in the recipient can kill residual host leukemia cells and alloreactive cells that resist the marrow graft without causing GVHD. The alloreactive donor NK cells may also reduce susceptibility to GVHD by killing recipient APCs needed to activate donor T cells.424,425 While striking antileukemic effects of KIR mismatching were detected in heavily conditioned patients receiving high doses of haploidentical CD34 + stem cells,424 the effect of KIR incompatibility has been more variable in other clinical studies,426–431 and the antitumor benefit is most evident for acute myelogenous leukemia.432 The possible role of NK cells in rejecting solid organ grafts is somewhat controversial. NK cells are prominent among cells infi ltrating rejecting organ allografts and may be the earliest producers of inflammatory cytokines and chemokines and inducers of DC maturation.433–436 If NK cells make an important contribution to solid organ allograft rejection under normal circumstances, they must be dependent on T cells, as mice lacking T cells are unable to reject nonhematopoietic allografts. Furthermore, whereas bone marrow allografts from class I deficient donors (β2m−/−) are subject to potent NK-mediated rejection (because these cells cannot trigger inhibitory receptors on host NK cells437), β2m−/− skin grafts are not rejected by β2m + recipients.438 NK cells have recently been reported to play a critical role in cardiac allograft rejection in CD28 knockout mice,439,440 and

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NK cells can mediate a particular form of chronic allograft vasculopathy in a murine cardiac allograft model.435 This lesion may be triggered by viral infection.441 Inhibitory receptors on NK cells are quite broad in their class I specificity,442 and fully allogeneic class I MHC marrow is less susceptible to NK-mediated marrow destruction compared to class I–deficient marrow.437,443 Because of the increased disparity of xenogeneic compared to allogeneic MHC molecules, NK cells may receive fewer inhibitory signals from xenogeneic than allogeneic cells. Indeed, transduction of HLA molecules into porcine endothelial cells reduces NK cell–mediated xenogeneic cell adhesion and cytotoxicity.444–446 However, some inhibitory receptors, such as killer cell lectin-like receptor G1, do recognize xenogeneic ligands such as e-cadherin.447 NK cells may also be activated by interactions of activating receptors with ligands on xenogeneic cells,448,449 of which several examples have been identified.450,451 On balance, activating xenogeneic NK cell–target interactions are more effective than inhibitory interactions. Indeed, NK cells resist xenogeneic marrow engraftment to a greater extent than allogeneic marrow.421,452–454 NK cells have also been implicated in the acute vascular rejection455 that can destroy solid organ xenografts that have escaped hyperacute rejection (see following discussion) and in xenogeneic skin graft rejection.456 As one mechanism by which NK cells mediate cytolysis is antibody-dependent cell-mediated cytotoxicity, it is possible that immunoglobulin G natural antibodies play a significant role in initiating NK cell–mediated rejection. NK cells also release cytokines, such as IFNγ., and TNF-α, which activate macrophages and endothelial cells, and induce inflammation.455 Role of Natural Killer T Cells. While NKT cells have apparent inhibitory effects on graft rejection457–460 and GVHD,461,462 they have also been reported to participate in rejection of tissues and bone marrow in mice.463–465 The latter is due to the ability of NKT cells to activate NK cells.465 NKT cells promote skin graft rejection by cross-primed CD8 cells via their ability to produce IFNγ.466 Role of Monocytes/Macrophages and Eosinophils as Effectors of Rejection. Classical delayed-type hypersensitivity (DTH) responses are thought to depend on the activation of macrophages by helper T cells through production of IFNγ. It is likely that proinflammatory cytokines and chemokines produced by activated monocytes and macrophages play a role in endothelial cell activation and lymphocyte recruitment. Additionally, activated macrophages may damage tissue through the production of toxic molecules such as nitric oxide.467 Macrophages play an especially important role in the rejection of cellular xenografts such as islets468,469 in a T cell– dependent manner.470 Macrophages cause almost immediate rejection of xenogeneic bone marrow, even in the absence of adaptive immunity.471–473 Human macrophages can phagocytose porcine cells in an antibody- and complementindependent manner.474 Additional studies have implicated macrophages in solid organ and skin xenograft rejection.475–480 This prominent role for xenogeneic macrophages may reflect the combined ability of certain xenogeneic

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receptors to activate macrophages,481 whereas important inhibitory interactions, such as that between CD47 and its macrophage ligand SIRPα , are not effective.482 Surprisingly, a system for monocyte-mediated recognition of allogeneic non-MHC nonself has been described.483 Eosinophils recruited to allografts by Th2 T-cell responses have been reported to be effectors of graft rejection in some experimental models, and eosinophils are often found clinically in rejecting allografts.333 Th2-derived IL-4 and IL-5 recruit and activate eosinophils, which release highly cytotoxic substances from granules into the tissue.

Chronic Rejection and Chronic Graft-versus-Host Disease Most experimental studies of rejection are performed without immunosuppression and, therefore, graft destruction usually occurs within the first several days or weeks by one of the mechanisms described previously. In clinical practice, however, the use of immunosuppression usually allows graft survival for much longer periods of time. Nonetheless, clinical survival statistics reveal that even when 1-year graft survival has been achieved, the loss of transplanted organs continues to occur at a rate of about 3% to 5% per year, and a significant proportion of this delayed or late graft failure appears to be due to immunologic mechanisms. The term “chronic rejection” is commonly used to describe this later process of delayed graft destruction, although in kidney transplantation the Banff classification schema has proposed to replace this term with interstitial fibrosis and tubular atrophy.484 As immunosuppressive reagents have become more effective at controlling acute rejection, chronic rejection has emerged as one of the most important problems in clinical practice. Indeed, while there has been ongoing improvement over the past 30 years in the 1-year graft survival rates for kidney transplants, the halflife for organs that have survived for 1 year has not changed significantly over that entire period of time; as a result of this ongoing loss, only about 50% of transplants are still functioning 10 years later. Although almost every type of organ transplant suffers from deterioration in function over time, the pathologic manifestations are different in each case. Kidney biopsies tend to show interstitial fibrosis along with arterial narrowing from hyalinization of the vessels—hence the terminology interstitial fibrosis and tubular atrophy. In the heart, the process is manifested principally as a diffuse myointimal hyperplasia, proceeding to fibrosis of the coronary arteries that has often been referred to as “accelerated atherosclerosis” or “transplant arteriosclerosis.” Chronic rejection in lung transplants primarily affects the bronchioles with progressive narrowing of these structures and is referred to as “bronchiolitis obliterans.” The liver may be the one type of organ transplant that is relatively resistant to chronic rejection, but the progressive destruction of bile ducts referred to as the “vanishing bile duct syndrome” may be another manifestation of this process. Some of the causes of chronic graft destruction may not be immunologic in origin.485,486 Analysis of sequential

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kidney transplant biopsies suggests that chronic rejection represents cumulative and incremental damage to the graft from time-dependent nonimmunologic and immunologic causes.487,488 Potential nonimmunologic factors that have been considered to contribute to the development of chronic rejection include the initial ischemic insult, the reduced mass of transplanted tissue (especially in the case of kidney transplants leading to hyperfi ltration injury), the denervation of the transplanted organ, the hyperlipidemia and hypertension associated with immunosuppressive drugs, the immunosuppressive drugs themselves, and chronic viral injury, amongst others. Nonetheless, while these factors undoubtedly contribute to the process, there is a marked difference in survival between syngeneic and allogeneic transplants in experimental models. Thus, there is almost certainly an important immunologic component in most cases of chronic rejection. Several important observations regarding the pathogenesis of chronic rejection have emerged from clinical practice, particularly the analysis of biopsy samples. In kidney transplants, two distinctive phases of injury of chronic allograft nephropathy have been described.487 Previous studies have suggested that there is a high correlation between the onset of chronic rejection and a history of early acute and subclincial rejection episodes.489,490 Analysis of protocol biopsies has revealed that the onset of mild chronic rejection by 1 year after kidney transplantation is associated with an initial phase of early tubulointerstitial damage from ischemic injury that occurs before severe rejection is detected. Beyond 1 year, a later phase of chronic allograft nephropathy was characterized by microvascular and glomerular injury.487 Importantly for long-term outcomes, the clinical data show that the process of chronic rejection is usually refractory to increases in immunosuppressive therapy, in contrast to acute rejection episodes that almost always respond to treatment. The development of chronic rejection has also frequently been associated with the presence of antidonor antibodies,491,492 and the deposition of complement component C4d in the allograft. Taken together, these clinical observations have suggested to some that chronic rejection is the result of donorspecific alloantibody production.493 Moreover, there are also now data emerging to suggest that components of the innate immune system such as NK cells can also contribute,494 in some cases in conjunction with alloantibody.495 All of these suggestions may be correct, but neither the logic nor the evidence fully supports the exclusive involvement of one mechanism. The relationship between alloantibody, C4d deposition, and neointimal fibrosis is complex.496 In the first place, alloantibody production often reflects activation of indirect pathway T cells (see previous discussion), and hence it might equally well be a marker for other rejection mechanisms as opposed to a cause of chronic rejection. Moreover, the presence of alloantibody and C4d may be transient, and there are clear examples of chronic changes in the graft in their absence.496 In addition, early rejection episodes probably reflect primarily the degree of antidonor immunoreactivity, and there is no proven direct link with later deterioration of graft function. Therefore, even if sufficient

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immunosuppression were given to prevent acute rejection, chronic rejection might still occur when the levels of immunosuppression are reduced over the long term, even if acute rejection had never occurred. Finally, experimental studies have suggested that the mechanisms of chronic rejection are not absolutely dependent on either antibody formation or on the occurrence of acute rejection episodes. The uncertainties that arise from the interpretation of the clinical data make it important to develop experimental models for studying the mechanisms of chronic rejection. It is difficult in the laboratory, however, to mimic a process that may take 5 or 10 years to develop in patients treated with immunosuppressive drugs. Thus, the effort to study chronic rejection experimentally has depended on surrogate short-term pathologic markers that are thought to predict the long-term changes of chronic rejection. In particular, these studies have concentrated on the development of the myointimal proliferation that is thought to be the precursor of the chronic vascular changes typically observed in patients. In rodents, pigs, and primates, this has often been done with grafts after an initial period of immunosuppression that prevents acute rejection.497–499 All of these experimental studies are subject to the caveat that the surrogate pathologic lesion occurs much earlier than the typical changes of chronic rejection in patients. Thus, the process being studied experimentally may not be the same as the clinical process.

Pathologic Manifestations of Experimental Chronic Rejection The typical pathologic features of the experimental lesion associated with chronic rejection are shown in Figure 46.10.500 The marked narrowing of the vascular lumen is caused by the substantial proliferation of endothelial and then smooth muscle cells that can be host-derived.501 Associated with this proliferation is progressive destruction of the media. In time, the cellular proliferation becomes less pronounced and is replaced by concentric fibrosis that narrows the vascular lumen. Immunohistologic staining indicates that there is increased expression of several adhesion molecules,500

FIG. 46.10. Histology of Chronic Rejection.

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intracellular proteins such as vimentin,502,503 and easily detectable levels of several molecules, including nitric oxide synthase,504 acidic fibroblast growth factor, insulin-like growth factor, IFNγ,505 and endothelin,506 each of which may play a role. The abnormal expression of self-molecules in allografts undergoing chronic damage can lead to autoantibody formation.507 Ultimately, the ischemia resulting from vascular occlusion results in fibrosis in the parenchyma of the organ and consequent organ dysfunction.508 In the case of the lung or the liver, chronic injury may cause changes most prominently in the bronchioles or the bile ducts, but this is also associated with arterial lumen loss, which may be the primary lesion causing bronchiolitis obliterans or bile duct fibrosis, respectively.508

Immunologic Mechanisms of Chronic Rejection Rejection requires a dialogue between the innate and adaptive immune systems.509 Innate immunity is most likely involved at the outset of the process that leads to the development of chronic rejection, and there is evidence to suggest that NK cells494,510 and complement activation can be involved.511 At the level of the adaptive response, because it is assumed that stimulation of direct pathway T cells is likely to diminish over time as donor APCs are replaced by recipient APCs (see previous discussion), it is commonly assumed that the predominant immune response that causes chronic rejection occurs through the indirect pathway.512 Studies in pigs have suggested that the vascular changes of chronic rejection are more apt to develop when there are class I antigenic disparities than when there are only class II disparities and that the development of the lesion depends especially on CD8 + T cells.513 In mouse models, in contrast, there is evidence that either CD4 + or CD8 + T cells can produce the lesion and that either class I or class II antigenic disparities are sufficient to stimulate chronic rejection.514 The finding that class II antigenic disparities are themselves sufficient to induce this pathology is consistent with the observation of class II MHC expression on the vascular endothelium and medial smooth muscle cells of mouse cardiac allografts with these vascular lesions.515 Because class II MHC is not constitutively expressed by mouse vascular endothelial cells, indirect recognition of donor class II transferred from passenger leukocytes may be responsible for inducing an inflammatory response that leads to subsequent upregulation of class II on the donor vascular endothelium. In keeping with the prediction of many clinical studies, adoptive transfer experiments into mice with severe combined immunodeficiency have shown that alloantibodies in the absence of T cells can induce the typical pathologic vascular changes, and lesions can develop in T-cell– deficient mice.516 However, T cells without B cells have also been shown to cause the lesion, although there may be somewhat less tendency to progress to end-stage fibrosis.517 Several studies have indicated that the induction of donor-specific tolerance can prevent the development of the vascular changes of chronic rejection, although not all of the short-term manipulations that have been effective in preventing acute rejection have necessarily prevented the later onset of chronic rejection. Remarkably, mice rendered

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tolerant by neonatal injection of donor splenocytes, or by the induction of high levels of lasting, multilineage mixed chimerism with demonstrated central deletion of donorreactive T cells and permanent acceptance of donor-specific skin grafts, demonstrate graft vasculopathy in donor cardiac allografts.510 Thus in the complete absence of antidonor T-cell reactivity, other cell types such as NK cells may induce these types of lesions in cardiac allografts. In addition, T-cell recognition of cardiac-specific antigens presented by donor MHC and not shared by donor hematopoietic cells could play a possible role in the development of these lesions in immunocompetent, tolerant mice.494 From these data, it seems likely that multiple immunologic mechanisms may be capable of creating the graft arteriosclerotic lesions that are characteristic of chronic rejection, and that T-cell alloreactivity is not essential for their induction. Whether there is a critical final common mediator involved in all of these pathways is not currently known. However, IFNγ has been shown to play an important role in the development of lesions in several models,518,519 and signal transducer and activator of transcription (STAT)4-deficient mice, which do not respond to IL-12 and therefore cannot generate Th1 responses, show markedly reduced severity of graft vasculopathy compared to wild-type mice.520 TGF-β has been shown to attenuate the lesions, but has also been detected within the lesions and implicated in the development of fibrosis.521

Chronic Graft-versus-Host Disease cGVHD is the most common and severe complication among patients surviving for more than 100 days after allogeneic BMT. Clinically, acute and cGVHD can be distinguished on the basis of the time of onset, clinical manifestations, and distinct pathobiologic mechanisms. Acute GVHD usually occurs within 2 to 6 weeks following allogeneic BMT and primarily affects the skin, liver, and the gastrointestinal tract with T-cell infi ltration of the epithelia of the skin, mucous membranes, bile ducts, and gut. However, acute GVHD has been noted to occur later in recent protocols involving nonmyeloablative conditioning for HCT. In contrast, cGVHD involves a wider range of organs and clinical manifestations include scleroderma, liver failure, immune complex disease, glomerulonephritis, and autoantibody formation. The pathogenesis of cGVHD, like chronic rejection, is poorly understood. The disease involves T-cell responses to alloantigens or autoantigens.522 Because most BMT is performed between HLA-identical or closely HLA-matched pairs, alloreactivity may be directed against miHAs presented by shared MHC molecules, or against MHC alloantigens when present. T cells developing de novo in a recipient thymus that is damaged due to GVHD may result in the emergence of autoreactive T cells into the peripheral repertoire. The injury to target organs is poorly understood, but may involve inflammatory cytokines and fibrosis, as well as B-cell activation and production of autoantibodies. The main risk factors for the onset of cGVHD are HLA disparity, donor and patient age and sex, source of progenitor cells, graft composition and previous acute GVHD.

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cGVHD can be treated providing it is identified sufficiently early after initiation. Even with treatment, extensive skin involvement, thrombocytopenia, and progression are poor prognostic factors.

PHYSIOLOGIC INTERACTIONS THAT MODULATE GRAFT REJECTION AND GRAFT-VERSUS-HOST DISEASE Although graft rejection and GVHD often involve exceptionally strong immune responses, these responses are still accompanied by downregulatory components that can be manipulated to promote graft survival. While many of these manipulations are described in the section on tolerance, some of the regulatory components of the rejection response are briefly described here.

Downregulating Signals Following T-Cell Activation Interactions between Fas and Fas ligand (FasL), which is upregulated during rejection responses, can mitigate GVHD and graft rejection by killing activated T cells and APCs.306 FasLdeficient recipients are more susceptible than normal mice to the development of GVHD,523 and FasL can promote resistance to rejection of tissues transplanted to some “privileged sites,” such as the testis or the anterior chamber of the eye.524 However, forced overexpression of FasL causes a nonspecific inflammatory syndrome associated with prominent neutrophil infiltration525 and can promote graft destruction.526,527 Overexpression of FasL has, however, been reported to promote survival of heart allografts in recipients of FasL-expressing donor-specific transfusions (DSTs),528 and of bone marrow529 and islet530 allografts. CTL antigen (CTLA)4 helps maintain self-tolerance, as evidenced by the T-cell lymphoproliferative autoimmune syndrome that develops in CTLA4 knockout mice.531,532 CTLA4 has been shown to play a role in T-cell tolerance in many systems.384,533–546 Blockade of CTLA4 accelerates cardiac allograft rejection547 and increases GVHD.548 PD1, an additional downregulatory molecule expressed by activated T cells that recognizes the B7 family members PDL-1 and PDL-2, also mitigates graft rejection549–554 and GVHD.555–557 Interaction of one of the PD1 ligands, PD-L1, with its alternative receptor B7.1, also mitigates graft rejection.558 While PD1 plays an important modulatory role in the presence of extensive antigenic disparities between murine heart graft donors and recipients, the B- and T-lymphocyte attenuator (BTLA)herpesvirus entry mediator (HVEM) inhibitory pathway predominates in the presence of more restricted antigenic disparities.559 BTLA-HVEM interactions may also control GVHD,560 but the opposing effects of BTLA and HVEM expressed by T cells on T-cell activation complicate interpretation of experiments, which have produced conflicting results with respect to GVHD560,561 and islet allograft survival.562,563

Immunomodulatory Effects of Cytokines There is considerable evidence of roles for IL-10 and TGF-β in downmodulating graft rejection564–566 and GVHD,372,567–570 and these cytokines may have therapeutic utility.571–575 These

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activities may reflect the important role of these cytokines in regulatory T (Treg)-cell and B-cell generation and function. On the other hand, IL-10 can enhance cytolytic mechanisms of islet graft rejection,576 and high doses can accelerate GVHD.372 Moreover, TGF-β is an important mediator of fibrotic pathologies in both chronic rejection and GVHD.577,578 TGF-β, in concert with proinflammatory cytokines such as IL-1 and IL-6, also promotes the development of proinflammatory Th-17 cells.579 The inflammatory condition that develops in IL-2 knockout mice is clear evidence of the important immunomodulatory role of this cytokine, and studies in several graft rejection580 and GVHD581 models have confirmed such a role in transplantation. Much of this effect is due to the important role of IL-2 in survival and expansion of Treg cells,582,583 as is discussed elsewhere in this chapter. IL-2 can also promote activation-induced cell death of alloreactive CD8 cells.582,584 While IFNγ can also promote graft rejection, rejection is rapid or even accelerated in IFNγ knockout mice.383,564,585–587 IFNγ has also been shown to play a downregulatory role in GVHD.371,385,588 IFNγ has antiproliferative effects on T cells,384,587 increases activation-induced cell death via the Fas/FasL pathway,589–591 upregulates nitric oxide production,592–595 and is necessary for Treg-cell function in certain conditions.596–598 Consistently, the major cytokines that induce IFNγ, IL-12 and IL-18, can inhibit graft rejection382,384,599 and GVHD.385,600,601 IL-12 and IL-18 act in an IFNγ and Fas-FasL–dependent manner,385,601,602 which preserves or enhances GVL effects.588,603–606 IFNγ promotes the GVL effect of CD8 T cells,385,607,608 apparently by promoting lymphohematopoietic grat-versus-host responses while inhibiting tissue GVHD,609 perhaps due in part to increased PDL1 expression by APCs,610 promotion of donor Treg-cell expansion,610 and reduced Th17 differentiation.337 Despite interest in the notion that Th2 cytokines are antiinflammatory and may suppress rejection and GVHD,359–367 IL-4 deficiency does not accelerate graft rejection564,611 and can actually downmodulate GVHD.371 However, there clearly are situations in which an immunomodulatory effect is achieved by Th2 cytokines. For example, the use of total lymphoid irradiation can alter the balance between NKT cells and conventional T cells in BMT recipients, and IL-4 production by enriched recipient NKT cells downmodulates GVHD.612 This approach, which also apparently involves Treg cell enrichment when combined with antithymocyte serum,613 has recently been extended to clinical trials of HLA-identical HCT for treatment of hematologic malignancies and renal allograft tolerance induction.614–617

The Presence of the Transplanted Organ Vascularized organ allografts may be accepted spontaneously618–621 or with a short course of immunosuppression in rodents or pigs.621–629 The long survival of these transplanted organs can prevent rejection of other allografts from the same donor.618,630,631 In clinical transplantation, long survival of a transplanted organ may diminish the rejection response, as much less immunosuppression is required late after transplantation than in the early period. Studies in

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transiently chimeric monkeys and patients achieving tolerance with HLA-mismatched combined kidney and BMT strongly implicate a role for the kidney itself in promoting tolerance.632–636 Treg cells have been implicated in many of these models (see following discussion). The capacity of transplanted organs to regulate their own survival is often confused with the capacity of a treatment to induce tolerance. For example, many transient immunosuppressive regimens achieve vascularized allograft survival in rodents, but the role of the immunosuppression is to allow a strong Treg cell response induced by the graft to predominate under the controlled experimental conditions. These conditions do not usually apply to human transplantation, thus explaining the failure to translate the many tolerance regimens that succeed in rodents into clinical practice.

Role of Graft and Tissue Injury Graft injury, such as that associated with ischemiareperfusion and host tissue injury induced by conditioning therapy in the case of HCT, play an important role in promoting graft rejection and GVHD, respectively. Local inflammatory processes activate innate and consequently adaptive immunity and T-cell activation. Inflammation plays an important role in promoting APC migration from tissues to lymph nodes637–641 and also promotes trafficking of activated T cells into tissues, as is illustrated dramatically in HCT models. Administration of large numbers of nontolerant donor lymphocytes to established mixed bone marrow chimeras (ie, animals not recently treated with conditioning therapy) leads to a graft-versus-host response that attacks only lymphohematopoietic tissues and does not cause GVHD, a disease of epithelial tissues such as skin, intestines, and liver.642,643 In contrast, similar numbers of T cells cause rapidly lethal, severe GVHD in freshly irradiated hosts.642,644 Conditioning rapidly induces production of chemokines in the GVHD target tissues, promoting immigration of T cells that then elicit a further cascade of chemokines that amplifies the response.645 Upregulated adhesion molecules also promote leukocyte infi ltration through the microvasculature of these tissues. Lethal total body irradiation (TBI) and cyclophosphamide, for example, upregulate the proinflammatory cytokines IL-1, IL-6 and TNF-α ,646–648 which can upregulate endothelial cell E-selectin, P-selectin, ICAM-1, and vascular cell adhesion molecule-1.649,650 In the absence of such host target tissue inflammation, mature, activated graft-versus-host–reactive effector T cells are unable to traffic into skin and induce injury.644 Provision of a local tolllike receptor (TLR) stimulus promotes the entry of such cells into the skin and induces localized GVHD,644 indicating that tissue inflammation provides a critical checkpoint for T-cell recruitment to GVHD target tissues. A systemic TLR stimulus in this setting promotes severe, multiorgan GVHD.644 GVHD can also occur when very large numbers of nontolerant parental T cells are administered to genetically tolerant F1 hosts,651 indicating that, in the presence of sufficiently powerful graft-versus-host responses, the need for tissue inflammation to induce GVHD can be bypassed, possibly due to inflammation induced by high systemic cytokine levels.

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All forms of organ and tissue transplantation involve ischemic and traumatic injury to the donor tissue, which may be one of the reasons that rejection episodes occur most frequently early after transplantation. The surgical trauma associated with transplantation is associated with very early production of chemokines in the graft,433,652 promoting infi ltration of NK cells433 and neutrophils,653 which in turn perpetuate inflammation that promotes subsequent T-cell infi ltration.654 At least partly due to the influence of these cells of the innate immune system,433 chemokines are produced before T-cell infi ltration is seen. IFNγ, whose early production may require CD8 T-cell activation,655 also activates macrophages to become effective APCs and release chemokines.655–657 These phenomena, along with microbial exposures that drive innate immunity, contribute to “danger” signals that promote graft rejection.658 Nevertheless, skin and cardiac allografts that are allowed to heal before being exposed to alloreactive T cells are rapidly rejected if there is sufficient antigenic disparity between donor and host.659,660 Similarly, patients with long-standing allografts are rarely able to terminate immunosuppressive therapy without rejection. Therefore, “danger” signals are not a critical requirement for graft rejection, and it is better to picture the antigenic disparity and the recipient’s immune responsiveness as the dominant features controlling graft rejection, while danger signals may influence the timing, intensity, or character of the rejection response.

Role of the Innate Immune System The innate immune system comprises a group of cells and molecules (Table 46.5) that provide a first line of defense against pathogens and which also play an important role in allograft rejection. Primary adaptive immune system responses that rely on the activation and expansion of antigenspecific T and B cells take several days to reach maturity. In contrast, the innate immune system can be considered as a “preformed” defense mechanism that is immediately available to defend the host until either the dangerous stimulus is cleared or the adaptive immune system is able to mount an antigen-specific response.661 Clearly, this is a somewhat

TABLE

46.5

Components of the Innate Immune System

Cell

Primary function

Macrophage/ neutrophil

Phagocytosis, opsonisation, antigen presentation Release of inflammatory mediators Antigen uptake and presentation to lymphocytes Release of cytokines Cytotoxic to virally infected or mutated cells Release of inflammatory mediators Killing of antibody-coated entities Opsonisation, target cell lysis, and chemoattraction

Dendritic cell Natural killer cell Eosinophil Complement

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simplistic view; while many components of the innate immune system can be recruited very quickly after transplantation, their activity can be amplified after activation. The physical process of graft retrieval and implantation generates signals within the graft and the recipient that trigger rejection. The concept of “danger” triggering an immune response has evolved as an idea over many years.170,661 Pattern recognition receptors exist to detect the unwanted presence of bacterial or viral pathogen-associated molecular patterns, but after transplantation the TLRs that form part of the pattern recognition receptor family can also be used to detect the molecules produced as a result of implantation of the graft, so called damage-associated molecular patterns (DAMPS). These signals include heat shock proteins, reactive oxygen species, high mobility group protein B1, complement breakdown products, nucleic acids (deoxyribonucleic acid and RNA), mitochondrial components, and molecules associated with tissue fibrosis that activate cells of the innate immune system via TLR ligation. The immune system monitors the health of cells and responds to ones that have been injured and killed. Cell death is an inevitable consequence of the ischemia and reperfusion injury that is caused by organ and tissue retrieval. Dying cells expose intracellular DAMPs that can be recognized by components of the innate immune system.662 The role of DAMPs may vary depending on the type of transplant performed and the degree of injury resulting from cell isolation or organ retrieval. Some DAMPs may be expressed in a tissue-restricted manner (eg, one class of DAMPs called alarmins include molecules such as β-defensins that are expressed primarily by leukocytes and therefore may be more relevant after hematopoietic stem cell transplantation or BMT). In addition, the location or type of injury may contribute to the outcome of dying cells triggering a response. Thus, the contribution of different DAMPs to triggering the innate immune system may vary with different types of transplant. Macrophages and other phagocytic cells can ingest dying cells and necrotic tissue; when activated, they release cytokines such as TNF-α , IL-1, and IL-6, which all contribute to the local inflammatory environment. Production of active IL-1β requires proteolytic cleavage of the inactive form, pro-IL-1, a process linked to a multiprotein complex called an inflammasome that acts as a platform for caspase 1, the cysteine protease involved in the proteolytic maturation and secretion of IL-1β.663 A number of different inflammasomes exist, but the nucleotide oligomerization domain–like receptor protein 3 inflammasome is the best studied to date. IL-1β can be highly deleterious to the tissue in which is produced; therefore, inflammasome activity is tightly controlled. As a consequence of these events, the early infi ltration of macrophages into a graft at the onset of rejection has been suggested to be a poor prognostic sign for transplant survival. Macrophage colony-stimulating factor, produced by tubular and mesangial cells, promotes macrophage infi ltration and proliferation, and may play a pathogenic role in acute rejection. Damaged tissue can also trigger complement activation in the absence as well as the presence of antibody; complement has been demonstrated to contribute to ischemia reperfusion

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injury.664 Activated complement components constitute a proteolytic cascade that generates a range of effector molecules.170 The anaphylatoxins C5a and C3a are chemoattractant molecules that assist leukocytes to home to the graft while other soluble mediators are able to opsonise cells, targeting them for destruction by phagocytes.665 Recognition of C3b, C4b, or their fragments covalently bound to target cells by complement receptors on the surface of leucocytes facilitates antigen presentation and T-and B-cell activation.666,667 Generation of the terminal components of the complement cascade (C5b-9) results in formation of the membrane attack complex within the target cell membrane and initiation of target cell lysis. This has been demonstrated to play an important role in ischemia reperfusion injury.664 In addition to the potential of the damaged tissue itself to activate complement, there is also evidence that natural immunoglobulin M antibody can trigger complement activation via both the classical and mannose-binding lectin pathway.668 Studies in muscle reperfusion models initially identified natural immunoglobulin M as a major initiator of pathology through the activation of the complement system and recruitment of inflammatory cells. When the repertoire of natural immunoglobulin M antibodies was altered, significant protection of the myocardial tissue was observed with only limited apoptosis of cardiomyocytes and decreased neutrophil infi ltration compared to when natural antibody was present.669 As mentioned previously, there is also increasing evidence that complement can influence graft outcomes, contributing to the development of acute and/ or chronic rejection, either directly or through antibodydependent mechanisms.511,670 NK cells are innate immune mediators that express cell surface receptors, including activating receptors that bind to widely expressed carbohydrate residues on self-cells and inhibitory receptors that bind self-MHC class I molecules. Activating NK cell receptors including NKG2D recognize natural stress signal ligands, whereas the inhibitory receptors include the CD94-NKG2A complex, KIR family in humans, and Ly49 family in mice. The possible role of NK cells in graft rejection is discussed previously. Absence of an appropriate MHC class I ligand on an allogeneic cell informs the NK cell that the allogeneic cell should be killed. Some malignant or virally infected cells downregulate MHC class I expression or express altered class I molecules as a strategy to evade CD8 + T-cell cytotoxicity. As a result, they are unable to stimulate inhibitory receptors and are vulnerable to NK cell killing. Thus, NK cells could contribute to tissue damage following cell and solid organ transplantation. While the role of NK cells in rejecting bone marrow (at least in mice) is clearer than for solid organ transplantation,671 NK cells can have a marked and lasting impact in this setting,672 particularly with a form of chronic cardiac graft vasculopathy in mice.494 NK cells likely contribute to acute rejection in certain donor-recipient combinations where, even if they are not the major drivers of the responses, they have a significant impact by secreting IFNγ. Nevertheless the precise role of NK cells requires further elucidation as NK cells have also been shown to promote tolerance induction (see the following discussion) by killing donor APCs.673

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Increasing evidence demonstrates the important role that components of the innate immune system play in activating the adaptive immune system. In particular, ligation of TLRs on DCs induces maturation, as defi ned by upregulation of costimulatory molecules and MHC class II, enhancing their ability to act as a bridge between the innate and adaptive immune systems.674 TLRs are critical sentinels of the innate immune system and contribute to the early response after transplantation. Using HY grafts, Goldstein and colleagues showed that rejection was not triggered in the absence of MyD88 TLR signaling pathway due to the migration of a reduced number of mature DCs to the draining lymph nodes that resulted in the impaired generation of anti–graft-reactive T cells and Th1 immunity.675 Further studies using fully allogeneic grafts have shown that multiple TLR signaling pathways contribute to the initiation of rejection.676,677 There are also data indicating that TLR2, TLR4, and/or TLR9 can play a role in the ischemiareperfusion injury in heart, brain, liver, intestine, and kidney.678,679 Haptoglobin, a serum acute-phase reactant protein released by necrotic donor skin grafts, has recently been shown to promote graft rejection via a MyD88-dependent pathway.680 As well as stimulating rejection, TLR signaling and activation of the innate immune system may also prevent or increase the difficulty of induction of tolerance to alloantigens681 (see following discussion).

MANIPULATIONS TO PREVENT GRAFT REJECTION Methods of preventing graft rejection can be divided into nonspecific immunosuppression, which reduces the overall immunocompetence of the recipient, and specific suppression of the response to the graft, leaving the immune system otherwise intact. The latter, known as tolerance, is a major goal of transplantation research.

Nonspecific Techniques Immunosuppressive Medications Reviewing the pharmacology of the nonspecific immunosuppressive drugs commonly used in clinical transplantation is beyond the scope of this chapter. It is important to note, however, that most of the major advances in clinical transplantation that have occurred over the past four decades have been made possible largely because of these agents. Most recipients of allogeneic organs today receive exogenous immunosuppression in the form of combination therapy with several drugs (Table 46.6), including steroids, a calcineurin inhibitor (cyclosporine or tacrolimus), and an antimetabolite (eg, azathioprine or mycophenolate mofetil). Newer drugs, including sirolimus and several other agents tested as subsitutes for the calcineurin inhibitors in some regimens and both steroid-free and single-drug regimens, are being tested,682–685 as are strategies incorporating immunosuppressive antibodies.686 In general terms, both standard and experimental immunosuppressive drugs suppress immune responses either by depleting immune cells, by blocking costimulation, or by inhibiting lymphocyte gene transcription (eg, cyclosporine, tacrolimus), cytokine

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TABLE

46.6

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1177

Immunosuppressive Medications

Type of Drug Antirejection medication First generation Second generation

Polyclonal antibodies Monoclonal antibodies

Examples

Mode of Action

Prednisone Azathioprine Cyclosporin FK-506 MMF Rapamycin ATG Thymoglobulin Anti-CD3 Anti-CD25 Anti-CD2 Anti-CD154 CTLA4-Ig Anti-CD40

Anti-inflammatory; inhibition of cytokines Antimetabolite (purine analog) Calcineurin inhibition (downregulates IL-2) Calcineurin inhibition (downregulates IL-2) Inhibitor of nucleotide synthesis Inhibitor of signal transduction T-cell depletion T-cell depletion T-cell sequestration Activated T cells T-cell depletion and costimulation blockade Costimulation blockade Costimulation blockade Costimulation blockade

ATG, anti-thymocyte globulin; CD, cluster of differentiation; CTLA, cytotoxic T-lymphocyte antigen; MMF, mycophenolate mofetil; IL, interleukin.

signal transduction (eg, rapamycin), or nucleotide synthesis (eg, azathioprine, mycophenolate mofetil).

Anti–T-Cell Antibodies The first therapeutic anti–T-cell antibodies used clinically were heterologous antisera prepared against human lymphocytes or thymocytes. These powerful immunosuppressants are still used today, both as induction agents and for the treatment of rejection episodes. Their major side effects include serum sickness and infectious complications. A variety of monoclonal antibodies (mAbs) are also being used actively in clinical transplantation or are being tested in clinical trials687 (see Table 46.6). These include OKT3, Campath-1, and numerous other, newer mAbs. In general, these antibodies are now either “humanized” from the mouse versions by genetic engineering or prepared in mice carrying human immunoglobulin genes688 to avoid immunization of the recipients. mAbs to the α chain of the IL-2 receptor (CD25) have been used in an effort to achieve greater antigen specificity with anti–T-cell antibodies, based on the hypothesis that these mAbs might selectively eliminate only those T cells activated by the transplant. Clinical results using humanized anti-CD25 mAbs have so far demonstrated immunosuppression, but not tolerance induction. Some mAbs have also been used in attempts to block the effector mechanism of graft rejection, including anti-ICAM antibodies and anti-TNF antibodies. Agents that suppress T-cell costimulation, including anti-CD154, anti-CD40, and CTLA4-immunoglobulin, have shown promise,687 and will be discussed further with regard to their potential value in tolerance induction protocols (see following discussion). CTLA4-immunoglobulin (belatacept) in particular, has recently been reported to have better cardiovascular and metabolic risk profiles than a calcineurin inhibitor in the control arm of a recent clinical trial.689 While mAb therapy has been extremely effective, several problems still exist, including toxicities related to the release of cytokines and immunogenicity, leading to antibody

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production against both constant region and idiotypic determinants of the antibody molecules.

Donor-Specific Tolerance Induction The Need for Tolerance-Inducing Regimens Immune tolerance denotes a state in which donor-specific nonresponsiveness is maintained without immunosuppressive agents. For the purpose of this discussion, we will define tolerance as acceptance of a donor organ by an otherwise intact immune system, and will qualify the term as “systemic” when it applies to the entire immune system so that the immune system fails to respond to cells or other grafts from the same donor. Transplantation tolerance is a desirable goal for three major reasons. First, while improvements in immunosuppressive therapy have dramatically increased the success of clinical organ transplantation, these drugs are associated with increased risks of infection and malignancy as well as metabolic and organ toxicities that can result in additional organ failure. Second, despite improved immunosuppression, chronic rejection still contributes to constantly down-sloping long-term survival curves for organ allografts. Third, a critical shortage of allogeneic organs has increased interest in the use of other species as organ and tissue sources. However, immune barriers to xenografts are greater than those to allografts, and the induction of both B-cell and T-cell tolerance may be essential to the ultimate success of xenotransplantation.

Mechanisms of Transplantation Tolerance Other chapters in this book describe the mechanisms by which tolerance to self-antigens is achieved (see Chapters 13 and 32). Rather than reiterate these in detail here, we will mention each and provide examples in which they have been implicated in transplantation. Mechanisms for inducing Tand B-cell tolerance include deletion, anergy, and suppression. In addition, a graft may simply be ignored by recipient lymphocytes but in most transplant settings this is unlikely

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to be a reliable mechanism for sustaining tolerance. The thymus is the central organ for T-cell development. Induction of tolerance among developing thymocytes is referred to as “central,” as distinguished from the “peripheral” tolerance that may develop among already mature T cells when they encounter antigen in the peripheral tissues. The marrow is the central organ for B-cell development and, as for T cells, B-cell tolerance may also be thought of as central or peripheral. Clonal Deletion B Cells. When a developing B cell with autoreactivity encounters self-antigen, it undergoes developmental arrest. Recombination activating gene–dependent light chain receptor editing then takes place. If this results in the formation of a nonautoreactive immunoglobulin receptor, the B-cell survives; if not, the B cell undergoes apoptosis.690 B cells are susceptible to tolerance at particular stages of development upon recognition of membrane-bound antigen.691 Several checkpoints for elimination of autoreactive B cells during development in the marrow and after migration to the periphery have been identified.692 Deletion, rather than receptor editing, eliminates autoreactive peripheral B cells,693 which may also become anergized, depending on the nature of the antigen.694,695 In the context of transplantation, B-cell responses to allogeneic MHC molecules are largely T cell–dependent, so an absence of alloantibodies may not necessarily reflect intrinsic B-cell tolerance; an absence of T-cell help would produce a similar outcome. However, MHC-alloreactive B cells may be intrinsically tolerized by encounter with antigens in the absence of T-cell help, apparently by a deletional mechanism.696,697 Elimination of natural antibody (eg, xenoantibody) responses must target B cells in addition to T cells, as these antibodies are produced even in the absence of T cells.698 Nevertheless, T-cell responses can clearly augment these antibody responses.699–701 Cells of the B-1 subset, which produce natural antibodies responsible for xenograft hyperacute rejection,702 can be deleted when their receptors are crosslinked by cell-bound antigen.703 Initial anergy followed by deletion of preexisiting B cells and deletion/receptor editing is responsible for the long-term tolerance of natural antibody-producing B-1 cells in mice given hematopoietic cells expressing an antigen for which natural antibody-producing cells preexist in the recipient.153,704,705 T Cells. Central T-cell tolerance involves deletion of developing thymocytes that recognize self-antigens presented by hematopoietic cells and thymic epithelial cells. In addition, antigens presented only by nonhematopoietic thymic stromal cells can induce T-cell anergy706 or drive Treg-cell differentiation.707–709 Several hematopoietic cell types, including DCs,710 B cells,711 and thymocytes,712,713 as well as thymic stromal cells,714,715 can induce intrathymic deletion. Medullary epithelial cells producing tissue-specific antigens can mediate thymocyte deletion by presenting antigens directly or transferring them to DCs.716 Transplantation tolerance induced by intrathymic deletion should be very

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reliable, as donor-reactive T cells would be largely absent. Durable mixed hematopoietic chimerism achieved with regimens that completely deplete preexisting T cells in the thymus and periphery results in donor- and recipient-specific tolerance through this central deletion mechanism.717,718 Mature peripheral T cells can also be deleted.719–724 Peripheral CD8 cells may undergo “exhaustion” in the presence of persistent antigen.725,726 Persistent antigen encountered on recipient nonhematopoietic cells leads to deletion and exhaustion of graft-versus-host–reactive CTLs following HCT.556,727,728 Peripheral deletion of donor-reactive T cells has been demonstrated in mice receiving DSTs and/or BMT with costimulatory blockade,533,729–734 and has been implicated in a model involving tolerance induction with anti-CD154 mAb and rapamycin.735 While peripheral deletion in the BMT/ anti-CD154 model is not simply explained by exhaustion or “activation-induced cell death,”551,733,736 activation-induced cell death was implicated in tolerance induction with antiCD154 mAb plus rapamycin.737 In addition, veto cells (see the following) can delete alloreactive CTLs. A non-veto mechanism mediated by CD4–CD8– cytotoxic regulatory cells has been reported to delete alloreactive CD8 + T cells with the same specificity as the regulatory cells.738 Anergy. Anergy is a state that may result when lymphocytes recognize antigen without adequate accessory or costimulatory signals706,739–742 or when they encounter ligands for which they have low affinity.743,744 Anergy is an important tolerance mechanism for the many self-reactive B cells that escape deletion in the bone marrow,745,746 particularly if they recognize abundant but low-avidity antigens.691,747 Anergy requires persistent antigen and is characterized by immunoglobulin receptor downregulation,691 altered signaling patterns, and increased apoptosis upon antigen encounter.747 T-cell tolerance and the resulting absence of help is important for the maintenance of B-cell anergy, as anergic B cells can be activated in the presence of high avidity antigen and T-cell help.747 Anergy is responsible for the early tolerance of natural antibody-producing B-1 cells in mice rendered mixed chimeric with bone marrow cells expressing an antigen recognized by natural antibodyproducing cells in the recipient.153,704,705 A T cell is considered to be “anergic” if it cannot proliferate or produce IL-2 in response to the antigen for which it is specific. T-cell anergy has been associated with altered signaling and tyrosine phosphorylation patterns.748–751 T-cell anergy can often,752 but not always,753,754 be overcome by providing exogenous IL-2. Thymocytes are also susceptible to anergy induction by antigens presented on hematopoietic711 or nonhematopoietic stromal755–757 cells. Anergy is generally reversible in vivo and can be overcome by infection758 or by removal of antigen,759–762 and therefore may not be reliable in transplant recipients, in whom infections may perturb a nondeletional state of tolerance by activating innate immunity.763,764 While deletion may follow the induction of anergy of T cells in the continued presence of antigen,765,766 anergic T cells may also persist.767,768 In a transplantation model involving BMT under cover of costimulatory blockade, peripheral donor-reactive CD4 and CD8 T cells are rendered

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CHAPTER 46

antigen-unresponsive prior to their deletion.550,734,769 Anergic T cells may also have a suppressive effect on other T cells,770,771 which may involve conditioning of suppressive APCs.772 Immunoregulation Regulatory T Cells. The concept of antigen-specific suppression and the existence of a specialized population of T cells, known initially as suppressor cells and more recently as Treg cells, that could control immune responsiveness, originated in the 1970s from the studies of Gershon and Kondo,773 among others, who demonstrated the existence of complex regulatory networks involving several levels of T-cell–mediated suppression. In the specific setting of transplantation, a role for suppressor cells in tolerance induction was also identified.774 However, despite the efforts of many groups through the 1980s, the cellular and molecular characteristics of these suppressor cells were never defined with any precision, even though the phenomenon of suppression was highly reproducible in vivo, particularly in transplant models. In 1990, Hall and colleagues reexamined the function of T-cell subpopulations present in rats with long-term surviving cardiac allografts after cyclosporine treatment. Interestingly, they reported that tolerance could be adoptively transferred by CD25 + CD4 + cells.775 However, interest in characterizing suppressor/Treg cells was not fully revived until Sakaguchi and colleagues followed up on data showing that susceptible strains of neonatally thymectomized mice developed multiorgan autoimmune disease. These workers found that neonatal thymectomy resulted in a loss of T cells with suppressor or regulatory properties that could be enriched amongst T cells expressing CD25, the alpha chain of the IL-2 receptor.776 Naturally occurring or thymus-derived CD25 + CD4 + T cells with regulatory properties are generated in the thymus through interaction between the developing thymocytes with an intermediate affinity ligand, too low for negative selection, expressed on nonhematopoietic cells of the thymus for their positive selection.777,778 Cortical thymic epithelium has been found to be the site of MHC class II expression required for the development of functional Treg cells.779 In addition to the naturally occurring populations of CD25 + CD4 + T cells with regulatory activity, populations of T cells with regulatory properties that also express CD25 can be induced in the periphery following exposure to antigen under certain conditions.780 These populations are known as antigen-induced or adaptive Treg cells. It is likely that following transplantation or exposure to alloantigen, Treg cells that can control the immune response and prevent rejection will be derived from both the naturally occurring and induced populations (see the following). In addition to the wealth of data in mice, CD4 + T cells expressing high levels of CD25 in the peripheral blood and thymus have also been shown to have suppressive activity in humans781–786 and are present in transplant recipients with long-term surviving liver allografts.787,788 CD25 is by no means an exclusive marker for T cells with regulatory activity. Although the kinetics of expression are different, CD25 is also expressed by activated T cells. Indeed in transplantation, mAbs targeting CD25 are licensed for use as antirejection therapies. Thus, there is a need to define

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other markers for Treg cells. The list of potential cell surface markers has been growing steadily, and includes CD152 (CTLA4), CD103, CD127, LAP, and glucocorticoid-induced TNF receptor (GITR) related proteinl amongst many others. However, as yet, a Treg cell–specific “perfect cell surface marker” has not been identified. Intracellular proteins and transcription factors can also potentially provide a tool for identifying committed cell lineages. In both humans and mice, forkhead box protein 3 (foxp3), a forkhead/winged-helix transcription factor, has been shown to be a master regulator for the development and function of Treg cells. In humans, mutations in the Foxp3 gene in patients with the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome results in uncontrolled activation/expansion of CD4 + T cells, resulting in severe immune deficiency.789,790 Scurfy mice also have mutations in foxp3 and a related immune profi le.791 A direct link between foxp3 expression and regulatory activity came from retroviral gene transfer studies where transduction of naïve mouse T cells with foxp3 resulted in acquisition of regulatory function.792 This correlation between foxp3 and regulation has been strengthened by the generation of foxp3 transgenic mice where cells expressing FOXP3 protein express either green793 or red fluorescent protein,794 allowing such cells to be identified and their function explored. While in mice the relationship between foxp3 expression and regulation may be tightly linked, the situation may not be so straightforward in humans, as FOXP3 protein has also been found to be expressed transiently in activated T cells.795 The methylation status of the Foxp3 gene turns out to be a marker of the stability of FOXP3 expression.796,797 Recipient-derived CD25 + CD4 + T cells were shown to have potent regulatory properties in both the induction and maintenance phases of tolerance to alloantigens in vivo in mice.798–801 In BMT, donor CD25 + CD4 + T cells present in the bone marrow inoculum were found to protect from GVHD.802–804 In humans, CD25hiCD4 + T cells have been found in the peripheral blood of immunosuppression-free allograft recipients,787 and their presence may contribute to long-term graft survival.805 As mentioned previously, Treg cells with capacity to control immune responses to a transplant most likely comprise naturally-occurring and alloantigeninduced Treg cell populations.806 Naturally occurring Treg cells present in naïve, unmanipulated adult mice can prevent rejection of allografts mismatched for a single minor histocompatibility antigen (eg, H-Y). However, they appear to be much less potent than Treg cells induced following exposure to alloantigen, as studies comparing the activity of naturally occurring and induced Treg cells directly suggest that 10 times fewer induced Treg cells are needed to prevent allograft rejection. These findings may explain why in situations where grafts are mismatched for multiple major and MiHAs it has sometimes been difficult to demonstrate that naturally occurring Treg cells can control the rejection response. Another situation where Treg cells are less effective at controlling allograft rejection arises when donor alloantigen-reactive memory T cells preexist in the recipient. In this case, the kinetics of activation of the memory cells is very rapid and, unless very high numbers of Tregs are present, the

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balance between rejection and regulation is overwhelmingly in favor of rejection.807 Importantly, this critical balance can be shifted in a number of ways, notably by employing strategies that increase the relative frequency and/or the activation status and consequently the functional activity of induced Tregs that can then respond to donor alloantigens before or in the early period after transplantation801,808 or by inhibiting the activity of the effector cells. After transplantation, the ability to generate Tregs in vivo may be influenced by the microenvironment. The balance between the presence of IL-6 and TGF-β in vivo can have a dramatic impact on which T-cell population develops. Importantly, Treg generation can be prevented by inflammation,809,810 which is commonly present in vivo after cell or organ transplantation, leading instead to the generation of cells capable of mediating tissue damage, including TH17 cells, which produce IL-17.809–811 Treg stability in the face of an inflammatory response remains a controversial issue.812,813 The mechanisms of regulation effected by Tregs to prevent allograft rejection involve a variety of different pathways. When Tregs are reactivated by their cognate antigen through the TCR, they can rapidly and transiently produce IFNγ, which is needed to mediate their regulatory activity and prevent graft rejection.814 IFNγ triggers the STAT-1 signaling pathway that also influences the functional activity of Tregs.815 Interestingly, IFNγ has been shown previously to be an essential mediator for the induction of tolerance in mouse models where costimulation blockade was used to prevent graft rejection.816 The transient production of IFNγ by Tregs can have multiple effects on cells with which they interact,817 including other T cells, mediated either directly or indirectly through effects on APCs.818 Treg activity can result in inhibition of cytokine production and secretion, downregulation of costimulatory and/or adhesion molecule expression, inhibition of proliferation, induction of anergy, elimination of the effector population by promoting cell death, or even conversion of naïve and/or effector T cells to a regulatory phenotype, a process known as “infectious tolerance.”783,819–821 IL-2, IL-10, and TGF-β may have a role in this phenomenon.822 If this process is effective, any new T cells entering the repertoire after transplantation where immunoregulation is the dominant mechanism of tolerance will be converted to Tregs, thereby propagating and reinforcing the tolerant state throughout the posttransplant course. Indoleamine 2,3 dioxygenease (IDO), an enzyme that catalyses the initial and rate limiting step of the kyneurinine pathway of tryptophan catabolism, is known to be subject to transcriptional regulation by IFNγ823,824 in a manner that is dependent on STAT-1 signaling.825 IFNγ released by Tregs can lead to the development of IDO-competent DCs that may acquire the capacity to control T cells locally through tryptophan depletion, inhibiting alloimmune responses.826–828 The molecular mechanism responsible for the inducing DCs to become IDO producers requires further investigation, but roles for TGF-β and IL-10 have been suggested.820,829 In addition, the activity of Tregs in transplant models has been shown to be dependent on IL-10,541,798,799,830–834 TGF-β, and CTLA-4.799 CTLA-4 (CD152) is constitutively expressed by Tregs, and engagement of CD80/86 on DCs can also induce

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IDO.835 In addition, CTLA-4 has been found to modulate T-cell motility by activating leukocyte function–associated antigen (LFA)-1 clustering and adhesion,836 thereby altering the threshold for T-cell activation.836 While the mechanistic links between each of these mediators still requires clarification, it is important to note that many of the same molecules have also been found to be required for Treg activity in other settings, including autoimmune diseases, suggesting that Tregs capable of controlling disease states have many properties in common. One property of Tregs that can potentially be exploited in the context of transplantation is their ability to mediate bystander regulation in a defined microenvironment in vivo. To elaborate their functional activity, Tregs need to be activated through their TCRs. Data obtained by Thornton and Shevach in vitro showed that reactivation of Tregs with a defined antigen specificity through their TCR enabled the cells to suppress the response of other cells present in the same cultures.837 Bushell and colleagues have shown that it is possible to exploit this observation in vivo to enable Tregs that are generated and respond to an unrelated protein antigen, such as human gamma globulin, to control rejection when the cells are restimulated through TCR immediately before they are asked to function in vivo.838 There is evidence that the location in which Tregs function in vivo may change with time after transplantation. Jones and colleagues have shown that while Tregs are active in the draining lymph nodes in the first few days following skin transplantation, later in the posttransplant course Tregs are found within the allograft itself.839 Indeed, there is increasing evidence that an important site of immune regulation is within the allograft itself, where Tregs function to create an environment that is permissive of control.801,840 Moreover, reexposing Tregs to antigen in a tissue may enable them to become more potent suppressors.841 Despite the current interest in the role of CD25 + CD4 + Tregs, it is important to remember that regulatory activity is not exclusive to CD4 + T cells, and CD8 +,842–844 CD8 + CD28–,845 TCR+ CD4–CD8– (“double negative”),846 and NKT cells847,848 have also been shown to have regulatory activities in different situations after transplantation. In fact, regulatory mechanisms in both the innate and adaptive immune systems will most likely contribute to the overall outcome after transplantation. This is highlighted by the finding that both NKT cells and CD25 + CD4 + Tregs play a role in preventing acute GVHD after allogeneic BMT,802– 804,847 and by a recent study showing that inhibitory NK cell receptors can play a regulatory role in T-cell homeostasis.849 It may be possible to exploit Tregs to control the immune response after transplantation either by developing strategies using immunosuppressive agents that promote Treg generation and/or do not inhibit their function or by generating Tregs ex vivo and using them as a cellular therapy at different stages in the posttransplant course to prevent rejection and reestablish control. Understanding the impact of immunosuppressive drugs on Treg generation and function is an important part of using the potential of Tregs in vivo. Data from mouse models suggest that calcineurin inhibitors have the capacity to inhibit T-cell apoptosis after activation, thereby

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CHAPTER 46 Day -28

Day -14

Tolerance induction

Anti-CD25

TRANSPLANTATION IMMUNOLOGY

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1181

Day 0

100 80 No CD25 depletion

% Grafts surviving

60 Depletion of CD25+ cells

40 20 0 0

20

40

60 Days

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100

preventing a reduction in the frequency of T cells that mediate rejection that would allow Tregs to assume a more dominant functional role850,851 (Bushell and Wood, personal communication). Similarly, administration of anti-CD25 monocloncal antibodies after the induction of Tregs can prevent long-term graft survival (Fig. 46.11). On the other hand, other classes of immunosuppressive drugs, such as the mammalian target of rampamycin inhibitors sirolimus and everolimus, may enhance the generation of Tregs. Strategies to generate and/or expand Tregs ex vivo for therapeutic purposes are being developed with potential application in cell and organ transplantation. A variety of different approaches have been shown to be successful for generating and/or expanding T cells with regulatory capacity in both mice and humans, including exposure to TGF-β, IFNγ, and stimulation with anti-CD3/CD28 in the presence of IL-2 to name just some.852–854 The development, maintenance, and expansion of Tregs is critically dependent on IL2.673,855 Small molecule inhibitors such as phosphodiesterase inhibitors can be used to generate populations of T cells with regulatory activity. In each case, at the end of the culture period, the T cells that emerge have been shown either in vitro and in some studies in vivo to have regulatory activity that can prevent allograft rejection854 or GVHD.856,857 Strategies for expanding human T cells with regulatory activity ex vivo usually rely on selection of a population of CD4 + T cells that contains regulatory cells, either by enriching for cells that express high levels of CD25 alone or low levels of CD127 in addition.854,858,859 In proof-of-concept studies using a humanized mouse transplant model, such ex vivo expanded Tregs have been shown to be capable of controlling rejection.854,860,861 Antibody-Mediated Suppression. Idiotypes are unique antigenic determinants that characterize the binding sites of antibody or TCRs. These determinants can be antigenic and induce the production of anti-idiotypic antibodies.862 Antibody-mediated suppression could theoretically occur through the recognition of idiotypes of antidonor immunoglobulin receptors. Anti-idiotypic antibodies can

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FIG. 46.11. Administration of Anti–Cluster of Differentiation 25 Monocloncal Antibodies after the Induction of Regulatory T Cells can Prevent Long-Term Graft Survival.

suppress antibody reactivity by directly binding to the antigen-binding site of the antibody, and the development of such antibodies has been suggested as one of the possible benefits of pre–kidney transplant blood transfusions. Such antibodies have also been suggested to contribute to the apparent hyporesponsiveness to noninherited maternal antigens in renal allograft recipients. In the past, it was also considered possible that anti-idiotypic antibodies might inhibit T-cell recognition of antigen, but this now seems unlikely, given that T cells recognize peptide/MHC complexes, whereas antibodies recognize epitopes of intact molecules. It continues to be an intriguing question whether normal regulatory mechanisms for B-cell responses might include anti-idiotypes, as suggested by Jerne.862a However, efforts to control transplantation using exogenous anti-idiotypic antibodies to either TCRs or B-cell receptors have been disappointing.863 Antibodies can also induce tolerance through a process known as enhancement. Enhancement is defi ned as prolongation of graft survival achieved by the presence of antigraft antibodies.864 This phenomenon was first described in experiments involving allogeneic tumor growth. Subsequently, enhancing regimens using anti-MHC antibodies and/or soluble antigen were shown to produce long-term tolerance for rodent allogeneic kidney transplants.865 The simple interpretation was that anti-MHC antibodies bind to the antigen and thereby block the immune response, but this explanation has not turned out to be sufficient. For example, tolerance following enhancement can be transferred by cells and not serum from enhanced recipients. Apparently, the administered antibody sets up a host reaction that leads to specific immunosuppression. An idiotype/anti-idiotype network would be an attractive explanation for this phenomenon. Unfortunately, the spectacular success obtained using enhancement for kidney graft survival in rats has not been observed for grafts in other species, and this approach has largely been abandoned, at least for the present. Ignorance and Immune Privilege. Peripheral antigens may be ignored by T cells866–868 or B cells691 that recognize them. This may be due to the presentation of these antigens by

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“non-professional APCs,” or it may reflect a failure of recipient T cells to migrate to the antigen-bearing tissue, as in murine solid tumor models.869 The level of peripheral antigen expression, how recently the responding T cell has emerged from the thymus,866,870 and the presence of proinflammatory cytokines871,872 and costimulatory molecules873 may all influence the decision of a T cell to ignore or respond to peripheral antigens. However, “ignorance” may be a precarious state, which can be upset by additional immunologic stimuli provoked by inflammation that may be induced, for example, by infections868 or by presentation of antigen on professional APCs, as observed for endocrine allografts that are depleted of APCs prior to transplantation.874 Immunoprotection in specific niches may protect allografts from immune attack, either by making antigen invisible to the immune system or by providing cell types in the microenvironment that protect the allogeneic cells. An example of the former is corneal transplantation, which is done without systemic immunosuppression due in part to the sequestered status of the anterior chamber of the eye.875– 878 However, a number of active mechanisms also contribute to this immune privilege.876,879 An example of a cell that may be protected largely by other cells in its niche is the hematopoietic stem cell, which can survive in an allogeneic stem cell niche without immunosuppression.880

Strategies for Inducing Transplantation Tolerance Strategies to Achieve Central Tolerance Hematopoietic Chimerism. It has been known for 60 years that hematopoietic chimerism induced in utero is associated with transplantation tolerance.881,882 The capacity of hematopoietic cells to induce intrathymic clonal deletion achieves tolerance to the most immunogenic allografts, such as fully MHC-mismatched skin and small bowel grafts.452,883,884 However, HCT has not yet been routinely applied to the induction of tolerance in man. HCT for tolerance induction in rodents originally involved recipient treatment with lethal whole-body irradiation. Removal of mature donor T cells before transplantation prevented GVHD.885–888 However, MHC-mismatched allogeneic HCT in humans has proved to be less successful and more dangerous than in rodents because of the toxicity associated with myeloablative conditioning, and the high risk of GVHD, and of engraftment failure when donor T-cell depletion is used to prevent GVHD.889 Thus, more specific and effective methods of overcoming the barriers to marrow engraftment with minimal GVHD risk are needed for the application of HCT for organ allograft tolerance. Achievement of a state of mixed rather than full allogeneic hematopoietic chimerism has several advangtages for allograft tolerance induction: 1) Mixed chimerism can be achieved with less toxic (nonmyeloablative) conditioning regimens than those that lead to full donor chimerism. Nonmyeloablative regimens preserve some host hematopoiesis so that life-threatening marrow failure is not a risk if donor marrow is rejected. 2) Mixed chimeras, unlike full chimeras, contain abundant recipient APCs in the periphery, allowing optimal antigen presentation to T cells that have developed in the host thymus, and which therefore

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preferentially recognize peptide antigens presented by host-type MHC molecules.126,890 Antiviral CTL responses in mixed chimeras demonstrate exquisite specificity for recipient-derived MHC-restricting elements.891,892 3) Mixed chimeras contain hematopoietic cells from both the recipient and the donor in the thymus and hence delete both hostreactive and donor-reactive T cells.717,893 Thymic stromal cells are less effective at inducing deletion, so intrathymic deletion of host-reactive cells is more complete in mixed compared to full chimeras.717,894 Tolerance achieved with durable mixed chimerism is “systemic,” as evidenced by specific unresponsiveness to the donor and recipient in mixed lymphocyte response and cell-mediated lympholysis assays, and the acceptance of donor skin grafted at any time postBMT.452,895–898 In contrast, tolerance approaches that do not lead to durable chimerism generally do not induce systemic tolerance, suggesting that tolerance might be less robust. Several nonmyeloablative approaches have been recently developed to permit the use of BMT to achieve mixed chimerism and specific tolerance, bringing the approach toward clinical application. In mixed chimeras prepared with a nonmyeloablative regimen consisting of low dose (3 Gy) TBI, T-cell depleting mAbs, and thymic irradiation,452 intrathymic deletion was shown to be the major mechanism maintaining long-term donor-specific tolerance.717 Administration of donor MHC-specific antibody to eliminate donor chimerism from established mixed chimeras led to loss of tolerance and to the de novo appearance in the periphery of T cells with receptors that recognize donor antigens. However, if the recipient thymus was removed prior to elimination of chimerism, specific skin graft tolerance was preserved and donor-reactive TCR did not appear.718 Thus, chimerism is needed only in the thymus and not in the periphery in order to ensure persistent tolerance of a peripheral repertoire that is deleted of donor-reactive cells. In contrast, peripheral anergy and suppression generally require persistent antigen to maintain tolerance. Because thymic APCs are continually turning over, this observation emphasizes the need for hematopoietic stem cell engraftment at sufficient levels to ensure an uninterrupted supply of donor APCs in the recipient thymus for the life of the mixed chimera. The absence of suppressive tolerance mechanisms makes these animals particularly vulnerable to breaking of tolerance when non-tolerant T cells emerge from the thymus after intentional depletion of donor antigen or after exogenous administration of nontolerant host-type T cells.718,734 The universal requirements for achievement of lasting chimerism are summarized in Figure 46.12. T-cell alloreactivity in both the thymus and periphery must be overcome in order to permit allogeneic stem cell engraftment and early seeding of the thymus with allogeneic APCs. Intrathymic alloreactivity can be overcome with irradiation,452 high doses of T cell–depleting antibodies,899 or costimulatory blockers.731,896 Peripheral T cell–mediated alloreactivity can be overcome with T cell–depleting mAbs452,900 or with costimulatory blockers.731 The need for low-dose TBI or busulfan to facilitate engraftment of donor hematopoietic stem cells899,901 can be eliminated by administering very high marrow doses897,900,902 or by adding expanded recipient Treg cells to donor marrow.903

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cells (e.g., anti-T thymic irradiation).

intravenously.

The mechanisms of peripheral tolerance achieved with the combination of costimulatory blockade and allogeneic BMT are discussed in the section on peripheral tolerance. Additional methods for achieving durable mixed chimerism for tolerance induction include the use of total lymphoid irradiation plus BMT904,905 and various combinations of anti–T cell antibodies, irradiation, and immunosuppressive drugs in both large906–908 and small909–912 animals. It has been difficult to achieve T-cell depletion with antibodies in large animals and humans that is as exhaustive as that achieved in the previously mentioned rodent models, perhaps due to the use of inadequate doses or suboptimal reagents. A second concern is that, if sufficiently exhaustive T-cell depletion could be achieved in humans, T-cell recovery from the thymus might be dangerously slow, especially in older individuals.913 The ability to replace some898 or all731,897,902 T cell–depleting antibodies with costimulatory blockade is therefore of considerable interest. GVHD does not occur in the rodent models discussed previously, despite the inclusion of T cells in the donor bone marrow graft, probably because of the presence of the T cell–depleting or costimulatory blocking antibodies in the serum of the hosts at the time of BMT.914 Extension of the Mixed Chimerism Approach to Xenotransplantation. Host treatment with mAbs to T cells and NK cells along with a low dose (3 Gy) of TBI has also permitted rat marrow engraftment in mice, resulting in mixed xenogeneic chimerism and donor-specific tolerance.453 Both γδ T cells and NK cells play an important role in resisting xenogeneic marrow engraftment.454 Anti-CD154 mAbs can overcome the requirement for CD4 cell depletion.915 Mixed chimerism in the rat-to-mouse species combination was associated with tolerance of both T cells and natural xenoantibody-producing B cells.453,916–919 Mixed xenogeneic chimerism thereby prevents HAR, acute vascular rejection, and cell-mediated rejection of vascularized cardiac xenografts.154

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FIG. 46.12. Requirements for Induction of Long-Term Mixed Hematopoietic Chimerism. GVHD, graft-versus-host disease.

Pigs are widely believed to be the most suitable xenogeneic donor species for transplantation to humans, but transplantation from this species is impeded by the presence in human sera of Natural Antibodies (Nab) that cause hyperacute rejection of porcine vascularized xenografts. The major specificity recognized by human Nab on porcine tissues is a ubiquitous carbohydrate epitope, Galα1-3Galβ1-4GlcNAc-R (αGal). Humans lack a functional α1-3Gal transferase (GalT) enzyme, as do GalT knockout mice, which also make anti-αGal Nab. Both preexisting and newly developing B cells recognizing anti-αGal are tolerized by the induction of mixed chimerism in GalT knockout mice receiving αGal-expressing allogeneic or xenogeneic marrow.153,704,920,921 The major immunoglobulin M Nab-producing cells in mice are splenic B-1b cells,702 and these are tolerized rapidly by an anergy mechanism that is followed later by clonal deletion and/or receptor editing of Gal-specific B cells.154,704,705,918,922 Splenic B cells, possibly of a similar subset, are also, along with immature plasma cells, the major anti-Gal immunoglobulin M–producing population in nonhuman primates and baboons.923 Studies in mixed allogeneic chimeras show that recipient NK cells are tolerized to the donor,924 and studies in the ratto-mouse combination also indicate a state of NK-cell tolerance.925 While this tolerance is specific for the donor in mixed allogeneic chimeras, whose NK cells retain the ability to reject class I–deficient bone marrow,924 the NK-cell tolerance in mixed xenogeneic chimeras is associated with global NK-cell unresponsiveness.925 The most plausible explanation for these divergent results is that the presence of a xenogeneic cell population lacking any inhibitory ligand for recipient NK cells results in chronic activation of all NK cells and hence global anergy. Such “disarming” of NK cells could also explain dominant hyporesponsiveness in mixed chimeras involving class I–deficient donors or mosaic class I MHC transgenic mice.926,927 In general, inhibitory NK receptors are less functional between species than activating receptors.444–446,449,450,928–936 In mixed xenogeneic chimeras,

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therefore, the xenogeneic cells may lack ligand that inhibit recipient NK cells, which become chronically activated and hence anergic. In contrast, allogeneic cells in mixed allogeneic chimeras express MHC molecules that will inhibit some subsets of recipient NK cells expressing relevant inhibitory receptors. Thus, in mixed allogeneic chimeras, only the subset of recipient NK cells lacking an inhibitory ligand for donor cells will chronically receive unopposed stimulation and hence become anergic; the consequence is a functional NK-cell repertoire that is inhibited by recognition of the donor (in addition to recipient) cells. The dominant tolerance exerted by mixed allogeneic or xenogeneic chimerism favors the “disarming” concept927 over the “licensing” model, which proposes that inhibitory interactions license NK cells to become functional,937 as the mechanism for NK-cell tolerance. Consistent with the disarming model, NK cells have been shown to tune their responsiveness in direct proportion to the number of inhibitory interactions they experience in vivo.938 Moreover, continuous engagement of an NK cell–activating receptor renders NK cells hyporesponsive.939,940 The complete lack of inhibitory interactions with xenogeneic cells could thereby render all NK cells anergic. Despite this T-, B-, and NK-cell tolerance in mixed xenogeneic chimeras, the levels of donor chimerism decline gradually over time, due to a competitive advantage of recipient marrow over xenogeneic rat marrow as the host recovers from low-dose TBI.941 Species specificity or selectivity of cytokines,942,943 adhesion molecules, and other interactions that regulate hematopoiesis944 probably account for this advantage. Achievement of xenogeneic hematopoietic repopulation has proved to be an even more formidable challenge in highly disparate (discordant) species combinations. Using immunodeficient mice transgenically expressing porcine hematopoietic cytokines to promote chimerism,945 proof of principle was obtained that human T cells can be centrally tolerized in a human thymus to porcine xenoantigens via induction of mixed xenogeneic chimerism.946 Macrophages pose a major obstacle to engraftment of xenogeneic marrow from highly disparate species,471,472 probably due to the failure of inhibitory ligands on xenogeneic cells to interact with macrophage inhibitory receptors, as observed for the porcine inhibitory ligand CD47 and the mouse482 and human macrophage receptor SIRPα .482,947,948 Xenogeneic Thymic Transplantation. An alternative approach to achieving xenogeneic T-cell tolerance involves replacement of the recipient thymus with a xenogeneic donor thymus after host T-cell depletion and thymectomy. Immunocompetent mice treated in this way reconstitute CD4 + T cells in xenogeneic porcine thymic grafts.949 These cells repopulate the periphery, are competent to resist infection,950 and are tolerant of porcine donor antigens.949,951 Tolerance to both donor and host develops, at least in part, by intrathymic deletional mechanisms in these animals, reflecting the presence of class IIhigh APCs from both species within the thymic graft.951,952 Studies using TCR-transgenic mouse recipients showed that positive selection in such

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grafts is mediated only by porcine thymic MHC, with no influence of mouse MHC.953,954 However, excellent immune function is achieved in these mice. Importantly, human T cells can also develop normally and be rendered specifically tolerant of the porcine donor by developing in xenogeneic porcine thymus grafts.955–958 This approach has demonstrated promise in pig-to-primate xenograft models.959–961 Transplantation of allogeneic962 and concordant xenogeneic963 thymic epithelial tissue obtained from fetuses before seeding with hematopoietic cells can also induce tolerance by generating donor-specific regulatory cells.964,965 Transplantation of xenogeneic thymic tissue into congenitally athymic recipients has frequently resulted in the development of a multiorgan autoimmune syndrome, possibly due to the lack of recipient-type thymic epithelium needed for the development of regulatory cells with specificity for certain recipient antigens.779,966–970 This complication occurs much less frequently in thymectomized, T cell–depleted mice than in congenitally athymic mice receiving porcine thymic transplants,971 probably due to the persistence of regulatory cells derived from the host thymus prior to thymectomy and T-cell depletion. This autoimmunity can be avoided by adding recipient thymic epithelial cells to the thymic xenograft, resulting in improved deletion of recipient-reactive T cells and generation of Treg cells that suppress autoimmune disease.972 Development of Chimerism without Host Conditioning Developmentally Immunoincompetent Recipients. The first demonstration of allogeneic tolerance was obtained in Freemartin cattle, which are fraternal twins that share a placental circulation and develop mixed chimerism spontaneously in utero.881,882,973 Injection of allogeneic hematopoietic cells to preimmune human fetuses has been used successfully to correct immunodeficiency diseases diagnosed in utero.974–976 Low levels of chimerism have also been achieved in preimmune normal mouse and sheep fetuses.977–980 While the ability of in utero HCT to induce transplantation tolerance has been somewhat unpredictable,981,982 durable chimerism and renal allograft tolerance were achieved in pigs receiving in utero transplantation of T cell–depleted adult bone marrow.983 Acquired immune tolerance was first demonstrated by Medawar and colleagues, who injected allogeneic cells and tissue lysates into neonatal mice.984 Untreated neonatal rodents can achieve skin graft tolerance if they are given allogeneic hematopoietic cells shortly after birth. Lasting microchimerism and sometimes sufficient levels of chimerism for flow cytometric detection (macrochimerism) have been observed.985–987 Both intrathymic and extrathymic deletional mechanisms of tolerance have been implicated,985,986 and regulatory cells probably play a role, as tolerance cannot be easily broken by the infusion of nontolerant host-type lymphocytes.985,988,989 Neonatal mice tend to produce Th2 responses, which have been implicated in tolerance.362,363 The ability of allogeneic spleen cell infusions to induce tolerance may also reflect the high ratio of non-costimulatory APCs (T and B cells) in donor inocula to recipient T cells in the neonate.990

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Adult Recipients. Very low levels of chimerism (“microchimerism”) can exist for many years in the tissues of human recipients of solid organ allografts who did not receive HCT.991 This observation led to the hypothesis that microchimerism, resulting from emigration of passenger leukocytes from the graft to recipient tissues, leads to donor-specific tolerance.992 Microchimerism, which requires highly sensitive techniques for its detection, should be distinguished from the mixed chimerism discussed previously, in which multilineage chimerism is readily measurable by flow cytometry. Mechanisms by which microchimerism might promote peripheral T-cell tolerance include nonprofessional APC function of donor-derived B or T cells993–995 and “veto” activity of T cells and NK cells.996 However, microchimerism has not been shown to be required for or even necessarily associated with tolerance in a variety of models.997–1001 Donor bone marrow cell infusions without specific myelosuppressive conditioning have been evaluated in efforts to achieve tolerance in patients receiving organ transplantation with standard chronic immunosuppressive therapy.1002,1003 Such transplants were initially associated with significant risks1004,1005 without having a measurable impact on acute rejection episodes or immunosuppressive medication doses in kidney1006,1007 or liver1008 transplant recipients, despite the persistence of and gradual increase in low levels of chimerism.1007 A similar approach failed to achieve islet allograft tolerance in patients.1009 However, long-term kidney allograft survival was favorable in donor marrow–infused compared to noninfused kidney graft recipients,1010,1011 and immunologic assays suggested that bone marrow promoted donor-specific unresponsiveness.1012 Immunosuppression withdrawal was not achieved. Macroscopically detectable, though transient, chimerism has been observed in a nonhuman primate model that includes sublethal TBI.1013 Myelosuppressive host conditioning may promote marrow engraftment by creating physical niches due to the destruction of host hematopoietic cells and by upregulating cytokines that promote hematopoiesis. Upregulation of chemokine SDF-1, which binds to stem cell CXCR4, and complement (C3) cleavage fragments are implicated in radiation-induced marrow injury and promotion of stem cell homing to that microenvironment.1014 In mice, myelosuppression is required to promote engraftment of syngeneic marrow cells given in conventional doses,899 but this requirement can be avoided using very high doses of marrow.1015,1016 Engraftment of high doses of allogeneic marrow can be achieved without myelosuppressive treatment in mice and pigs receiving T cell–depleting mAbs,900,1017 or in mice receiving costimulatory blockade.897,902 The ability of expanded recipient Treg cells to promote chimerism in nonmyelosuppressed recipients of conventional marrow doses903 and advances in the ability to expand hematopoietic stem cells ex vivo1018,1019 hold promise for the achievement of mixed chimerism without host myelosuppression in the future. Strategies to Achieve Peripheral Tolerance Antigen Infusion. DST/gene therapy with autologous cells: In rodents, DSTs have the ability to prolong allograft survival,1020 and in certain donor/recipient combinations

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have been shown to induce operational tolerance.1021 In most other species, DST alone has a less dramatic impact on graft outcome, but there is no doubt that it can influence graft survival in a positive manner and even in humans has been shown to be beneficial to graft outcome.1022,1023 However, alongside these potential positive effects, infusion of cells expressing alloantigens from the organ donor can also have negative effects, including sensitizing the recipient to donor alloantigens, thereby increasing the risk of HAR. Moreover, with the introduction of erythropoietin into clinical practice, there was no longer a medical need to use transfusions to treat dialysis patients on transplant waiting lists; thus, the practice has largely stopped. Investigations into the mechanisms by which donor alloantigen, either following infusion or as a result of donor antigens released from the graft at the time of transplantation, modifies the immune response to a subsequent transplant have provided important insights into how the adult immune system can be manipulated in vivo. Studies in rodents have shown that the level of induced unresponsiveness varies considerably depending on the quantity and source of alloantigen infused, as well as the immune status of the recipient.1024 High doses of donor alloantigens expressed by cells of haematopoeitic origin can result in deletion of donor-reactive leukocytes, even in a host that is otherwise naïve. Lower doses of antigen resulted in the development of T cells with suppressive properties (see the following discussion). Interestingly, it was found not to be necessary to pretreat recipients with every donor major and MiHA they would subsequently encounter on the allograft. Exposure to a single donor alloantigen was sufficient to induce some graft prolongation as long as the allograft also expressed the same alloantigen.1025,1026 This phenomenon was demonstrated in a number of experimental systems involving both transplantation and autoimmune disease, and is referred to as linked unresponsiveness or suppression.1027–1029 The mechanisms underpinning this effect have subsequently been elucidated and involve Treg cells1030 that once activated can mediate so-called bystander suppression, a mechanism identified initially in vitro837 that also operates in vivo, thereby influencing the functional activity of other leukocytes present in the same microenvironment.838 An alternative strategy to using allogeneic cells for inducing specific unresponsiveness is to manipulate recipient cells to express defined MHC molecules that are subsequently expressed by the graft. The first proof-of-concept study using this approach transfected a fibroblast cell line of recipient origin with a mouse MHC class I gene, H2K b.1026 When the transfectants were used to pretreat recipient mice before transplantation, graft survival was prolonged. Obviously, cell lines are not applicable to the clinical situation, and therefore recipient or autologous bone marrow cells and hematopoietic stem cells were investigated as alternatives. This approach, using either replication defective retroviral or adenovirus constructs, has permitted markedly prolonged survival of class I–disparate skin grafts bearing the class I gene that was introduced into the autologous marrow1031 and fully allogeneic heart allografts.1032 In some settings, tolerance to the transduced antigens can be induced either by infusing

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the transduced cells alone1033 or by combining the transduced cells with other immune-modulating agents.1034,1035 As with other approaches to tolerance induction, the persistence of the antigen, either in the form of the transduced cells, or after transplantation, from the graft, is critical to the maintenance of the unresponsive state in vivo.1036 Tolerogenic Antigen-Presenting Cells. DCs are central to the activation/priming of an immune response, but paradoxically they can also promote the development of tolerance.173,1037,1038 One of the keys to both effects is the state of maturation of the DC when it functions in vivo and/or its lineage. Initially, immature myeloid DCs that express low levels of MHC class II and costimulatory molecules at the cell surface were identified as the dominant form of DCs that had the capacity to induce T-cell tolerance. In contrast, mature myeloid DCs expressing much higher levels of both MHC and costimulatory molecules were required for priming T-cell responses. However, mature DCs have subsequently also been shown to have the capacity to induce tolerance; therefore, the relationship between the state of maturity of a DC and its tolerogenic potential is now less clear. In addition, plasmacytoid DCs (pDCs) have also been found to have a role in tolerance induction. pDCs were originally defined by their capacity to secrete large amounts of type I IFNs in response to viruses and to play an essential role in protection against inflammatory responses to harmless antigens, but they have now also been shown to be able to induce human Treg cells in vitro that produce significant amounts of interleukin IL-10, low IFNγ, and no IL-4, IL-5, or TGF-beta.843 pDCs, unlike myeloid DCs, can rapidly express inducible T-cell costimulator-L upon maturation, which has been found to play a key role in the generation of IL-10–producing Treg cells.1039 In mice, pre-pDCs appear to be capable of facilitating hematopoietic stem cell engraftment, thereby promoting donor-specific skin graft tolerance in allogeneic recipients.1040 However, the exact role and practicality of using pre-pDCs in this setting requires clarification as T cells are more abundant and hence more powerful in promoting engraftment. Significantly, donor-derived pre-pDCs infused 7 days before transplant were found to prolong subsequent heart allograft survival (from 9 to 22 days) in the absence of immunosuppressive therapy,1041 but this effect was markedly enhanced by anti-CD154 mAb administration.1042 Immature DCs have been shown to promote tolerance to solid organ allografts and bone marrow grafts. For example, a single injection of immature donor-derived DCs 7 days before transplantation of an MHC-mismatched heart allograft extends1043 or prolongs survival indefinitely1044 in a donor-specific manner. The potential tolerogenic effects of immature DCs can be potentiated by the coadministration of immune-modulating agents such as costimulation blockade.1045 Among several distinct approaches to generate stably immature DCs, pharmacologic manipulation may offer a promising and clinically applicable option. For example, sirolimus has been found to inhibit DC maturation and their effector functions.1046 “Alternatively activated” or “regulatory” DCs, which have low costimulatory ability, were also found to protect MHC-mismatched skin grafts

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from rejection and to protect mice from lethal acute GVHD when administered 7 days before transplantation.1047 Reports showing that mature DCs can induce tolerance despite expressing high levels of MHC and costimulatory molecules include in vitro data with human cells demonstrating that maturation of human monocyte-derived DC with TNF-α and prostaglandin E2 triggered cross-priming and proliferation of CD8 + T cells with tolerogenic properties1048 and that mature but not immature DCs can prime CD4 + T cells that inhibit allogeneic mixed leukocyte reactions.1049 In vivo, bone marrow–derived DCs matured with TNF-α , but not lipopolysaccharide or antibody to CD40, protected mice from CD4 + T cell–mediated experimental autoimmune encephalomyelitis, despite the expression of high levels of MHC class II and costimulatory molecules.1041,1042,1050 The molecular mechanisms used by pDCs to promote tolerance are complex,1051 including evidence that they can promote the differentiation of Foxp3 + Tregs. In transplantation models, tolerizing pDCs can acquire alloantigen in the allograft and then migrate through the blood to home to peripheral lymph nodes where they induce the generation of CCR4 + CD4 + CD25 + Foxp3 + Tregs.1052 Infusion of donor-derived pDCs 7 days before transplant were significantly found to be capable of prolonging subsequent heart allograft survival (from 9 to 22 days) in the absence of immunosuppressive therapy,1041 but this effect was markedly enhanced by anti-CD154 mAb administration.1042 In mice, pre-pDCs appear to be the principal cell type that facilitates hematopoietic stem cell engraftment and induction of donor-specific skin graft tolerance in allogeneic recipients.843 Although the concept that pDCs have the potential to promote transplantation tolerance has been suggested, the role of pDCs in experimental transplant tolerance remains poorly characterized and may be context dependent. Taking the data using different populations of DCs together, it seems that both myeloid and pDCs can promote tolerance, and that maturation by itself is not the distinguishing feature that separates their immunogenic from their tolerogenic function. Indeed, maturation is more of a continuum than an “on-off” switch, and a “semimature” state, in which DCs are phenotypically mature but remain poor producers of proinflammatory cytokines, appears to be linked to tolerogenic function.1053 The combination of DCs administered with costimulatory blockade may be the most promising approach identified thus far. T-Cell–Depleting Antibodies. Many tolerance induction strategies that have been investigated in small and large animals have used depletion of leukocytes (antithymocyte globulin, anti-CD52) or T cells (anti-CD3 with or without immunotoxin, anti-CD2, CD4, and CD8) to create an environment that allows reprogramming of the immune system.1054 In small animals, the short-term depletion of T cells appears to be sufficient in some situations for tolerance to develop and be maintained in the long-term. The success rate can be enhanced by removing the thymus before transplantation to prevent repopulation of the periphery with T cells after transplantation.1055 Initial data from primates

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using anti-CD3 immunotoxin conjugated alone before transplantation or in combination with deoxyspergualin, a drug that inhibits NF-κ B and therefore monocytes and macrophages, at the time of transplantation suggested that T-cell depletion can be used to induce tolerance to donor alloantigens.1056–1058 However, follow-up trials in humans undergoing renal transplantation and T-cell depletion with the anti-CD52 mAb alemtuzemab revealed that neither profound T-cell depletion alone or in combination with deoxyspergualin induced tolerance in humans.1059,1060 Nevertheless, clinical results, across organ systems, reveal that acute rejection is lower1061 and that steroid-free regimens with reduced maintenance doses of immunosuppression may be used after alemtuzemab therapy.1062–1064 Depletion of leukocytes at the time of transplantation creates a transient immunodeficiency in the recipient, compromising the recipient’s ability to reject the transplant. The degree and duration of leukocyte depletion achieved in adults and pediatric transplant recipients determine how effective and for how long the graft is protected from immune attack.1065–1067 However, memory lymphocytes, including those capable of cross-reaction with allogeneic tissues, are not as readily depleted. Thus, while the intense immunosuppression provided by leukocyte-depleting antibodies prevents rejection in the rejection-prone early transplant period,1061 active treatment can promote the homeostatic proliferation expansion of memory cells. The downstream events that occur once leukocytes begin to reappear in the recipient’s circulation are not well understood. An increased proportion of Treg cells after alemtuzumab induction has been demonstrated and found to be most pronounced in calcineurin inhibitor–sparing protocols with early introduction of sirolimus.1068 Interestingly, Treg cells present in the peripheral blood of kidney transplant recipients who had received alemtuzumab induction were able to control the functional activity of Th17 cells that were also present.1069 Marked enrichment of regulatory cells is observed in blood of patients receiving humanized anti-CD2 T cell– depleting therapy in conditioning regimens for BMT.1070,1071 Enrichment for Treg cells has also been demonstrated in mice after treatment with antithymocyte globulin.1072,1073 Studies using TCR-transgenic recipients have shown that when leukocytes are depleted, the maintenance of tolerance depends on transplantation of the graft within a time window of depletion of donor-reactive cells in the thymus and periphery.1074 If the organ graft is transplanted at the appropriate time, donor-specific cells fail to repopulate from the thymus, whereas cells with reactivity to other antigens repopulate the periphery of recipients with long-term surviving organ grafts. These data can be used to suggest a mechanism for the long-term survival observed in primates treated with anti-CD3 immunotoxin complex. In this case, one can argue that the CD3 + T cells are depleted by the immunotoxin before transplantation. A window of opportunity is created such that when a renal allograft is transplanted, donor-reactive cells are absent from the periphery. As cells repopulate the periphery with time after the transplantation, donor-reactive cells are deleted or eliminated as a result of the presence of the surviving graft.

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Although clinical trials utilizing T-cell depletion strategies alone have yet to demonstrate true immunologic tolerance, they have allowed for further larger-scale trials and have fueled combination of this strategy with administration of donor antigen in an attempt to achieve some level of mixed chimerism.1075 Costimulatory Blockade and Other Biologic Proteins With Solid Organs. T-cell activation, and hence rejection, is dependent upon multiple signals. Cell surface costimulatory molecules provide “signal 2” which, when combined with “signal 1” through TCR, triggers the activation of naïve T cells.1076,1077 When signal 1 is forced to act on its own, T cells have been shown to undergo anergy or apoptosis.1078 The original concept of a single costimulatory pathway has long been superseded, and it is now clear that there are multiple levels at which costimulation can participate both in initiating and determining the direction that an immune response takes.1079–1081 Nevertheless, targeting costimulatory pathways with either mAbs or recombinant fusion proteins specific for the costimulatory molecule itself or the ligand with which it interacts can be very effective at suppressing immune responses and in some cases may have the capacity to promote tolerance to donor antigens in vivo. Members of the immunoglobulin and TNF/TNF receptor superfamilies make up many of the costimulatory molecules that are integral to positive costimulation in the pathway of T-cell activation.1082 Two pairs of ligandreceptor interactions that seem to play key roles in positive costimulation are CD40/CD40-ligand (CD154), which are members of the TNF:TNF receptor superfamily and CD80/ CD86 and CD28, which belong to the immunoglobulin superfamily.1083 Although the precise mechanisms that each of these costimulatory pathways plays during rejection and tolerance is still not completely understood, the complete abrogation or attenuation of either of these pathways can modulate the immune response to an allograft in vivo. Data from clinical trials using CD28 blockade as part of an immunosuppressive drug strategy has led to the licensing of CTLA-4-immunoglobulin (abatacept) for clinical use in patients with rheumatoid arthritis and of the mutated version of CTLA-4 immunoglobulin LEA29Y (belatacept) for use in kidney transplantation.1084 Interestingly, anti-CTLA4 therapy (ipilimumab) has also been developed for clinical use, but in this case the approach is to augment immune responsiveness to tumour antigens.1085 The B7:Cluster of Differentiation 28/Cytotoxic T-Lymphocyte Antigen-4 Pathway. CD80 (B7-1) and CD86 (B7-2) are expressed as cell surface molecules by APCs and are responsible for delivering additional signals to T cells when they interact with CD28. CD86 and CD80 can also interact with a second molecule, CD152 (CTLA-4), which is expressed by T cells later in the activation process and is expressed constitutively by Treg cells. CD86 and CD80 can exhibit preferential binding to CD28 and CD152, respectively.1086 In contrast to CD28, CTLA-4 negatively regulates T-cell activation when it engages its ligand on the APC and, as described previously, is implicated in the control of clone size to maintain normal

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homeostasis in the immune system.1087 CTLA-4 is pivotal in regulating the threshold of signals during T-cell activation, and recent findings suggest that upregulation of CTLA-4 expression increases T-cell motility and overrides the TCRinduced stop signal required for stable conjugate formation between T cells and APCs. This results in reduced contact periods between T cells and APCs, leading to decreased cytokine production and proliferation.1088 Utilizing the B7:Cluster of Differentiation 28/Cytotoxic T-Lymphocyte Antigen-4 Pathway for Therapeutics. When CTLA-4-immunoglobulin, an immunoglobulin fusion protein of CTLA-4, was produced, it was shown to inhibit graft rejection in xenogeneic and allogeneic systems1089,1090 and to promote engraftment of allogeneic stem cells.731 In rodent models, CTLA-4-immunoglobulin therapy alone promoted tolerance to human islet xenografts in mice and, in combination with anti-CD154 (see the following), to vascularized allografts,1089,1091 an effect that was enhanced when donor antigen was included in the treatment protocol.1091–1093 The mechanism by which CTLA-4-immunoglobulin promotes long-term graft survival has been investigated in a mouse model. Blockade of CD80 and CD86 at the time of alloantigen recognition has been suggested to trigger T-cell apoptosis in the early phase after transplantation.850,851 However, it should be noted that specific markers for alloantigen-specific T cells were not incorporated into this analysis, and therefore this mechanism has not been definitively demonstrated. When an antiapoptotic gene, bcl-x, was expressed in the responding lymphocytes, deletion did not occur, and graft prolongation was prevented. This finding suggests that CTLA-4-immunoglobulin facilitates graft survival by reducing the number of donor-reactive cells that have to be controlled after transplant. Although primate studies failed to achieve tolerance with CTLA-4-immunoglobulin,1094 the theoretical foundation of blocking this pathway to promote graft survival continues to intrigue researchers.1095 Additionally, it was known that the binding properties of CTLA-4 could be manipulated to optimize the ligation of both CD80 and CD86, a crucial component to experimental efforts at tolerance induction.1083 Belatacept. Experiments using CTLA-4-immunoglobulin laid the groundwork for further development of therapeutic agents targeting the B7:CD28/CTLA-4 pathway. Belatacept, LEA29Y, was originally derived from the fusion protein CTLA-4-immunoglobulin, or abatacept.1096,1097 It differs from CTLA-4-immunoglobulin by two amino acids, conferring an approximately twofold increased binding capacity to CD80 and CD86. This increase in avidity allows for a 10-fold increase in the in vitro suppression of T-cell activation when compared to CTLA-4-immunoglobulin; in nonhuman primate studies, belatacept was found to prolong renal allograft survival and inhibit donor-specific alloantibody production both alone and in combination with other traditional immunosuppressive regimens.1096 These and other findings allowed for the translation of LEA29Y to renal transplant patients. Results from clinical trials comparing belatacept to cyclosporine in partially randomized

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studies across 22 centers in North America and Europe of over 200 patients demonstrated that patients treated with belatacept-based therapy had improved renal function, reduction in chronic allograft nephropathy, and decreased calcineurin-related toxicity.1098 Additionally, recent experiments in nonhuman primates using neonatal porcine islet grafts have revealed long-term xenograft survival under the cover of CD28-CD154 blockade with maintenance immunosuppression of sirolimus and belatacept.1099 The use of belatacept in clinical transplantation has revealed some safety concerns, particularly when high doses were used.1100 Cluster of Differentiation 40-Cluster of Differentiation 154 Pathway. The CD40-CD154 pathway has been targeted to inhibit graft rejection using mAb therapy either alone1101–1103 or in combination with alloantigen infusion.1104 CD154, or CD40-ligand, is a type 2 membrane protein of the TNF family and is expressed predominantly by activated CD4 + T cells and by a small proportion of CD8 + T cells, NK cells and eosinophils,1105 and, significantly, on platelets as well.1106 Structural models predict that CD154 forms a homotrimer that binds to CD40 on the surface of APCs. CD40 is expressed by B cells, macrophages, DCs, and thymic epithelium, and is inducible on the surface of endothelial cells and fibroblasts. The CD40-CD154 pathway interaction is pivotal for the induction of humoral and cellular responses to nominal antigens as well as alloantigens. A CD40-immunoglobulin fusion protein and a blocking mAb to CD154 were shown to inhibit B-cell cycling, proliferation, and differentiation into plasma cells in response to T cell–dependent antigens.1107 In vivo studies using CD154 mAb or CD40 or CD154 knockout mice1108,1109 demonstrated a crucial role for this interaction in the generation of primary and secondary humoral responses to T cell–dependent antigens, class switching, and development of germinal centers. The lack of a humoral response in the absence of CD40-CD40L interaction is due not only to a lack of signaling through CD40 on the B-cell surface, but also to inhibition of priming of CD4 + T cells through CD40L.1110 The CD40-CD154 pathway is bidirectional. Not only does CD154 engagement on T cells augment T-cell activation, but also CD40 triggering on the APC primes the APC for stimulation. Signals through CD40 have been shown to upregulate expression of CD80 and CD86, as well as induce IL-12.1111 Activation of DCs through CD40 promotes their ability to present antigen to T cells; this may explain why targeting CD154 and blocking its ability to interact with CD40 has a profound effect on T cell–dependent immune responses in vivo. Blocking CD154-CD40 interactions may promote tolerance induction by altering both this interaction and signals between APCs and immunoregulatory and suppressor T cells.1112 Utilizing the CD40-CD154 Pathway for Therapeutic Intervention in Cell and Organ Transplantation. Long-term acceptance of cardiac, renal, and islet allografts in several mouse and nonhuman primate models has been achieved with CD40 blockade using anti-CD154 mAb as monotherapy or in conjunction with anti-CD28.1101,1104,1113,1114 However, with

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the exception of BMT, in which durable chimerism and tolerance can be achieved with anti-CD154 mAb,731,1115,1116 tolerance is not generated by anti-CD154 therapy alone in stringent models, in which therapy withdrawal leads to rejection.1117,1118 Further studies in the mouse revealed that only when rejection was dependent upon CD4 + T cells was CD154 blockade on its own effective at prolonging graft survival. In fact, several studies in donor-recipient combinations in which CD8 + T cells also play a role in rejection have shown that the CD8 + T-cell subset is unaffected by CD154 mAb therapy,1119,1120 such that CD8 + T cells become activated, proliferate, and home to the graft in the presence of high-dose continued anti-CD154 mAb therapy. The addition of low dose TBI with donor hematopoietic cells can overcome this CD8 + T cell–mediated resistance.1121 Interest in this approach was also reflected in reports that a humanized mAb specific for CD154 (hu5c8) was capable of prolonging the survival of renal and islet allografts in rhesus monkeys.1094,1102,1122 The initial data from these primate studies appeared encouraging, with rejection-free survival of the kidney grafts, provided that antibody therapy at a relatively high dose (25 mg/kg) was continued in the first 6 months after transplant. When anti-CD154 therapy was discontinued after the first month after transplant, rejection episodes did occur. Analysis of the status of recipients with long-term surviving grafts showed that peripheral lymphocytes from the monkeys failed to respond in vitro to donor antigens, but the recipients developed antidonor antibodies and biopsy samples from some of the long-term surviving grafts revealed T-cell infi ltrates. Together, these observations were sufficiently encouraging to initiate a pilot clinical study using Hu5c8 in renal transplantation. In this study, Hu5c8 was administered to seven patients with low-dose steroid alone, and five patients went on to experience episodes of rejection.1123 Moreover, the unexpected complication of thrombogenesis in some patients treated with anti-CD154 highlighted that CD154 plays a key role in coagulation and clotting, with some reports suggesting that CD154 acts to stabilize thrombi while others implicate CD154 in platelet activation.1106 Other variants of costimulatory blockade that target different epitopes of CD154 have been developed with hopes of improved efficacy in transplantation without promotion of thrombogenesis. Experimental results in cardiac allografts of cynomolgus monkeys treated with an inhibitor of CD154, IDEC-131, either alone or in combination with leukocyte depletion in the form of antithymocyte globulin prolonged allograft survival; however, tolerance was not induced, as alloantibody production and transplant vasculopathy, while delayed, still occurred.1124 Still other anti-CD154 antibodies, such as ABI793, have been developed, but have been plagued with continued thromboembolic complications.1125 Whatever the role that CD154 may play in prolonging allograft survival, it is clear that this molecule acts via independent pathways in a variety of cascades unrelated to tolerance induction.1083 Further studies have been undertaken to evaluate antibodies targeting CD40 in order to bypass the potential

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ramifications of CD154 blockade. Initial animal knockout models revealed a propensity of CD154 knockout mice to develop unstable thrombi, a phenomenon not seen in CD40 knockout mice.1126 Studies in primate renal allograft models reveal promising results with a short course of low-dose calcineurin inhibitor administered concomitantly with anti-CD40/anti-CD86 costimulation blockade1127 or with anti-CD40 therapy alone.1128 Trials using a fully humanized anti-CD40 antiagonist are currently in phase II clinical development. Targeting Cluster of Differentiation 3 and Accessory Molecules. Initially, administration of depleting anti-CD4 and anti-CD8 mAbs was shown to result in prolonged graft survival.1129–1131 That this treatment strategy resulted in antigen-specific tolerance was first shown most clearly when a protein antigen was administered in conjunction with a depleting anti-CD4 mAb.1132–1134 Refinements of these types of protocols have resulted in the ability to achieve long-term T-cell unresponsiveness to protein and alloantigens in the absence of T-cell depletion in experimental models.1135–1137 In fact, many other accessory molecules, other than anti-CD4 and anti-CD8, have been targeted in an attempt to induce tolerance in models of bone marrow,1138,1139 islet,1140 renal,1141,1142 and cardiac allografts,1139,1143 to name a few.1144 OKT3, a murine antihuman CD3 mAb, received approval for human use in 1986 in kidney transplant patients undergoing rejection and eventually for liver and cardiac transplant recipients as well.1145 Although widely used, OKT3 brings with it the undesired complications of the human antimouse antibody response as well as a first dose reaction characterized by fevers, chills, and gastrointestinal, respiratory, and cardiac complications.1146,1147 These ramifications are thought to be the result of CD3 cross-linking activating the signaling pathways downstream of the TCR, leading to transient T-cell activation and subsequent cytokine release.1145 Therefore, investigators have attempted to construct pharmacotherapeutics that mimic the efficacy of OKT3 with less immunogenicity.1148 These designer antiCD3 monoclonals retain T-cell stimulatory capacity, albeit greatly impaired, as a consequence of their failure to readily cross-link targeted CD3 molecules on the cell surface. Interestingly, these antibodies stimulate T cells to express TGF-β, a potent, immunosuppressive cytokine, an effect crucial to immune tolerizing effects achieved in autoimmune models.1148 Early clinical studies using the humanized “nonstimulatory” anti-CD3 antibodies, teplizumab and otelixizumab, showed some benefit in patients with new onset type 1 diabetes,1145,1149–1151 though lasting insulin independence was not achieved. While anti-CD3 mAbs have the potential to tilt the balance of immunity toward tolerance, adjunctive agents may be required to enable use safe nontoxic doses in attempts to reinstate tolerance. As yet, neither has been introduced into clinical transplantation. Along with anti-CD3, antibodies to CD11a (LFA-1) and its ligands, ICAM-1, -2, and -3, have been investigated and have led to prolonged graft survival in many of the aforementioned models. LFA-1 has been implicated as an essential molecule for cellular trafficking and motility as well

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as T-cell activation.1152,1153 Additionally, the interaction of LFA-1 and the ICAM molecules may serve as a costimulatory pairing for T-cell activation.1154 Preclinical studies in transplant models have shown that combining anti–LFA-1 with costimulatory blockade may be efficacious1155 ; these findings are supported by results of a pilot clinical study using efalizumab in islet transplantation.1156 Operational tolerance induced in animals by these strategies has been shown to develop over several weeks after the initial antigen encounter.1157,1158 When a combination of donor antigen and mAb therapy targeting accessory molecules is used, the precise mechanism of tolerance induction in part depends on the amount of antigen infused.1137 Deletion also may be one of the initial mechanisms of tolerance,1024,1159 but in the absence of complete deletion of donor-reactive cells, immunoregulation develops. When antibodies targeting accessory molecules are used as therapeutic agents at the time of transplantation, immunoregulation is the dominant mechanism that comes into play to maintain tolerance in the longer term. In these systems, tolerance to donor antigens is induced and/or maintained as a result of the development of a population of Treg and suppressor T cells that can mediate unresponsiveness to the initiating donor antigen as well as other antigens present on the graft (linked unresponsiveness).1160 In mice and rats, this type of tolerance has been shown to be infectious1161; it can be transferred from one generation of cells to another, provided that there is sufficient period of contact between the two populations. The maintenance of tolerance in these systems requires the persistent presence of antigen in the form of the organ when the thymus is still functional.1162 In the absence of donor antigen, tolerance is eventually lost, presumably as a result of the export of naïve T cells from the thymus into the periphery. Quantitatively, if these cells fail to encounter antigen, they eventually outnumber the unresponsive T cells induced by the monoclonal antibody therapy.

Costimulatory Blockade with Infusion of Donor Cells While long-term tolerance is maintained by intrathymic deletion in mixed chimeras prepared with costimulatory blockade,731,897,898 initial tolerance of peripheral T cells involves specific deletion of donor-reactive CD4734 and CD8533 T cells. The deletion of CD4 cells achieved following BMT with anti-CD154 is not due to binding of anti-CD154 mAb to activated T cells, and only requires absence of the CD154-CD40 interaction.1163 Peripheral deletion of donorspecific CD8 cells occurs within 1 to 2 weeks. CD25-negative CD4 cells are required for CD8 tolerance only during this initial 2-week period.533 Deletion of peripheral donor-specific CD4 and CD8 cells is preceded by specific unresponsiveness toward the donor.550,734,1163 Regulatory cells do not appear to play a major role in maintaining the long-term tolerance in this model, as tolerance and chimerism are obliterated by the infusion of relatively small numbers of nontolerant recipient-type lymphocytes and linked suppression is not observed.734 Because hematopoietic stem cell engraftment ensures complete central deletional tolerance in these longterm chimeras,731,732,897,898 and specific peripheral deletion is

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quite complete, the absence of donor-reactive T cells may preclude the expansion and maintainence of specific regulatory mechanisms. Administration of DST with anti-CD40L mAb prevents islet allograft rejection,1164 but in contrast to BMT, does not achieve central deletion896,897 and is most effective in thymectomized mice.540,729,1165 The inability to resist breaking of tolerance by new thymic emigrants in this model argues against powerful peripheral regulatory mechanisms, but CD4 cells are needed to promote skin graft acceptance.540 DST promotes peripheral deletion of donor-reactive CD8 T cells729 and can be replaced by CD8 depletion with mAbs.1166 The combination of anti-CD154, BMT, and DST seems to be particularly effective in achieving mixed chimerism, largely because of the capacity of DST to overcome residual CD8 T cell–mediated alloresistance.730,1167 Costimulatory blockers can also be used to prevent GVHD, using B7/CD28 blockade or anti-CD154 mAb,1168–1170 or preBMT exposure of donor T cells to recipient alloantigens in the presence of anti-CD40L.1171 Clinically, HLA-mismatched BMT has been attempted with T cells anergized by exposures to recipient alloantigens in the presence of CD28/B7 blockade, with apparently reduced GVHD.1172,1173

A Large Animal Model MHC-defined, inbred miniature swine have provided an instructive model for delineation of the role of various histoincompatibilities in tolerance and rejection in large animals. Studies of pig renal transplantation have demonstrated that spontaneous tolerance can be induced by organ grafts when MHC antigens are matched. The ability to achieve such tolerance is dependent on one or possibly two non–MHC-linked genetic loci in the recipient animals. The presence of the “acceptor” phenotype also permits the spontaneous acceptance of single haplotype class I–mismatched kidney grafts.7 Graft acceptance is associated with donor-specific CTL unresponsiveness, apparently due to a deficiency in help for these CTL, and not due to a deletional mechanism. Thus, in class II–matched, class I–mismatched porcine donor–recipient pairs, a 12-day course of high-dose (10 mg/kg/day) cyclosporine (CsA) permits long-term renal allograft acceptance in 100% of cases.7 The requirement that class II antigens be matched between donor and recipient in order for this tolerance to be achieved may reflect the influence of a major difference in class II antigen expression that exists between large and small animals. Unlike large animals and man, in which class II antigens are expressed constitutively on vascular endothelial cells, the corresponding endothelial cells of rodent species do not express MHC class II molecules.18,19 Consistent with this interpretation, the use of a short course of CsA can facilitate the ability of renal allografts to induce tolerance in rodents across fully MHC-mismatched barriers, but tolerance induction in swine requires class II matching between donor and recipient for uniform success. Animals accepting class II–matched allografts are systemically tolerant to the donor’s class I and minor antigens, as indicated by the fact that the accepted graft can be removed and replaced by a second donor-matched graft,

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which is accepted without immunosuppressive therapy. This ability of CsA to facilitate tolerance induction, and the ability of exogenous IL-2 to prevent the induction of tolerance in this model,7 is consistent with the interpretation that induction of tolerance of donor class I–reactive CTLs is due, at least in part, to the absence of adequate T-cell “help” during the time of initial exposure to antigen. A selective decrease of expression of the Th1-associated cytokine IFNγ relative to the Th2-associated cytokine IL-10 has been observed in these accepted grafts.1174 The thymus appears to play a role in the induction of tolerance among preexisting peripheral T cells in this model, as removal of the host thymus prior to kidney allotransplantation leads to rejection.1175–1177 The possible mechanisms responsible for this role of the thymus in inducing peripheral tolerance phenomena are discussed elsewhere in this chapter. The kidney allograft itself clearly plays an important role in the tolerance induced in this model. Class II–matched cardiac allografts are not accepted after a similar short course of CsA, but they are accepted if grafted to animals that are tolerized in this manner to kidney allografts bearing the same mismatched class I alleles as the donor heart.631 The mechanisms of this tolerance has been demonstrated to involve Treg cells that can specifically suppress antidonor CTL responses.1178–1180

The Relationship between Peripheral T-Cell Tolerance and Central Tolerance The thymus can play an active role in tolerizing peripheral T cells. For example, in the pig model involving a short course of CsA with kidney transplantation, the recipient thymus is required to achieve tolerance among preexisting peripheral T cells.1176 In rats, peripheral tolerance can be achieved by intrathymic injection of donor antigens combined with peripheral organ transplantation.1181–1183 The capacity of T cells that are activated in the periphery to migrate to the thymus,1184–1186 migration of donor graft APCs to the thymus where they induce thymic T-cell deletion,1187,1188 or the development of Treg cells that migrate to the periphery and promote tolerance1189,1190 may contribute to this role for the thymus in peripheral tolerance induction. Tregs have indeed been implicated in both the pig model and the rat intrathymic injection model.1179,1191,1192 The intrathymic injection approach has not been successful in “high responder” rat strain combinations1193,1194 and has not successfully prevented xenograft rejection or chronic rejection of cardiac allografts.1195 While one attempt to use this approach in nonhuman primates was discouraging,1196 donor-specific skin graft prolongation was reported in three animals receiving allogeneic or xenogeneic (human) CD34 + cells intrathymically.1197 From Animal Models to Clinical Transplantation Tolerance Almost every transplant clinician has treated patients who have chosen to withdraw immunosuppressive therapy for a variety of reasons. While the majority of such patients reject their allograft, rare cases achieve “spontaneous” tolerance in this manner, demonstrating that tolerance can be achieved in humans. Blood biomarkers have been described

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that identify tolerant patients who have been successfully weaned from immunosuppression,1198–1200 but so far none have been used to identify such patients before immunosuppression withdrawal and thereby shown to have predictive value. In general, short-term results of most organ allograft transplants are excellent, making it essential to have reliable methods of inducing tolerance in order to ethically justify their use in place of conventional chronic immunosuppressive therapies. Because the risk of rejection due to immunosuppression withdrawal is difficult to accept in patients, it is our opinion that extension of results from animal models to humans should only be attempted after 1) rodent studies have demonstrated robust tolerance in multiple strain combinations using extensively histoincompatible, highly immunogenic grafts such as skin; 2) efficacy has been demonstrated in large animal models; and 3) acceptable toxicity has been demonstrated in large animal models. When complete removal of immunosuppression is likely to be achieved, the level of toxicity accepted in the short term may be somewhat higher than that which could be acceptable on a longterm basis. Most efforts to achieve peripheral tolerance in larger animals have not been as effective as in rodent models. The extensive animal data that have been accumulated on the ability of mixed chimerism induced with reduced intensity conditioning to achieve robust transplantation tolerance, including its demonstration in large animal models,1013,1201 combined with clinical data obtained in patients with a more conventional indication for HCT, have allowed clinical evaluation of this approach for tolerance induction. Mixed chimerism can be achieved with reduced toxicity using nonmyeloablative conditioning in patients with hematologic malignancies,1202 and lymphohematopoietic graft-versus-host reactions induced by donor lymphocyte infusion (DLI) can be used to achieve graft-versus-tumor effects.1203 These observations provided an opportunity to evaluate the potential of this approach to induce transplantation tolerance in patients with a hematologic malignancy, multiple myeloma, and consequent renal failure. Recipients of a simultaneous nonmyeloablative BMT and renal allograft from HLA-identical siblings achieved either transient or durable mixed chimerism or, in a few cases, full allogeneic chimerism (some after DLIs). All three subsets of patients accepted their kidney grafts without any immunosuppression for follow-up periods of up to 14 years, with very good tumor responses.1204,1205 In vitro studies suggested that, in patients who lost their chimerism, tolerance may be specific for donor antigens expressed by the kidney, while responses to antigens expressed on hematopoietic cells but not the kidney may even be sensitized.1204 Treg cells may also be involved.634,1204 These promising efficacy results, combined with safety data in patients receiving HLA-mismatched transplants for hematologic malignancies,1206 cleared the way for trials of HLA-mismatched haploidentical bone marrow and kidney grafts in patients with no malignant disease (ie, BMT was performed solely for allograft tolerance induction), using a regimen that had led to transient mixed chimerism without GVHD in an initial cohort of patients with hematologic malignancies. Donor kidneys were accepted off immunosuppression in 7 of 10 patients, with

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follow-up for as long as 10 years in the first patient at the time of this writing. These studies were the first intentional protocol to achieve transplantation tolerance across HLA barriers in humans. Mechanistic studies implicated regulatory cells and the kidney graft itself as possible contributors to long-term, systemic, donor-specific tolerance that may involve eventual peripheral deletion of donor-reactive T cells.635,636,1207,1208 Several additional protocols have recently used different nonmyeloablative HCT protocols to achieve allograft tolerance across minor histocompatibility615,617 or HLA1209 barriers. The different mechanisms involved with these various approaches have recently been reviewed.1210,1211 Both Tregs and inducible NKT cells have been implicated in the HLA-identical combined kidney-BMT protocol developed at Stanford.615,617,1211 A poorly understood “facilitating cell” population was suggested to be involved in the full chimerism achieved across HLA barriers, reportedly without GVHD, in five patients at Northwestern University. However, the safety of this latter protocol remains to be established, and a life-threatening complication led to infusion of recipient lymphocytes for rescue, resulting in graft rejection in one of the patients.1209 Animal studies would predict that these full chimeras with extensive (up to five of six) HLA mismatches might not regain full immunocompetence.891,892

TRANSPLANTATION OF SPECIFIC ORGANS AND TISSUES Kidney Transplantation Kidneys are the most frequently transplanted human organs, with approximately 10,000 kidney transplants being performed annually in the United States. Renal allograft survival has increased steadily since its inception, and at present there is over 95% patient survival and over 91% organ survival at 1 year.1212 However, even well-matched recipients of renal transplants must continue to take immunosuppressive medications for the rest of their lives, with complications including an increased risk of infection, cancer, hypertension, and metabolic bone disease. In addition, even when immunosuppression is well managed, there is an inexorable loss of kidneys to chronic rejection at a rate of approximately 5% to 7% per year. Both of these problems might be eliminated by the induction of tolerance, for which clinical trials have recently begun in earnest615,635,1204,1208,1213,1214 (see previous discussion). As for other organs, a major obstacle in kidney transplantation is the shortage of organs, which has been a paradoxical consequence of the success of this field. Unlike hearts and livers, where the inadequate supply of cadaveric organs spells death for many potential recipients, candidates for renal transplants are instead faced with long periods on dialysis while they wait for a kidney. This waiting time can be 4 or more years even for unsensitized candidates and even greater for those with high levels of sensitization. To avoid this delay, many patients are now being offered kidneys from living donors, and in many transplant centers the number of living donor renal transplants performed per year now exceeds the number of transplants from cadaver donors. Sensitization of renal transplant candidates usually results from prior antigen exposure, either by blood transfusions

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or previous transplants. Such highly sensitized patients are said to have high “panel reactive antibody”; they may wait many years to obtain a kidney that is cross-match negative, and some never receive a transplant at all, despite the recent development of antibody desensitization procedures.1215 Indeed, as discussed in the following, these patients may be among the first to benefit if and when xenotransplantation becomes a clinical reality.1216 Finally, late failure of kidney transplants, or “chronic rejection,” is a continuing problem to the field. This form of graft loss may occur many years after transplantation and it probably involves both immunologic reactivity and other factors, including the effects of the early ischemic injury and the ongoing effects of drugs and metabolic abnormalities in the recipient.1217 This process leads to an inexorable loss of organs. Thus, although the 1-year survival rate for kidney transplants has improved from roughly 40% to over 90% over the past 40 years, at 5 years there is only 71% graft survival. Indeed, the half-life for kidney transplants has barely increased during the same period and remains less than 10 years.

Liver Transplantation Transplantation of the liver represents a major technical challenge. For this reason, the organ and patient survival rates have not been as high as those for renal transplantation, but have nevertheless increased yearly, with a current 1-year organ survival rate of about 80% and patient survival rate of about 86%.1212 There are currently about 6,000 liver transplants carried out in the United States per year. The severe shortage of available organs has recently led to use of living donors, although the number is still small, accounting for only about 5% of liver transplants, or about 300 cases per year. Immunologically, the liver is relatively resistant to early antibody-mediated rejection, allowing successful transplantation even across blood group barriers and in the face of a positive cross-match, although a negative cross-match is generally considered preferable.1218–1220 In addition, longterm survival of liver transplants does not appear to be improved by HLA matching between donor and recipient,1221 and many centers do not even type recipients and donors. Both of these immunologic features may be responsible for the fact that the long-term results of liver transplantation are almost as good as those for kidney, with 65% graft survival and 71% patient survival at 5 years. Finally, transplantation of the liver carries with it large numbers of donor lymphoid cells, thus creating the potential for inducing GVHD.1222 The symptoms can range from antibody-dependent hemolysis of recipient red blood cells across a blood-group incompatibility to severe or even fatal, full-blown GVHD.

Heart and Lung Transplantation There are approximately 2,000 heart transplants performed annually in the United States. As might be expected for this vital organ, the 1-year statistics for heart transplantation are approximately the same for patient (87%) and graft (86%) survival.1212 One of the immunologic issues of particular importance in heart transplantation is the high rate of

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atherosclerotic disease in the coronary arteries of the transplanted organ. This atherosclerotic disease is generally considered to be a manifestation of chronic rejection, although the causes appear to include more than just the immune response. Nevertheless, patient and graft survivals are about 70% for heart transplants at 5 years. The number of lung transplants performed annually in the United States is approximately 1,200, while only about 30 combined heart and lung transplants are performed. The statistics for lung transplant survival are about the same as for liver (82% graft survival and 83% patient survival) over the first year, but then decrease much faster, with 5-year survivals of only about 45% for grafts and 46% for patients.1212 A major cause of graft loss is a process called bronchiolitis obliterans, thought to be the pulmonary manifestation of chronic rejection.1223 However, these patients are also highly susceptible to pulmonary infections, which undoubtedly contribute to the poor long-term statistics.

Pancreas and Islet Transplantation Transplantation of the whole pancreas was almost without success until about 1980, largely for technical reasons. More recently, successful pancreas transplantation to treat diabetes mellitus has been achieved using new technical approaches, with success rates of about 92% at 1 year, approaching those for kidney transplantation, as long as the two organs are transplanted together. There are approximately 850 simultaneous kidney/pancreas transplants per year in the United States. The lower survival rates (77% graft survival at 1 year) achieved when pancreas transplantation is performed alone (about 500 cases annually) probably reflect the difficulty in diagnosing rejection episodes involving this organ. By the time blood sugar levels begin to rise, destruction of the pancreas is generally so far advanced that it cannot be reversed by immunosuppressive therapy. Measurement of the serum creatinine, reflecting early dysfunction of a simultaneous kidney transplant, allows much earlier detection of rejection and, thus, better outcomes. Simultaneous transplantation of both a kidney and a pancreas from a single donor has demonstrated, surprisingly, that rejection activity in one organ is not always associated with rejection activity in the other. It is not known whether this occasional dichotomy reflects tissue-specific antigens or localized inflammatory (or regulatory) events in one but not the other organ. Most pancreas transplantation is carried out as a curative treatment for diabetes mellitus. For this purpose, the potential also exists of transplanting only the insulinproducing islet cells rather than the whole pancreas. In rodent models, both allogeneic and xenogeneic islet transplants have successfully achieved normoglycemia in diabetic animals.1224,1225 Attempts to extend these results to patients, however, have met with limited success,1226,1227 and until recently, the results of whole organ pancreas transplantation in correcting the hyperglycemia of diabetes have been far superior to those of islet transplantation.1228–1230 In 2000, the “Edmonton Protocol”1231 showed much improved survival of islet transplants at 1 year in 4 of 12 patients, using a new

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combination of immunosuppressive drugs that appeared to cause less damage to transplanted islets. However, this protocol required use of two or sometimes three pancreases for a single recipient, and by 5 years, in a much larger series of patients, only 10% of patients have remained insulin independent.1232–1234 Thus, these results, while representing an improvement, remain much inferior to the results of whole organ pancreas transplantation.

Hematopoietic Cell Transplantation BMTs, and more recently, transplants of hematopoietic stem cells and progenitors mobilized from the marrow into peripheral blood by treatment with granulocyte-colony stimulating factor, are used most commonly for the treatment of otherwise incurable leukemias and lymphomas, aplastic anemia, and congenital immunodeficiency states. Additional applications include hemoglobinopathies and inborn errors of metabolism. Autologous HCT is used for hematologic rescue following high-dose chemo-/radiotherapy for the treatment of malignancies and is currently being explored as a treatment for autoimmune diseases. However, autologous HCT will not be considered further here as it does not involve the broaching of any immunologic barriers. One fundamental difference between HCT and the transplantation of all other organs is that the recipient’s treatment for his or her malignancy usually results in ablation of the immune and hematopoietic systems prior to transplantation (ie, the “conditioning” for transplantation is myeloablative). Originally, hematopoietic cell allografts were administered only as a means of replacing ablated host hematopoiesis. However, clinical experience revealed that one of the main therapeutic benefits of allogeneic HCT is due to the GVL effect of donor lymphocytes.1235 With the recognition of this immunotherapeutic benefit of allogeneic HCT, clinicians began to evaluate less toxic, nonmyeloablative conditioning as a means of allowing allogeneic marrow to engraft so that donor lymphocytes can mediate GVL effects.1202,1203,1236,1237 In contrast to HCT for malignancies and other indications in immunocompetent recipients, transplantation for immunodeficiency states does not require myeloablation or immunoablation in order to achieve alloengraftment. Another major distinguishing feature of HCT is that the recovering immune system in durable chimeras is tolerant of the donor alloantigens, so there is no requirement for immunosuppressive therapy to prevent allograft rejection once the initial immune resistance to the allograft has been overcome. However, initial rejection must be prevented in order to achieve this state. Mechanisms of hematopoietic cell rejection have been discussed previously. In humans, while the importance of class I mismatching, and particularly that of HLA-C,1238 might suggest a role for NK cell–mediated rejection, clear evidence for NK cell–mediated rejection has not yet been obtained.1239 In contrast, a role for classical CTLs is well established in the rejection of even HLA-identical donor marrow following myeloablative conditioning,1240 particularly when the donor product is T cell depleted1241,1242 or lowintensity conditioning is used.1243 Expanded CTLs and or Th against donor MiHAs have been detected in association

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with expanded recipient T cells in patients losing chimerism after nonmyeloablative HLA-identical HCT.1204,1244 Rejection occurs more frequently in the setting of HLA-mismatched related or unrelated donor transplants1238,1245 and increases even further with T-cell depletion.1246,1247 Antidonor CTLs specific for the mismatched donor class I1248 or class II28,634 allele and class II–specific helper cells634 have been associated with HLA-mismatched marrow rejection. Donor-specific alloantibodies in presensitized patients are predictive of rejection of mismatched transplants.1249 A third unique feature of HCT (as well as transplants of other organs that are rich in lymphoid tissue, such as small intestinal grafts and, to a lesser extent, liver grafts) is the ability of T cells in the allograft to mount an immunologic attack on the recipient’s tissues, resulting in GVHD. While GVHD rates and severity are reduced using prophylaxis with nonspecific immunosuppressive drugs, GVHD still affl icts 30% to 50% of recipients of HLA-matched sibling HCT, though this may be reduced by substituting a mammalian target of rampamycin inhibitor for calcineurin inhibitors.1250 HCT can be extended to additional patients by including umbilical cord blood registries in the donor search, as HLA mismatching is better tolerated in cord blood, possibly due to the naivety/immaturity of the immune cells.1251,1252 The frequency and severity of the GVHD that develops when extensive HLA barriers are traversed has essentially precluded the routine performance of such transplants from adult donors, though some recent clinical trials using nonmyeloablative conditioning have achieved acceptable GVHD rates.1206,1253 The establishment of large marrow donor registries and the recognition of common HLA haplotypes has permitted the performance of closely matched transplants from unrelated donors, but these transplants are also associated with a high incidence of severe GVHD, due to the existence of subtle HLA mismatches and more extensive minor histoincompatibilities13,1251,1254–1256 (see section on Donor Antigens Responsible for Graft Rejection). The GVL effect of allogeneic lymphocytes, especially T cells, transferred with the donor graft, is due largely to recognition by donor T cells of host alloantigens, which are also expressed on malignant cells. Therefore, while T-cell depletion of the donor graft decreases the incidence of GVHD, this benefit is offset by increased relapse rates1256 and increased risk of engraftment failure due to rejection.1246 Graft rejection can be offset by more intensive host conditioning and high doses of donor hematopoietic stem cells, even with extensively (one haplotype) HLA-mismatched stem cell grafts,1257 but at the expense of delayed immune reconstitution.1257,1258 While T cell–mediated GVL effects due to graftversus-host reactivity are greatly diminished with such an approach, this may be compensated for by the ability of NK cells to mediate GVL when the host lacks inhibitory MHC ligands recognized by the donor, at least for acute myelogenous leukemia.424 Such alloreactive NK cells may also help to promote donor engraftment by eliminating alloresistant host T cells.424 A major and elusive goal in the HCT field has been to separate the GVL effect of donor T cells from their potential to cause GVHD. Recently, several new approaches

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for inhibiting GVHD have been attempted.1259 The use of costimulatory blockade has been discussed previously. Blockade of proinflammatory cytokines such as TNF-α and IL-1 has shown some efficacy in animal models but were less effective in clinical trials.386 Many other strategies, such as immune deviation and the use of NKT cells, have been discussed elsewhere in the chapter. Another approach to separating GVL from the GVHDinducing capacity of MHC-directed alloreactivity is to separate the HCT and the administration of donor T cells in time, so that the T cells are given after some host recovery from the initial conditioning regimen has occurred. Established mixed hematopoietic chimeras are immunologically tolerant of their original marrow donor’s antigens. As expected, a graft-versus-host reaction occurs after administration of nontolerant DLIs, resulting in conversion of mixed hematopoietic chimerism to full donor chimerism. Remarkably, this powerful graft-versus-host alloreaction against lymphohematopoietic cells is not associated with GVHD in mice, even though donor T cells are given in numbers that would cause rapidly lethal GVHD in freshly conditioned recipients.642,643 Although antihost MHC alloreactivity mediates the most potent GVL effects268,1260 and GVH-reactive donor T cells can clearly be shown to be activated and proliferating in mixed chimeric recipients of DLI,268,644 these cells do not migrate to GVHD target tissues (epithelial tissues such as skin, intestines, and liver) unless there are inflammatory signals in those tissues.644 Such inflammatory signals can be induced by conditioning therapy645 or by TLR activation,644 which may be induced by infection or gut translocation of bacteria. The observation that graft-versus-host reactions can be confined to the lymphohematopoietic system suggests an approach to separating GVHD from GVL reactions, as hematologic malignancies reside largely in the lymphohematopoietic system. Proof of principle has been obtained that similar results can be achieved in patients receiving nonmyeloablative BMT with in vivo T-cell depletion of the donor and recipient, followed by delayed DLI for the treatment of lymphomas.1203,1206 In addition to recovery of epithelial GVHD target tissues from conditioning-induced injury, increasing resistance to GVHD with time may be conferred by recovering Treg-cell populations that downregulate graftversus-host reactions.1261–1263 Delayed DLI is somewhat variably associated with GVHD in patients, but generally to a lesser degree than would be expected in freshly conditioned recipients of similar cell numbers.1235,1264 Graft-versus-host reactions can also be confined to lymphohematopoietic tissues to prevent GVHD by preventing their egress from lymphoid tissues with FTY-720,1265 preventing attraction to epithelial target tissues by blocking chemokines,1266–1268 or by reducing tissue inflammation with keratinocyte growth factor,1269,1270 TLR or MyD88 inhibition, adenosine triphosphate inhibitors, or the nucleotide oligomerization domain/CARD15 axis.1259 The surprising protective effect of IL-2 against GVHD581 may be due in part to its ability to promte Treg expansion, an effect that can be enhanced by administering rapamycin.1271 Additional approaches to expanding Tregs in vivo include manipulation of DCs with histone deactylase inhibitors,

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which results in numerous immunomodulatory effects in addition to Treg expansion.1259 A clinical trial has been initiated using expanded third-party Tregs in patients receiving cord blood transplantation, but efficacy was not determined in the phase I trial.1272 A variety of promising inhibitors of T cells are being explored clinically, such as IL-21 blockade,1273 protein kinase C theta blockade, and proteasome inhibitors.1259 Imatinib, a tyrosine kinase inhibitor with strong activity against Philadelphia chromosome–positive leukemias, also inhibits platelet-derived growth factor receptor (PDGF-R) signaling and has promising activity against the fibrosis associated with cGVHD.1259 Additional strategies for separating GVHD from allogeneic graft-versus-tumor effects include the transduction of donor T cells with suicide genes so that the alloresponse can be turned off at will, hopefully after residual tumor has been eradicated.1210,1274,1275 Another approach is to avoid the graftversus-host alloresponse and try to target the donor immune response to tumor-specific antigens.1276 Minor histocompatibility alloantigens expressed by lymphohematopoietic cells (including leukemias and lymphomas) may also be targeted using in vitro expanded CTLs.75,1277 Immunization of HCT donors with tumor antigens could help to overcome the very limited frequency of T cells with these specificities,103 as memory CD4 cells may have reduced GVHD-inducing capacity compared to naïve cells.1278,1279

Xenotransplantation Over the past two decades, the increasing shortage of allogeneic donor organs has evoked a worldwide resurgence of interest in xenotransplantation, that is, the replacement of human organs or tissues with those from a donor of a different species. Routine clinical application of this therapeutic modality is still in the future. However, recent progress, which is reviewed briefly here, offers cause for optimism.

Concordant versus Discordant Xenotransplantation Xenotransplants have been classified into two groups— “concordant” and “discordant”—on the basis of phylogenetic distance between the species combination, speed of the rejection, and levels of detectable preformed antibodies.1280 Animals that are evolutionarily closely related and that have minimal or no preformed natural antibodies specific for each other are called “concordant,” whereas animals that belong to evolutionarily distant species and reject organs in a hyperacute manner are termed “discordant.”

Choice of Donor Species for Clinical Xenotransplantation From a phylogenetic viewpoint, nonhuman primate organs would undoubtedly be the most similar to allotransplants immunologically. However, due to considerations of size, availability, and likelihood of transmission of infectious disease, most investigators have decided against the use of primates as a future source of xenogeneic organs. Instead, the discordant species, swine, has been chosen by many as the most suitable xenograft donor. The pig has essentially unlimited availability, as well as favorable breeding

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characteristics, and many of its organ systems are similar to those of humans. Partially inbred miniature swine are a particularly attractive choice, because of their size (adult weights of approximately 120 kg), their physiology (also similar to humans for many organ systems), and their breeding characteristics, which have permitted inbreeding and genetic manipulation.1281

Mechanisms of Xenograft Rejection Xenografts are subject to all four of the rejection mechanisms described previously in this chapter and give rise to more powerful immune responses than allografts, probably for each type of rejection. Primates have large amounts of “natural antibodies,” so-called because they are present even though a primate has never been exposed to tissues from pigs. The reason for the presence of these antibodies is that during evolution, at the level of Old World primates, the gene for a particular enzyme was apparently lost due to mutation. This enzyme, α-1,3-galactosyltransferase, puts the sugar αgalactose (αGal) onto cell surface proteins in all species except for Old World primates and humans.1282 Because the αGal antigen is found on bacteria and other environmental antigens, humans and Old World primates make a large amount of antibody against αGal. It is these anti-Gal antibodies that then react with pig tissues after xenotransplantation, causing vigorous rejection. In addition, the hyperacute rejection that occurs with pig-to-primate transplantation is more vigorous than in the case of allogeneic blood group disparities. This is true, at least in part, because the complement regulatory proteins expressed by pig endothelium are less efficient in controlling human complement activation than are the human regulatory proteins expressed by human organs. Thus, these molecular incompatibilities also contribute to the increased intensity of the hyperacute rejection mechanism. Similarly, the factors responsible for accelerated graft rejection are more prominent in xenogeneic than in allogeneic transplantation, probably also because the process is magnified considerably by the failure of regulatory molecules to function effectively with human coagulation factors, thus increasing the tendency toward intravascular thrombosis.161 The available evidence also suggests that cellmediated immune responses to xenografts are more powerful than those directed to allografts.1283 Initially, there was some uncertainty about this point as cell-mediated immune responses to xenogeneic stimulating cells were first studied using mouse T cells, for which molecular incompatibilities with human cells lead to weaker direct recognition of xenogeneic than allogeneic stimulators in vitro. In this case, the incompatibilities turned out to involve the accessory molecules that are required for T-cell activation rather than a lack of antigens that stimulate TCRs. Thus, it seemed that cellmediated rejection in vivo might also be weak. However, cell-mediated xenograft rejection, even by mice, has consistently been found to be extremely powerful in vivo, apparently initiated by CD4 + T cells responding to the many additional antigenic peptides through the indirect pathway. For reasons of potential clinical applicability, greatest attention has been directed at investigation of the

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human-antipig cellular response. In contrast to the murine studies, direct responses by human CD4 and CD8 T cells to pig stimulators can be readily measured in vitro.1284 In addition, the cell-mediated reaction in vitro has been found to include a significant contribution by NK cells that can lyse pig targets. Most of the other molecular interactions involved in cellular immunity that have been examined appear to be at least partially functional in primate antipig responses. Therefore, human-antipig T-cell responses are likely to be as great or greater than those in allogeneic combinations.

Therapeutic Strategies for Xenotransplantation There are three main strategies that have been pursued to achieve long-term survival of xenogeneic transplants. The first has been to seek nonspecific immunosuppressive drugs that might prove especially effective for xenotransplantation. Unfortunately, none of the new drugs that have contributed to improved outcomes for allografts have proven sufficient alone to make xenografting possible, and considering the immunologic barriers to xenotransplantation discussed previously, it is unlikely that any such drug exists. Furthermore, the heightened immune response to xenografts compared to allografts suggests that larger amounts of exogenous immunosuppression will be required to achieve xenograft survival comparable to that of allografts. Given the narrow therapeutic window that already exists in allogeneic transplantation, most investigators believe that more than just immunosuppressive drugs will be needed to accomplish widespread clinical application of xenogeneic transplantation. Data so far for long-term survival of functioning pig-to-primate transplants of organs960 support this impression. However, for islets, recent results with immunosuppression have become sufficiently promising that clinical trials are planned in the United States1285 and are apparently

already underway in New Zealand, although precise details are so far unpublished. The second therapeutic approach has been to use genetic engineering of donor animals to lessen the immunologic barriers to xenografts. Because the two features that distinguish xenografts from allografts are the larger number of antigens and the molecular incompatibilities between species, these genetic modifications have been aimed primarily at correcting these two disadvantages of xenografts (Fig. 46.13). The first transgenic pigs produced by genetic engineering for xenotransplantation attempted to overcome the species selectivity of complement regulatory proteins. Transgenic pigs were produced that expressed human genes for several of these proteins. Organs from animals expressing one of these molecules (hDAF) have been studied extensively and appear to be more resistant to hyperacute rejection than are those from wild-type pigs,366 but are nevertheless susceptible to acute humoral rejection.1286 Numerous other transgenes (see Fig. 46.13) have been tested in attempts to further alter the primate host’s humoral response to pig xenografts, but none to date has prolonged the survival of organ transplants beyond the survivals achieved with hDAF organs.161 The alternative genetic engineering strategy to the addition of transgenes is the removal or inactivation of genes through knockout technology.1287 The advent of cloning through nuclear transfer technology, first demonstrated in the famous sheep “Dolly,”1288 has made knockout technology possible in several large animal species, including pigs. As, as described previously, the major stumbling block in pig-to-primate xenotransplantation has been the large amount of natural antibody in primates directed toward the Gal determinant, investigators have utilized this technology to eliminate this same enzyme from pigs.1289–1292 These new knockout pigs (called GalT-KO), like humans and Old World primates, do not put αGal on to the surface of their Transgenics

•

Complement inhibition – DAF

DNA

– CD46

Fertilized egg

Knockouts •

α 1,3 Gal Transferase

•

PERV (via knockout or RNAi)

– CD59 •

Growth factors – pIL-3, pSCF – Human GF receptors

•

MHC genes – Class I (NK inhibition)

•

Macrophage inhibition

•

Inhibition of coagulation and thrombosis

– CD47 – CD39 – Thrombomodulin – Hirudin – Tissue Factor Pathway Inhibitor

FIG. 46.13. Genetically Modified Pigs as Xenograft Source Animals. GF, growith factors; NK, natural killer; PERV, pig endogenous retroviruses.

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cells. As a result, xenotransplants can now be performed without the powerful rejection previously caused by natural anti-Gal antibodies. The results have been remarkable, with increased survivals of both heart and kidney transplants from pigs to baboons.961,1293,1294 Using immunosuppressive drugs, organ survivals were prolonged using these new Gal knockout (GalT-KO) pigs, but new antibodies soon appeared, causing rejection.961,1293,1294 However, using a regimen directed toward induction of tolerance (see the following), organ survivals were prolonged markedly, and no rejection was seen.960 There are still problems to be resolved before such transplants will be attempted in patients, but survivals are now being measured in months rather than in days, as they were only a few years ago. The third strategy to achieve successful xenotransplantation is the induction of tolerance of donor antigens. Potential applications of this strategy have been described previously in this chapter, with reference mainly to transplantation in rodent models. There have been attempts to utilize either mixed chimerism or thymic transplantation to induce tolerance across xenogeneic barriers in primates. So far, long-term success by the mixed chimerism approach has been attained only for concordant cynomolgus monkey to baboon renal transplants.1295 Both mixed chimerism and thymic transplantation approaches had been attempted for the discordant pig to baboon combination before the availability of GalT-KO swine, but with limited success.1296–1298 However, combining the thymic transplantation approach with the use of the GalT-KO as a source of donor kidneys has extended survivals to over 3 months.960 Thus, it seems possible that elimination of the natural antibody problem along with tolerance induction could make discordant xenotransplantation as successful as allogeneic transplantation in providing a long-term solution for patients waiting for transplants. Of course, it is possible that when these barriers are overcome, other obstacles, not yet apparent, will still limit the survival of xenogeneic transplants, and additional measures will be required to achieve success.

Nonimmunologic Barriers to Xenotransplantation In addition to the immunologic mechanisms that prevent successful xenografting, there are two other potentially important obstacles to clinical application. First, molecular incompatibilities between species may cause physiologic dysfunction of xenogeneic organs. This kind of incompatibility is least likely for the heart, for which the function, albeit vital, is relatively simple. It could certainly become an obstacle, however, for the liver, as the liver is known to produce so many different products, including serum proteins and enzymes, that it is likely that at least some of these products may not function properly in a primate host. On the other hand, if the number of incompatibilities leading to physiologic dysfunction is limited, they could be correctable through knockout and transgenic technology. Thus, the physiologic dysfunction of xenogeneic organs is unlikely to be an insurmountable barrier to xenotransplantation. The other nonimmunologic barrier to xenotransplantation is the risk of cross-species transfer of infectious agents, potentially creating a health hazard, not only for the

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recipient, but also for society as a whole. This possibility has gained significant attention, both in the scientific literature and in the lay press, and the issue has become confused by enormous uncertainties about the true risks that are involved. “Zoonosis” is a term that has been used for some time to describe the general process of cross-species infection, and the term “xenozoonosis” has been developed to describe infection transmission that might occur as a result of xenotransplantation.1299 It is important to realize that from the point of view of the individual recipient, the risk of transmitting infection by xenotransplantation is likely to be less than by current clinical allotransplantation, both because of the natural resistance to cross-species transmission of infectious diseases and because it should be possible to screen for and eliminate the presence of known pathogens from the herd of donor pigs. The major infectious concern, therefore, is that endogenous retroviral sequences from donor cells might infect the recipient’s cells, giving rise to previously unrecognized pathogenic viruses.1300 The concern has been raised that such new viruses might prove hazardous to other human beings in addition to the xenograft recipient.1301 However, to date there is no evidence that such cross-species transfer after a pig-to-human transplant would generate a virus that would be infectious or pathogenic. Indeed, studies of humans exposed to pig tissues have not revealed any cases of detectable pig endogenous retroviruses.1302,1303 Nevertheless, the concern about infections from xenotransplantation involves fear of the unknown, for which it is impossible to assign an accurate level of risk. At this time, therefore, public health agencies and members of the transplant community are attempting to design rational approaches for identifying the true risks of xenotransplantation and detecting untoward events rapidly, while at the same time allowing further progress in this potentially enormously important field of transplantation.1304

Clinical Progress in Xenotransplantation Early clinical efforts in xenotransplantation took place in the 1960s and involved organ transplants from nonhuman primates. One of the patients survived for 9 months with normal renal function provided by the kidney of a chimpanzee.1305 Additional clinical trials thereafter, using baboon hearts and livers, were considerably less successful. More recent clinical trials have involved fetal pig cells transplanted into the brains of patients with Parkinson or Huntington diseases. Survival of pig tissue 8 months after the transplant was documented in a patient taking only moderate doses of immunosuppression.1306 These studies suggest that cellular xenotransplantation may be achieved more easily, and thus may be performed sooner, than solid organ transplants, especially because free cellular transplants lack the vascular endothelium that is the target for both hyperacute and accelerated rejection. Indeed, xenogeneic islet transplants have already been performed in New Zealand (see www.sciencemediacentre.co.nz/2011/03/22/nzbio-the-potential-of-livingcell-therapies/), and the recent long-term successes reported for porcine islet transplants into nonhuman primates1307 has made it likely that clinical trials will soon be initiated in the

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United States. For organ xenotransplantation, most investigators believe that further clinical trials should await a reasonable expectation of success on the basis of pig-to–nonhuman primate experimental studies, a goal that is becoming closer but has not yet been realized.

SOME IMMUNOLOGIC ISSUES IN CLINICAL TRANSPLANTATION The Effect of Antigen Matching on Organ Graft Survival: Clinical Data In the absence of transplantation antigen disparities, graft rejection does not occur. However, the importance of antigen matching is one of the controversial issues in clinical transplantation. The evidence from transplantation of kidneys using living related donors provides a clinical demonstration of the importance of HLA antigen matching in subsequent graft survival. Data from a large international database on kidney allograft survival from 1985 to 1999 showed a survival half-life of HLA-identical sibling allografts of 23.4 years, as compared to 12.8 years for haploidentical-related allografts. These data support the basic concept that antigen matching matters and that the use of HLA-matched sibling donors is advantageous. The advantage of complete HLA matching for unrelated donors is also clear, although less compelling than for siblings, and has led to the United Network for Organ Sharing (UNOS)-mandated shipping of kidneys to distant centers for fully matched transplants from deceased donors. Whether or not there are advantages to partial HLA matches over full mismatches remains a subject of controversy, and most surgeons consider the length of the ischemic time necessitated by shipping of deceased donor organs as more important than the degree of HLA mismatch, as long as there is a negative crossmatch (see the following).

Human Leukocyte Antigen in Hematopoietic Cell Transplantation In contrast to the results described previously for solid organ transplants, the importance of HLA matching for unrelated HCT is unquestioned. Although restriction to HLA-matched donors has severely limited the availability of HCT (only 25% of individuals have an HLA-identical sibling; another 5% has a single antigen-mismatched–related donor), severe GVHD and rejection have been prohibitive limitations in the presence of extensive HLA disparity, as is discussed previously in this chapter. High-resolution, polymerase chain reaction–based HLA typing methods have improved the ability to identify mismatches in unrelated donors, and matching at this level has improved outcomes.1308,1309 While genotypic matching with unrelated donors is possible for patients with major conserved HLA haplotypes,13,14 this level of typing reduces the chance of fi nding a fully matched unrelated donor for patients with uncommon HLA types, even with the availability of large registries such as the National Marrow Donor Program in the United States.1255 Already, many patients do not succeed in finding an unrelated donor, and thus the ongoing effort to identify “acceptable” mismatches in the unrelated donor setting is important.1310,1311

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Crossmatch There are several tests designed to detect preexisting antibodies with specificity for donor antigens in the serum of potential recipients. First, recipients and donors must be matched for ABO blood type, because, with the exception of organs from donors of the A 2 blood group, transplantation across blood group barriers may cause hyperacute rejection (see previous discussion). Second, immediate pretransplant sera from prospective recipients are tested for reactivity against lymphocytes of potential donors. This “crossmatch” is generally performed by a two-step, antibody-mediated, complement-dependent cytotoxicity assay, although methods utilizing flow cytometry and luminex beads are now becoming more popular in many centers. However, whether a positive crossmatch detected only by the highly sensitive luminex bead assay should be considered in allocation of deceased donor kidneys is controversial.1312

“Sensitized” Candidates for Organ Transplantation Because kidneys and many other vascularized organs cannot be transplanted safely into recipients with preexisting antibodies, the clinical goal is to avoid transplantation of organs that express HLA antigens against which the recipient has been sensitized. Except for blood group antibodies, recipient sensitization to transplantation antigens generally occurs by prior exposure to alloantigens as a result of blood transfusion, previous organ transplantation, or, in women, by exposure to paternal antigens on fetal cells during pregnancy and parturition. The degree of sensitization against a potential kidney donor is measured by testing recipient serum on a panel of lymphocytes expressing a broad representation of HLA antigens, thereby assessing panel reactive antibody, as defined previously. Highly sensitized candidates may wait many years to receive a kidney transplant. The level of sensitization may decrease over time, leading to a negative crossmatch with recently obtained serum, but a positive crossmatch using previously collected sera. Transplantation in the face of this “historical positive crossmatch” has been performed successfully. Until recently, obtaining crossmatch-negative donors by searching for well-matched organs or waiting for a decline in the level of sensitization represented the primary solutions available for sensitized patients. Recently, however, several centers have begun to transplant kidneys in the face of antidonor antibodies by a variety of “desensitization” procedures, including plasmapheresis,1313 treatment with anti-CD20 mAb (rituximab),1314–1316 intravenous immunoglobulin,1317 and/or bortezamib (Velcade, Millenium Pharmaceuticals, Cambridge, MA),1318 followed by immunosuppression geared toward preventing antibody responses.130,158,1319

The Diagnosis of Rejection In clinical organ transplantation, the most obvious manifestation of the rejection process is usually diminished function of the transplanted organ, but it is important to confirm the immunologic origin of the event before increasing immunosuppression. The measurement of urinary perforin and granzyme B levels may be useful in this diagnosis.1320,1321

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Nevertheless, the “gold standard” remains histologic examination of the biopsy of the transplanted organ itself. Pathologists have been able to identify the abnormal lymphocytic infi ltrate within grafts, to grade the intensity of the infi ltrate, and, for some organs, to describe histologic findings characterizing the effects of immunologic injury.165 Some pathologic changes, including a lymphocytic infiltrate of the vascular wall, seem to be well correlated with rejection activity. In addition, pathologic changes suggesting nonimmunologic causes of renal dysfunction may be helpful in patient management. The immunohistologic finding of deposition of the complement component C4d in the peritubular capillaries is generally considered to be indicative of antibody-mediated rejection, especially in the kidney.1322 Despite the widespread reliance on the biopsy to define episodes of rejection, however, rejection is sometimes difficult to differentiate from drug toxicity and/or viral infection. Furthermore, when routine “protocol” biopsies of well-functioning transplanted organs have been performed, histologic findings have often revealed cellular infi ltrates similar to those of rejection. These results are consistent with several experimental models of tolerance induction that have shown intense lymphocytic infi ltrates in organs that go on to survive indefinitely and/or in recipients who develop tolerance to the donor antigens.1323,1324 These studies suggest that the amount of lymphocytic infi ltrate detected pathologically may not be helpful in diagnosing rejection episodes and determining the need for treatment.

How Much Immunosuppression is Enough? While the majority of transplant recipients respond immunologically to their new organ despite immunosuppression, some patients seem never to generate any rejection activity and maintain their transplanted organ with very small doses of immunosuppressive drugs. Indeed, a few patients have been known to stop all of their medications but have kept their transplant for years without rejection. On the other

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hand, some patients seem to require and tolerate very high doses of exogenous immunosuppression, while others seem to be severely immunocompromised by low doses of these drugs. These observations make it clear that the amount of immunosuppression that is required or that is safe is not the same for every individual or for all grafts. Unfortunately, there is no well-established assay to determine the amount of immunosuppression an individual requires and can safely tolerate for their particular transplant.

CONCLUSION The great danger in any textbook chapter is that the need to summarize what we think is known will obscure the much greater amount still left to be learned. For example, we have recently gained important insight into the role of APCs in T-cell sensitization, but we still have not explored adequately the role that indirect presentation of alloantigens plays in graft rejection. During the past two decades, we have learned much about the generation and function of CTLs and about their likely role in some mechanisms of graft rejection and GVHD; however, our understanding of noncytolytic mechanisms of rejection and GVHD, and of the role of B cells and alloantibodies, is much more limited. Finally, this chapter has outlined several techniques for the generation of immunologic tolerance to alloantigens in experimental systems; however, the first human beings have only recently been transplanted with tolerance-inducing regimens that allow the early discontinuation of nonspecific immunosuppression and the mechanisms of tolerance, which involve the kidney graft itself, are incompletely understood. The encouraging initial results raise hopes that routine tolerance induction may soon become a broader clinical reality that can be extended to other organs besides the kidney. It is, of course, the great fascination of transplantation immunology that new insights into basic immunologic issues will likely have important consequences for clinical transplantation in the future.

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Cancer Immunology Hans Schreiber

INTRODUCTION The goal of this chapter is to provide foundations and key facts in tumor immunology to students, immunologists, and oncologists in order to stimulate critical thinking and experimentation so that we may better prevent, cure, or at least control cancer. The study of cancer has had a tremendous impact on all fields of science (eg, molecular biology, virology, genetics, and immunology). For example, the discovery of the major histocompatibility complex (MHC) by Peter Gorer7,8 clearly separated cancer immunity from autoimmunity. His successful search for specificity led subsequent generations of investigators to search for cancer-specific changes (eg, the consistent chromosome translocation in chronic myelogenous leukemia9). However, conclusive evidence that rejection antigens on cancer cells are cancer-specific came in 1995 when it was shown that they were encoded by somatic cancer-specific mutations.10–12 Inherent in understanding the field of cancer immunology is the need to understand that immunology and cancer are two very large, different, and rapidly evolving complex fields of research. Thus, in order to make the immune system effective to prevent or treat cancer, an understanding of cancer and cancer models is required. The discussion of the important roles of cytokines in many aspects of tumor immunology is integrated in the various parts of this chapter.

CANCER Despite all the progress in molecular biology and genetics, the pathologist using histologic or cytologic criteria13 usually makes the only reliable diagnosis of cancer. Although tumor means “swelling,” the term is usually meant to include cancer cells and the stroma supporting the cancer cells; together they are often referred to as neoplasm, which literally means “new growth.” A neoplasm is an abnormal mass of cells that persists and proliferates after withdrawal of the stimulus that initiated its appearance. Leukemias are cancers caused by neoplastic proliferations of blood cells, but usually do not form tumor masses. There are two types of neoplasms: benign and malignant. The common term for all malignant neoplasms is cancer. Cancers of epithelial tissues (carcinomas) break through the basement membrane to invade adjacent tissues by infi ltrative destructive growth. Invasion may or may not be followed by cancer cells entering the lymphatics, bloodstream, or fluid of the coelomic cavities to implant at sites discontinuous with the original tumor (metastasis ; Greek for “emigration”). With

extremely rare exceptions, metastasis defi nes a tumor as malignant; benign tumors do not metastasize. Invasion usually precedes metastases and suffices as diagnostic criterion of cancer, although this criterion cannot be used for leukemia and mesenchymal tumors. With the development of inbred mouse strains many decades ago, transplantability of tumors from one syngeneic animal to another became (and still is) a diagnostic criterion for the malignant phenotype of experimental tumors.

Cancer Cells Many lines of evidence show that cancer is not a single disease. However, there are important principles that apply to many, if not all, cancers. There is substantial evidence that cancers in mice and humans are the result of multiple sequential mutations. As a result, certain molecules in cancer cells are mutant, up- or downregulated, or no longer expressed. In addition, most if not all cancers show epigenetic changes in gene expression. An estimated 15% of the worldwide cancer incidence is attributed to infections,14,15 but chemical and physical carcinogens are involved in the induction of most human cancers in industrialized countries.16 Many of these carcinogens are mutagens.17–19 While most mutations in cancer cells seem to be acquired (somatic mutations), increasing numbers of germline mutations are being discovered that make individuals prone to develop cancer.20 A cancer may require as many as 10 or more mutations to develop full malignancy. The first stage of cancer development is called tumor initiation,21 which is generally assumed to be irreversible due to somatic mutations or germline mutations in various oncogene, tumor suppressor gene, or deoxyribonucleic acid (DNA) repair pathways. Initiated cells do not form tumors. However, initiated cells clonally expand to premalignant lesions evolving over many years, often decades. This second protracted stage is driven by tumor promotion (ie, exposure to promoting conditions or chemicals22–24 [see Cancer and Inflammation]). The most advanced stage of these premalignant lesions is referred to as intraepithelial neoplasia also carcinoma in situ, often abbreviated as, for example, CIN for cervical intraepithelial neoplasia, VIN for vulvar intraepithelial neoplasia, or PIN for prostatic sites. The premalignant process ends with invasion, the appearance of the first cancer cells. Remarkably, however, there is now clear molecular evidence for premalignant cells spreading to distant sites where these cells remain premalignant unless promoted to become malignant.25–27

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A cancer cell that survives treatment may cause recurrence of the entire malignancy. Such cells are referred to as cancer stem cells or also tumor-propagating cells.28–30 The elimination of these cancer stem cells is critical to prevent relapse. The sweeping assertion that these cells represent an extremely small percentage of the cancer cells in a tumor is not supported by rigorous experimentation or clinical experience. Some of the misconception comes from the observed rarity of only a few human tumor cells able to adapt to growth in a foreign (mouse) milieu.30 Furthermore, flow cytometric analysis of cancer cells may detect a rare population that results from intraclonal and nonheritable heterogeneity in cancer cell populations.31,32 Tumor progression originally meant the change from a benign neoplasm to cancer33,34 but is now usually used to describe the third phase of the multistep process. Invasive growth of a lesion usually ends with a highly aggressive, widely metastatic cancer that ultimately kills the host.35,36 There is compelling evidence that most cancers are clonal in origin and that in cancer progression, new subpopulations of cells arise continuously due to Darwinian selection of genetic variants that have a growth advantage, escape homeostatic controls, or resist destruction by defense mechanisms or treatment.37 During this evolution, sequential mutations result in changes in rate of growth, morphology, hormone dependence, enzyme and cytokine production, and expression of surface antigens. Importantly, by the time cancer is first detected in a patient, it measures usually at least 1 cm in average diameter, contains ∼109 cancer cells, and has already undergone about 30 generations.13 Thus, most of the diversity of a cancer has already occurred at time of detection with only 10 generations left before death of the individual unless treatment intervenes. The term primary indicates the tumor from which cancer cells emigrate to secondary sites (ie, metastatic growth in tumor-draining [sentinel] lymph nodes or more distant organs). Experimentalists use the term spontaneous metastases to describe metastases that occur without experimental manipulations; artificial metastases are caused by cancer cells injected into systemic or portal veins of a tumorfree mouse to cause lung or liver metastases, respectively. Cancer cells can disseminate without further cell division (microdissemination) or they divide only minimally causing micrometastases, conditions that can only be recognized by microscopy and immunohistochemistry but are potential sources of relapse, the central problem of cancer therapy. Similarly, residual microscopic foci of cancer cells may remain at sites of incompletely excised cancer and cause local recurrence. Efforts are ongoing to develop sensitive markers and assays for determining the need for additional therapy or determining the effectiveness of a therapy before relapse is detected clinically.

Cancer Stroma Definition Most of the cells in tumors may not be cancer cells but nonmalignant cells, referred to as stromal cells. Some of the most aggressive cancers, such as pancreatic cancer,

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mostly consist of nonmalignant stromal cells.38 Virchow39 believed that compression of the growing cancer cells induced a structural fibroblastic framework (now generally referred to as “stroma”) in which the cancer cells grew. He thought that cancer cells and stroma both developed from the same primitive precursors. This concept changed with Ehrlich stating clearly that the host provided the stroma of solid tumors.40 Borst41,42 was the fi rst to clearly point out the essential mutual relationship between cancer cells and tumor stroma by stating that the question of whether the epithelium or the connective tissue has the leading role in carcinogenesis was difficult to answer, because stroma of tumors is dependent on the presence of cancer cells, and cancer cells are dependent on stromal cells. Thus, cancer cells release factors that attract stromal precursor cells, and stromal cells in turn produce factors that support cancer cell growth.43–45 Interestingly, Rous 46 emphasized the importance of vascularizing stroma for successful tumor transplantation and that immune reaction to nontumor cells led to rejection of the inoculum. Rous, however, was working with noninbred animals. It therefore remained unclear from his experiments whether an immune reaction just to stroma sufficed to cause tumor rejection until 199247 when similar experiments done in inbred mice showed that immune reaction to the stroma of transplanted tumor fragments led to the eradication of the inocula.

Components Willis, in a careful survey of his own studies and published literature,48 subdivided tumor stroma into just two major components: connective tissue, which usually represents the bulk of stroma, and vasculature, which is usually a smaller fraction. However, we should distinguish at least four critical components: 1) fibroblasts, 2) vasculature, 3) extracellular matrix (ECM), and 4) cells of myeloid and lymphoid lineage, such as macrophages, neutrophils, natural killer (NK) cells, and T and B cells, all derived from hematopoietic stem cells. Fibroblasts are a prominent cell type in tumor stroma as well as in healing wounds and embryonic connective tissues. Stromal fibroblasts in cancers are metabolically active making matrix substances; the degree of activation of stromal fibroblasts correlates with aggressiveness of the cancer and inversely with survival of patients.49,50 Unfortunately, we still lack reliable fibroblast-specific immunologic markers for these cells despite repeated assertions to the contrary.51–61 Therefore, fibroblasts are still mostly defined by morphology and function.62–66 Characteristically, they synthesize, secrete, and modulate proteins of the fibrous ECM, particularly alpha-collagen.62,63,67 Macrophages are an essential and prominent part of stroma of every tumor. They lack Gr-1, express low levels of MHC class II, and express F4/80. These macrophages are “alternatively” activated (ie, typically have a tumor-promoting immunosuppressive M2 differentiation phenotype, particularly in hypoxic areas of the tumor68). Neutrophil granulocytes, also referred to as neutrophils or polymorphonuclear leukocytes, are an equally pivotal component of tumor stroma. Like the tumor-associated

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macrophages, neutrophils in cancers are “alternatively” activated (ie, “N2”69). Unlike classically activated neutrophils, N2 do not cause tissue damage. These neutrophils express cluster of differentiation (CD)11b, Ly6-C, high levels of Gr-1 (defined by monoclonal antibody RB6-8C570), and high levels of Ly6-G (recognized by the monoclonal antibody 1A8).71 RB6-8C5 (anti–Gr-1) recognizes primarily Ly6-G71 but apparently also binds weakly to Ly6-C,71 although this is controversial.72 In any case, RB6-8C5 (anti–Gr-1) not only eliminates neutrophilic and eosinophilic granulocytes effectively (though only transiently),43–45,70,73,74 but also markedly decreases cells of the subset of blood monocytes that express high levels of Ly6-C but lack Ly6-G.74 Because of their suppressive effects on cultured T cells,75 these Gr1+ CD11b + leukocytes are also referred to as myeloid-derived suppressor cells (MDSCs).76–79 MDSCs are increased in the circulation of tumor-bearing individuals but also found in the stroma of autochthonous80 and transplanted tumors.68

Sources It is presently unknown what percentage of bone marrow– derived stromal fibroblasts originates from hematopoietic versus mesenchymal lineage, what percentage of stromal fibroblasts is derived from circulation rather than local adjacent sources, and whether bone marrow–derived fibroblasts are functionally different.81 As suggested over a century ago, some cells in the stroma come from progenitor cells entering the site of tumor growth either via the blood circulation or from adjacent normal tissues.81–102 The relative contribution of the two sources (circulation or adjacent tissues) is still hotly debated,103,104 but it is questionable whether circulating endothelial precursors contribute significantly to tumor vasculature in well-established tumors. Similarly, it needs to be determined whether local tissue reservoirs are a major source of alternatively activated M2 macrophages in autochthonous or transplanted cancers (> 2 weeks after transplantation) as it appears to be the case in other pathologic conditions of “type 2 inflammation.”105,106 Type 1 inflammation in tumors is an artifact clearly visible during the first 2 weeks after tumor transplantation.68,107 An alternative major source of macrophages in tumor stroma may be monocytes or MDSCs from blood circulation.68

Function Lack of tumor stroma drastically reduces tumorigenicity.47,108,109 All cancers depend on stromal support and establish some type of paracrine loop.43–45,52,110–113 Signals initiating the loop seem to be intrinsic to the cancer cells (ie, endogenous) and depend on oncogenic mutations in the cancer cells114–117 (see Cancer and Inflammation). The proinflammatory mediators attract mesenchymal, endothelial, myeloid, and lymphoid progenitors to the stroma from adjacent and systemic reservoirs; the mediators also induce these cells to make factors that stimulate the growth of the cancer cells.43,45,118–125 Cytokines released by transfected cancer cells can have powerful local effects on all components of tumor stroma including fibroblasts.73,126–143 Angiogenesis is a fundamental necessity for tumors to grow by allowing oxygenation and nutrients to diffuse from

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the blood into the mass. Myeloid cells, including mast cells, neutrophils, eosinophils, Gr-1+ CD11b + MDSC/monocytes, and tumor-associated macrophages, can promote tumor angiogenesis.144 However, cancer cells and tumor-associated fibroblasts can produce proangiogenic as well as growth stimulatory factors such as vascular endothelial growth factor (VEGF) and Bv8.145 Several studies have shown the importance of neutrophils and granulocyte– colony-stimulating factor (G-CSF) production by cancer cells in causing refractoriness to anti-VEGF therapy.43–45,146–149 Metalloprotease released from neutrophils and from Gr-1+ CD11b + MDSC/ monocytes catalyzes the release of preexistent VEGF and transforming growth factor (TGF)-β from the ECM and activates latent TGF-β.150–153 Neutrophils are essential for mobilizing various types of stromal progenitor cells including the macrophages from bone marrow and other reservoirs in the body.154 Finally, neutrophils and Gr-1+ CD11b + monocytes in the tumor stroma themselves can produce large amounts of TGF-β1.69,80 ECM is an essential stromal component providing the cancer cells not only with a scaffold for adherence and structure but also with growth and antiapoptotic signals, thereby preventing anoikis.155–160 TGF-β1 stimulates fibroblasts to produce ECM proteins, including collagen, fibronectin, and proteoglycans, and TGF-β1 prevents ECM degradation.161 Accordingly, transfecting cancer cells to produce TGF-β1 makes them more aggressive.128 ECM is also a major reservoir for binding and releasing growth factors, chemokines, and cytokines.162,163 Cancer cells may release the ECM proteoglycan versican that helps attract and activate myeloid cells via toll-like receptors (TLRs) to release interleukin (IL)-6.164 At later stages, neoplasms often replace paracrine with autocrine loops, notably IL-6 activating signal transducer and activator of transcription (STAT)3.123,165,166 However, all evidence suggests that even the most aggressive cancers still depend on some factors and ligands provided by tumor stroma. Knowing how essential stroma is for cancers to grow, it is somewhat surprising that cancer cells would not generate their own stroma. Indeed, epithelial cancer cells can form a “pseudo-stroma” by assuming a mesenchymal phenotype at the invading edges of the cancer.167,168 However, there is no evidence that this so-called epithelial-to-mesenchymal transition can replace the host-derived stroma (ie, the need of cancer cells to establish paracrine stimulatory loops with nonmalignant stroma169). Fusion of cancer cells with stromal cells, particularly macrophages, has been proposed as a major mechanism of cancer development and progression.170–175 However, we still lack conclusive experimental evidence supporting this attractive hypothesis formulated over a century ago.176,177

Reaction to Cancer Cell Inoculation Careful studies showed decades ago that the many cancer cells that die on inoculation play a critical role in the establishment of the cancer. It was found that adding lethally irradiated cancer cells to an inoculum of viable cancer cells at a 100:1 or larger ratio can increase the take of a cancer cell inoculum by more than a 100-fold.178 The dead cancer

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cells have potent thromboplastic activity,179 and death of the cancer cells is required for this activity to form fibrin at the site of inoculation.178 In addition, hypoxia induces VEGF-A and CXCL12 (stroma-derived factor [SDF]-1) in many types of cells, but many cancer cells produce VEGF even in normoxic conditions.180 VEGF-A is a most potent inducer of vascular leakage of plasma proteins, including fibrinogen, that rapidly form a fibrin-fibronectin clot as a provisional tumor stroma.181 Fibrin deposited at the site of inoculation serves as primitive ECM for cancer cells to escape anoikis. The ECM then undergoes major remodeling during the first 2 weeks after inoculation. Remodeling the ECM microenvironment requires the activity of ECM-degrading enzymes such as matrix metalloproteinases.182 Thereby, these transplanted tumors acquire later the harder consistency typical for autochthonous tumors that evolve with stroma.

EXPERIMENTAL CANCER Key Principles Number of Cancer Cells Targeted A critical determinant for any cancer therapy is the number of the targeted cancer cells that have proliferative potential (“cancer stem cells”). That number determines the likelihood of recurrence/relapse after most cancer cells have been destroyed by treatment. Even microscopic parts of a cancer left behind by a surgeon often lead to recurrence. Nevertheless, the size of tumor–stem cell population is not the only factor determining the likelihood of relapse as other cancer cells, even when dead or lethally irradiated, can increase the tumorigenicity of the remaining cancer cells by orders of magnitude.178 Most human tumors are not detected until they are 0.5 to 1 cm in diameter, have a volume of ∼500 mm3, and contain ∼109 cancer cells.13 In leukemia, malignant cells generally do not form tumors, but the patient has also ∼109 cancer cells when the disease is clinically detected. It is not appropriate to adjust for the difference in host size when comparing cancer in man and mice. Not the size of the species but the size of the cancer cell population determines the chance of relapse because the latter correlates with the number of therapy-resistant variants causing relapse after therapy. Skipper, who pioneered combination chemotherapy of childhood leukemia in the mouse model of L1210 leukemia, targeted 109 cancer stem cells as the starting population. This was one major reason why the principles he established in an animal model were clinically relevant and led to the cure of most childhood leukemias. Cellular heterogeneity within a tumor becomes much more relevant when the tumor accumulates 1 billion (109) cancer cells, equivalent to a tumor with a diameter of 1 cm.183–185 At this point, the accumulation of so many cancer cell variants makes it improbable that all cancer cells are susceptible to a single chemotherapeutic agent or specific T cell. Cancer cell variants can be considered analogous to drug-resistant bacteria or viruses (ie, the nature of the problem is fundamentally the same in both cases). Unfortunately, in most experimental studies on immunotherapy, almost fivefold smaller populations of cancer cells are being treated4 (Fig. 47.1).

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FIG. 47.1. Precipitous Drop in the Number of Publications Reporting the Effects of Experimental Cancer Immunotherapy with Increasing Size or Duration of Growth of Tumors Treated by Immunotherapy. A search of PubMed for year 2010 publications using the keywords “immunotherapy” AND “cancer” recovered 195 experimental studies listing tumor volume or size and 158 studies listing duration of growth at start of treatment. Seventy-five percent of tumors treated in these studies were smaller than 121 mm3. Only 9 of the 158 tumors had grown for 2 weeks or longer. Modified from Wen et al.4

Duration of Growth Duration of growth of a cancer greatly influences experimental results. Summarizing three decades of studies on immunity to cancer, Woglom concluded in 1929 that immunotherapy is futile against an established tumor, and “nothing may accordingly be hoped for at present in respect to a successful therapy from this direction.”186 Eighty-two years later, the outlook is not quite so grim, but unfortunately, the vast majority of research in animal models is still concentrating at treating malignant cell populations grown for an average of only 5 days after cancer cell inoculation4 (see Fig. 47.1). The word “established” is not a scientific term, yet is frequently used to describe neoplastic lesions caused by recently implanted cancer cells,186 conveying the message that the malignancy being treated does not differ from what would be found in a human cancer patient. Just the opposite is true; transplanted tumors must grow for at least 2 weeks before they are histologically indistinguishable from autochthonous murine or human cancers.107 Most human cancers have resided in the patient for months if not years before being detected and treated, whether primary, metastatic, dormant, or relapsing. An additional problem of experimental models is that growth of many serially transplanted “standard” cancer lines is so rapid that death may occur so early that treatment has to be started before solid tumors have established a microenvironment even vaguely comparable to that of an autochthonous tumor.

Measuring Growth and Destruction Tumors are masses and have weight and volumes best approximated by the formula of an ellipsoid (V = πlwh/6 or V = 0.5236 lwh or V∼ ½ lwh); length l, width w, and height h are the orthogonal diameters in the three perpendicular

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axes. As was carefully documented, the third dimension, height (or depth), has an inordinately large effect on errors in volume187 but can usually be determined accurately for subcutaneous tumors.187–189 Describing tumors as areas is incorrect and illogical despite widely practiced misuse. By definition, areas have no volume nor contain a single cancer cell. Area measurements as commonly used by radiologists frequently disagree with volumetric measurements.187,188,190 Depth of a tumor can greatly vary and determines malignancy and prognosis (eg, in melanoma). There are important variables when extrapolating the number of cancer cells from a given tumor volume; for example, the human or murine adenocarcinoma of the pancreas usually consists predominantly (∼90%) of nonmalignant stromal cells yet is highly aggressive, whereas in most tumors stromal and cancer cells are more balanced in numbers. Furthermore, while it takes weeks for a tumor to disappear completely even after all cancer cells have been eradicated, it is usually impossible to determine from volume measurements how many viable cancer cells are left. Certain cancers release hormones that have systemic effects such as insulin; even microscopic growth can have major systemic effects.191 While many experimental models utilize subcutaneous tumors, some cancers, particularly human tumor xenografts, may require transplantation to sites where it is more difficult to assess growth objectively.192 Cancer cells have been transduced with luciferase to give a signal proportional to the tumor size when substrate is injected; this allows whole body scans with a luminometer, though sensitivity decreases for fewer than 1,000 cancer cells, and there is absorption by overlying tissues.193

Cure and Dormancy The effects of the immune system on cancer can be read out in different ways. Certainly, cure of the cancer is the objective. There should be clear prolongation of survival and absence of relapse, but proving that all cancer cells have been eradicated is often difficult. Unfortunately, for most cancers, we lack assays sensitive enough to detect and quantify remaining dormant cancer cells. This information would indicate whether or not a patient requires further treatment. Only in a few types of cancers194–196 does a negative diagnostic polymerase chain reaction analysis of the tissue reservoir from where a cancer may relapse suggest—though not prove— complete eradication. Experimental evidence for dormant cancer cells may come from provoking relapse by treating the animal with antibodies neutralizing factors or cells suspected of causing tumor dormancy. Experimentation should include waiting for minimally 30 days after the tumor has disappeared completely (it is best to wait several months or longer). A common unacceptable practice is using the word eradication or cure when there is no follow-up after the cancer becomes undetectable. Relapse of cancers may occur within days, weeks, months, or even years after complete disappearance, and is one of the most important problems of cancer therapy. Nevertheless, experimentalists commonly describe treatments as effective even when followed by rapid relapse.197 Eradication means tearing a tree out with its roots so it cannot regrow, and the term is synonymous with cure.

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Thus, using the term eradication is only appropriate if the host does not harbor dormant cancer cells, comparable to “sterilizing immunity” in infections.198–201

Inhibition, Arrest, Regression, and Equilibrium Dependent on the extent and type of destruction as well as when it occurred, tumors may be found to have smaller or larger volumes after treatment compared to controls. It is important to determine whether the rate of tumor growth has been altered or the rate of growth remained unaltered but the onset of growth has changed. Altered rates of growth require an ongoing process, whereas altered onset of outgrowth is usually caused by an event that happened at times of inoculation. Slower growth rates (growth inhibition) should be distinguished from shrinkage (regression) of a cancer. A steady size of a treated tumor compared to controls is referred to as growth arrest, equilibrium, or progression-free survival in cancer patients, an important goal when cure cannot be achieved.169,202 Specificity Controls It is completely inappropriate yet quite customary to study immunologic therapies specific for a self-antigen on a human cancer in mouse models (human tumor xenograft) when the murine host does not express the same target. These irrelevant models often yield impressive results and mislead the reader that the targeted molecule is tumor-specific when in the real situation it is not.203,204 If someone subscribes to the widespread highly questionable perception that many, or even most, useful tumor antigens are self-antigens, then this investigator should also use appropriate experimentation. Either a mouse model must be used in which normal tissues express the target molecules closely resembling expression patterns in man or there should be a clear warning to the reader. For example, anti–carcinoembryonic antigen (CEA) immune responses caused toxicity in mice that expressed the target antigen also in normal tissues, as humans do,205 predicting the severe toxicity later observed in a clinical study.206 Side effects often become apparent only when the treatment is intense enough to provide clinical efficacy.207 It is therefore questionable to dismiss results in proper animal models.208,209

Selection of Tumor Model General Considerations Experimentalists have to be able to translate their fi ndings to clinicians and vice versa. There is no single human cancer, let alone single animal model, that can serve as appropriate model for all human cancers.28 Organ site as well as histologic type of a cancer may greatly influence the results. Thus the complexity of cancer makes it extremely important that the experimental model used to study cancer immunity be relevant to the question asked; a single model, if carefully chosen, may be appropriate to answer a specific question. Nevertheless, it is essential that we uncover the broader principles underlying cancer-host interactions. For example, the extensive clinical and experimental research on immunotherapy of melanoma has failed to answer the

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central question: why are we struggling to make similar advances in the immunotherapy of more common cancers such as breast, colon, prostate, and lung cancers? Weiss argued in 1980 that the failures of clinical immunotherapy were due to using irrelevant laboratory tumor models for extrapolating results for clinical application.210 One of the major reasons for translating results from animal models to clinical cancer immunotherapy being frustratingly evasive is the disregard of using truly established tumors for experimental therapy (see Fig. 47.1).4 Current experimentation in cancer immunology mostly uses young hosts to study the effects of the immune system on small, recently implanted inocula of cancer cells derived from tumors once induced in inbred mice (with homozygous genetic loci) and then passed in vivo for decades from animal to animal, whereas human cancers are already well established when first detected, have a very high probability of heterozygous loci, have never been transplanted, and develop in mostly older individuals.

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manipulations are driven by tissue- or cell lineage–specific promoters that are either constitutively active or inducible locally or systemically. While these mice become prone to develop cancer, the tumors they develop are autochthonous but by no means “spontaneous” despite widespread misuse of terminology.

Carcinogen-Induced (Physical, Chemical, or Viral). Many of the physical and chemical carcinogens involved in the induction of cancers are mutagens.16,17 Since repeated application of coal tar was used to induce the first chemically induced cancers,214 a very large number of chemicals such as polycyclic hydrocarbons and nitroso compounds has been identified with remarkably potent cancer-inducing activity.215 Cancers have been induced in many tissues and organs and several animal species. Ultraviolet light (UV) has potent skin cancer–inducing activity in man and mice,216,217 and many well-defined models of UV-induced tumors are now available.218,219 The potential of ionizing radiation to cause cancer in humans was recognized soon after Roentgen’s discovery of x-rays in 1895.220,221 But study of radiation carcinogenesis in animals mostly occurred after World War II when large-scale tumor-induction studies were carried out in many species over the succeeding three decades in response to the threat of irradiation from nuclear reactors or bombs.221–223 Finally, many models of viral cancer induction have been developed after Rous224 showed that viruses can also induce cancer in animals.

Autochthonous Tumors Autochthonous tumors originate in the place where they were found (autochthonous, in Greek, means indigenous). The antonym is transplanted tumors. Autochthonous tumors can be spontaneous, carcinogen-induced, or transgeneinduced. Even when induced in the same experiment by the same mode of induction, autochthonous tumors will differ genetically, biologically, and antigenically from one another, because additional but individually differing genetic changes are required for each tumor to develop. Thus autochthonous tumors lack the uniformity of well-defined transplantable models. An advantage of autochthonous over transplanted tumors, however, is that the host’s immune system has been neither artificially primed nor altered by an inoculum. Unlike autochthonous tumors developing after exposure to physical, chemical, or viral carcinogens, transgeneinduced autochthonous tumors have the disadvantage that transgene expression in the thymus during development usually causes systemic tolerance to the transgenic proteins. Nevertheless, several excellent transgenic cancer models have been developed.225,226 Some models are based on immunologic findings first made in patients.12 For example, the R24C mutation in the cyclin-dependent kinase 4, first identified as tumor-specific antigen by T cells of a melanoma patient, causes familial melanoma when in the human germline.227 Introducing this mutation in the germline makes mice highly susceptible to develop melanoma225 ; tumor development, however, requires additional carcinogenic insults followed by prolonged chemical promotion. Certain oncogenes such as SV40T are powerful because they inactivate several important suppressor pathways and may therefore require fewer additional mutations. In some transgenic cancer models, the transforming genetic event can be controlled, sometimes reversibly, by topical or systemic application of an inducer such as tamoxifen or tetracycline.193 Alternatively, systemic or topical application of Cre-recombinase may excise a “floxed” blocking element or attenuator of gene expression.228 These temporal controls appear to be helpful for answering important questions on tolerance, because expression at birth causes neonatal tolerance to a highly antigenic oncogene such as SV40 T antigen. An approach that more closely mimics the sporadic nature of human cancer relies on a spontaneous mutational event activating an introduced floxed oncogene. The sporadic nature of this event unfortunately also makes time and site of tumor development less predictable.228

Transgene-Induced. Experimental cancers are produced artificially by inserting oncogenes into the germline of mice or by manipulating the mouse genome to allow excision of tumor suppressor genes. Genes used for these

Most current experimental work in tumor immunology uses transplantable tumors. However, there are substantial differences among transplantable tumors, and it is

Modes of Induction Spontaneous . The term spontaneous cancer is defined as cancers arising “in the absence of any experimental manipulation.”211 Spontaneous murine cancers develop without any known exposure to carcinogens or genetic (often transgenic) manipulations introducing oncogenes or eliminating tumor suppressor genes. For example, spontaneous tumors can be observed in many mouse strains with advancing age. All genes have a spontaneous rate of mutation, and genetic instability, though often a consequence, may not be a requirement for tumor development.212,213 As would be expected from the sporadic occurrence of spontaneous mutations, the occurrence of spontaneous tumors is random and unpredictable.

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important that the chosen cancer is appropriate for the particular question being asked. It is important to know that 24 hours after inoculation, the majority of the injected cancer cells usually have died,178 leaving only a shallow outer rim of viable cancer cells at the oxygenated margin.107 Histology or in vivo imaging reveals pronounced edema at the injection site. In fact, much of the “tumor growth” early after inoculation is due to tissue swelling caused by the inflammatory reaction. Cancer cells may well be invading surrounding normal tissues and be vascularized within 48 to 72 hours after transfer, thereby meeting standard criteria for malignancy. But experienced pathologists will immediately spot the abnormal inflammatory reaction to the transplantation injury and the necrotic center of the early inoculum. Thus, common terms such as “three-day established cancer” or “three-day established metastases” are highly misleading. Such necrosis would not be found in an autochthonous cancer of similar size. The inflammatory reaction progressively decreases with time, and at 14 days, the necrotic material has usually disappeared. The tumor then contains ∼109 cancer cells, measures ∼1 cm diameter, and is histologically indistinguishable from primary autochthonous tumors. Ideally, therapy uses tumors 2 weeks or more after inoculation (see Fig. 47.1). Even though the terms tumor transplantation and tumor injection are old and commonly used, they are quite misleading, as they usually refer to inoculation of tumor fragments or a suspension of cancer cells. A common error is the belief that tumors can be injected or “transplanted” like a vascularized organ such as kidney or heart. Instead, much of the inoculum—whether a cancer cell suspension or fragments of a tumor—dies initially and needs about 2 weeks before it becomes histologically indistinguishable for autochthonous tumors. Tumors can be induced in animals by injecting cancer cell suspensions prepared from cultures or by mincing tumors into 1-mm3 pieces and injecting the tumor fragments using a 12-gauge trocar. Cancer cells in fragments are 10to 100-fold more tumorigenic than stroma-free cancer cell suspensions.47,108 It had long been known that certain cancers would only grow in mice when transplanted as tumor fragments. This was erroneously thought to be due to more cancer cells being inoculated with fragments.229 Later analyses revealed that fragments contained fewer cancer cells than injected cell suspensions, yet produced a higher take or larger tumors earlier.47,108 Another erroneous explanation was that the stroma of tumor fragments provided a physical barrier preventing cancer cells from migrating to draining lymph nodes and priming a protective T-cell response.108,230 More likely, more cancer cells remain viable when embedded in tumor stroma (by preventing anoikis160) and therefore release less antigen than suspended cancer cells, most of which die. In any case, as long as cancer cells express sufficient levels of antigen, professional antigen-presenting cells in the tumor stroma pick up the antigen and travel to the draining lymph nodes where they present the antigen to naïve T cells.231 It is important to know that whether suspensions of cancer cells or tumor fragments are being used, a threshold

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number of cells or fragments must be inoculated for tumors to develop. The threshold in T cell–deficient mice may be due to innate immunity or nonimmunologic mechanisms.178,232 The increase in threshold in immunocompetent mice is probably due to adaptive immunity. Cell Lines from Tumors Serially Passed in Mice. When choosing tumors that have been serially passed in animals for transplantation, it is critical to be aware of how such tumors were altered by the serial transplantation. As was discovered decades ago, even a single in vivo passage of a cancer can select for heritable cancer variants.45,219,233–237 Even half a century ago, investigators already noted that serial transplantations of these cancers “inevitably result in progression toward more rapid growth rate, loss of functional and histological differentiation, loss of responsiveness to extraneous stimuli”238(p.522) and a diminution of strain specificity, a problem shared by B16.239 These serially transplanted cancers, such as B16, can easily be transferred in mice using cancer cell suspensions rather than tumor fragments. As a result of hundreds of passages in mice, 239 these cancer cells have become resistant to anoikis caused by lack of stroma by acquiring alternate signaling pathways that replace the prosurvival signals of ECM.240 Many of these tumors grow and kill so fast that they must be treated early before they are truly established. Certainly, many of these tumors no longer resemble primary mouse or human tumors that virtually always grow at much slower rates and have been established for months or years before being treated. B16 melanoma was derived in C57BL/6J mice in 1954 and is the parent of many available B16 sublines.239 B16 had been transplanted serially through 328 mice for 13 years before it became available as a standard cancer cell line. Nevertheless, B16 still is arguably the “Escherichia coli ” of tumor immunology with almost 1,000 entries in PubMed in 2011. The A/J-derived Sa1 originated in 1947 in an A albino mouse and was serially transplanted in A/J mice for 1,017 generations for 19 years. Similarly, the A/J-derived neuroblastoma 1300 derived in 1940 was serially transplanted for decades from mouse to mouse. The Lewis lung carcinoma, isolated by Lewis,241 arose spontaneously in 1951 at the Wistar Institute in a black C57 mouse (not a C57BL/6 mouse), was serially transplanted extensively, and is still being used in numerous studies as a model for exploring the immune responses to lung cancers and their metastases. When genetically inbred mouse strains became available as sources for murine cancers over half a century ago, serial transplantation was necessary for maintaining a tumor. Dependable long-term cryopreservation did not become available until the late 1960s and early 1970s.242–244 Even after dependable cryopreservation became available, investigators still continued to use serial transplantations to propagate newly derived cancers such as the BALB/c-derived CT26 colon cancer,245 the C57BL/6-derived MC38 colon cancer,246 and the BALB/ cCr-derived RENCA renal cancer.247 Many cell lines are renamed sublines of old parental cell lines. Thus, it is often overlooked that RMA, RMA-S, MBL-2, and EL-4 tumors are derived from the same single tumor line,248 most likely

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E.L.4 induced by 9:10-dimethylbenzanthracene in 1945 by Gorer249 and serially transplanted for many decades from mouse to mouse. Importantly, referring to EL-4 as a C57BL/6 tumor is incorrect as this strain did not exist in 1945. Similarly, Neuro2a is a subline derived in 1969 from the A/J-derived neuroblastoma 1300 derived in 1940. Unfortunately, organizations providing these lines and/or publications often fail to cite the fact of long-term in vivo passage before cryopreservation. Cell Lines from Autochthonous Mouse Tumors. Autochthonous cancers never need to be serially transplanted. Standard lines can be created by adapting the autochthonous cancer cells to culture and/or by freezing fragments or cells in liquid nitrogen.218 To prove that antigens expressed by the malignant cancer are truly tumor-specific, nonmalignant cells, tissue and DNA from the host of tumor origin must be available.218,219 This approach is commonly used by researchers studying the antigenicity and genetics of human cancers. Carefully controlled experimental cancers exist.218,219 Human Tumor Xenografts. For certain purposes, human tumor xenografts are useful models. Human cancer cell lines grown in vitro can cause tumors when injected into T-, B-, and/or NK-deficient mice. It is important to remember that the stroma of such cancers is entirely of mouse origin and the cancer cell–stromal loops are defective because of multiple mismatches in ligand-receptor signaling.250 This problem can be partially alleviated by “humanizing” the recipient mouse. Humanizing usually refers to expressing certain human molecules such as receptors/cytokines in mice that lack murine T, B, and/or NK cells, and/or transferring human mesenchymal and/or hematopoietic cells to such mice. Interestingly, human T- and B-lymphocytes and fibroblasts contained in human tumor fragments may coengraft thereby “humanizing” the mouse192 (eg, when injecting cell clusters or aggregates from human fresh ovarian cancers into nonobese diabetic–severe combined immunodeficiency IL2γR null mice). Progressive growth of these xenografts leads with great regularity to ascites formation, and pleural metastasis closely simulating classical tumor progression observed in patients with ovarian cancer.192 Despite continuous improvement, mice can never be completely humanized. Only very few cancer cells of the xenografts may be able to progress in the chimeric milieu.30 Nevertheless, once a tumor grows, its sensitivity to potential therapeutic agents might reveal the sensitivity of the original cancer growing in the patient.

Selection of Recipient/Host The vast majority of human cancers develop in later midlife and old age,251–254 and there is clear evidence that, at comparable ages, mice have difficulties rejecting immunogenic cancers.255–258 Yet most experimental studies use young mice. Mice should have a clinically relevant tumor burden or be selected for treatment when the bulk of cancer cells has been removed to undetectable levels by surgery or chemotherapy, but dormant cancer cells stay behind to cause later relapse. T cell–deficient mice have been used extensively

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as models for adoptive transfer of T cells.259 However, cancer patients are usually T cell competent and capable of generating regulatory T (Treg) cells that may not allow an effective “take” of the transferred T cells unless the recipient is lymphodepleted.

CANCER ANTIGENS No term in cancer immunology is more important and confusing than the term cancer antigen (or tumor antigen). Any molecule detected with T cells or antibody on the surface or within cancer cells is commonly referred to as cancer antigen. The usefulness of a cancer antigen for detection and destruction depends on its specificity (ie, that the antigen is not common to normal cells). The first well-defined tumor antigens encoded by the MHC were discovered by Gorer.8 Immunity to MHC antigens will kill the cancer cells but also the host because of the ubiquitous expression of MHC on normal cells.

General Aspects Early History In the late 1800s,260,261 it was discovered that in some instances tumors developing spontaneously in experimental animals could be transplanted into other animals of the same species and in this way could be propagated continuously. This finding provided an important experimental tool for cancer research.40,262 Immediately, scientists began to investigate the possibility of immunizing against such transplantable cancers. Rodents exposed to a small nonlethal challenge of certain tumors became immune to subsequent challenge with large transplants of the same tumor that regularly killed nonimmunized recipients. Also, complete removal of the transplanted tumors, after initial growth, immunized animals against that tumor. These early results seemed to suggest that immunization against cancer was possible. Furthermore, there were certain other spontaneous tumors that were not readily transplantable, and this was taken as evidence for “natural resistance” or “natural immunity” to the cancers. Many years later, it became clear that no such conclusions could be drawn from these early studies, because outbred, or incompletely inbred, rats or mice had been used. The problem became apparent when it was realized that the immunization with tumor would also immunize the host against normal tissue of the donor and that normal tissues of the donor could also immunize the host against the tumor.186 These experiments brought the idea of tumorspecific antigens into disrepute but also started the search for antigens that caused rejection of normal transplanted tissue. This research eventually led to the discovery of the MHC and to the development of inbred mouse strains.8,263,264 Once inbred mouse strains became available, it was found that cancers transplanted within an inbred mouse strain usually grew so well that the existence of tumor-specific antigens seemed very unlikely. In fact, transplantability of tumors in syngeneic animals became (and still is) a diagnostic criterion for the malignant phenotype of an experimental tumor. This criterion was especially useful because many rodent

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tumors are cancers of nonepithelial origin (sarcomas) and also because a clear histologic demonstration of local invasive growth can be especially difficult in such cancers.

Proof of Specificity After discovery of the MHC and the development of inbred mouse strains, the gloom over tumor immunology ended with the discovery that inbred mice could be immunized against syngeneic sarcomas induced by the chemical carcinogen methylcholanthrene (MCA). The first demonstration of induced immunity to a transplantable MCAinduced sarcoma was by Gross in 1943265 ; however, it was not until the 1950s that more complete experiments provided unequivocal evidence for “tumor-specific” rejection of transplanted cancers.229,266–270 In particular, the experiments of Prehn and Main268 in 1957 made it likely that the rejection antigens on the MCA-induced sarcomas were functionally tumor-specific, because transplantation assays could not detect these antigens in normal tissues of the mice used. However, irrefutable evidence for the existence of tumorspecific antigens in autochthonous unmanipulated cancers only came after a 50-year-long search3,5,218,219,236,267–282 when in 1995, it was proven that tumor-specific antigens on cancer cells were encoded by somatic cancer-specific mutations absent in the normal cells of the host of tumor origin.10–12 Even though it is clear that cancer-specific antigens are encoded by somatic cancer-specific mutations, the terms “tumor-specific” or “cancer-specific” are frequently used inappropriately, sometimes even used in conjunction as “relatively” tumor-specific. The discussion is far from being semantic. In reality, an antigen either is or is not tumor-specific. Germline controls are absolutely critical for proving mutations are somatic and tumor-specific.283–286 Unfortunately, germline controls are missing from virtually all tumors used for experimental work today. Nevertheless, mouse tumors with proper autologous controls have also been used and are available for distribution.218,219 Notorious problems are genetic polymorphisms. For example, even after 20 backcross generations when a strain is arbitrarily pronounced “inbred” (because it is then more than 99.9% genetically identical), about 373 polymorphic proteinencoding loci remain allogeneic.287 Any one of these loci could encode a pseudotumor-specific antigen when a tumor is transplanted into a mouse misperceived to be fully syngeneic. Mice respond preferentially to nonself- or mutant– self-antigens whether caused by genetic polymorphism or tumor-specific somatic mutation. Peptide Antigens and Major Histocompatibility Complex Affinity T cell–mediated destruction of cancer cells requires the interaction of T-cell receptor (TCR), peptide, and MHC molecules. In this “three body problem,” two affinities simultaneously determine the interaction288,289 : the peptide to the MHC and that of the TCR to the peptide-MHC complex. Even when the complex cell-cell interaction is reduced to the three molecules interacting in vitro, biochemical analysis is still too complex for analyzing physiologic interactions. TCR affinity to peptide-MHC is therefore usually

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measured by plasmon resonance in the presence of saturating nonphysiologic amounts of the peptide, and TCR affinities measured this way range between (Kd) 1 to 100 μM.288,289 This is a narrow range considering that affi nities of peptides to the MHC range from 1 to more than 20,000 nM.290 This difference points at the peptide-MHC affinity probably being the greatest variable and emphasizes the paramount importance of choosing target peptides with highest possible affinity to the presenting MHC. Proper selection is particularly important as the amount of peptide produced by the cancer cell may be relatively small and always must compete for binding with all other peptides naturally present in the cancer or in the cells cross-presenting the antigen. Affi nity of a peptide to a given MHC molecule is best measured empirically and many empirical affinities are already available (eg, http://tools.immuneepitope.org or www.syfpeithi.de).290–292 However, even when a peptide binds with high affinity to MHC, it will only be expressed on the cell surface when it is naturally processed and present in sufficient amount.293,294

Antigens Revealed by “Reverse Immunology” “Reverse immunology” is the attempt to predict T-cell epitopes within a given amino acid sequence. Traditionally, “reverse immunology” has focused on finding optimal T-cell antigens, properly referred to as peptide epitopes, on infectious agents for generating vaccines. More recently, self- or mutant proteins recognized by T cells or antibodies from cancer patients have been analyzed in the search for peptide epitopes that may be effective targets of T-cell immunity. Computerized algorithms (eg, http://tools.immuneepitope. org295,296 and www.syfpeithi.de297) have been developed to predict the affinity of peptide-MHC binding, appropriate proteasomal cleavage, and transport by the transporter associated with antigen processing (TAP).290–292,297 Though these algorithms are useful, it appears that these tools cannot replace empirical biochemical measurements. Many of the antigens proposed by reverse immunology have affinities to MHC insufficient to serve as effective targets when tested in appropriate animal models. Together, high peptide-MHC affinity is essential but not sufficient to predict that a given peptide serves as an efficient target. Complete sequencings of cancer cell genomes from individual patients is becoming increasingly affordable and has revealed up to many thousands of mutations per cancer cell.298–300 However < 1% of these mutations cause amino acid substitutions. For example, of the 33,345 nucleic acid base substitutions found in a human melanoma, most of the mutations were intergenic, intronic, noncoding, silent, or truncating. Only 187 caused amino acid substitutions in coding genes,300 and only a few of these 187 coding substitutions are expected to lead to mutant peptides that serve as effective targets. To be effective, the mutant peptide 1) must bind with a high affinity (IC50 in the nM range) to the particular MHC molecules of that individual and 2) be naturally processed, 3) escape destruction by proteasome cleavage, and 4) be present in sufficient amounts. Only a few of the myriad of tumor-specific mutations identified in human or murine cancers give rise to antigens that fulfill these requirements. However, the frequency of unique tumor-specific targets in

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a cancer cell may be larger than predicted when considering only mutations in coding sequences, because mutations in intron sequences can be translated and also encode tumorspecific antigens.11 These mutations are over 50-fold more frequent than those in protein-encoding loci.

Antigens Recognized by Patients’ T cells In the search for the best antigens to eradicate the cancer, T cells infi ltrating tumors (TILs) have been recovered, expanded, and used therapeutically. However, TILs are removed from cancers that have not been destroyed. It is, therefore, a widespread misperception that the TILs were necessarily fighting cancer growth. Three questions become obvious: 1) Are strong antigens retained because the host has been tolerized during very early stage of cancer growth? 2) Can T cells specific for the strongest antigens be recovered from the TILs? 3) Can T cells specific for weak antigens promote rather than inhibit tumor growth? It is possible that 1) potent rejection antigens are retained because they tolerize the host very early during cancer development,228,301 2) a few T cells have not been tolerized and can be propagated for therapy, and 3) tolerization is reversible, and competent effector cells can be obtained from TILs. Clinically, the patients who fare best have T cells in their peripheral blood that are specific for antigens encoded by somatic tumor–specific mutations.12,302,303 Whether the same T cells were infi ltrating these cancers as TILs is unknown. In any case, it is likely that only a few (if any) of an

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endless array of antigens recognized on cancer cells by antibodies or tumor-infi ltrating T cells may have significance as targets or diagnostic markers.

Cancer-Specific Antigens (Encoded by Mutant Genes) Prevalence All cancers in man and mouse that have been analyzed carefully express bona fide tumor-specific antigens that could be targeted by T cells. Because tumor-specific antigens arise from mutations,10–12 they are usually unique. Each patient’s cancer seems to have a unique set of mutations, and unique antigens can provoke powerful immune responses. In mice, immunization with one tumor protects only against the same tumor (Fig. 47.2).5,274 Shared cancer-specific antigens do exist when the same mutations occur in several cancers, but by comparison, such antigens are relatively rare. Most cancer-specific mutations affect intracellular proteins that may be recognized by T cells as mutant peptide-MHC complex on the surface of viable cancer cells. Very few cancer-specific mutations affect surface proteins, such as the mutant epidermal growth factor receptor (EGFR), a shared tumor-specific antigen on glioma cells.304,305 Oncogenicity A major misconception is that unique cancer-specific antigens are caused by random mutations and are incidental to the oncogenic process. For example, a group of experts

FIG. 47.2. Demonstration of the Individual (Unique) Specificity of Rejection Antigens on Independently Derived Methylcholanthrene-Induced Tumors in Transplantation Experiments. For details, see Basombrio.3 Modified from Basombrio and Prehn.5

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stated in 2009306(p.5326) that unique antigens come “from random mutations presumed to be present in all patients,” implying mutations encoding unique tumor antigens have no functional significance in the malignant process. Nothing is further from the truth. Of course, all mutations caused by chemical and physical carcinogens are random, but virtually all of them are lost during cancer evolution except those that promote the neoplastic process. When it was first shown in 1995 that cancers harbor cancer-specific antigens caused by somatic tumor–specific mutations,10–12 every one of the three antigens represented a mutation in a tumor suppressor locus.307–311 For example, the mutation in the cycline-dependent kinase 4 reduces the binding to its inhibitor and tumor-suppressor protein p16INK4a,12 and the same mutation is found in the germline in cases of familial melanoma.227 The fact that selection for a tumorspecific antigen commonly represents a mutation in genes with functional significance in the malignancy of that cancer (oncogenes or tumor suppressor genes) has been shown in many other studies.219,312–315 Remarkably, T cells against unique tumor antigens identify mostly novel, functionally important tumor-specific mutations and oncogenic proteins that would not have been easily detected by other technologies.10–12,219,307–309,316 Importantly, several of these unique tumor-specific antigens are excellent targets because they cannot be lost by immune selection. The reason is that some of these mutant proteins are not only oncogenic but are also needed to provide an essential household function no longer provided by the second allele due to Knudson-type loss317 or mutational inactivation.302,307–309

Therapeutic Significance Another misperception surrounding unique tumor-specific antigens is that they have remained unexploited clinically, because truly personalized therapy would be required. Again, nothing is further from the truth. One of the most effective immunologic treatments of cancer today is the adoptive cell transfer of autologous tumor-infi ltrating lymphocytes from patients with metastatic melanoma: response rates are in the range of 50% to 70% of the patients, and a few of these patients are cured.318 This truly personalized therapy involves reinfusion of the patients’ own lymphocytes isolated from the patients’ own melanomas and expanded in vitro. Because the TILs response is dominated by T cells to unique tumor-specific antigens,303 it is likely that the success of the reinfused T cells depends on their reactivity to unique tumor-specific antigens. Similarly, clinical studies show that therapeutic vaccinations with autologous cancer cells are likely to be much more effective,319,320 confirming decades of experimental work.229,234,268,269,271,321,322 Self-antigens may serve as useful and effective targets in a very few instances (eg, CD20 and CD19), but industry supported by government panels and organizations focuses almost exclusively on self-antigens,306,323,324 so far with very modest results.325 Finally, it is a misperception that immunizing each patient to the unique antigen of the individual’s tumor is impractical because of cost and should not be pursued. This is incorrect compared with other highly individualized treatments such as those used in renal transplantation. Thus, individualizing

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cancer therapy could be affordable as other strategies used in the clinics.

Shared Tumor-Specific Antigens Ideally, antigens targeted on cancers would be expressed exclusively on malignant cells but be shared by cancers of the same type or at least subtype. Several cancer-specific mutations have been identified that are shared between cancers,9,304,326,327 but very few of them have so far been found to be an effective immunologic target. The main reason probably is that, aside from specificity, additional requirements for effective recognition by T cells are 1) high-affinity binding to the patients’ MHC molecules and 2) expression of the protein at sufficient amounts to compete with other peptide for presentation. Unique tumor-specific antigens must fulfi ll the same requirements but, because these unique mutations are more abundant, chances are much higher to yield an effective antigen. A few examples of shared tumor-specific antigens are discussed in the following. Mutant Epidermal Growth Factor Receptor. See further under Immunotherapy. Fusion Proteins. Fusion proteins found in cancer cells are the result of internal deletions (see mutant receptor EGFRvIII under Immunotherapy) or chromosomal translocations.9,326 New antigenic determinants can result from the juxtaposition of previously distant amino acid sequences, resulting in a mutant peptide sequence at the breakpoint and possibly a change in conformational structure. The same chromosomal breakpoints consistently recur in different individuals with the same cancer, therefore result in shared tumor-specific antigens. Fusion proteins encoded by these translocations are usually essential for cancer maintenance, making them ideal targets for pharmacologic and immunologic intervention because tumor cells may not easily escape therapy by losing expression of the fusion proteins.328 Pharmacologic approaches are exemplified by the drug imatinib targeted to the fusion protein of the 9;22 translocation in chronic myelogenous leukemia.9,294,329 The antikinase drug imatinib does not selectively inhibit the catalytic activity of the BCRABL fusion protein, and this results in toxicity to normal cells in the patient. The intracellular BCR/ABL fusion proteins can be recognized specifically by antibody but only in fi xed cells.330 BCR/ABL fusion peptides can also be recognized by human CD4 + T cells in the context of MHC class II331 and serve as targets for CD8 + human cytolytic T cells.332 Why do we then still lack effective immunologic therapies for targeting this and other fusion proteins? As explained previously, the predicted epitopes may 1) not be generated naturally by cells,293 2) lack sufficient affinity to the particular MHC of that patient, or 3) not be produced in sufficient amounts. Mutant RAS. Point mutations in oncogenes can also be shared by several cancers and could encode useful antigens. For example, a valine for glycine substitution at position 12 of RAS is one of the most common mutations in human cancers and can be recognized by human CD4 + T cells.333 The region of the mutant RAS protein from which

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the peptide was derived is identical for all the three members of the RAS proto-oncogene family, namely, H-RAS, K-RAS, and N-RAS, which have different prevalence in different cancers. In addition, about 90% of pancreatic adenocarcinomas, a very aggressive human cancer, have one of three to four different single amino acid substitutions in codon 12 of the cellular Kirsten RAS gene.334 Despite remarkable clinical and experimental efforts,335–340 targeting mutant RAS-derived epitopes has so far met with little if any success, the likely reasons being the same as those mentioned under the section titled Fusion Proteins. Immunizing cancer-prone mice harboring “initiated” mutant ras-expressing cells with mutant ras peptide resulted in mutant ras-specific CD4 + T cells, antibodies to the mutant ras protein, and more tumors and faster tumor development.341 Possibly, the antibody, produced in response to antigen, activated myeloid cells via FcR-γ to become tumor-promoting, and/ or antibody carrying TGF-β downregulated CD8 + effector responses.342,343

Self-Antigens (Encoded by Normal Genes) All antigens listed in this section are encoded by nonmutant cellular genes and expressed not only by cancer but also by at least some normal adult cells. Therefore, these antigens are not tumor-specific and are commonly referred to as tumor-associated antigens. The level of expression of these antigens can vary from widespread expression to restriction to a small population or a subset of normal cells. However, self-antigens should not be called “quasi–tumor-specific” because even very low levels and/or selective expression of a self-antigen may cause severe even lethal toxicity when targeted.204,206 Self-antigens expressed by tumor cells are used for destruction, inhibition, or detection of cancerous growth. When used as targets for destruction, T cells or antibodies must eliminate the cancer cells while not destroying normal cells expressing the same self-antigen, or destruction of selfantigen–expressing cells must be tolerated. Thus, no serious toxicity must occur even when the immunity is strong enough to destroy the cancer cells.195,196 Antibodies against growth factors or their receptors can inhibit growth of cancer cells without destroying them. When self-antigens are used for diagnosis, background levels of antigen generated by normal cells complicate use of these antigens for early detection of cancer. However, changes in the amount of circulating self-antigens may indicate relapse of cancer after therapy. All normal individuals have so-called natural autoantibodies as well as T cells to a wide spectrum of self-antigens without causing pathology, but those B-cell receptors and TCRs have usually very low affinity. In fact, any immune receptor binds with some affinity to any particular antigen344 and immune receptors may bind to several molecularly unrelated structures,345 making the discussion of specificity seemingly useless. However, the antibody response of an individual to self- and nonself-antigens differs in affinity by many orders of magnitude, and therapeutically effective antiself-antibodies or TCRs are commonly raised in a nonself, usually xenogeneic, setting. Whether removing

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natural mechanisms that prevent autoimmunity is a general approach to achieve truly effective antitumor immunity needs to be substantiated. The problem of B-cell unresponsiveness can be overcome by immunizing mice with xenogeneic human antigen. Misleadingly, these high-affinity antixenogeneic antibodies are usually advertised as “fully human” when they were made in mice in which the murine immunoglobulin (Ig) gene locus had been inactivated and replaced with the human Ig gene locus.346 The toxicities of such “fully human” antibody will still be those of high-affinity antiself antibody: severe to lethal toxicity. These reactions may occur when the variable region of such antibodies (eg, anti-CEA or anti– human epidermal growth factor receptor [HER]-2204,206) is fused with the transmembrane and signaling domains and then transduced as chimeric antibody receptors (CARs) into T cells.347 T-cell tolerance to self can be overcome by making T cells in a host that is allogeneic to the MHC class I molecule presenting the targeted peptide.348 However, recent studies exposed that such allo–human leukocyte antigen (HLA)– restricted high-affinity T cells can have severe “off-target” reactivity likely to cause toxicity when used in patients.349 T cells transduced with these TCRs kill each other (fratricide) when the self-antigen (eg, wild-type p53 or survivin) is expressed by the T cells.350,351 High-affinity TCR cells can also be generated in knockout mice lacking the targeted selfpeptide (eg, wild-type p53 peptide sequences352), but T cells expressing these TCRs are lethal when given to normal mice because wild-type p53 is expressed in bone marrow and can also be expressed by most other normal cells.350

Overexpressed Molecules Growth Factors and Their Receptors. See Immunotherapy. Survivin. Although survivin is overexpressed in many cancer cells, it is a problematic antigen particularly as a vaccine because it is widely expressed on lymphocytes causing fratricide.351 Not surprisingly, this antigen has been ineffective in vaccine trials. p53. Research using p53 as target for immunotherapy is exemplary of the problems and persistent misconceptions of research on tumor-specific (unique or shared) and selfantigens. Mutations in the p53 suppressor gene are among the most common found in human and experimental cancers.353,354 These mutations tend to cluster in evolutionarily conserved regions of the gene, but the exact locations are highly diverse in individual cancers.355 Targeting these mutations would require an individualized therapy considered by many impractical. Therefore, researchers have attempted to exploit the fact that mutant p53 is usually overexpressed in cancers. Thus, major efforts have been made to target conserved, nonmutated regions of the p53 protein hoping for “relative tumor specificity” based on the usual belittlement for low-level expression of this protein by every normal cell including lymphocytes. As a result, T cells expressing highaffinity anti-p53 TCRs commit fratricide unless the anti-p53 TCRs are transduced into T cells from p53 knockout mice.

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However adoptive transfer of these T cells causes lethal hematopoietic ablation in normal mice.350 Mortality can be prevented by reconstitution of the recipients with bone marrow from p53 knockout mice,350 an option unavailable in humans. Not surprisingly, there is no evidence for clinical efficacy of targeting wild-type p53 in humans. Prostate-Specific Membrane Antigen. Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein more correctly referred to as glutamate carboxypeptidase II. PSMA is an inappropriate misnomer because it is anything but specific for prostate; it is also expressed by duodenal mucosal cells, proximal renal tubule cells, and a subpopulation of neuroendocrine cells in colonic crypts,356 and has important functions in the brain.357 Vaccination of mice with human PSMA induces antitumor effects without causing toxicity but because the mouse model lacks expression of human PSMA on normal tissues, the fi ndings in this inappropriate model are meaningless.358 Similarly, a “fully humanized” anti-PSMA antibody had potent antitumor xenograft activity in mice that lacked expression of this antigen on normal cells.359 Therefore, this antibody may have severe, possibly lethal, toxicity in patients.

Differentiation/Lineage-Specific Molecules Some antigens expressed on tumor cells are also expressed during at least some stage of differentiation on nonmalignant cells of the lineage from which the tumor developed. Differentiation antigens may therefore help to determine the organ or cell type of origin (lineage) of a cancer.360 For example, B-cell tumors express surface Ig, and T-cell leukemias can be separated into helper and suppressor cell leukemias using T-cell subset–specific monoclonal antibodies to surface and intracellular antigens including transcription factors. For metastatic cancer of unknown origin, differentiation antigens can be important indicators of the histologic and organ site of the primary tumor.361 Careful diagnostic delineation of different subtypes of cancer is important because different tumor subtypes may have different prognoses and may be susceptible to different therapies. However, the use of differentiation markers for histologic or cytologic tumor classification has pitfalls.361 Lineage-specific antigens represent a very diverse group of proteins: glycoproteins (including mucins) and glycolipids (carbohydrate, peptide, glycopeptide, or glycolipid epitopes). Several of these antigens are being explored as potential immunotherapeutic targets. However, the normal cell types that express these antigens will determine toxicity and usefulness of any antibody or agent for which these are targets. Cluster of Differentiation 20 and Cluster of Differentiation 19. CD20 is a signature B-cell differentiation antigen targeted by a genetically engineered monoclonal antibody that is relatively effective in the treatment of B-cell lymphoma. Lineage restriction of this marker limits the cytocidal effects to long-term depletion of normal B cells. This depletion is usually well tolerated because patients can be protected by intravenous administration of IgG. While

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antibodies to CD20 antibodies are therapeutically effective, relapse is common. CD19, but not CD20, appears to be expressed on the more immature malignant cells causing the relapse. Therefore, anti-CD19 Fv has been used in the killing of these cancer cells, either as a fusion protein with an antiCD3 Fv to engage T cells195 or as chimeric antibody receptor inserted into the patient’s T cells.196 These treatments may prevent relapse. Melanocyte-Specific Differentiation Antigens. Several differentiation antigens (such as tyrosinase, the related brown locus protein or tyrosinase-related protein 1 (Trp-1), gp100, and Melan A/MART-1362,363) appear to be restricted to melanocytes, and all of them are being explored as immunotherapeutic targets in melanoma.364–366 Immune recognition of the melanocyte differentiation antigens can lead to rejection of a tumor challenge but this self-antigen–specific immunotherapy not only targets the tumor cells but also normal cells expressing the shared antigens,365,367 resulting in the depigmentation of normal skin (vitiligo) and possibly other, more serious toxicities (see following discussion). For example, mice immunized with syngeneic Trp-1 in various adjuvant settings fail to produce a T- or B-cell response.368 Xenogeneic or altered Trp-1, however, induces responses that cause vitiligo and protect against challenge with melanoma cells.368 Passive transfer of Trp-1–specific antibodies from these mice causes vitiligo and protects against metastatic spread of melanoma cells in mice when given at the time of seeding of the malignant cells.367 Similar antibodies may have analogous beneficial effects in human melanoma patients,369 but evidence is lacking for such antibodies eliminating bulky human melanoma. Trp-1–deficient mice respond to Trp-1 as a foreign antigen and Trp-1–specific CD4 + T cells from these mice can eradicate large B16 melanoma 10 to 14 days after inoculation,370,371 cause autoimmune vitiligo, and damage the retina.370 Designating these T cells as “tumor-specific” when they are clearly not is incorrect and misleading.370 Trp-1 is expressed in all neurocrest-derived pigmented cells, not only those of the skin but also those of the eye (uvea), inner ear, and brain (substantia nigra, forebrain, and midbrain).372–375 Other targets include gangliosides GD2 and GD3 that are also not only overexpressed in melanoma but are also found in other cells of neurocrest origin and in other tissues.376,377 Prostate-Specific Antigen. Prostate-specific antigen (PSA), also called kallikrein-3, is a chymotrypsin-like protease that digests semenogelin I and II to release motile sperm.378 Elevation of PSA above the normal range occurs in inflammation (prostatitis) and benign hypertrophy of the prostate as well as in prostate cancer. Using PSA levels for early detection of prostate cancer is controversial. The U.S. Preventive Services Task Force no longer recommends this test in healthy man.379 However, detection of any PSA following complete surgical removal of the prostate indicates residual tumor cells and/or recurrence.380 Prostate-specific phosphatase is selectively expressed and secreted by the epithelial cells of the prostate gland.378,381 Sipuleucel-T, a vaccine targeting prostate-specific phosphatase

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and approved by the U.S. Food and Drug Administration for the treatment of castration-resistant prostate cancer, results in only a very modest improvement in overall survival.323 Furthermore, a recent analysis of internal documents that became available after the U.S. Food and Drug Administration approval questions the adequacy of the trial and correctness of the conclusions drawn.382 Epithelial Cell Adhesion Molecule. Normal and malignant cells of epithelial origin express the transmembrane glycoprotein epithelial cell adhesion molecule (Epcam).383 Anti-Epcam antibody 17-1A 384 showed promising results in patients with colon cancer in several early studies,385 whereas subsequent randomized clinical trials consistently failed to show any benefit.386 A high-affinity engineered anti-Epcam antibody caused cases of acute pancreatitis and was discontinued.387 Other anti-Epcam antibodies in the form of bi- and trifunctional constructs may be more effective, but finding efficacy while keeping lethal toxicity under control presents a major hurdle.

Tumor Antigens Caused by Altered Glycosylation Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers,388,389 and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy. Mucins. Mucins are normally heavily glycosylated glycoproteins (ie, containing complex O-glycans) that protect the luminal mucous epithelial surfaces. Mucins of cancer cells often show decreased expression of the complex O-glycans and increased expression of short oligosaccharides, the TF, sialyl-Tn, and Tn antigens390 (see following discussion). Thus, human adenocarcinomas of the pancreas, breast, and colon express mucins391 that can be recognized on cancer cells by MHC-unrestricted cytolytic T cells. These T cells apparently react specifically with repeated epitopes on the protein core of the mucin molecules,392 exposed because of deficient glycosylation in the malignant cells.393 The epitopes are expressed at high density, do not require processing, have a stable conformation, and can directly bind to certain TCRs without being presented by MHC molecules.394 Hypoglycosylated MUC1 is expressed on about two-thirds of newly diagnosed cancers. However, multiple types of vaccines using the nonglycosylated tandem repeat peptides or tumor-associated saccharide antigens conjugated to carriers failed to immunize effectively.395,396 This finding is consistent with multiple lines of evidence that humans and MUC1-transgenic mice are tolerant to the unglycosylated long MUC1 peptide. Numerous approaches have been developed capable of overcoming this tolerance such as using short and long synthetic glycopeptides or plant-expressed MUC1.397–400 Many different mucin genes have been identified401 and antigens have been defined, but evidence for clinical efficacy with controllable toxicity is lacking. T and Tn Antigens. Tn antigen on human erythocytes, first described by Moreau et al. in 1957,402 is the cause of a hemolytic autoimmune disorder, Tn syndrome.403 Tn (“T antigen nouvelle”) is a Ser/Thr-O-linked N-acetylgalactosamine

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monosaccharide distinct from the disaccharide T (or TF) antigen galactose-β1-3-N-acetylgalactose O-linked to a Ser or Thr (Galβ1–3GalNAcα1-Ser/Thr) described earlier by Huebner, Thomsen, and Friedenreich.404 Human cancers very frequently express TF and Tn antigens.405,406 Recently it was shown that the Tn syndrome is caused by somatic mutations in the chaperone COSMC.407 While TF antigen is an oncofetal antigen highly expressed in the embryo and fetus,390 there is no evidence that Tn is also an oncofetal antigen.404 Mutational deletion of the chaperone COSMC leads to expression of the Tn antigen on virtually every embryonic cell and leads to early embryonic death.408 Most adults naturally have anti-Tn as well as anti-TF antibodies due to antigenic stimulation by Tn and TF antigens expressed on the bacterial flora.409,410 Tn antigen is also expressed on human immunodeficiency virus-1 and pathogenic parasites. Thus, evidence is lacking for any expression of Tn antigen on adult or embryonic human or murine cells except by patients suffering from Tn syndrome and cancers that may or may not show a tumor-specific deletion of COSMC. There is no evidence that Tn is an effective therapeutic target. This may be due to anti-Tn antibodies being usually IgM or IgA of very low affinity, though recently developed IgG antibodies reduced slightly the growth rate of human cancer cells in vitro and in vivo.411 Glycopeptide Antigens Resulting from Tumor-Specific COSMC Mutations. The remarkable characteristic of this new class of antigens is the exquisite tumor specificity of the antigen412–414 even though it is encoded by normal genes. Appearance of the antigen, however, depends on a tumor-specific somatic mutation that destroys the chaperone COSMC that is essential for any functioning of the core 1β (1–3) galactosyl-transferase, T-synthase. COSMC protects the newly synthesized T synthase from aggregation and subsequent endoplasmic reticulum–associated degradation.404 T-synthase is essential for extending O-linked glycosylation beyond a single O-linked N-acetylgalactosamine (ie, Tn antigen). Importantly, the antigen recognized by the high-affinity, tumor-specific antibody does not bind Tn alone but contains the Tn hapten (ie, the single O-linked Nacetylgalactosamine on a threonine or serine). X-ray crystallography shows that the antibody completely envelops the carbohydrate moiety while interacting with the unique sequence of the peptide moiety in a shallow groove.413 Because Tn does not exist on normal embryonic or adult human or murine cells, there is no central or peripheral tolerance to these antigens allowing high-affinity destructive immune reactions to occur. This also explains the severity of the autoimmune disease associated with the Tn syndrome caused by somatic (not germline) mutations of the COSMC gene.407 Importantly, somatic tumor–specific mutations disabling the X chromosome encoded COSMC gene (for which one copy is naturally silenced) seem to be more common than originally assumed and occur in a wide variety of spontaneous or virally induced human and murine cancers.415 Nevertheless, it must be shown that high-affinity receptors when used as CARs on T cells or linked to anti-CD3 are effective against tumors but have little if any toxicity to

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normal tissues. This may be expected from the fact that the epitopes depend on somatic tumor-specific mutations.

Oncofetal, Carcinoembryonic, and Cancer-Germline Antigens Over 50 years ago, human cancer cells were found to express antigens that serologically cross-react with normal embryonic tissue.416 Since then, it has been postulated repeatedly that certain normal genes that are completely silent in all nonmalignant cells may be activated exclusively in malignant cells.417 Alternatively, it has been postulated that cancer cells may express proteins (or immature forms of a protein) that are only expressed in fetal but not in nonmalignant adult cells.418 There certainly is profound hope for finding a universal tumor rejection antigen that can be used for vaccination, prevention of cancer, and antitumor therapy. However, repeated claims of selective activation of normal genes or selective expression of immature forms of these proteins in cancer cells leading to tumor-specific antigens have not been substantiated. Expression of the same antigen by at least one normal cell type in the adult was later often discovered.419–422 Cancer-Germline/Cancer-Testis Antigens. Changes in gene methylation lead to the expression of a large number of genes encoding a group of antigens referred to “cancer-testis,” “cancer-germline,” or “cancer-spermatogonal” antigens. Many cancer-testis antigens have been described in humans such as MAGE, BAGE, GAGE, LAGE/NY-ESO-1, SAGE, HAGE, and BORIS.422–430 All cancer-testis antigens are expressed at high levels in spermatocytes in the testis. And most, if not all, are also expressed in the thymus.430–432 Many, but not all, cancer-testis antigens are encoded on the X chromosome.433 Most of cancer-testis antigens can be recognized by autologous cytotoxic T-lymphocyte (CTLs), but there is no evidence for these antigens being generally effective targets for cancer cell destruction in human.434,435 P1A was the first cancer-testis antigen to be identified in the mouse mast cell tumor line P815.436,437 P1A can be induced in a broad range of tumors of diverse histologic origins with the demethylating agent 5-aza-2’-deoxycytidine.438 Various normal tissues including thymus and premeiotic spermatocytes also express P1A.437,439 While active immunization has only modest protective effects against cancer cell inoculation,440 adoptively transferred T cells (monoclonal or polyclonal) targeting only the P1A antigen shrink large tumors (> 1 cm in diameter) for several weeks followed by relapse caused by epitope-loss variants.441 For eradication, cancer cells must first be transfected to express costimulatory molecules, which probably leads to the induction of T cells to recognize other tumor antigens.442 Remarkably, autoimmunity has not been reported to be associated with a response to this antigen. In humans, the HLA-A2–restricted NY-ESO1 peptide has very poor affinity to its presenting MHC class I molecule, but high-affinity TCRs to this antigen have been generated and their efficacy in therapy is being explored.443 While many cancer-testis antigens have been described and studied extensively for two decades, their usefulness as targets eradicating human cancers still remains to be shown. Despite these uncertainties,

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occasional successes have been observed. The hypothetical explanation: the relatively few T cells induced against the cancer-testis antigen served to elicit additional antitumor T-cell clones directed against other antigens that are actually responsible for the tumor regression by a poorly understood phenomenon referred to as epitope spreading.434,444 Numerous cancer-testis antigens have been detected using sera of cancer patients for identifying antigens by recombinant expression cloning (SEREX). Importantly, sera from mice infected with cytopathic or noncytopathic viruses or injected with tumor cell lysates also show an autoantibody response of broad specificity, and intriguingly the majority of the identified autoantigens have been previously described as autoantigens in humans.445,446 This suggests that human SEREX antigens may have to be regarded as afterglows of infection-associated immunopathology and/or tissue damage. This is consistent with the assumption that the human adult IgG autoantibody repertoire is the result of lifelong encounters with bacterial and viral agents and tissue damage. Together, usefulness of the SEREX-defined selfantigens remains to be demonstrated. Carcinoembryonic Antigen. CEA is a 200-kDa membraneassociated glycoprotein that is expressed not only in fetal but also in adult nonmalignant tissues such as normal colonic mucosa, lung, and lactating breast tissue.447,448 It is released into surrounding fluids. At one time, it was hoped that CEA could be used as a marker for early diagnosis of gastrointestinal and other malignancies449 ; however, elevated serum levels of CEA are also found in the absence of malignancy (eg, in smokers and in inflammatory bowel diseases such as ulcerative colitis). Though serum levels of CEA are not useful for detecting early cancer, the level of CEA in the blood can be used to monitor the effects of therapy to indicate whether a cancer has been successfully eradicated or has recurred.450 Using CEA as immunotherapeutic target is highly problematic because of severe toxicity. Mice expressing the target antigen in normal tissues closely resembling expression in humans showed severe toxicity from anti-CEA T-cell responses,205 a result that predicted the severe adverse effects later observed in patients treated with anti-CEA CARs.206 Alpha-Fetoprotein. Alpha-fetoprotein (AFP) was the first defined oncofetal protein.451 Though produced by fetal liver and yolk sac cells, it is also present in small amounts in cells and the serum of normal adults. The amount of this protein is elevated in some patients with cancer of the liver or testis and also in some patients with various nonmalignant liver diseases. Therefore, similar to CEA, using AFP as a marker for the early diagnosis of cancer is of questionable use.452 Nevertheless, assays of AFP can detect primary liver cancer at a time when the cancer is treatable, and AFP assays are also used for monitoring patients after therapy.452 The use of AFP as target for active or passive immunotherapy is complicated by lack of specificity.417,453

Clonal Antigens Clonal antigens are expressed only on the clone of cells from which the cancer originated.454 Except for idiotypes of surface Ig-positive B-cell and T-cell malignancies, there

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are presently no candidates for other clonal antigens. Immunization of animals against the idiotype induces an idiotype-specific transplantation immunity against the growth of cancers (myeloma, lymphoma, and leukemia) expressing the idiotype.455,456 Furthermore, idiotype-specific antibodies prevented the growth of surface idiotype-positive murine malignant B cells in mice and guinea pigs in vivo and in vitro,457–460 and occasionally induced the cancers to go into a long-lasting dormant state.461 Finally, idiotypespecific monoclonal antibodies have caused several partial remissions and one complete remission in patients with B-cell lymphoma,462 and targeting the idiotype on B-cell malignancies continues to be explored.463–465 It is necessary to generate different monoclonal antibodies or idiotypic vaccines for each individual cancer to be treated, but the advantage of using such clonal antigens over less restricted tumor-associated antigens is that eliminating the few normal cells bearing the same antigen would probably not adversely affect the patient.

Viral Antigens (Encoded by Viral Genes) Cancer-causing tumor viruses such as SV40, polyoma viruses, human papillomavirus (HPV), hepatitis B virus, hepatitis C virus, human T-lymphotropic virus 1, Epstein-Barr virus (EBV), and other herpes viruses and their antigens are discussed in other parts of the chapter. For discussion of a

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70-kDa glycoprotein (gp70)419,420,466,467) and other important antigens468–470 encoded by ribonucleic acid (RNA) tumor viruses (eg, murine leukemia virus, maize streak virus,471–474 and mouse mammary tumor virus475), see Coffin476 and Schreiber.477

IMMUNOGENICITY OF AUTOCHTHONOUS CANCERS There is the widely held misconception that human cancers are usually less immunogenic than are murine tumors.478 However, convincing evidence indicates that many, probably all, human cancers are antigenic. Similarly, even the most antigenic murine UV-induced “regressor” tumors grow progressively and invariably kill the primary host218,479 (Fig. 47.3). Only transplantation of primary murine tumors into young, syngeneic, immunocompetent, tumor-free recipients reveals their antigenicity and immunogenicity. UVinduced regressors are so immunogenic that they are rejected by naïve immunocompetent hosts. Many experimental cancers require preimmunization to be rejected upon transplantation, and the degree of immunogenicity usually refers to the relative strength of antigen-specific protection a tumor can induce against rechallenge with that tumor. Obviously, the resistance of the host to its autochthonous tumor cannot be great at the time it is clinically apparent and progressively growing.228 Only weak reactions can be detected321 because resistance of the autologous host depends on vaccinations

FIG. 47.3. Autochthonous Cancers are Indistinguishable in the Primary Host Regardless of Whether They are Highly Antigenic or Not. Only subsequent transplantation into young syngeneic immunocompetent hosts defines these tumors as regressors or progressors, and shows their antigenicity and immunogenicity.

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with the autochthonous tumor following complete tumor removal.269 Antigenicity refers to the antigens on a cancer cell. However, a cancer may have strong antigens yet fail to induce a response (ie, lack immunogenicity). A cancer may also be antigenic and immunogenic but resistant to destruction by the immune response it induces. Thus immunogenicity, antigenicity, and immunosensitivity must be distinguished. Progressively growing autochthonous cancers differ greatly in immunogenicity as determined by responses of syngeneic, tumor-free hosts. Autochthonous tumors induced in mice with UV are among the most immunogenic cancers; transplantation resistance to these so-called regressor tumors appears to be absolute rather than relative. Rejection by normal syngeneic mice is observed without prior immunization, even when the largest testable doses of tumor cells or fragments are used.275 Most MCA-induced fibrosarcomas, unless induced in immunodeficient mice (see following discussion),480 display an intermediate degree of immunogenicity in normal mice.268,269,481 This is shown by the fact that induction of immunologic resistance to most chemically induced tumors requires prior immunization because the initial graft of the tumor generally produces progressive lethal growth.268 Importantly, failure of a cancer to induce a tumordestructive immune response does not mean it lacks either antigenicity or immunogenicity. (See below discussion of sporadic SV40-induced tumors under Immune Surveillance of Cancer228,301.)

Age and Latency Experimentally, the length of the latency period of a tumor is usually inversely proportional to the dose of carcinogen. In humans, the doses of (ie, the levels of unintentional exposure to) carcinogens and promoters are believed to be usually relatively low. This means usually a long time is needed for the initiated cells to accumulate the multiple genetic events essential for a premalignant or cancerous lesion. Once the neoplastic cells have reached sufficient numbers to stimulate a response, the host may be too old to respond vigorously. The overwhelming majority of human cancers arise in individuals past 60 years of age,254 and it has been shown repeatedly in humans and mice that the immune response to new antigens declines with age258,482–484 ; this should include antigens on the developing tumors. Thus we do not know the extent of which cancers arising in old individuals may have been selected or retained antigenicity and immunogenicity. In stark contrast, most experimental cancers are induced at an age correlating to that of young middle-aged humans. If 2 years of mouse life roughly correlate to 60 years of human life, then most experimental cancers mirror cancers developing in humans 30 years old or less. Tumors induced by MCA or transgenic oncogenes are usually produced in mice less than 1 year old.485–489 The length of the latency period of these MCA-induced tumors correlates inversely with the degree of immunogenicity.229,490–492 There is no such correlation in UV-induced tumors275,479 that begin to develop past 9 months of age, mostly in mice more than 1 year old. Old mice beginning at about 9 months of age

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fail to reject various types of highly antigenic cancer cells that are regularly rejected by young mice.255–258 Even though UV irradiation is immunosuppressive, young UV-irradiated mice remain immunocompetent long enough to select for antigen loss variants.235 This indicates that advanced age must contribute to allowing the developing cancers to remain immunogenic. An intriguing question is how many of the tumors that spontaneously arise in older animals would grow in younger syngeneic hosts.480 Considerable but only indirect evidence makes it very unlikely that all tumors would grow.

Spontaneous versus Induced “Spontaneous” cancers that develop without any known exposure to carcinogens tend to be less immunogenic than cancers induced by DNA tumor viruses or by deliberate exposure to carcinogen.266,267,493–495 Unfortunately, serially transplanted tumors were used for most of these comparisons, and transplantation may have selected for less immunogenic variants, thereby confusing the results. If spontaneous murine cancers more closely resembled human cancers, this could suggest that human cancers are poorly immunogenic.495 However, most human cancers are not “spontaneous” but induced by environmental carcinogens, have never been transplanted, and develop mostly in old individuals that may not select for cancer variants. Most, if not all, carcinogens are mutagens17 and probably always cause the expression of tumor-specific antigens. However, it is not clear why tumors induced with the same dose of chemical or physical carcinogen may exhibit quite different degrees of immunogenicity.268,479 One reason might be that the actual local dose of carcinogen that is delivered to a particular target cell or target tissue may vary greatly from animal to animal. Another reason might be that mutations are selected that favor malignant behavior irrespective of the degree of immunogenicity of that mutant protein. This is consistent with the observation that considerable differences in immunogenicity of primary UV-induced tumors only become apparent after transplantation into secondary hosts218,479 (see Fig. 47.3).

“Regressors” from Immunocompromised Hosts Immunocompetence of the host may not affect the cancer incidence but still influence the immunogenicity of the developing tumor. Conversely, immunosuppression or immune deficiency of the host during carcinogenesis should allow growth of highly immunogenic tumors in the absence of such selection.

Clinical Evidence Clinical support comes from the appearance of highly antigenic EBV-associated lymphomas in immunosuppressed transplant recipients that are virtually unknown to occur in immunocompetent humans.502 In addition, renal transplant patients experience a reduced risk of developing UVinduced skin cancer after immunosuppressive medications are stopped.503 It is tempting to suggest that many of these

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cancers developing in transplant patients are actually “regressor” tumors that have arisen only because host defenses were damaged.

Experimental Evidence In normal adult mice, Moloney sarcoma virus496 induces tumors larger than 2 cm in diameter that then regress while tumors continue to grow and kill immunodeficient adult or newborn mice.497–499 The first experimental evidence for UV-induced regressors came from Kripke in 1974,275 showing that UV irradiation made mice immunodeficient and induced cancers that were often “regressors,” meaning that they were rejected with any testable size inoculum by naïve syngeneic immunocompetent mice. Tumor transplants will grow for about a week and then disappear, though small numbers of the same tumor cells will grow and kill athymic nude mice. The same regressors grew after inoculation of few cancer cells in UV-irradiated mice. So, it may be highly relevant that some carcinogens are immunosuppressive.500,501 Repeated exposures of mice to UV induce persistent immune suppression,502–504 leading to the development of highly immunogenic regressor tumors. In contrast, the single injection of MCA induces tumors in 100% of mice and only a short-lived state of immune suppression,501 thus allowing a fully immunocompetent host to select for less immunogenic variants. The concept that immunocompetence of a host influences the immunogenicity of the developing tumor has been tested experimentally by Roberts and Daynes480 comparing the MCA-induced tumors occurring in immunodeficient UV-irradiated mice with cancers induced in normal mice. Indeed, cancers induced with MCA in mice immunosuppressed with UV were frequently regressor tumors, whereas none of tumors induced with MCA in immunocompetent mice were regressors.480 Decades later, this concept was confirmed with tumors induced with MCA in nude, severe combined immunodeficiency, or Rag − / − mice.487,505,506

Selection by the Immunocompetent Host Immunocompetent hosts can select for cancer variants. Thus, when regressors are transplanted into normal immunocompetent hosts, heritable progressor variants can escape. Some of these variants show antigen loss,218,219,507–509 but many others retain their antigenicity and grow faster than the parental tumor when their growth is compared in T, B, or NK cell–deficient hosts,43,45,218,307,505,508,510 suggesting that mechanisms other than antigenicity must participate determining the differences in growth behavior between regressors and progressors.

“Regressors” from Immunocompetent Hosts Human cancers are sporadic (ie, occur at irregular times and locations); this is in part due to the occasional genetic events that can cause malignant transformation in a single cell that expands to become a detectable tumor. A murine model of sporadic genetic events is the recombinational activation of a normally silenced transgene encoding the strongly oncogenic and strongly antigenic molecule SV40 T,

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thereby causing sporadic appearance of cancers.228,301 These tumors are regressors. The tumors “snuck” through immune surveillance and immunoselection despite expressing such a powerful antigen and oncogene, but not unnoticed. The developing cancers did induce antibodies and proliferation of antigen-specific T cells during their early dormant phase of growth, but developing tumor-specific T cells were anergized in that they failed to produce interferon (IFN)γ and kill tumor cells in vivo. In contrast, titers of the T antigen-specific IgG response increased with progressive tumor growth.

EFFECTOR MECHANISMS IN CANCER IMMUNITY Because cancer is not a single disease, it is not surprising that findings using one tumor model may not apply to other tumor models. Considering the antigenic diversity found in tumors, it is also not surprising that different types of innate and adaptive, humoral and cell-mediated immunity have been shown to play different roles in the destruction or enhancement of malignant cells in one or another of the numerous tumor models.

Assays to Study Effector Mechanisms in Vivo In principle, five different assays have been used to evaluate the importance of different effector mechanisms in vivo. The fi rst type of assay involves transfer of effector cells, cytokines, or antibodies into sublethally irradiated, cyclophosphamide-pretreated, or normal animals challenged with tumor cells. There are certain limitations of this assay. Effector cells or molecules may not reach or localize in the tumor unless both the effector cells and cancer cells are injected intravenously, and both may be trapped in the lungs. Furthermore, if transferred cells or reagents are effective, the assay does not rule out that other effector mechanisms of the host may have been activated by the procedure. In a second procedure, called the Winn assay, 229,269,511 tumor cells are mixed with effector cells or serum in vitro; the mixture is injected subcutaneously into an animal to determine whether tumor growth in vivo is prevented. Tumor cells may be killed within minutes before or shortly after the injection, though the readout takes much longer. Therefore, the Winn assay is, in part, an in vitro cytotoxicity assay, even though the host is used as a readout for viable cancer cells. A third method involves elimination of specific lymphocyte subsets or cytokines in vivo by treatment with antibodies specific for different lymphocyte subsets or cytokines. Failure of the host to resist a tumor challenge indicates that the particular subsets or cytokines are an essential component of the host resistance. A fourth method is to use mice genetically deficient in a certain effector mechanism, cell, or cytokine. An analysis of tumor variants that have escaped tumor destruction by the host provides a fi fth way for determining the importance of immunologic effectors in vivo.43–45,218,235,236,507,512 The phenotypic changes observed in these variants may indicate which effector mechanism was responsible for the selection. Therefore, the type of phenotypic change

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may give insight into the relative importance of a naturally occurring defense mechanism that may function in immunocompetent mice (analogous to deducing the action of an antibiotic from the type of change found in the bacterium that has become drug resistant). Host-selected variants from experimentally induced regressor tumors are used extensively for this approach.

Antibodies and B Cells The role of B cells in regulating tumor immunity continues to be studied extensively yet remains poorly understood. In a tumor model of leukemia in which CD4 + helper T cells were required for successful treatment, B cells were necessary for efficient T-cell priming.513 Conversely, in other tumor models, elimination of CD4 + T cells promoted tumor rejection by CD8 + cells, and an absence of B cells improved CTL responses and tumor rejection.514–516 Antibodies and B cells have been associated with enhanced papilloma formation and autochthonous cancer development.341,489,517–519 The mechanism recently postulated is that regulatory B cells help the macrophage become tumor-promoting,520,521 or that they suppress surveillance by T cells that normally eliminate neoplastic cells with oncogenic mutations.519 There are other possible mechanisms.342,343 Also, none of these experiments identified the antigens involved, except one study showing that induction of mutant-ras specific–antibodies correlates with much more effective papilloma development in an autochthonous tumor model.341 Human antisera and monoclonal antibodies reactive with autologous tumors have been isolated.420,522 However, a strong humoral response to tumor antigens does not correlate with demonstrable resistance of the host to the tumors. TL + leukemias induce high titers of TL-specific antibodies that are cytotoxic to TL+ leukemia cells in vitro in the presence of heterologous complement,420 but TL + leukemias grow equally well in immunized mice having high titers of TL antigen–specific antibody and in nonimmunized mice.523,524 Similarly, humoral immune response to MCA-induced sarcomas does not provide protective immunity against a tumor transplant.525 Obviously, the presence of antibodies for these kinds of tumors has no relevance in predicting whether the host will reject the tumor. Normal or malignant cells of hematopoietic origin are generally lysed quite effectively by antibody and heterologous complement in vitro; however, normal cells such as fibroblasts or malignant cells derived from solid tissues may be much less affected, even when expressing high levels of antigen. The reasons for this striking difference are still unclear. In vitro, some tumor cells are killed by a process involving coating with antibody, opsonization, and subsequent phagocytosis by macrophages; this process may be enhanced by the presence of heterologous complement. Alternatively, antibody-coated tumor cells may be killed in the absence of phagocytosis by antibody-dependent cellmediated cytotoxicity when cocultured with macrophages, NK cells, or neutrophils. The general relevance of these mechanisms for killing tumor cells in vivo is unclear. Exogenous antibodies that block growth factors and/or their

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receptors or activate costimulatory molecules are useful for some kinds of immunotherapy (see Immunotherapy).

T-Lymphocytes It has been demonstrated convincingly that T cell–mediated immunity is critical for rejection of virally526–528 and chemically induced tumors,229,269,529 or for the rejection of allogeneic530 and UV-induced tumors.218,275,503,508 For example, in the model of murine MCA-induced tumors, it was shown that intravenous injection of immune cells, but not of immune serum, could transfer systemic tumor-specific immunity into sublethally x-irradiated mice.229 These findings were consistent with earlier work with allogeneic tumors showing that lymphocytes not serum were effective in transferring transplant resistance.531–533 In another study, transfer of immunity to a plasma-cell tumor was abolished by pretreatment of the immune cells by anti–T-cell antibodies and complement.529 The relative importance of various T-cell subsets in tumor rejection (ie, TC1 [type I CD8 + cytotoxic T cells]; TC2 [type II CD8 + cytotoxic T cells]; TH1 [type I CD4 + helper T cells]; TH2 [type II CD4 + helper T cells], TC17 [IL-17–producing CD8 + T cells], etc.) have been the subject of repeated, and probably unnecessary, controversies.534,535 Different tumors are dissimilar enough so that differences would be expected in T-cell subset requirements. Also different therapeutic settings may require different subsets (eg, to prevent cancer development or destroy premalignant lesions), established solid cancers, malignant effusions, microdisseminated cancer cells, or leukemic cells. Dependent on their subtype, T cells produce and induce various cytokines and chemokines that may destroy tumors by direct effects on tumor vasculature or recruit neutrophils, macrophages, and NK and other innate effector cells that are needed for tumor eradication.536 Many recent studies employ overexpressed model antigens and TCR-transgenic T cells not available in humans; thus, results need to be confirmed using non–TCRtransgenic models and genuine tumor-specific antigens. Interestingly however, adoptive transfer of TCR-transgenic CD8 + T cells specific for a model antigen can eliminate large established tumors536–540 or artificial pulmonary metastases541 without the T cells needing perforin.536,541 Release of IFNγ and tumor necrosis factor (TNF) by the T cells seems to be critical; receptors for both cytokines must be expressed on bone marrow– and non–bone marrow–derived tumor stroma to kill T cell–resistant cancer variants as bystanders or relapse may occur.537,538,540,542 CD4 + T-cell subsets can influence antitumor immunity, and truly tumor-specific CD4 + T cell–recognized tumor antigens exist.10 CD4 + T cells seem to be critical at the effector phase of CD8 + T cells for cancer cell destruction in vivo, 543,544 for sustaining CD8 + T-cell memory, 545 and for the survival of adoptively transferred CD8 + T cells.546,547 However, CD4 + T cells do not necessarily require recruitment of CD8 + T cells for eliminating cancer cells in vivo,10,371,548–551 even when the cancer cells are MHC class II negative and killing as well as presentation must be indirect.10,549,552,553 Destruction of the cancer cells

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in vivo requires IFNγ, but occurs even when cancer cells lack the receptor for it.552 Thus the effects of CD4 + T cell-released IFNγ must be on stroma, most likely tumor vasculature.554,555 Certain CD4 + subsets may also suppress tumor rejection, because elimination of the CD4 + T-cell subset may increase tumor resistance in certain tumor models.514,556 Now termed Treg cells, these Foxp3 + CD25 + T cells mostly suppress the induction of immune response to new antigens, reduce anticancer immunity particularly to self-antigens, and suggest poor prognosis when prevalent in excised cancers.557 However, it is uncertain whether these Tregs can prevent adoptively transferred tumor-specific memory T cells from eradicating cancers.

Natural Killer, Lymphokine-Activated Killer, and Natural Killer T Cells NK cells are distinct subpopulations of lymphocytes that, without prior sensitization and without the requirement for MHC restriction, can kill some cancer cells, particularly while circulating in the bloodstream, as well as nonmalignant nonself cells558–565 (see Chapter on NK cells in this book). NK cells occur as 1) “resting” NK cells that nevertheless kill very sensitive targets such as YAC, 2) “activated NK” cells induced within hours by IFNαβ to become cytolytic but without proliferation (many conditions and microbial agents rapidly induce IFNαβ), and 3) lymphokine (IL-2)activated killer (LAK) cells developing after days of culture in high doses of IL-2 and requiring proliferation. NK cells can “recognize” the absence of self566 (ie, the missing MHC allele fails to provide an inhibitory signal to prevent the activation of NK cells to kill the target567). Therefore, cancer cells that fail to express at least one of the MHC class I alleles of the host are killed more effectively.568,569 Transformed cells often have decreased or lost MHC class I surface expression or have induced expression of ligands for activating receptors on NK cells and thus are targets of NK cells. In vivo, NK cells or NKG2D-mediated effects inhibited autochthonous and transplanted tumor formation and tumor recurrence, and reduced metastatic dissemination of intravenously injected cancer cells.570–574 However, NK cells were effective usually only at incipient stages.575,576 In patients, intratumoral NK cells may reduce metastatic seeding, thereby leading to longer survival,577 and NK cells may be a key factor in the occasional cure of acute myeloid leukemia and childhood acute lymphoblastic leukemia by allotransplantation.578 Activation of peripheral blood cells in vitro with high doses of IL-2 induces LAK cell.579 Cancer cells, even when resistant to NK cells, are usually susceptible to killing by LAK cells in vitro, whereas most nonmalignant target cells have been reported to be resistant to killing by LAK cells.580 Intravenous injection of LAK cells early after intravenous seeding of cancer cells into mice reduces the metastatic tumor cell growth in the lungs; however, with this procedure, both LAK and cancer cells are trapped in the lungs.581,582 Antitumor responses have also been reported in humans after adoptive transfer of LAK cells in

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patients with renal cell carcinoma and melanoma.583 This selectivity is difficult to explain, considering the general susceptibility of cancer cells to LAK cells in vitro. In more recent studies, adoptive transfer of NK cells activated in vitro with IL-2 failed to cause tumor regression in patients with melanoma.584 The cells that mediate the killing in vitro of a broad range of malignant cells are more than 90% activated CD16 + /CD3 − NK cells, 585,586 but which cells have antitumor activity in vivo is not fully established. Even though murine LAK cells can be generated from nude mouse spleen cells, 587 it has not been demonstrated that LAK cells from nude mice or normal mice have similar therapeutic effects against tumor cells in vivo. Other cell types, such as CD3 + lymphocytes, which are regularly present in every preparation of LAK cells, may contribute significantly to the killing of tumor cells in vivo, particularly because activated CD8 + T cells express the stimulatory lectin-like NKG2D receptor and can kill tumor cells expressing the ligands for the receptor.588 Many surface receptors originally discovered in NK cells are expressed by subsets of T cells such as NKT cells, defined by the invariant Vα14/24Jα18 TCR α chain.589 NKT cells, when treated with IL-12 at the time of cancer cell inoculation or after 1 day, inhibited tumor development and metastasis.590,591 Also, the development of tumors induced by MCA was found to be reduced in some but not other studies.486,489,592 In any case, clinical studies are exploring the usefulness of NKT cells in certain cancers.593–596 NKT cell infi ltration of neuroblastoma is associated with a favorable outcome; NKT cells may target the tumor-promoting stromal macrophages rather than the neuroblastoma cells directly.597 Together, previous studies have failed to provide evidence that NK, LAK, or NKT cells can eliminate well-established solid tumors casting continuing skepticism about the clinical usefulness of these cells in the therapy of advanced solid tumors.575,576,598 A recent study, however, shows even very large solid tumors being eradicated by NK cells alone when the NK cells are properly activated by IL-15 in vivo at the site of the tumor.599

Macrophages and Granulocytes Neutrophils rapidly appear at the site of injury and respond very rapidly to diverse chemotactic and inflammatory stimuli.200 Furthermore, neutrophils often appear to pave the way for the influx of inflammatory monocytes that then mature to “angry” classically activated macrophages (or M1 macrophages) (eg, after “classical” activation by intraperitoneal/pleural injection of thioglycolate105,106,600–602). However, such inflammatory stimuli are usually absent in cancers. As a result, neutrophils can be relatively rare (0.1% to 0.4% of the total cells in solid tumors).603 Macrophages, however, are regularly found in the microenvironment of solid tumors and are usually at least 10 times more abundant than neutrophils,604 sometimes a third or more of the tumor mass. These tumor-associated macrophages differ from “angry,” tissue-destructive, “classically activated” macrophages (or M1) but are “alternatively” activated macrophages (M2).

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“Alternatively” activated macrophages seem to be derived from resident tissue macrophages that proliferate.105,106 Cancers may not recruit tissue-destructive inflammatory cells (N1 neutrophils and M1 macrophages); apparently, cancer cells lack the signals necessary for activating innate immunity,605 but cancer cells transfected to express CD95L induce cancer cell–destructive neutrophils.606 Therapeutic interventions may aim at converting cells of innate immunity to a tumor-inhibitory “type 1” response pattern. IL-4, the prototypical “type 2” cytokine, can inhibit growth of cancer cells transfected to produce this cytokine by activating granulocytes73,127,137,138 and can activate macrophages to kill cancer cells in vitro,607 but may also interfere with eventual clearance leading to outgrowth of escape variants.142 IL-4–secreting tumors are heavily infi ltrated by eosinophils,73,127 and many clinical and experimental studies reported eosinophilic tumor infi ltrates.535 Nevertheless, the contribution of eosinophils to tumor destruction and tumor growth is still incompletely understood.138,535,608 Macrophages and neutrophils from normal donors are generally not cytotoxic to tumor cells or normal cells in vitro; however, macrophages and neutrophils can be activated by bacterial products and IFNγ (or other cytokines) in vitro to cause selective cytolysis or cytostasis of malignant cells.609–617 Fully activated macrophages require long-term 16- to 72-hour assays to demonstrate in vitro tumoricidal activity in isotope-release assays or cytostatic activity in growth-inhibition assays. Some of the cytolytic or cytostatic effects of macrophages on tumor cells involve cell contact and/or the secretion of various cytotoxic substances, but phagocytosis may also play an important role.618 TNF-α619,620 produced by thioglycolate-activated macrophages can account for all of the classical tumoricidal effects of macrophages against some cancer cells in vitro.621–624 However, as might be expected because of the plethora of cytotoxic molecules that can be released by activated macrophages,200,625 other mechanisms such as reactive nitrogen intermediates can also be important mediators of killing of cancer cells in vitro.626,627 Systemic activation of macrophages reduces metastatic seeding,628 while a (nonactivated) colony-stimulating factor 1–dependent macrophage subset seems to help metastatic seeding.629 Because of the rather selective cytotoxicity of thioglycolate-activated macrophages against malignant cells, numerous studies have considered the potential role of this cell type in cancer (see Cancer and Inflammation, Immunotherapy, Factors Limiting Tumor Immunity for discussion of myeloid-derived suppressor cells). As would be expected, these in vitro–activated leukocytes mixed with cancer cells and injected into an animal prevent cancer development from the inoculum “in vivo” (a Winn assay, see previous discussion).630 Interestingly, dependent on the role neutrophils have in a particular cancer model, their transient elimination in vivo can either abrogate or enable T cell–mediated transplantation resistance.44,631 However, there is a major difficulty of effectively eliminating macrophages or neutrophils long-term in vivo. Therefore, there is, at present, no critical evidence to establish or refute the idea632 that macrophages and or granulocytes activated

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in vivo destroy nascent tumors and therefore play a role in immune surveillance.

FACTORS LIMITING CANCER IMMUNITY Various mechanisms allow cancers to escape innate or adaptive host immunity, principally by 1) inducing a protective and supportive stromal microenvironment, 2) increasing resistance to direct attack, and 3) inducing T-cell anergy. Often, cancer cells seem to use a combination of the three, but numerous lines of evidences indicate that the first is key and virtually always part of tumor progression and escape. Unfortunately, most experimental tumor immunology depends on observations using cancers serially transplanted for decades. This is surprising, knowing that a single transplantation of an autochthonous tumor into an immunocompetent host dependably results in heritable variants that avoid destruction by the host43–45,218,219,508 (see above under Experimental Cancer, Key Principles, and Selection of Tumor Model).

Tumor Microenvironment Many escape variants keep their antigens but induce a stroma more effectively than the parental cells for better support of growth and protection against destruction by the host (also see Cancer Stroma). Experiments have shown that stroma is critical for preventing or permitting immunologic destruction of cancer,47 and it is likely that cancer stroma is also an important factor in causing very early cancers to resist therapeutic immunization.633 Local factors must explain why tumor-bearing mice, while failing to reject a primary tumor transplant, reject a later implant of small numbers of the same cancer cells at second sites, a phenomenon called concomitant immunity (see discussion below under Cancer and Inflammation, Facilitation of Inhibition of Metastasis). Local factors particular to the tumor environment must also explain why mice bearing malignant grafts fail to reject the established tumors but reject nonmalignant grafts expressing the same rejection antigen634 ; T cells in these tumorbearing mice are neither clonally exhausted nor systemically anergic.635 Antigenic stroma, as it exists in nonmalignant allografts635 or when strong tumor-specific antigens are crosspresented by tumor stroma,537–540,542 can help T cells to eradicate cancers. In addition, recent work in two tumor models suggest that lack of “help” at the site of cancer growth may be an important reason for the failure of cancer cells to be rejected by CD8 + T cells, thereby stressing the importance of CD4 + T cells cooperating with CD8 + T cells in the effector phase.543,544 The situation might be somewhat analogous to transgenic mice that express allo-MHC class I molecules as self-antigen on islet cells and have autoreactive T cells that infiltrate the islets,636 but even after priming, the autoreactive cells fail to destroy the islet cells unless local help is provided in the form of IL-2.636 Antigen-specific T cells can infiltrate even tumors growing in immunologically privileged sites, but proper differentiation of the infiltrating T cells is prevented.301,637–639 Several different models have shown that the

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milieu of large tumors can inhibit the function of adoptively transferred T cells.640–646 Lack of costimulatory molecules or expression of Fas ligand by the cancer cells may lead to peripheral anergy.647–649 Suppression in the tumor microenvironment may also be the result of macrophages long known for abrogating immune responses when prevalent in culture.650 In the stroma, the macrophages are alternatively activated and produce indolamine-2,3-dioxygenase, inducible nitric oxide synthetase, lactate dehydrogenase-A, and myeloid cell– derived arginase.651–653 Autochthonous cancers transplanted once and reisolated are usually heritable variants that grow more progressively, more effectively attract macrophages and neutrophils that are pro-angiogenic (see Cancer Stroma), and more rapidly induce a protective and supportive stromal microenvironment.43–45 Treatment with RB6-8C5 allows mice to reject a lethal tumor cell inoculum,43–45 thereby indicating the profound role that Gr1+ CD11b + leukocytes have in helping tumors to escape. The significance of tumor-infiltrating Treg cells is discussed separately in the following. Finally, tumor-bearing mice and cancer patients may have alterations in the signal-transduction machinery in T cells, particularly in those infiltrating the tumors but also in T cells from draining lymph nodes or, at later stages, even in circulating T cells.654–656 NK cells can be similarly affected.654 A decrease in NF-κB p65 at an early stage is followed by loss of TCR ζ chain and p56 lck after continued tumor growth.657–662 Activated macrophages can secrete substances that induce these structural abnormalities.663 As another example of subversion of host defenses in the tumor microenvironment, tumors are a privileged site for bacterial growth.664,665 Together, numerous immunosuppressive mechanisms have been shown to be functioning in the tumor microenvironment and the tumor draining lymph node. Their relative significance is difficult to judge because there is rarely a real “positive control” (ie, destruction of a truly long established large solid tumor in the absence of the implicated mechanism).

Resistance to Direct Attack Cancer variants can become resistant to direct attack by T cells by 1) loss/downregulation of MHC class I and II molecules, 2) losing expression of the rejection antigens, or 3) increased resistance to the killing pathways. Selective loss or downregulation of MHC class I molecules or the associated antigen-processing machinery allows cancer cells to resist direct attack by T cells.237,666–692 These mechanisms allow cancer cells to escape while retaining the antigen that might be essential for cancer cell survival and malignant behavior.10,302,307,693 As noticed already decades ago, MHC-heterozygous F1 tumors can escape destruction when transplanted into either parent by loss of the mismatched MHC antigens.694,695 This agrees with the commonly observed loss of the mismatched HLA after haploidentical hematopoietic stem cell transplantation.696,697 MHC homozygous tumors rarely lose MHC antigens and require MHC compatible strains for serial transplantation, and they were therefore an essential tool to define the MHC.698 Two cancers are known to be naturally transmitted

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within a species as cells.699 The devil facial tumor disease, first observed in 1996, is a lethal cancer cell line serially transmitted by facial bites during fights among Tasmanian devils.700,701 These marsupial carnivores in Tasmania have very little MHC diversity and are in rapid decline due to spread of the cancer. Canine transmissible venereal tumor, first described in 1876,260 is passed between dogs through coitus and bites.702–704 The tumor cells express very little β2-microglobulin and MHC class I during the progressive growth phase followed by regression after ∼6 months unless the dog is immunosuppressed. Some cancer cells express low levels of MHC, but levels can be upregulated by cytokines. At least some of these changes seem to be due to epigenetic mechanisms.705,706 In other cancer cells, irreversible loss of MHC class I expression is caused by several molecular mechanisms, including mutations in the gene coding for β2-microglobulin.672,674,675,707 Loss of a single HLA class I allele was found more commonly than loss of all class I alleles.706 The E1A gene of the adenovirus (Ad) strain Ad12 suppresses MHC class I expression in transformed cells that are tumorigenic and escape T cell–mediated destruction,708 but there was no correlation between the level of MHC class I expression and tumorigenicity of Ad2 and Ad5 transformed cells. Oncogenes such as myc can cause locusspecific suppression of MHC class I antigen expression.709 Importantly, MHC class I molecules have now been shown to act as tumor suppressor genes.710 Thus, some of the observed changes in MHC expression may not be the result of immunoselection. Cancers also escape by loss of expression of the rejection antigens.218,219,236,507,508 Even when the antigen is necessary for cancer cell survival,10,302,307,693 point mutations might preserve the oncogenic potential while rendering the protein nonantigenic. Mutations in SV40 large T that preserve the transformed phenotype in vitro have been selected for with CTL clones, but, in vivo, evidence is lacking for tumorigenicity or selection of such clones.711 Interestingly, mutational changes in E6 and E7, the transforming genes of HPV, have not been observed in cervical cancer while total or allelic loss of HLA class I expression is commonly observed in this cancer.712 Immune selection has been reported in patients at later stages of malignancy and after therapy.713–715 Partial immune suppression may lead to a higher yield of antigen loss variants.235,716 UV-irradiated mice show a partial immune deficiency so that the generation of cytolytic T cells is delayed.235,717 Incomplete therapy of bacterial infections with antibiotic drugs also favors the outgrowth of variant bacterial strains that show heritable resistance to these drugs; by analogy, partial or incomplete immunotherapy of cancer-bearing individuals may lead to selection of antigenloss variants. When targeting cross-presented tumor antigen on tumor stroma only, T cells do not select for cancer cell variants.169 Thus, direct (or very short-range) cell-cell interactions appear to be essential for selecting antigen or MHC-loss variants. Antigenic modulation, a reversible antibody-induced loss of surface antigen,523,524,718,719 and cellular resistance to perforin/granzyme,720 FAS/CD95,721 and other pathways to evade destruction are not discussed.

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Anergy/Unresponsiveness/Neonatal Tolerance Antigens newly expressed on cancers in the adult may be ignored at the earliest stages of autochthonous cancer development because of insufficient amounts of antigen.191,228,301,722 But once the tumor grows and the amount of antigen increases, the host responds.723 Thus, immunologic ignorance is unlikely in cancer patients. Tumor transplantation induces acute inflammation that may influence the immune response to the transplanted tumors.724,725 By contrast, the antigens on sporadic autochthonous tumors fail to induce tumor destruction.228,301 Importantly, these cancers grow progressively though they are not resistant to direct immune attack (eg, by adoptively transferred T cells). Furthermore, tumor antigen–specific CD8 + T cells are remarkably increased in number in mice bearing autochthonous cancers but failed to lyse antigen-specific targets in vivo and the production of IFNγ was low whereas the production of TGF-β1 was increased and high titers of IgG antibody specific for the rejection antigen were observed.228,301 Thus, antigen-specific B and T cells in mice bearing the autochthonous tumors responded to the rejection antigen but in a way that resulted in failure to reject the cancers. This altered responsiveness726,727 must be distinguished from “peripheral” or “extrathymic” tolerance that occurred under certain conditions when an antigen was presented to an adult in highly immunogenic form with the consequence of temporary expansion of mature T cells followed by clonal elimination.728 The important distinction is that clonal deletion would require adoptive T cell transfer whereas retention of antigen-specific T cells in the host bearing autochthonous cancers may allow rescue or induction of a tumor-specific destructive T-cell response. Thymic tolerance to self may explain why oncofetal and carcinoembryonic or oncospermatogonal self-antigens, all expressed in the thymus, induce weaker protection than is found in animals immunized with tumor-specific antigens.364,453,729,730 Nevertheless, at least certain cancers seemingly do not follow the rules that prevent immune recognition of normal self-tissues. Thus, some tumor-bearing hosts may readily recognize normal differentiation antigens on certain cancers such as melanomas. In this type of cancer, it appears to be possible to uncouple the mechanisms of autoimmunity from tumor immunity.731,732 However, it is unclear how effectively these T cells can control tumor growth.733 Epigenetic memory can prevent self-reactive CD8 + T cells from escaping their tolerant fate.734 Because the thymus “selects the useful, neglects the useless and destroys the harmful” T cells,735(p.57) only self-antigen–reactive T cells with too low avidity to cause destruction escape deletion and prominently infiltrate tumors.736,737

Age As already discussed (see Immunogenicity of Autochthonous Tumors), most cancers developing in older individuals may be fully sensitive to tumor-specific T cells but for age-related reasons fail to induce an effective immune response. Immunotherapy in older individuals may require rescuing the age-dependent immune deficiencies of the host

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environment738 as well as the T cells484 because adoptively transferred lymphocytes from young, but not old, immunized mice eradicate large solid tumors.258

Regulatory T Cells/B Cells/Antibodies/ Blocking Factors Passively administered antibody can prevent the rejection of tumor allografts (homografts).739–741 These antibodies are also referred to as “enhancing antibodies.” “Blocking factors” are complexes of antigen and antibodies that can suppress cell-mediated immunity in vitro.742,743 Complexes of tumor antigen and antibody can induce suppressor T cells744 (now generally referred to as Tregs) that can suppress specific immune responses in vivo. Treatments of recipients with donor-type cells as antigen and homologous antibody to donor-type cells enhances acceptance of rat renal allografts.745,746 The mechanisms responsible for these fi ndings are incompletely understood. The induction of Tregs required active TGF-β ; latent TGF-β linked to IgG causes the suppression of sensitization/proliferation of CD8 + effector T cells. Recent and earlier experiments have pointed at a joint role of CD4 + T cells and B cells/ antibodies in preventing the rejection of transplanted tumors514,515 and enhancing the development of autochthonous cancers in mice.341,517,520 Eliminating B cells or CD4 + T cells can have virtually identical effects in preventing tumor rejection in the same tumor model, 514,515 pointing at the fact that B cells are required for multiple functions of CD4 + T cells and vice versa747–750 and that Tregs may not necessarily be generated or act independent of B cells and antibodies.751,752 “Suppressor T cells” can suppress tumor rejection.514,556,753–755 Residual cancer cells remaining after incomplete tumor removal can be sufficient for continuing the suppression.756 However, unlike what has been reported,756 suppression is short-lived after complete tumor removal and may give way to specific immunity without further immunization.757 Unlike CD4 + helper T cells, Tregs usually express CD25 and Foxp3.758–762 The Foxp3 + CD25 + T cells are essential in preventing autoimmune destruction of the host759 and suggest, as discussed previously, poor prognosis when prevalent in excised cancers.763 Tregs are sensitive to low-dose gamma irradiation and cyclophosphamide pretreatment.764,765 Preirradiation, cyclophosphamide pretreatment, T-cell deficiency, or CD4 + T-cell depletion of the tumor-bearing host facilitates tumor destruction by adoptively transferred immune T cells.766,767 However, these treatments also cause homeostasis-driven expansion and activation of the transferred T cells,768 which may be sufficient or at least synergize with the effects of Tregs depletion. However, whether Tregs can suppress memory T cells needs further study. In any case, these proposed mechanisms and observed effects confi rm the early pivotal discovery that low dose whole-body x-irradiation and/or chemotherapy are an essential adjunct to adoptive tumor immunotherapy in mice.766,767 “Nonmyeloablative lymphodepleting chemotherapy” is now standard treatment in melanoma patients before adoptive T-cell transfer.769

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Myeloid-Derived Suppressor Cells 75–79

68

MDSCs are a subset of monocytes that express CD11b and Ly6C (resulting in intermediate levels of Gr-1 (RB6/8C5) staining), but lack Ly6G (defined by 1A8) (see Components, under Cancer Stroma). They differ from alternatively activated tumor-associated M2 macrophages in well-established tumors 3 weeks after transplantation and from neutrophils. Neutrophils express high levels of Ly6G and Gr-1 and intermediate levels of Ly6C. Cells of myeloid origin are increased in numbers in the peripheral blood of tumor-bearing mice and cancer patients, especially in later stages. In the mouse, MDSCs come from bone marrow as well as spleen, which is a natural extramedullary blood-forming organ in this species. MDSCs and blood monocytes seem to be the predominant precursors of intratumoral macrophages not only in transplanted but also autochthonous tumors.68,770 A vast number of studies indicate that MDSCs isolated from the peripheral blood of cancer-bearing individuals are profoundly immunosuppressive in vitro. How this correlates to immune suppression in vivo remains an open question.77,78

IMMUNE SURVEILLANCE OF CANCER Types of Surveillance Mechanisms The term surveillance should be restricted to its meaning (ie, protection from cancer development771). Early in the last century, Ehrlich wrote, “I am convinced that the development of aberrant [mutant] cells occurs very frequently during the extremely complicated fetal and postnatal development but that these foci luckily remain completely latent in most humans because of protective mechanisms in the host. If these mechanisms were not existent, cancer would probably develop with an incredible frequency.”261(p.95),262(pp.289–290) Ehrlich’s statement was based on experimental and clinical findings which included Xeroderma pigmentosum, an inherited defect in repair of DNA damage caused by UV leading to countless skin cancers. Ehrlich distinguished two basic types of surveillance: 1) immunologic responses (acquired or natural) and 2) cellular (cell-intrinsic) resistance. The latter, attributed by Ehrlich to deprivation of nutrients (athrepsia), is now best explained as cell-intrinsic controls such as DNA repair, checkpoint functions, programmed cell death, and epigenetic controls. With great foresight, Ehrlich emphasized his “firm conviction” that cell-intrinsic protective mechanisms were critical for resistance to cancer development, but discussing these cell-intrinsic mechanisms772–774 is beyond the scope of this chapter. Today, the microenvironment produced by stroma should be added as third important type of surveillance. Numerous cytokines and cell-cell interactions in the stromal microenvironment can be highly effective in preventing tumor development from transformed cells.139,555,775–779 Diet, host microbial flora, and chemicals affect stroma and microenvironment, and can thereby promote or prevent cancer development depending on the agent and the condition. However, these factors have little or no effect once full-blown cancer has developed. This means therapy directed at stroma to prevent cancer or to cure cancer must

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be fundamentally different, though recent reviews do not make this distinction.780 Of the three principle types of surveillance mechanisms, focus here is on the immune system that can be a powerful restraint against the development of certain cancers.

Adaptive Immunosurveillance The concept of surveillance by adaptive immunity was especially attractive in the 1950s and 1960s because it provided evolutionary significance to T cell–mediated cellular immunity, which previously seemed to have no use other than to cause rejection of experimental allografts.781–783 Thus, Burnet’s hypothesis of immunologic surveillance suggested that the primary reason for development of T cell– mediated immunity during the evolution of vertebrates was defense against altered self- or neoplastic cells.784 We now know that T cell–mediated immunity is necessary for resistance to many viral and other infections and therefore may be important in the host response to cancers induced by these agents.

Congenital Immunodeficiency Diseases Patients with certain congenital immunodeficiency diseases may have a markedly (several thousand-fold) increased incidence of cancer,785 but mostly of lymphopoietic and reticuloendothelial or certain virally induced cancers (Table 47.1).786,787 Most common forms of cancer (eg, lung, breast, colon, and prostate) do not occur earlier or at a significantly higher rate than in the general immunocompetent population. It is therefore possible that the congenital abnormality itself contributed to the high incidence of lymphopoietic and reticuloendothelial cancers. Mice with congenital immunodeficiency diseases also have a higher incidence of cancers of hematopoietic origin.788–791

Virally Induced Cancers While gene-targeted mice have demonstrated the influences of innate immunity on tumor induction,792 these defects are usually absent in human transplant patients that therefore have intact innate immunity to exert cancer surveillance. This would also explain why patients do not show an increased incidence of nonvirally related cancers, and we need more information how innate effector mechanism might influence cancer incidence in humans. It is important to realize that resistance to tumor induction by oncogenic viruses is also genetically determined, as illustrated by the example of the lymphotropic herpes virus saimiri.793 In its natural host, the Old World squirrel monkey, the virus is an innocuous inhabitant probably due to Darwinian selection. However, some New World monkeys (such as the marmoset or owl monkey) do not harbor the virus, and experimental inoculation of the virus regularly causes malignant lymphomas. The susceptible monkeys do respond immunologically to the virally encoded antigens but too late and only at a time when lymphoma development has already occurred. These results suggest that viruses with oncogenic potential survive because lethal tumors would eliminate the virus along with the host. A further example is

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SECTION VIII

47.1

IMMUNOLOGIC MECHANISMS IN DISEASE

Cancers with Increased Incidence in Patients with Immunodeficiency

Type of Immunodeficiency

Cancer

Cause/Carcinogen

Reference

Primary (inherited)

B-cell lymphoma Hepatocellular carcinoma, biliary tumors Gastric cancer* Hematologic malignancies B-cell lymphoma Kaposi sarcoma Skin cancer (nonmelanoma) Vulvar, oral, or anal carcinoma Merkel cell carcinoma Cervical carcinoma B-cell lymphoma Kaposi sarcoma Hodgkin lymphoma Anal, vulvar, or oral carcinoma Merkel cell carcinoma Cervical carcinoma Hepatocellular carcinoma Skin cancer (nonmelanoma)

EBVa HBV Helicobacter pylori Germline EBV HHV-8 HPV, UV HPV MCV HPV EBV HHV-8 EBV HPV MCV HPV HBV, HCV HPV, UV

786,1100

Secondary (drug-induced) (patients with or without allograft)

Acquired immunodeficiency syndrome

787,1101 1102 785,786,1103 909,1103,1104 1105 806,808,818,819 1106 796,1107,1108 909 909 909

909 796,1108,1109 909 909 909

EBV, Epstein-Barr virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HHV, human herpesvirus; HPV, human papillomavirus; MCV, Merkel cell polyomavirus; UV, ultraviolet light. *Patients with primary hypogammaglobulinemia.

lymphotropic EBV, which causes a self-limiting lymphoproliferative disease called mononucleosis in humans, the natural host of EBV.771 Infection usually occurs at very early age usually without any symptoms or, rarely, at later ages (10 to 35 years old) when it may cause disease (mononucleosis). After infection, EBV becomes latent. About 90% of adults are latently infected. Only immunosuppressed individuals appear to develop EBV-associated lymphoproliferative disease and malignant lymphomas. EBV-encoded, CTLrecognized antigens must be important for host recognition and tumor rejection because lymphomas expressing these antigens in immunocompetent individuals have not been found. Instead, EBV-associated lymphomas express the full array of CTL-recognized antigens only in immunosuppressed patients.794 In principle, adaptive T cell–mediated immunity may protect against the development of primary virus-associated cancers by either preventing or shortening the duration of infection with the oncogenic viruses or by eliminating virally transformed cells expressing the virus. As example for the latter, cells malignantly transformed by oncogenic DNA viruses often do not produce (in contrast to oncogenic RNA viruses) viruses; only the tumor formation, not infection by these viruses, may be prevented by the immune system. Such a resistance to tumor induction by DNA tumor viruses is consistent with the fact that polyomavirus is a common harmless passenger virus in adult mice and is commonly found in wild mice without inducing malignancies.795 But even a generally harmless polyomavirus when it integrates and acquires the mutations needed to inactivate viral production may induce cancer, as in the Merkel cell carcinoma in immunosuppressed humans.796 By contrast, in the case of high-risk human papilloma viruses 16 and 18,

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T cell–mediated responses may prevent or at least shorten the duration of viral infection. This may reduce the chance that the initially mostly episomal virus integrates its oncogenic E6/E7 sequences into the host’s epithelial cells15,797,798 (also see Immunoprevention). Cells transformed by certain DNA or RNA viruses can be very immunogenic.771 For example, SV40 and polyomaviruses usually do not induce tumors in adult animals799 or humans800 because the viruses induce rejection antigens on the transformed cells that are immunogenic enough to elicit rejection without prior immunization. Therefore, the use of immunoincompetent animals, such as neonatal animals or nude mice, is required for tumor induction, a fi nding that led to a breakthrough in studying the tumorigenicity of viruses and of cells transformed by viruses in vitro. It is important to note that this immunologic resistance of the natural host is directed against the oncogenes expressed by the virally transformed cells, not against the virus itself. Interestingly, when the oncogenic SV40 large-T antigen is expressed as a sporadic extremely rare event in single cells (rather than as part of a systemic infection alerting the host to respond), this strongly antigenic and oncogenic protein can cause cancer in mice.228,301 Transplants of these autochthonous cancers are regularly rejected by naïve syngeneic normal hosts, indicating that during development, the autochthonous cancers managed to sneak through all host controls in the euthymic normal individual without losing their highly antigenicity and immunogenicity.228,301 Even a progressor tumor may be highly antigenic, immunogenic, and sensitive to immune destruction (immunosensitive), yet induce, for example, a strong Treg cell and B cell “type 2” immune response that downregulates destructive T-cell responses and promotes tumor growth.

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MHC genes may regulate immune responses to cancer cells or cancer-causing viruses, and since the discovery that the MHC profoundly influences the susceptibility of mice to leukemia caused by Gross virus,801,802 investigators have searched for a possible association between MHC type and cancer susceptibility in humans and mice.803 However, no firm association between HLA haplotype and the occurrence of any major human cancer has been established except for virally induced nasopharyngeal, cervical, and liver cancers.804,805

Ultraviolet Light–Induced Cancers Renal transplant patients show a 65- to 250-fold increase in skin cancers virtually restricted to UV-exposed sites of the body.806,807 The increase is independent of whether the immunosuppressive agent is mutagenic or not,808 although cyclosporine enhances transformation in vitro.809 Skin cancers in transplant patients are often more aggressive with a mortality of 7% to 9% due to metastases.810 The increased incidence in transplant patients could be in part due to an increase of mutations due to impaired repair of DNA damaged by UV.811,812 Immunosuppressive drugs can decrease local production of IFNγ,813 and this decrease may favor tumor development.555,814–817 UV-signature mutations in oncogenes and suppressor genes play a critical role for skin cancer development. Nevertheless, viruses of the beta HPV group may play an essential role as cocarcinogens.818,819 This would make these cancers virally related and more similar to the other cancers increased in transplant patients. In any case, two-thirds of these transplant patients experience reduced skin cancer development after immunosuppressive medications are stopped820 ; it appears that, similar to murine UV-induced tumors,218,479 many human UV-induced cancers are actually “regressor” tumors and have arisen only because host defenses had been damaged. Chemically Induced Cancers We lack evidence that the incidence of chemically induced cancers is increased in immunosuppressed humans.821,822 Experimentally, loss of adaptive immunity increases the incidence of virally induced cancers in virtually all studies and all species tested; however, the increased incidence or a shortened latency of chemically induced cancers due to lack of T cells has been found only in some experiments and not in others.487–489,506,792,823–828 Transporter associated with antigen processing–deficient mice are defective in presenting intracellular mutant peptides (the key targets of adaptive immunity to nonviral cancers) to αβ TCR-bearing T cells (the key effectors of adaptive T-cell immunity). Nevertheless, transporter associated with antigen processing knockout mice do not have an increased or accelerated incidence of chemically induced cancers whether or not these mice are also nullizygous for p53.829 Clearly, immunosurveillance by adaptive T-cell immunity is effective against the development of several virally induced cancers, but it is still uncertain how effectively adaptive T-cell immunity prevents the development of forms of cancers that are induced by chemical or physical carcinogens.

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Innate Immunosurveillance The innate immune system uses nonrearranging germline receptors to trigger responses of cellular effectors that can recognize and kill cancer cells or normal cells. Many studies using gene-targeted mice have demonstrated the important influence of various components of innate immunity on tumor induction.792 When it comes to the mechanisms of protection against cancer, the effects of innate immunity are not easily delineated from cancer cell–intrinsic and/or microenvironmental/stromal control mechanisms mentioned briefly at the beginning of this section. For example, mice lacking the transcriptional activator interferon regulatory factor (IRF)-1 show several immunologic disorders, most notably a severe defect in the development of NK cells,830 but the tumor-prone phenotype of these mice was shown to be directly attributable to the cell-intrinsic lack of protective IRF-1.815 IRF-1 regulates DNA repair and is also an activator of IFNα and IFNβ transcription, and is required for double-stranded RNA induction of these genes. Many cell types secrete IFNα and IFNβ. Furthermore, IFNα / β signaling contributes to p53-mediated tumor suppression.831 Thus, cooperation between innate and cellintrinsic mechanisms is likely to be common. IFNγ induces the IRF-1 that functions as a tumor suppressor,139,814 and mice with a defective IFNγ signaling pathway (IFNγ receptor– or STAT1-deficient mice) have an increased incidence of MCA-induced tumors.485,487,777 The spontaneous485,487 but not MCA-induced777 tumor incidence was increased when these mice also lacked p53. Thus, the IFNγ pathway may help eliminate somatic cells harboring spontaneous DNA damage. Stimulation of the IFNγ pathway by IL-12 reduces oncogene-driven tumor development in HER-2/neu transgenic mice.832 This surveillance effect of IFNγ may be complemented by cytolytic γδ T cells or NKT cells. These cells may use their NKG2D receptors to eliminate somatic cells exposed to the initiating chemical carcinogen followed by the tumor promoter TPA.573,574,827 While gene-targeted mice have demonstrated the influences of innate immunity on tumor induction,792 these defects are usually absent in human transplant patients that therefore have intact innate immunity to exert cancer surveillance. This would also explain why patients do not show an increased incidence of nonvirally related cancers; we need more information about how innate effector mechanism might influence cancer incidence in humans.

CANCER AND INFLAMMATION Key Principles Rudolf Virchow was among the first to stress the importance of local irritation for neoplastic proliferation, but central parts of his “Reiztheorie” (irritation theory) were incorrect.833,834 Instead Virchow’s contemporary, Carl Thiersch, may have been the first to state clearly that epithelial cancer cells invaded adjacent tissues and were not derived from connective tissue or inflammatory cells.835 (An exception may be gastric cancer induced in Helicobacter felis–infected mice and proposed to originate from bone marrow–derived

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cells recruited to sites of injury and inflammation836.) In this section, we will discuss the evidence that local inflammatory reactions can stimulate or destroy cancerous growth, and that stroma plays an integral role in permitting or forbidding these interactions. It seems confusing that in one and the same organ (eg, urinary bladder), inflammation can promote cancer development in humans as well as prevent cancer relapse. Schistosomiasis, a parasitic helminth infection, increases the incidence of cancer whereas instillation of live Bacille Calmette-Guérin (BCG) prevents relapse of superficial bladder cancer. Schistosomiasis causes a chronic, growth-promoting, IL-4–driven “type 2” inflammation characterized by tissue repair and angiogenesis, alternatively activated neutrophils (N2) and macrophages (M2), and TH2 and Treg leukocytes driven by active TGF-β and STAT3 signaling. In contrast, BCG causes an acute, bacteriocidal, tissue-destructive “type 1” inflammation with classically activated neutrophils (N1) and macrophages (M1), and TC1, TH1, TH17, and other killer leukocytes driven by STAT1, IFNγ, TNF, and IL-17. Dependent upon the antigen, cell, and cytokine involvement, the result may be either inhibition or

stimulation of growth of the premalignant or malignant cells by acquired and/or innate immunity (Fig. 47.4). There is little knowledge of how differences in amount, kinetics, and local concentration of cytokines determine these different biologic outcomes.619,837–842

Exogenous Promotion It is generally assumed that most human and animals harbor initiated mutant cells from exposure to low levels of chemical or physical carcinogens. Experimentally, this can be mirrored by exposing a small area of mouse skin to very small doses of the polycyclic hydrocarbon carcinogen 7,12-dimethylbenz[a]anthracene. No cancers develop over the life of the animal unless this site is exposed to prolonged inflammation caused by wounding or a nonmutagenic proinflammatory promoter23,34,843–845 (eg, croton oil, a now obsolete laxative,21,22 or its active component, phorbol ester [12-O -tetradecanoylphorbol-13-acetate, also referred to as phorbol-12-myristate-13-acetate] 846). Phorbol esters are strong inducers of NF-κ B in keratinocytes and many other cell types. NF-κ B, as a crucial communicator of innate immunity and inflammation, normally receives exogenous

FIG. 47.4. Both Acquired and Innate Immunity can Stimulate or Inhibit All Stages of Cancer Development and Progression.

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FIG. 47.5. Opposing Effects of p53 and NF-kB. p53 not only inhibits cell cycle progression, cellular proliferation, and the replication of deoxyribonucleic acid, but also leads to apoptosis. By contrast, NF-κB promotes cellular proliferation and blocks apoptosis. Thus, any substance that chronically upregulates the NF-κB pathway may promote cancer development and therefore be dangerous.

signals from the proinflammatory cytokines TNF-α and IL-1β, TLRs signaling tissue damage and microorganisms. The major force opposing NF-κ B is p53 that is upregulated by sensing DNA damage caused by the carcinogens (Fig. 47.5).119 Upregulated p53 induces apoptosis and blocks cell proliferation and the replication of cells with damaged DNA. Evasion from p53-induced cell death is essential for mutant cells to survive and for cancers to develop.774 The prosurvival signal exerted by NF-κ B is therefore essential for the premalignant cells to escape apoptosis-mediated cell death.119 Wounding, used by Rous,844 also seems to be effective if there is a prolonged healing.847–849 Tumors have been compared with “wounds that do not heal.”850(p.1650) Mouse strains that have low or poor inflammatory reaction to wounding or phorbol esters are more resistant to tumor promotion.851 Until recently, few experimental studies have critically examined whether the microbial flora and chronic infections can exert substantial tumor-promoting effects, as suggested by clinical evidence. In one early study, the sensitivity to chemical carcinogen of germfree or specific pathogenfree rats was compared with that of chronically infected animals.852 It was shown that chronic respiratory infection enhanced cancer development. Clearly, the microbial flora is a major regulator of innate and adaptive immunity,853 and certain bacteria that are part of the normal gut flora exert a protective effect against inflammatory bowel disease that is strongly associated with the development of colon cancer.854,855

Switch to Paracrine Loops When the chronic application of an exogenous chemical promoter is stopped during experimental skin carcinogenesis, some papillomas regress spontaneously while others persist

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and continue to grow.848 Tumor persistence and progression appears to depend on heritable changes in premalignant and cancer cells to establish a “paracrine stimulatory loop”43–45 : cancer cells producing chemokines that attract and activate leukocytes. These leukocytes in turn produce cytokines and growth factors that stimulate tumor angiogenesis or the growth of the cancer cells.110,629,856 Thus, several experiments have shown that, during tumor progression, cancer cells can switch from being inhibited to being stimulated by inflammatory cells43–45 or cytokines (eg, TGF-β or IL-6).857–861 It has also now become clear that cause of the heritable switch is cancer cell-intrinsic (ie, the expression of mutant oncoproteins that causes an activation of NF-κ B signaling pathways).114,115,862,863 This allows the neoplastic cells not only to escape apoptosis-mediated cell death but also to produce proinflammatory signals that initiate the paracrine loop.80,114–116,862,864–870 Interestingly, the signals coming from the same oncogenic mutation can diverge into separate proinflammatory NF-κ B and transforming RAS/PI3K signaling cascades.862 The K-RAS pathway can lead to the secretion of MCP-1 (CCL2), KC (CXCL-1), MIP-2 (CXCL-2), and IL-8 (CXCL-8).864,870 These more recent insights give a mechanistic basis for much earlier data showing that cancer cells release numerous inflammatory mediators and growth factors such as MCP-1 (CCL2), KC (CXCL-1), MIP-2 (CL-2), SDF-1 (CXCL12), IL-1β, IL-6, IL-8 (CXCL-8), IL-10, MIF, CXCL5, NFκ B, TGF-β, osteopontin, versican, PDGF, FGF-2, VEGF, G-CSF, and granulocyte–macrophage colony-stimulating factor (GM-CSF).164,780,869,871–884

Regression Following Acute Inflammation By the mid-1800s, physicians observed that occasional cancer patients who developed erysipelas, a serious acute bacterial infection of the skin, had remarkable regression, sometimes cures, of cancers that would have been inoperable today.885 This led to intentional infection of patients,886,887 later with cloned Streptococcus pyogenes, the causative agent of erysipelas.888,889 Stunning successes occurred, in some cases regression,888,889 and a decade later in 1893, the work was confirmed in the United States.890 The variability of success was suspected to be a function of the different types of cancers treated, and the successes in occasional patients were marred by serious complications including death in the preantibiotic era. Today, topical use of live bacteria remains restricted to treating residual superficial bladder cancer, which typically recurs following surgery.891,892 Repeated instillation of the live BCG mycobacteria into the bladder by a catheter after surgery has become a treatment of choice for this cancer. The repeated local infection with BCG leads to prolonged acute inflammation in the bladder wall. This is invariably associated with local production of type I and type II IFNs and a significantly reduced cancer recurrence. Consistent with this idea, topical application of imiquimod, a TLR7 agonist, can induce immunologic destruction of premalignant actinic keratoses and basal cell and squamous cell carcinomas.893,894 Imiquimod also enhances IFNγ production and T-cell effector function when topically applied to UV-induced skin cancers in transplant recipients.895

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Increased Incidence Following Chronic Inflammation Chronic “type 2” inflammation by infectious agents is associated with development of some kinds of cancers896 (eg, hepatitis B and C virus and hepatocellular carcinoma, Helicobacter pylori and gastric cancer, HPV infection and cervical cancer, and schistosomiasis and bladder cancer). Also, chronic tissue damage caused by physical or chemical agents is associated with development of some cancers (eg, reflux esophagitis and esophageal cancer), chronic pancreatitis and pancreatic carcinoma and inflammatory disorders of unknown etiology (such as ulcerative colitis or Crohn disease), and colon cancer.118,780,897 Substantial clinical, epidemiologic, and preclinical data indicates that nonsteroidal anti-inflammatory drugs (eg, aspirin) are effective in reducing colorectal polyps and colon cancer.898,899 Western diet is another highly significant risk factor to developing colon cancer.900,901 Diet has major effects on shaping the intestinal microbiome902 and may be associated with chronic inflammation,853 but the mechanisms of this important link remain to be discovered. Chronic inflammatory responses to impaired glycosylation of intestinal mucosa has been suggested to be tumor promoting.903 At least some patients with ulcerative colitis have impaired glycosylation due to lost T synthase function as a result of somatic mutations in COSMC in colonic Tn antigen–positive crypt cells.904 Such mutations have also been found in human colon cancer.412 Mice selectively deficient in T synthase in colonic mucosa express the Tn antigen and develop massive inflammatory infi ltrates and abscesses in their distal colon, similar to patients with the cancer-predisposing ulcerative colitis.904

Prognostic Significance of Tumor Infiltrates Melanoma Despite some claims,905 the infi ltration of lymphocytes into melanoma remains a histopathologic variable of unproved prognostic significance. In a recent study, absence of TILs in melanomas was associated with a sixfold higher probability of metastases in the sentinel lymph nodes906 ; however, depth of invasion of the primary melanoma, not the TILs status, was prognostic of survival of the patients.

staging. Different expression profi les may reflect differences in cancer cell–intrinsic oncogenes/regulatory pathway usage. This may result in different cytokines, chemokines, and other factors being released that attract different types of lymphocytes and innate immune cells. The incidence of primary colon cancer virtually remains unchanged in renal transplant patients compared to virally related cancers.909 In ovarian cancers, the presence of intratumoral T cells is correlated with delayed recurrence of cancer and delayed death.910 Absence of intratumoral T cells is correlated with increased levels of VEGF. Furthermore, recruitment of Treg cells predicts reduced survival.557,763 Again, differences between different ovarian cancers could be due to the use of different oncogenes/regulatory pathways resulting in different aggressiveness. Ovarian cancers are not increased in immunosuppressed organ transplant patients.909

Prognostic Significance of “Regression” and Vitiligo Much research continues to be stimulated by the observation that regression of melanomas can occur, spontaneously fostering the idea that immunosurveillance or immunologic destruction is responsible.911,912 However, regression of a melanoma is usually partial, rarely total, and usually involves its horizontally growing (radial), intraepidermal part while the major predictor of survival is the extent of vertical growth (ie, depth of invasion). The prognosis is excellent for melanomas < 1 mm in depth (thickness) but much worse if the cancer is ≥ 1 mm in thickness. In a recent analysis of over 2,000 melanomas, regression neither predicted the likelihood of lymph node metastases nor survival of the patient.913 Possibly, partial destruction of a malignant lesion may result from infi ltrative growth into the surrounding tissue and destruction of that part of the tumor’s blood supply. Vitiligo (a depigmentation of normal skin) has many causes including an autoimmune etiology.914 Though vitiligo is frequently induced experimentally or clinically as during treatment targeting melanocyte differentiation antigens,364,731,733,915,916 it is not predictive nor needed for success of eradicating the melanoma.917,918

Colorectal and Ovarian Cancers

Inflammatory Responses to Tumor Growth

Relapse of colorectal cancer (stage Duke B or C) ranges between about 25% to 70% in patients who had the cancer surgically removed. Histopathologic and gene expression analysis of leukocyte infi ltrates in primary colorectal cancers can apparently predict recurrence as well as survival. Cancers with dense CD45R+ CD8 + T-cell infi ltrates have a better prognosis at any stage, whereas patients with low numbers of T cells regardless of stage have more recurrences.907 Inflammatory cells, such as macrophages or lymphocytes, may look morphologically identical, but secrete different cytokines that have opposite effects on tumor growth. This may be the reason why gene expression analyses can serve as a better indicator of the functional properties of immune infiltrates in cancers.908 Current work suggests that these analyses may provide better prognostic indicators in colorectal cancers than conventional TNM

Experimental as well as clinical studies are now producing a rather coherent mechanistic picture of the significance of systemic inflammatory responses in tumor growth. About 30% of patients with solid tumor have elevated granulocytes in their peripheral blood (> 8,000/μl), and granulocytosis is common in tumor-bearing mice.871,883,919–925 It is long known that systemic effects, such as splenic enlargement,926 frequently accompany local growth of autochthonous or transplanted murine tumors. Although many tumors commonly used in experiments have been serially passed hundreds or more times through euthymic mice,239 a similar enlargement is also observed in response to growth of primary autochthonous tumors in nontransgenic mice.927 Enlargement is mostly caused by increased myeloid hematopoiesis with macrophages, monocytes, and neutrophils accounting for more than half of the splenocytes.770,928 G-CSF,

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GM-CSF, and/or IL-6,871,876,878,883,929,930 released by cancers, may be particularly important in inducing the systemic effects. STAT3 is persistently activated in many types of cancers, particularly in cancer cells at the invasive edge of tumors adjacent to inflammatory cells.849 IL-6 preferentially activates STAT3,931 causing cancer cells to resist apoptosis, sustain angiogenesis, and suppress acute STAT1-driven inflammation849,931,932 and destructive T-cell responses. G-CSF plays a central role in neutrophil production. Importantly, G-CSF also induces stem cell mobilization by upregulating CXCR4 and decreasing its ligand SDF-1 (CXCL12) in the bone marrow,154,933 spleen, and local86,88 reservoirs for progenitors. Degradation of SDF-1 in the progenitor/stem cell reservoirs is probably a result of G-CSF–induced granulocytosis that causes an increase of the neutrophil-derived proteases (TIMP-free matrix metalloproteinase-9 and neutrophil elastase, specific products of neutrophils that degrade SDF-1934–936). Mobilization of progenitors expressing CXCR4, the receptor for SDF-1, includes the mesenchymal, hematopoietic, and angiopoietic progenitors and Treg cells.146,937 Attraction of these cells into the tumor stroma of a neoplastic lesion occurs along a chemotactic gradient and depends on high local concentrations of SDF-1 produced by cancer cells879,880,938 and tumor-stromal myofibroblasts.939,940 Given these mechanisms, it is not surprising that elevated neutrophil blood counts, neutrophil/lymphocyte ratio, matrix metalloproteinase-9, and plasma levels of C-reactive protein have been described as prognostic indicators of recurrence and reduced survival.941–945 However, systemic responses to cancer vary greatly between individuals depending on stage and type of cancer. Even when they are not detectable, the mechanistic loops outlined previously are likely needed locally for every malignant growth.

Facilitation or Inhibition of Metastasis A systemic increase in circulating factors such as IL-6 and progenitor cells may facilitate metastasis by creating “metastatic niches” (ie, a stroma in which disseminated cancer cells can successfully engraft).629,946–954 Experimentally, there is little evidence that facilitation of metastasis (or secondary tumors developing at distant sites) is antigenspecific.757,884,955 However, a primary transplanted tumor may suppress the growth of a second inoculation with the same tumor.261 The phenomenon can be caused by immunologic as well as nonimmunologic mechanisms. The phenomenon is therefore properly referred to as “concomitant tumor resistance.”956 Following surgical removal of the primary tumor, accelerated seeding and/or growth of metastases will occur, obviously a troubling observation for patients undergoing cancer surgery. This phenomenon, first described a hundred years ago,957 occurs in different species and in several tumor models.958 As would be expected, the primary tumor during its growth must inhibit metastasis possibly by usurping most available progenitor cells and/ or consuming growth factors and nutrients (similar to what Ehrlich described nutrient deprivation or athrepsia261) and/ or by producing antimitotic factors.959 There is little evidence whether or not cancer patients inhibit metastasis by

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antigen-specific immunity. Concomitant immunity refers to the observation that an individual bearing a primary transplanted tumor may be resistant to secondary challenge with the same tumor at a different location because of an antigen-specific immune response.958 However, this old extensively studied observation958,960–962 may well be the result of artificially priming the host by tumor transplantation.

IMMUNOPREVENTION There is convincing evidence that immunosurveillance can prevent or reduce the incidence of cancers associated with certain viruses. Therefore, active immunization against viral capsid proteins may prevent infection and thereby cancer induction. While this is expected, it is still too early to confirm this with results. However, a federally funded, extensive vaccination program began in Australia in April of 2007 with a quadrivalent HPV vaccine to provide protection against the high-risk HPV types 16 and 18, which cause cervical cancer, and also low-risk types 6 and 11, which cause genital warts.963,964 There has been a highly significant decline in the diagnoses of genital warts and a significant decrease in high-grade cervical abnormalities only 3 years after implementation of the program.963 The latency period for cervical cancer is 15 to 25 years; therefore, evidence for a decline in cancer incidence cannot be expected until about 2027. Furthermore, only 70% of cervical cancers are caused by the HPV types that are in the present vaccine; thus, this vaccine can be expected to prevent only about 70% of cervix cancers. The effect might be somewhat higher because of cross-reactivity with the other HPV types, which may provide cross-protection with viral types 31, 33, and 45. Therapeutic HPV vaccines for treating already existent persistent infections or advanced cervical lesions are needed.965 The current vaccine has no effect against HPV infection once it has been acquired and must therefore be given before onset of sexual activity. Remarkably, there is a therapeutic vaccine that seems to be effective against HPV-induced vulvar preneoplastic lesions.966 The same vaccine has not been shown to be effective against high-grade premalignant cervical lesions. In general, premalignant lesions often persist for a very long time; destroying these lesions should prevent the development of cancer. After introduction of hepatitis B virus vaccines, a decline in the incidence and prevalence of hepatitis B virus infection occurred967; this should eventually lead to a decline of chronic hepatitis and hepatocellular carcinoma. Several strategies for vaccination against hepatitis C virus, human T-lymphotropic virus 1, and human herpesvirus 8 are being developed. Also, developing vaccines against Helicobacter pylori and Schistosoma infections remains extremely important. The major inf luences of diet and microbial f lora on the incidence of colorectal cancer and probably other cancers suggest new approaches for immunoprevention of these cancers. It will be important to determine whether cancer can also be prevented by active immunization of cancer-prone individuals with predisposing inherited or acquired antigens resulting from mutation (eg, in K-ras968). An ever-increasing

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number of predisposing inherited or acquired mutations are being identified; however, inducing immune responses against them may be problematic. For example, active immunization against an oncogenic viral protein became ineffective in preventing cancer when the immunization was begun in the later part of the latency period228,799,969,970 or after the oncogenic protein was expressed in premalignant host tissues301,722,971 for reasons that are poorly understood. Finally, we need to avoid stimulating cancer development when vaccinating cancer-prone individuals.341

IMMUNOTHERAPY General Aspects Multiple immunotherapeutic strategies involving innate or acquired immunity have been developed to control cancer; they include 1) local application of a live bacterial vaccine, BCG (see discussion under Cancer and Inflammation); 2) use of cytokines; 3) active immunization; 4) passive therapy with antibodies; and 5) adoptive transfer of effector cells. Some of these strategies are being combined with other forms of cancer treatment. It is important to realize that chemo- and radiation therapy can synergize with or antagonize innate or adaptive immunity dependent on timing and

sequence of the treatments. These important issues have been described and discussed elsewhere539,972–976 and previously in this chapter. But why are so few immunotherapies effective or the treatment of choice, except for melanoma and superficial bladder cancer? Extensive recent reviews of immunotherapy of cancers expose some complementary but several differing viewpoints.306,323,324,977–980 There is also an astounding repetition of findings made decades ago, and a tendency of overstating the translational potential of new findings.4,143 As already discussed (see Fig. 47.1), the focus of current experimental research remains treating very small transplanted lesions in mice < 2 weeks after cancer cell inoculation.4,633,981 Very few publications report immunotherapy in animals with cancers of clinically relevant size (≥ 109 cancer cells and ∼1 cm average diameter) and duration of growth (> 14 days). Adoptive T-cell therapy and anti-CD20 antibody treatments were singled out as experimental therapies effective in causing regression at this stage (Fig. 47.6). Indeed, adoptive transfer of T cells (with endogenous or transduced TCRs) may be effective with longer established tumor loads.977,982,983 Because both of these therapies can also be effective in humans, there is no “disconnect” between preclinical models and clinical experience.4,107 Relapse after therapy, the main problem of cancer treatments, is rarely

FIG. 47.6. Analysis of All Experimental Cancer Immunotherapy Publications Listed in PubMed for April, June, and November of 2010 and of the Entire Year 1980. Most experimental immunotherapies published treat small tumors (< 500 mm3) yet succeed only at slowing or delaying tumor growth. In several 2010 reports, larger tumors are being treated than in 1980, and a few reports indicate tumor regression. An effect size (E) of 1 indicates the treatment arrested tumor growth. An E < 1 indicates that the treated tumor still grew progressively but slower than control. An E > 1 indicates tumor regression. Regression of tumors > 200 mm3 is observed only after passive antibody or adoptive T-cell therapy. Modified from Wen et al.4

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considered; in fact, most animal studies break off before relapse could occur.

Therapeutic Vaccinations Active immunization of cancer-bearing mice or humans is a heroic approach that was abandoned in the clinical management of infectious diseases, except for rabies, which has very long incubation period.984,985 Cancer cells have a much slower generation time than most infectious organisms, and most of the bulk of the tumor load can usually be removed by other therapy (eg, surgery). At the time when the antigen load is lowest, the suppressive environment may be removed, and conceivably active immunization might lead to an effective immune response. However, persistent antigen appears to be a major reason why cancers and chronic infectious diseases are ineffectively treated by therapeutic vaccination. Possibly, combining active immunization with blockade of immunoinhibitory pathways may lead to more effective therapeutic vaccinations.965 Certain cancer cells may be fully sensitive to tumorspecific T cells but for various reasons fail to induce a response. The reasons for the poor immunogenicity may vary; therefore, different methods must be used for different cancers to immunize effectively.492 Currently, most methods have been developed in tumor-free mice (preventive/prophylactic vaccination).986 Immunization with small numbers of viable tumor cells may cause cancer, but, if not, it can stimulate solid long-lived memory immunity that prevents growth. Dead and disrupted tumor cells, membrane fractions, or cell extracts may enhance the growth of the cancer, although they stimulate “immune” (ie, lymphocyte) responses. Destroying the proliferative potential of the tumor cells, while leaving the cells viable and metabolically active, may result in a prolonged exposure to the antigen that allows T-cell immunity to develop.987 There are no general rules for accomplishing this, but exposing cancer cells to gamma radiation or certain cytostatic chemicals, such as mitomycin C, can work. However, chemotherapy makes cancer cells more immunogenic when they can undergo autophagy.986 In any case, chemotherapy of transplantable tumors is more efficient in immunocompetent than in immunodeficient mice.988 Because these methods alone are often insufficient to elicit a cytolytic T-cell response to cancer cells, many strategies including genetic engineering have been designed to increase the immunogenicity of the tumor-cell inoculum and/or stimulate innate immunity at the site of vaccinations by the use of chemical and/or bacterial agents. However, irrespective of which particular genetic engineering of the tumor cells is used, rejection of the modified tumor cells is often followed by T cell–mediated immunity against the unmodified tumor cells. Methods include infecting cancer cells with certain viruses989–991; somatic cell fusion with various nontumorigenic cells992–994 ; transfection of self- or foreign MHC class I or class II molecules995–997; hapten conjugation998,999 ; exposure to mutagens1000 ; transfection of tumor cells to express the B7 ligand that can provide a costimulating signal to T cells1001,1002 ; attracting secondary lymphoid structures

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to the cancer1003 ; targeting herpesvirus entry mediator pathways965 ; combining tumor cells with killed bacteria, such as Corynebacterium parvum1004 ; recombinant vaccines of antigen expressed by vaccinia, listeria, or virus-like particles2,1005 ; transfection of tumor cells to produce certain cytokines such as IL-2, IFNγ, IL-4, IL-6, IL-7, G-CSF, GM-CSF, or TNF-α134,138,141,535,1006 ; injecting naked DNA constructs encoding the tumor antigen (whereby the gene for GM-CSF may also be used to recruit dendritic cells)456,1007–1009 ; vaccination with anti-idiotypic antibodies, which bear the internal image of a tumor antigen1010 ; inhibiting extracellular adenosine triphosphate–degrading enzymes to increase autophagy 986 ; transfection of tumor cells to express antisense RNA of a required growth factor thereby inducing terminal differentiation1011; peptide vaccines combined with blocking IFNγ action1012,1013 with peptides long enough not to tolerize987,1013,1014 ; loading peptides to heat-shock protein1015 ; siRNA-mediated inhibition of nonsense-mediated messenger RNA decay,1016 which may trigger innate immunity1017 or the expression of new epitopes to which the host is not tolerant; and finally dendritic cells323,1018,1019 that can be loaded with 1) synthetic antigenic peptides, 2) recombinant proteins, 3) native peptides stripped from tumor cell surfaces, 4) tumor-derived, peptide-loaded heat-shock proteins, 5) tumor-derived messenger RNA, or 6) by fusion of tumor cells. One advantage of the latter three strategies is that immunity to (unique) individually distinct tumor antigens, as well as tumor-associated antigen may be induced without having to identify the antigens. The limitation is that the antigen dose cannot be standardized. Vaccines are also being used that target the mutant epidermal growth factor receptor EGFRvIII, a truly tumor-specific antigen on the surface of human malignant glioma cells capable of inducing strong B- and T-cell responses. The goal is to prolong patients’ relapse-free survival because this cancer, the most common primary brain malignancy, is untreatable by conventional therapy.305 The mutation in the EGFRvIII occurs in about 40% of the glioblastomas and represents an internal deletion in the gene encoding the receptor.1020 About half of the patients with this variant receptor have the same deletion, which generates a new amino acid at the fusion point of the resulting fusion protein and new antigenic determinant recognized specifically by a monoclonal antibody.304 A healthy skepticism is needed to stimulate experiments determining whether therapeutic effects of active immunization are found when the experimental tumor is not in early stages of malignant growth (see Figs. 47.1 and 47.7). Until then, it remains uncertain whether any of these procedures will be effective against longer established or advanced stages of cancer including microdisseminated cancer cells.382,985

Therapy with Engineered Antibodies Targeting Cancer Cells Directly General Considerations. The major alternatives to therapeutic vaccinations are antibody therapy and adoptive transfer of tumor-specific T cells. As already mentioned, there is little evidence that antibody produced by the host

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FIG. 47.7. Tumors can Escape from Therapeutic Vaccinations Once Truly Established. Then, therapeutic vaccinations become ineffective even against small tumors. Note the volumes of the tumors at start of therapeutic vaccinations, at day 7, or at day 29, respectively, are virtually the same size. However, only when vaccinations against were started early after TC1 cancer cell inoculation, tumors are eradicated and outgrowth prevented. †, mice in the naïve group were sacrificed. For human papillomavirus-16 E7 listeria, vaccine LLO-E7 leads to production of a fusion protein containing the complete E7 sequence.1,2 TC-1 cancer cells are HPV16 E6 and E7 and Ha-Ras transduced cell.6 Unpublished; courtesy of Zhen-Kun Pan and Yvonne Paterson, University of Pennsylvania, Philadelphia, PA, USA.

in response to autochthonous or transplanted cancers has any beneficial effects on the growing tumor. However, B-cell tolerance to self-antigens on human cancers can be overcome by immunizing mice with xenogeneic human antigen.1021 Thus, high-affinity antixenogeneic antibodies to selfantigens such as human Epcam, FAP, CD20, CD19, HER-2, and various other receptors and growth factors have been made.1022 Misleadingly, some of these high-affinity antixenogeneic antibodies are usually advertised as “fully human” when they were made in mice in which the murine Ig gene locus had been inactivated and replaced with the human Ig gene locus.346 The toxicities of such “fully human” antibodies will still be those of high-affinity anti–self-antibody. Severe toxicity and lethal reactions may only occur once the destructive capabilities of the antibody are increased further by engineering (see following discussion).204,206 Some antibodies can be relatively effective and useful in the therapy of selected human cancers, but rarely curative. To increase the clinical efficacy, scientists have searched to combine the specificity conferred by antibodies with the destructive potential of T cells. Two ideas, published decades ago,347,1023,1024 led to reagents that are in clinical use195,196,1025 and have little toxicity, but only if appropriate targets are used. The first approach makes bispecific molecules linking the cancer-reactive antibody (or its variable regions) with an antibody (or its variable regions) binding to and activating T cells (eg, anti-CD3).1023,1024 The second approach uses CARs consisting of the cancer-reactive variable region fused with transmembrane and signaling domains that are then transduced into T cells.347

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Considerable technologic efforts are being made to enhance the ability of antibodies to kill tumor cells by using them as carriers for cytokines or cytotoxic agents, such as radiochemicals or natural toxins.1026–1031 The recombinant antibody-cytokine or antibody-toxin fusion proteins may be useful to concentrate these agents in the stroma surrounding the tumor cells, but some of these coupled antibodies may have serious toxicity unless selective delivery of the conjugates to the tumor is achieved,1032 a problem shared with bispecific or trispecific antibodies that bind effector cells as well as tumor antigens.1033 Cluster of Differentiation 20 and Cluster of Differentiation 19. Monoclonal antibodies against B-cell lineage–specific differentiation antigens such as CD20 or CD19 are highly effective in preclinical models and can destroy large established B-cell lymphomas in patients,1034 though relapse is common. However, when anti-CD20 or anti-CD19 are combined with anti-CD3 antibody or their variable regions are chimerized and used as CARs, patients with chronic lymphocytic leukemia or other B-cell malignancies may be cured.195,196 Depletion of normal B cells occurs regularly but can be tolerated by giving intravenous Ig to the patient. Unfortunately, the antigens CD20 and CD19 are currently the exceptions rather than the rule. Other self-antigens exclusively expressed on cancer cells and only dispensable normal cells of the patient have not been identified. HER-2. Anti–HER-2 antibodies are effective and useful in the therapy of some cancers (see following discussion). Treatment may be tolerated relatively well because the most

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important effect may be inhibition by blocking signaling rather than destruction of cells.1035,1036 Thus, antibodies to HER-2 (anti–HER-2/erb-B2/neu antibody; trastuzumab) can inhibit tumor growth independent of perforin or FasL in mice but inhibition requires type I and II IFNs released by NK and CD8 T cells.1037 The role of antibody-dependent cell-mediate cytotoxicity in the trastuzumab efficacy in HER2-positive breast cancer patients is unclear, because a recent large study1038 failed to confirm the previously published association between therapeutic effectiveness of anti–HER-2 antibodies and FcR genetic polymorphisms reflecting high- or low-affinity Fc receptors. In mice, the effects of anti–HER-2 antibodies seem to depend also on the participation of innate and adaptive immunity of the host that may be abrogated when the antibody is combined with chemotherapy1039 ; the relevance of this finding for the clinic is still unknown. However, HER-2 is expressed at low levels in several vital human tissues, and severe toxicity and death have occurred when the antibody receptor was inserted as antigen-receptor for T cells as CARs.204 GD2 and Carcinoembryonic Antigen. Some therapeutic effects have also been observed in patients treated with other murine monoclonal antibodies,1040,1041 and anti-GD2 ganglioside-specific CARs had moderate antitumor effects in patients with neuroblastoma.1042 GD2 is expressed in the human cerebellum and peripheral nerves; serious toxicity as observed with anti–HER-2 CARs may have not been observed with anti-GD2 CARs because of a lesser therapeutic potency of the latter. Certainly, another CAR targeting CEA expressed on cancers and normal epithelium of the gut caused severe toxicity in patients.206 Other Receptors and Growth Factors. Antibodies to the IL-2 receptors can cause tumor regression in patients with cutaneous T-cell lymphoma.1043 Other examples of overexpressed growth factors and their receptors are VEGF and receptors and epidermal growth factor and its receptor-1 (EGFR-1). Treatments with antibodies to these factors and receptors are fraught with complications (eg, anti-VEGF [bevacizumab] therapy for example has been linked to cardiac failures, digestive tract perforations, and bleedings in the lungs1044).

Targeting Immunoregulatory Molecules Major clinical and preclinical efforts utilize monoclonal antibodies specific for regulatory and activation molecules expressed on T cells (anti-CTLA4, anti–PD-1 and antiCD137 [4-1BB]1045–1047) and on cancer and normal host cells (eg, antibodies to ligands of PD-1, PD-L1, and L21048). Monoclonal antibodies to the regulatory molecule CTLA-4 counteract tolerance and have antitumor effects in some but not in all tumor models.1045,1049,1050 Severe autoimmunity may be a complication of this treatment. This subject is extensively reviewed elsewhere.980,1051,1052

Cytokines The action of cytokines is essential for immunotherapy. Cytokines act in diffusion-limited spaces, but, when they act systemically, they may be toxic or lethal as in the

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Shwartzman-like shock reaction caused by systemic release of TNF-α .837,1053,1054 Mice surviving systemic TNF-α show a remarkable necrosis in the center of very large solid tumors of many different types of cancers.619 Unlike the tumor neovasculature, the preexisting vessels at the tumor margin are resistant to TNF-α , and residual cancer at the viable margins gives rise to relapse. “Cytokine storm” caused by systemic high-dose anti-CD3 antibodies is mediated largely by TNF-α and can be lethal. Intratumoral injections of genetic vectors encoding TNF-α or other cytokines have some restricted applicability.1055 Intravenous infusions of high doses of IL-2 induce clinical responses in a very small number of patients with a few types of cancers but have substantial side effects that need close medical supervision.1056 Infusions of IL-2 are also used to amplify the effects of adoptively transferred T cells; the effect of IL-2 is indirect through action on T cells, NK cells, endothelial cells, and by inducing other cytokines. IL-2 effects on endothelial cells may cause vascular leakage syndrome that may allow better extravasation of transferred T cells into tumors. Clinical trials with another powerful cytokine, IL-15, are beginning in patients with cancer and human immunodeficiency virus.1057

Adoptive T-Cell Therapy Seminal studies by Mitchison showed adoptive transfer of lymphocytes, not serum, produced resistance to allogeneic tumor transplants.531–533 This was confirmed using syngeneic tumor transplants.229,269,321,1058 Lymphocytes, either injected systemically or mixed with the cancer cell suspension and inoculated subcutaneously (Winn-type assay; see Assays to Study Effector Mechanisms in Vivo), protected against outgrowth of cancers while serum was ineffective. However, lymphocytes were as ineffective as serum in a therapeutic setting. Adoptive T-cell therapy of cancers was pioneered by Fefer, later working with Cheever, Greenberg, and Gillis, using established maize streak virus–induced tumors or Friend leukemia as a models.471,766,767,1059–1062 Critical for the advance was recognizing the need for transient lymphodepletion of the recipient and transiently culturing and expanding the T cells in vitro with the help of IL-2 before transfer to recipients. The use of TILs for adoptive T-cell therapy was pioneered by Rosenberg and coworkers at the National Institutes of Health.1063 In preclinical models, TILs were effective even when given about 2 weeks after intravenous cancer cell inoculation.1063 Adoptive cell transfer of autologous tumor-infi ltrating lymphocytes was effective in ∼50% of the patients with metastatic melanoma, but only few of the patients qualified for the treatment.318,1064 Melanomainfi ltrating lymphocytes were expanded in vitro with IL-2 and then infused into patients who receive IL-2 as well. Because the TIL response is dominated by T cells to unique tumor-specific antigen,303 it is likely that the success of the reinfused T cells depends on their reactivity to unique tumor-specific antigens. Unfortunately, it is unclear how effective this personalized therapy is in patients with cancers other than melanoma. Allogeneic EBV nuclear antigen–specific T cells sharing major MHC allele restriction with the patient can be used

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to treat successfully posttransplant lymphoproliferative disease1065 and EBV-positive lymphomas.1066,1067 Such antigen-specific allogeneic T cells proved to be safe1068 and effective.1069 However, detailed knowledge of the molecular biology and immunology of EBV were critical to establish procedures that drive out EBV-specific T cells while losing alloresponsiveness.1070 The donors did not need to be immunized with the patient’s cancer cells. Instead, donor T cells containing EBV-specific precursors were primed specifically to EBV antigens using autologous EBV antigen– positive stimulators in vitro. Thus, donor T cells were never primed to the patient’s alloantigens and were exclusively EBV-specific, although, as mentioned previously, donors and recipients need to be partially matched so that the EBVspecific T cells will be appropriately restricted. However for other cancers, immunization of the donor with the patient’s cancer cells will be essential but not acceptable until proven to be without hazard to the donor and recipient. Much more work remains. Rather than immunizing T-cell donors or cancer patients, current preclinical and clinical research focuses on the transfer of TCR or CAR genes into a patient’s own T cells.1071–1073 These gene-modified T cells thereby gain specificity for cancer antigens and are reinfused. Recent studies in humans and mice are promising but also raise serious concerns.204,208,1074–1077

Preventing Relapse and Targeting Variants As mentioned previously, experimental and human tumors have multiple tumor-specific CTL-recognized epitopes that are lost independently by selection with T cells in vitro.218,278 However, in vivo, the host fails to recognize all antigens simultaneously on a cancer cell.716,1078,1079 Recognition of a second antigen occurred only after the first antigen was lost by most of the tumor cells,716,1079 even though the subdominant antigen can serve as effective target.1080 This suggested that an immunodominant antigen prevents sensitization to other tumor antigens.237,716,1079 A hierarchy in the immune response to multiple independent antigens has also been described in the study of immune responses to multiple minor histocompatibility antigens expressed on a single cell.1081 The mechanism for the priority of the first response is unclear.342,1082 However, understanding how to break the hierarchy could help prevent immune selection and tumor escape. For example, studies in vitro using CTL clones suggest that the rate of mutation resulting in the loss of a single antigen from the tumor cells is less than 10 −6. Even if the frequency were as high as 10 −4, only one tumor cell that had lost four independent antigens would be expected in 1016 tumor cells (ie, in a tumor larger than the human body).278,1083 Thus, if the immune response of the host could be manipulated so that multiple antigens were attacked simultaneously, no escape of tumors should occur. Experimental evidence suggests that immunization with tumor-cell variants, selected in vitro and expressing selective antigenic components, can overcome immunodominance and prevent tumor escape.1084 This will be important for constructing vaccines.1085

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Targeting Tumor Stroma and Bystander Killing When complete surgical excision is no longer feasible, cancers usually contain variants resistant to numerous types of therapy. Whether killing of the overwhelming majority of antigen-positive cancer cells will result in the death of the few antigen-negative tumor cells “as bystanders” was studied over three decades ago with contradictory results.1086–1088 It is now clear that bystander killing of cancer variants can occur through stromal destruction and lead to the eradication of well-established solid tumors. This requires targeting tumorspecific antigens released from the cancer cells in the form of exosomes, microvesicles, and membrane fragments1089,1090 that are cross-presented by stromal cells in the immediate tumor microenvironment. Antigen-specific, adoptively transferred T cells eradicate large tumors including variants by also targeting and destroying the tumor antigen cross-presenting stroma.169,537,538,540,542 Pretreatment of the cancer by chemoor radiotherapy can temporarily increase stromal loading and thereby increase the efficacy of cross-presentation and tumor eradication by subsequent adoptive T-cell therapy.539 Thus, the therapy-resistant variants “caught” in the destroyed stroma are killed or die as bystanders in the necrotic stroma. Because cross-presentation depends on vicinity of stroma to the cancer cells, systemic toxicity has not been observed. No other antigens or molecules exist that are specific for stromal cells in cancers and not present on normal cells in the rest of the body. In contrast, other most intriguing and diverse approaches targeting antigens on stroma that are not cancerspecific1091–1096 must overcome likely limitations in terms of toxicity and/or efficacy.143

EPILOGUE Cancer immunology is at the interphase of two extraordinarily complex fields of research. Few fields of immunology have stimulated more emotional discussions. A healthy skepticism about the validity and relevance of experiments and concepts stimulates more experiments.800 Differences in opinion do not harm scientific progress and should not be branded as attempts to “destroy the field.” Any scientific field can only be strengthened by experiments in the search for truth.1097 Opinions, but not truth, can be refuted.1098 Many perceived “truths” have been refuted in the past, and this will likely also apply to parts of what is said in this chapter. The value of a scientist depends not on possessing real or perceived truth but his or her honest search for a better resolution of questions asked. Possession acquiesces and makes us proud and insensitive to opposing views of colleagues, but search for truth increases our strength to perfect our knowledge1099 and find answers to the many important questions remaining in tumor immunology.

ACKNOWLEDGMENTS The author thanks most sincerely D. A. Rowley, B. Engels, A. Arina, C. Idel and K. Schreiber for major critique, suggestions, and editing. The author acknowledges the support of National Institutes of Health grants PO1-CA97296, RO1-CA22677, and RO1-CA37516.Hans Schreiber

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Inborn Errors of Immunity Jean-Laurent Casanova • Mary Ellen Conley • Luigi D. Notarangelo

INTRODUCTION Louis Pasteur established the germ theory of infectious diseases whilst working near Alès, between 1865 and 1870, following his discovery that the two most prevalent diseases of silkworm were caused by different microbes.1 This groundbreaking discovery apparently settled a long discussion between two conflicting theories. The first, in which diseases were considered to be intrinsic, had long held sway over the second, in which diseases were considered to be extrinsic. It rapidly became clear, with the successive identification of numerous microbes, culminating in Koch’s discovery of Mycobacterium tuberculosis in 1882,2 that this new paradigm accounted for the heavy burden of childhood fever and death that had prevailed throughout human history. About half the children born died of fever before the age of 15, and this death toll could be attributed to microbes.3 This theory did not explain why rare children survived infectious diseases and assumed that healthy children had remained free from infection. However, the edifice on which this theory was constructed collapsed between 1905 and 1915, with the gradual realization that most infected individuals remained asymptomatic, often throughout their lifetime. Asymptomatic individuals were found to harbor latent microbes—nonreplicating microbes in a dormant state— such as M. tuberculosis.4 Even more strikingly, actively replicating microbes were found to cause silent infections in other individuals, such infections being termed “unapparent infections” by Charles Nicolle.5 The question of interindividual variability in the course of infection therefore became, and has since remained, a key question in the fields of infectious disease and immunology, and is, arguably, one of the most important questions in biology and medicine. The problem in itself suggests that there are, after all, some intrinsic determinants of disease. The first explanation put forward followed on naturally from another ground-breaking discovery by Pasteur in 1880 to 1881: the prevention of infectious diseases and the foundations of immunology, with the use of attenuated microbes to vaccinate against fowl cholera and sheep anthrax.6 This led to the hypothesis that related, less virulent microbes or smaller amounts of the same microbes had previously immunized the individuals who remained healthy in the course of infection with a microbe virulent enough to kill other individuals. This powerful idea can be seen as an immunologic or somatic theory of infectious diseases. We now know that this acquired immunity (often referred to as adaptive immunity) emerged twice in the evolution of vertebrates, by convergent evolu-

tion, that it is lymphoid, and that it involves both genetic and epigenetic components. However, although this theory was considered plausible in adults, and perhaps in teenagers, especially during secondary infections or reactivation from latency, it was less convincing for primary infections in early childhood, which comprised the majority of cases. In this context, a few human geneticists looking at the problem from the complementary standpoints of clinical and population genetics, including Archibald Garrod and Karl Pearson in particular, collected evidence between 1910 and 1930 for strong, germline, genetic determinism, controlling innate immunity against microbes.7,8 As stated by Garrod, “It is, of necessity, no easy matter to distinguish between immunity which is inborn and that which has been acquired.”7 The development of new vaccines and the discovery of sulfonamides and antibiotics during the 1930s rendered these questions obsolete; it became less pressing to understand a problem that everyone thought would soon be resolved. Paradoxically, antibiotics themselves triggered renewed interest in the question in the early 1950s, when a small group of pediatricians in Europe and America noted that rare children suffered from multiple, recurrent infectious diseases, each of which was treated with antibiotics, lacked a major leukocyte subset or gammaglobulins, and shared this phenotype with relatives, consistent with an inheritable trait.9–14 This was the birth of the field of primary immunodeficiency (PID), a term apparently first coined in 197115,16 that remains more frequently used than inborn errors of immunity, which was first used in 1966.16,17 These children had major immunologic abnormalities, such as neutropenia,11,14 alymphocytosis,12,13 or agammaglobulinemia.9,10 Moreover, their infectious and immunologic phenotypes often followed a Mendelian pattern of segregation within their families. It is, however, important to bear in mind that the defi nition of PID established in the 1950s was based on an artificial, as opposed to natural phenotype, because these children with multiple life-threatening infections would have died during their first episode of infection before the advent of antibiotics. Moreover, although the infectious phenotype of these patients led to their investigation and the pattern of inheritance was suggestive of a Mendelian trait, PIDs were classified and defined solely on the basis of the immunologic phenotype. There had been previous descriptions of inherited disorders associated with infectious diseases, such ataxia-telangiectasia in 1926 and 1941,18,19 WiskottAldrich syndrome in 1937,20,21 and epidermodysplasia verruciformis in 193322 (and its viral etiology in 194623), but

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these disorders were not recognized as PIDs until 1964,24,25 1959,26,27 and 2002,28 respectively. They were long considered from other angles, in the absence of detectable immunologic abnormalities. Autoimmune polyendocrinopathy syndrome with mucocutaneous candidiasis was probably first described clinically between the 1920s and 1940s, but the autoantibodies involved were not described until the 1970s.29,30 More surprisingly, Chediak-Higashi syndrome (CHS) was first described as an inherited predisposition to infections with detectable leukocyte abnormalities in 1943 to 1954,31–33 but was not recognized as a PID until 1962.34 Severe congenital neutropenia (SCN) and CHS were among the first PIDs to be described, but these and other inborn errors of myeloid cells, such as chronic granulomatous disease (CGD), which was fi rst described in 1966, and complement defects, first described in 1963 (hereditary angioneurotic edema due to an absence of C1 esterase inhibitor),35 were not included in the first classifications of PIDs, which were restricted to the lymphoid arm of immunity, in 1968,36 1970,37,38 1971,15 1972,39 1973,40 and 1974.41 Over this period, the fields of immunology and PIDs evolved in parallel, focusing on acquired or adaptive immunity, which is sometimes referred to as adaptive or lymphoid immunity. The lymphoid imprint was so strong that the first report on PIDs even distinguished explicitly between these disorders and myeloid abnormalities. Unsurprisingly, given that these PIDs affected the development or function of B- and/ or T-cell immunity, these patients displayed multiple, recurrent infectious diseases that were often opportunistic (ie, not seen in patients with apparently intact immunity). Patients with T- and B-cell PIDs also displayed noninfectious, autoimmune, allergic, and in some cases, cancer phenotypes. Despite the broad infectious phenotypes of the patients with complement and phagocyte disorders identified, the first mention of quantitative and qualitative disorders of phagocytes did not occur until 1974, when it appeared, somewhat amusingly, in a preliminary report,42 but not in the official report43 of the second World Health Organization (WHO) international workshop on PIDs. The weaknesses of this definition and classification of PIDs were apparent to some investigators, including Gatti, who ironically compared this system to the ancient Chinese classification for animals.44 The classification nonetheless evolved, as complement disorders were mentioned in 1976 but phagocyte defects were not,45 and qualitative phagocyte disorders (including CGD and CHS) appeared in the third (1978),46 fourth (1983),47 fifth (1986),48 sixth (1989),49 and seventh (1992)50 WHO classifications, although SCN did not. The full range of PIDs was not covered until the eighth WHO classification in 1999,51 and has since been dealt with in the 10th (2003),52 11th (2004),53 12th (2006),54 13th (2007),55 14th (2009),56 and the most recent (15th57) WHO International Union of Immunological Societies reports. Meanwhile, two prominent reviews published in 1984 and 1995 made no reference to phagocyte defects.58–60 Some aspects of the history of the field have been covered in at least two reviews.61,62 The common PID classification and the underlying definition of PIDs proved increasingly inadequate and unable to describe the situation in reality, which extended well beyond

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phagocytic disorders, from the 1990s onwards. The definition and classification of PIDs have always been engaged in an eternal game of catch-up with the reality of the situation on the ground, and this gap between the conservative words used to describe these conditions and the continual discovery of new PIDs, ever increasing the known spectrum of these diseases, has been maintained.63,64 Phenotypic studies in this field have progressed in at least two ways. First, multiple and diverse new phenotypes have progressively been attributed to PIDs, including, of course, autoimmunity,30 malignancy,65 and allergy,66 but also various other phenotypes, such as autoinflammation,67 angioedema,68 hemophagocytosis,69 and thrombotic microangiopathies.70 The underlying mutations involve multiple circuits affecting both myeloid and lymphoid cells. Second, patients with a single infectious disease, and often with a single infectious episode, have been shown to display PIDs.71 Again, the underlying mutations affect both lymphoid and myeloid cells, but they may also, in some cases, concern nonhematopoietic cells. PIDs were initially associated with multiple, recurrent, and often opportunistic infections with an early onset and fatal outcome. They were familial, recessive traits. Exceptions to these rules gradually emerged, beginning with the description of patients with autosomal recessive (AR) defects in the terminal components of complement (C5 to C9), who are specifically susceptible to Neisseria,72 patients with X-linked recessive (XR) lymphoproliferative syndrome, who are susceptible to Epstein-Barr virus (EBV),73 and patients with AR epidermodysplasia verruciformis, resulting in a selective predisposition to infection with skin-tropic, oncogenic papillomaviruses.74 These studies paved the way for the discovery of new PIDs underlying particular infectious diseases in children who were otherwise healthy and normally resistant to other infectious diseases. Children with mycobacterial diseases were found to carry inborn errors of interferon (IFN)γ immunity.75 Mutations in the toll-like receptor (TLR) and interleukin (IL)-1R pathway are associated with pyogenic bacterial diseases, whereas mutations in the TLR3 pathway are associated with herpes simplex encephalitis.76 Finally, inborn errors of IL-17 immunity underlie chronic mucocutaneous candidiasis.77,78 These discoveries indicated that otherwise healthy children with a single infectious disease can display inborn errors of immunity to primary (in cases of acute disease) or recurrent/latent (for chronic disease) infection.71,79 The definition of PIDs is evolving, thanks largely to the clinical delineation and genetic dissection of new phenotypes. These advances are also leading to changes in the classification of PIDs. PIDs were initially classified into two groups (defects of humoral and cell-mediated immunity), then into four major groups (T, B, complement, and phagocyte disorders). The 2011 WHO classification includes up to 10 (somewhat overlapping) categories of PIDs, despite the contentious omission of certain types of PID.79a However, there is no consensus about the definition and classification of PIDs.16 The classification of PIDs principally on the basis of immunologic phenotypes entails a risk of clinical and genetic overlap and of some disorders being ignored. A classification based on clinical phenotype would be more useful at the patient’s bedside, and a classification based

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on genotype would be more useful at the research bench. However, the apparent lack of a solution to this problem is not a major concern. Hopefully, the classification will improve with the characterization of more PIDs, the deciphering of their genotypes, the dissection of their molecular, cellular, and immunologic phenotypes. and the description of their clinical phenotypes. Like a jigsaw puzzle, every piece should start to fit into place as we approach the solution. This task may not be as vast as it might initially appear, as there are no more than 25,000 genes for 7 billion individuals reproducing every two or three decades. However, we may never reach the asymptote, as there is no such thing as a distinct disease entity, precisely because of the tremendous germline and somatic genetic variability that makes each disease in each patient unique: there are only patients. There is therefore unlikely to be any satisfactory definition and classification of inborn errors of immunity in the near future. This does not matter so long as rapid progress is made in this field, in terms of awareness, diagnosis, treatment, and above all, in explorations of the genotype and intermediate phenotypes of known and new clinical phenotypes. Why is it so important for the field of PIDs to thrive? This progress is above all important for the patients. As in other areas of medicine, strategies improving our understanding of pathogenesis constitute the only rational approach to improving the clinical management of patients, in terms of the quality of diagnosis, prognosis, and treatment. The availability of blood samples has made it possible to carry out very careful analyses of the relationships between genotypes, cellular phenotypes, immunologic phenotypes, and clinical phenotypes, as for inborn errors of erythrocytes and platelets, perhaps more thoroughly than in other fields of human genetics and pediatrics. For example, the discovery of new PIDs over the last two decades has made it clear that the 10 conventional warning signs used in PID awareness campaigns are completely out of date and require revision.80 Another clinical lesson learned in the last 50 years is that most PID-causing genes are associated with high levels of clinical heterogeneity. Remarkable examples include mutations in the NEMO gene, the effects of which range from death in utero to mild immunodeficiency in adults, reflecting the severity of the biochemical deficit caused by the morbid alleles,81 and mutations in RAG genes, the impact of which ranges from life-threatening severe combined immunodeficiency in infancy to combined immunodeficiency in adults.57 Several genes have even been found to harbor loss-of-function (LOF) and gain-of-function (GOF) mutations. These genes include WASP, LOF mutations, which underlie Wiskott-Aldrich syndrome (WAS), and GOF mutations, which underlie SCN,82 and STAT1, LOF mutations, which underlie mycobacterial or viral diseases, and GOF mutations, which underlie chronic mucocutaneous candidiasis (CMC).78 Similarly, most, if not all of the known clinical phenotypes are associated with high levels of locus and allelic genetic heterogeneity. For example, several agammaglobulinemia-causing autosomal genes were identified following the discovery of BTK mutations in boys with XR agammaglobulinemia.83 A large proportion of the patients with each PID, particularly for the most recently described

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conditions, do not carry mutations in known morbid genes. It is therefore highly likely that new genetic etiologies will be discovered in the future. The clinical implications of research in this field actually extend well beyond diagnosis and pathogenesis, as the first cases of successful immunoglobulin (Ig) substitution,9 bone marrow transplantation,84 transfusion-based enzymatic replacement,85 and gene therapy86 all concerned children with PIDs. These children were also among the first to benefit from PEGylated enzymatic replacement87 and treatment with recombinant cytokines.88 Perhaps of greater relevance to this book, this field has had extraordinary immunologic implications. One of the pioneers in this field, Robert Good, often referred to PIDs as “experiments of nature,” reviving a line of investigation that began with Harvey and was followed by Osler, Garrod, McQuarrie, and Burnet, among others.89,90 Indeed, physicians and scientists can learn much by deciphering the enigmas posed by the experiment of nature represented by each patient with a PID. No matter how rare a disorder, it can provide considerable insight into the fundamental laws operating in living organisms. More conventional experiments, designed by humans and carried out in animal models, benefit from being carefully thought out and executed in a controlled manner. However, they suffer from the limitations inherent to their experimental nature. Experimental protocols differ from natural processes in many ways (inbred animals, conditions of infection, microbes). Human genetics provides us with a unique opportunity to define the function of host genes in natural, as opposed to experimental, conditions: in a natural ecosystem governed by natural selection.91,92 Immunity in natura can be defined by the careful dissection of PIDs and by other related approaches, such as epidemiologic and evolutionary genetics. The differences between mice and humans are often discussed, and rightly so, as these two species differ in many ways, despite the similar architecture of their immune systems. Beyond these multiple, and in some cases large, differences between humans and mice, there may be major differences between the processes used to study phenotypes, infectious and otherwise, in the two models. For example, experimental infections in mice generally involve inoculation with microbes that have not coevolved with these rodents, via artificial routes of infection and at high doses. By contrast, most human infections are natural, although some experimental infections, such as those caused by live vaccines, have played an important role in the development of this field. The experimental triggers of autoimmunity in mice are also different from those operating in natural conditions in humans. The genetic dissection of PIDs thus provides us with a unique opportunity to cast new light on the function of human genes in a natural ecosystem. Over the years, these observations have provided invaluable insights sometimes at odds with the mouse model. It will not be possible to cover the entire field in this chapter. More than 200 inborn errors of immunity have been characterized genetically.57,93 Many other PIDs have been described clinically but have no known genetic etiology. Doctors in this field also know that a large fraction of the patients under their care suffer from exceedingly rare

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disorders, sometimes apparently limited to a single family, not reported in the medical literature. Moreover, many phenotypes might be due to new PIDs. These phenotypes form a large reservoir for the future dissection of new inborn errors of immunity, as previously illustrated by autoinflammation, hemophagocytosis, angioedema, thrombotic microangiopathies, and isolated infectious diseases.64 Finally, we know that most known PIDs are associated with tremendous locus and allelic heterogeneity. With about 25,000 known genes and only 8000 known inborn errors, including about 300 PIDs, we can predict that 10,000 inborn errors of immunity, defined by a specific causal relationship between a genotype and a phenotype, currently await discovery. An estimate of 1000 to 2000 PID-causing loci is not unrealistic. Even with the only 300 or so known PIDs, it is clear that we cannot cover even a substantial fraction of these diseases here. We will therefore focus our review on seven topics, focusing in more detail on a specific syndrome or disease for each and discussing its genetic basis. We will pay particular attention to the immunologic lessons that can be drawn from these experiments of nature.

INBORN ERRORS OF T-CELL DEVELOPMENT OR FUNCTION In 1950, Glanzman and Riniker described two infants who presented with severe infections, diarrhea, failure to thrive, and disseminated candidiasis, leading to early death. Severe lymphoid depletion tissue was found at the postmortem examination.94 Similar cases of early death due to severe infection (often with weakly virulent microbes), lymphopenia, and agammaglobulinemia were reported a few years later in Switzerland.95 Both male and female patients were affected by this disease, which was named “Swiss-type agammaglobulinemia”96 to distinguish it from Bruton agammaglobulinemia, which affects principally male patients and is characterized chiefly by recurrent bacterial infections.97 The cases described by Glanzman and Riniker94 and by Hitzig et al.95,96 constitute the first description of humans with severe combined immunodeficiency (SCID), a group of conditions characterized by a lack of autologous T cells and extreme susceptibility to infections caused by a broad range of pathogens, including weakly virulent agents. The genetic heterogeneity of SCID became apparent in the 1960s, with the identification of families in which the disease was inherited as an X-linked trait.98 In the 1970s and 1980s, the immunologic phenotype of SCID was shown to be heterogeneous, through the demonstration that all patients had low levels of autologous T-lymphocytes but that only some of these patients displayed an associated decrease in the numbers of B and/or natural killer (NK) lymphocytes.99 This has led to the identification of various immunological subtypes of SCID: a) with a complete absence of T, B, and NK lymphocytes (T− B − NK− SCID); b) T− B + NK− SCID; c) T− B + NK+ SCID; and d) T− B − NK+ SCID. Finally, genetic studies over the last three decades have shown that SCID is also highly heterogeneous in terms of its genetics57,100 (Table 48.1; Fig. 48.1). SCID has a prevalence of 1:50,000 to 1:100,000 live births. The study of SCID has played an essential role in

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the identification of the key mechanisms governing human T-cell development. In several cases, SCID-causing gene defects were identified in patients before the generation of animal models. Moreover, comparison of humans and mice with mutations in orthologous genes associated with SCID has often revealed differences in the immunologic phenotype in these two species, indicating the existence of speciesspecific differences in the mechanisms governing lymphoid development. Finally, the heterogeneity of mutations in individual SCID-causing genes is associated with variability of the clinical and immunologic phenotype, which may include manifestations other than SCID, such as various forms of immune deficiency and immune dysregulation, particularly in patients with hypomorphic mutations allowing residual development of T- and/or B-lymphocytes.101

Molecular Mechanisms Accounting for Severe Combined Immunodeficiency Defective thymus organogenesis is associated with a profound impairment of T-cell development. The genetic defects affecting the development of the thymic epithelium are extrahematopoietic in nature, but they may greatly disturb T-cell development, in some cases leading to a clinical and immunologic SCID phenotype. DiGeorge syndrome (DGS) results from defects of the third and fourth branchial pouches and is characterized by impaired thymic development, congenital heart disease, hypoparathyroidism, and facial dysmorphisms. Most patients carry a heterozygous interstitial deletion of chromosome 22q11.102 The molecular mechanism by which haploinsufficiency for the genes of the DGS critical region underlies defective thymic development remains unclear. The severity of the thymic development defect varies considerably between patients. Most patients with DGS have moderate T-cell lymphopenia, but a few have complete thymic aplasia (complete DGS) and no circulating T-lymphocytes.102–105 Another PID in which thymogenesis is impaired is caused by mutations of the gene encoding FOXN1, a transcription factor required for the development and maturation of the thymus stroma and eccrine glands.106,107 Biallelic FOXN1 mutations cause an AR SCID phenotype in which a lack of circulating T-lymphocytes is associated with alopecia.108–110 This disease is the human equivalent of the nude phenotype in mice, for which the underlying gene was identified before the discovery of human patients.111 The proliferation and survival of lymphoid progenitor cells are also essential for the generation of a normal number of mature lymphocytes. Some forms of SCID are associated with high rates of apoptosis. In 1972, while investigating polymorphic enzymes as genetic markers, Giblett discovered that the erythrocytes of two infants with SCID displayed no adenosine deaminase (ADA) activity.112 ADA converts adenosine to inosine (and deoxyadenosine to deoxyinosine).113 In patients with AR ADA deficiency, the accumulation of toxic phosphorylated derivatives of deoxyadenosine causes cell death, resulting in extreme lymphopenia, with an almost total absence of T, B, and NK lymphocytes.112,114,115 A few years after describing ADA deficiency, Giblett discovered

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CHAPTER 48

TABLE

48.1

INBORN ERRORS OF IMMUNITY

1239

Inborn Errors of T-Cell Development

Disease

Gene Defect

T Cells

B Cells

NK Cells

FOXN1 deficiency

FOXN1

↓↓↓

N

N

AR

DiGeorge syndrome

22q11del*

↓↓

N

N

Sporadic

Reticular dysgenesis

AK2

↓↓↓

N or ↓

↓↓

AR

Increased apoptosis

Adenosine deaminase deficiency†

ADA

↓↓

↓↓

↓↓

AR

Increased apoptosis

Nucleoside phosphorylase deficiency X-linked SCID†

PNP

↓↓

N

N

AR

Increased apoptosis

IL2RG

↓↓↓

N

↓↓↓

XL

JAK3 deficiency

JAK3

↓↓↓

N

↓↓↓

AR

IL-7R deficiency†

IL7R

↓↓↓

N

N

AR

RAG1 deficiency†

RAG1

↓↓↓

↓↓↓

N

AR

RAG2 deficiency†

RAG2

↓↓↓

↓↓↓

N

AR

Artemis deficiency†

DCLRE1C

↓↓↓

↓↓↓

N

AR

Defective signaling through gc Defective signaling through gc Defective signaling through IL-7R Defective V(D)J recombination Defective V(D)J recombination Defective V(D)J recombination

DNA ligase IV deficiency†

LIG4

↓↓

↓↓

N

AR

Defective V(D)J recombination

Cernunnos/XLF deficiency DNA-PKcs deficiency CD3γ deficiency

NHEJ1

↓↓

↓↓

N

AR

PRKDC

↓↓↓

↓↓↓

N

AR

CD3G

N or ↓

N

N

AR

CD3δ deficiency

CD3D

↓↓↓

N

N

AR

CD3ε deficiency

CD3E

↓↓↓

N

N

AR

CD3ζ deficiency

CD3Z

↓↓↓

N

N

AR

CD45 deficiency ZAP-70 deficiency†

CD45 ZAP70

↓↓↓ ↓↓↓ of CD8+ cells

N N

N N

AR AR

MHC class II deficiency

CIITA, RFX5, RFXANK, RFXAP CORO1A

↓↓ of CD4+ cells

N

N

AR

↓↓↓

N

N

AR

Defective V(D)J recombination Defective V(D)J recombination Defective TCR signaling Defective signaling through pre-TCR Defective signaling through pre-TCR Defective signaling through pre-TCR Defect of signaling Defect of signaling (impaired positive selection of CD8+ cells) Defect of positive selection of CD4+ cells Defect of thymocyte egress, impaired T lymphocyte survival

Coronin-1A deficiency

|

Inheritance

Pathogenesis Defective maturation of thymic epithelium Defective thymus organogenesis

Associated Features Other than Immunodeficiency Alopecia

Hypoparathyroidism, congenital heart disease Neutropenia, sensorineural deafness Bone abnormalities, neurologic problems, liver and lung involvement Neurologic problems, autoimmunity

Developmental defects, radiation sensitivity Microcephaly, dysmorphisms, radiation sensitivity Microcephaly, radiation sensitivity Most often, autoimmunity

(continued)

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SECTION VIII

48.1

IMMUNOLOGIC MECHANISMS IN DISEASE

Inborn Errors of T-Cell Development (Cont.)

Disease

Gene Defect

T Cells

B Cells

NK Cells

ORAI1 deficiency

ORAI1

N

N

AR

STIM1 deficiency

STIM1

N

N

AR

MAGT1 deficiency

MAGT1

N

N

XL

DOCK8 deficiency

DOCK8

N (but defective function) N (but defective function) ↓↓ of CD4+ cells, reduced proliferation ↓

↓

↓

AR

Defective activation

CD40L deficiency

CD40LG

N

N

N

XL

Impaired CD40Lmediated costimulation of B and dendritic cells by activated CD4+ cells

Associated Features Other than Immunodeficiency

Inheritance

Pathogenesis Defective calcium flux Defective calcium flux Defective magnesium flux

Autoimmunity, myopathy Autoimmunity, myopathy Chronic viral infections, lymphoma Severe atopy, severe cutaneous viral infections Neutropenia, Cryptosporidium infection, biliary tract disease, neuroectodermal tumors; IgM levels are often elevated

AR: autosomal recessive; CD, cluster of differentiation; DNA, deoxyribonucleic acid; gc, germinal center; Ig, immunoglobulin; IL, interleukin; MHC, major histocompatibility complex; N, normal; NK, natural killer; PNP, purine nucleoside phosphorylase; RAG, recombinase-activating gene; SCID, severe combined immunodeficiency; TCR, T-cell receptor; XL, X-linked; XLF, XRCC4-Like factor. *DiGeorge syndrome is most often associated with 22q11 chromosomal deletion; however, other cytogenetic and genetic abnormalities have been also reported in a minority of cases. † Hypomorphic mutations in these genes have been associated with Omenn syndrome or other conditions with immune dysregulation.

that a deficiency of another enzyme involved in purine metabolism, purine nucleoside phosphorylase, was also associated with impaired lymphoid development.116 SCID may also be caused by impaired cytokine signaling by T-cell precursors in the thymus. In 1993, two groups demonstrated that the newly cloned IL2RG gene encoding the γ chain of the IL-2 receptor was mutated in male patients with XR SCID, the most common form of SCID in western countries.117,118 XR SCID is characterized by a T− B + NK− phenotype, indicating that IL2RG mutations are deleterious to the development of both T- and NK lymphocytes. This was a surprising discovery, because disruption of the Il2 gene in mice had been shown to be associated with immune dysregulation rather than SCID.119 It soon became clear that the IL-2Rγ chain was also common to other cytokine receptors, including those for IL-4, IL-7, IL-9, IL-15, and IL-21,120–125 leading to its being renamed the common γ chain, γc. In all these receptors, the γc is coupled to the intracellular kinase JAK3, allowing intracellular signaling.126 These discoveries have helped defi ne the molecular basis of XR SCID and to identify other genetic defects causing SCID in humans. In 1995, two groups established that an AR variant of T− B + NK− SCID (a phenocopy of XR SCID) was due to mutations of the JAK3 gene.127,128 Furthermore, the observation of severely impaired T-cell development in Il7 − /− and Il7r − /− mice129,130 led to the discovery of IL7R mutations in patients with AR T− B + NK+ SCID.131,132 IL-7 provides important signals for the survival and proliferation of thymocytes and peripheral T cells and regulates the rearrangement of T-cell receptor (TCR) genes,133 thus accounting for the absence of circulating T cells in patients

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with SCID due to IL2RG, JAK3, and IL7R mutations. Interestingly, B-cell development is unaffected in these forms of SCID, but abolished in Il2rg−/−, Jak3−/ −, and Il7r − /− mice.134–137 These data highlight significant species-specific differences in the molecular mechanisms governing lymphoid development. Circulating NK lymphocytes are absent in patients with XR SCID and with JAK3 deficiency, but not in those with IL-7R deficiency, suggesting that impaired signaling through another γ c-dependent cytokine may occur. The cytokine concerned is probably IL-15, because IL-15–mediated signaling is essential for human NK cell development in vitro138 and Il15− / − and Il15r− /− mice lack NK cells in vivo.139,140 SCID can also result from impaired TCR gene rearrangement due to abnormalities in VDJ recombination, a process essential for the expression of both the pre-TCR and the mature TCR.141,142 The lymphoid-specific recombinaseactivating gene (RAG) 1 and RAG2 proteins initiate VDJ recombination by recognizing recombination-specific sequences flanking the variable (V), diversity (D), and joining (J) elements of the TCR and introducing deoxyribonucleic acid (DNA) double-strand breaks.143,144 These breaks are eventually repaired through the ubiquitously expressed nonhomologous end-joining (NHEJ) pathway.145,146 RAG1, RAG2, and the NHEJ pathway are also involved in the rearrangement of Ig genes, which is required for expression of the pre–B-cell receptor (BCR) and the BCR.141,143 Mutations of the RAG1 and RAG2 genes,147 and of the genes encoding Artemis,148 DNA ligase IV,149–152 and DNA-PKcs153 (all components of the NHEJ pathway) result in T− B − NK+ SCID. Mutations of Cernunnos/XLF, another component of the

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CHAPTER 48

INBORN ERRORS OF IMMUNITY

7

3

Thymus

4

pro-T/NK

DN

1241

mature NK

mature NK

imm. NK

|

5

SP4

SP4

SP8

SP8

DP

6

2 1 1 HSC

CLP

pro-B

pre-B

imm. B

mature B

Bone marrow FIG 48.1. Schematic Representation of Developmental Blocks in Combined Immunodeficiencies. Discrete stages in T-, B-, and natural killer (NK)-cell development are shown. Red bars identify stages at which developmental arrests are caused by specific gene defects. Numbers in boxes identify the molecular defects accounting for the developmental blocks. In particular: 1): adenosine deaminase deficiency; 2) defects of cytokine-mediated signaling (IL2RG, IL7R, JAK3 deficiency); 3) defects of V(D)J recombination (RAG1, RAG2, Artemis, LIG4, Cernunnos, and DNA-PKcs deficiency); 4) defects of pre–T-cell receptor signaling (CD3D, CD3E, CD3Z deficiency); 5) defects of positive selection of cluster of differentiation (CD)4+ thymocytes (major histocompatibility complex class II deficiency); 6) defects of positive selection of CD8+ thymocytes (ZAP-70 deficiency); and 7) defects of thymocyte egress from the thymus (Coronin-1A deficiency). The hatched bar indicates that NK-cell development is affected by mutations of IL2RG and JAK3 gene (that interfere with interleukin-15–mediated signaling) but not by IL7R mutations. Not shown is severe combined immunodeficiency due to FOXN1 mutations (that impair thymus organogenesis) and reticular dysgenesis, due to mutations of the AK2 gene, in which the exact stage at which T-cell development is blocked is not known. CLP, common lymphoid progenitor; DN, double negative; DP, double positive; HSC, hematopoietic stem cell; imm., immature; SP4, single positive CD4; SP8, single positive CD8.

NHEJ pathway, severely impair, but do not completely abolish T- and B-cell development.154,155 The genes encoding Artemis148 and Cernunnos/XLF154 were cloned from humans before they were cloned from mice, the availability of fibroblastic cell lines from SCID patients being essential to this achievement. NHEJ is involved in general mechanisms of DNA repair, including those occurring in nonlymphoid cells. Thus, patients with defects of this pathway also display an unusually high level of cellular sensitivity to radiation, have a higher than normal risk of malignancy, and may present with neurologic problems.146,156 Finally, hypomorphic mutations of genes involved in V(D) J recombination and NHEJ have been identified in patients with “leaky” forms of SCID, in whom the residual development of T- (and in some cases, B-) lymphocytes was associated with autoimmunity and a high risk of lymphoid malignancies.157–159 Signaling through the pre-TCR is essential to promote the progression from cluster of differentiation (CD)4 − CD8 − double-negative (DN) thymocytes to CD4 + CD8 + double-positive cells. This signaling is mediated by the CD3 complex, mimicking the requirement of this complex

Paul_CH48_final.indd 1241

for TCR signaling in mature T cells. Null mutations of the CD3D, CD3E, and CD3Z genes interfere with this process and result in AR SCID.160–163 By contrast, CD3G mutations are more often associated with a milder phenotype including autoimmunity.164

Other Combined Immune Deficiencies Due to Late Defects in T-Cell Development and Function The positive selection of CD4 + and CD8 + thymocytes requires the interaction of thymocytes expressing a functional TCR with thymic epithelial cells (and dendritic cells) expressing self-antigens bound to human leukocyte antigen (HLA) class II and class I molecules, respectively.165,166 Several gene defects account for HLA class II deficiency in humans,167–170 a condition characterized by severe CD4 + lymphocyte depletion and normal CD8 + T-cell development.171 Conversely, mutations of ZAP-70, a tyrosine kinase that binds to the CD3ζ chain and promotes TCRmediated signaling,172 cause immunodeficiency with a lack of CD8 + cells.173–175 The CD4 + lymphocytes of affected patients develop normally but are nonfunctional and fail to

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proliferate in response to mitogens and antigens. ZAP-70 deficiency in humans results in a phenotype different from that of Zap70− /− mice, which lack both CD4 + and CD8 + lymphocytes.176 The prolonged expression of SYK in human (but not in mouse) double-positive thymocytes seems to compensate for the lack of ZAP-70 protein, allowing the generation of CD4 + T cells.177,178 However, mature CD4 + lymphocytes from ZAP-70–deficient patients have no SYK and are therefore functionally impaired. The egress of mature thymocytes from the thymus to the periphery requires rearrangement of the actin cytoskeleton. Coronin-1A is involved in this process.179 Accordingly, Coronin-1A deficiency causes the retention of mature thymocytes and a failure to generate peripheral T cells, although impaired survival of newly generated T lymphocytes is probably another very important mechanism of disease.180,181 Finally, mature T cells in the periphery respond to mitogenic signals by releasing calcium from endoplasmic reticulum stores and opening calcium release–activating channels on the cell membrane, allowing calcium influx to occur.182 Mutations of STIM1 (which encodes a sensor of endoplasmic reticulum calcium stores) and of ORAI1 (which encodes calcium release–activating channels) cause a severe immunodeficiency in which T-lymphocyte generation in the thymus is not affected, but the function of peripheral T cells is impaired.181 Both conditions are also characterized by myopathy and the impairment of immune homeostasis, with autoimmunity.183,184

Clinical and Immunologic Features and Treatment Infants with SCID are susceptible to severe infections from shortly after birth. Infections may be due to bacteria, viruses, or fungi. Infections with weakly virulent microorganisms (eg, Pneumocystis jiroveci, cytomegalovirus) are common and frequently cause interstitial pneumonia and chronic diarrhea, leading to failure to thrive.101,185,186 Lymphopenia is present in 60% to 70% of affected infants.186 T-cell lymphopenia is even more common. In 30% to 50% of cases, a variable number of T-lymphocytes of maternal origin (that have crossed the placenta and not been rejected by the fetus with SCID) are detected.187 Maternal T-lymphocytes may cause alloreactive signs resembling graft-versus-host disease, with infiltration and damage to the liver, the skin, the gut, and the bone marrow.187,188 All patients lack autologous T cells. Alternatively, less severe (hypomorphic) mutations in SCID-causing genes may be permissive for residual T-cell development, but result in a lower level of TCR diversity in circulating T cells.101 The oligoclonal expansion of these T-lymphocytes is often associated with tissue damage, as in infants with Omenn syndrome.189 This damage resembles the severe inflammatory lesions resulting from the homeostatic proliferation of a few T-cell clonotypes in mice.190 In other patients, the mutations may underlie milder forms of combined immunodeficiency.159,191 However, the severity of the clinical and immunologic phenotype is not determined solely by the nature of the mutation, because different phenotypes (eg, SCID and Omenn syndrome) have been reported in different individuals harboring the same mutation and even in individuals from the same family.192

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Lymphocyte and lymphocyte subset counts are the mainstay of SCID diagnosis and may provide useful information about the underlying genetic defect.101 The presence of T cells does not exclude a diagnosis of SCID, as the T cells detected may be of maternal origin. However, the maternal T cells have a very limited in vitro proliferative response to mitogens (eg, phytohemagglutinin).186 Infants with SCID are unable to mount specific antibody responses.186 Genetic diagnosis is guided by the NK- and B-cell phenotype and the mode of inheritance. A SCID screening test for neonates has recently become available. This test is based on the polymerase chain reaction–mediated amplification of TCR excision circles (TRECs) from a dried blood spot collected at birth.193,194 TRECs are a byproduct of TCRα rearrangement that persist in newly developed T-lymphocytes expressing the αβ form of the TCR,195 but are diluted out during the subsequent proliferation of cells in the periphery. TREC quantification therefore provides important information about thymic function. Infants with SCID require the regular administration of Igs and antimicrobial prophylaxis. When indicated, blood products should be irradiated before transfusion to prevent transfusional graft-versus-host disease, and should be obtained from cytomegalovirus-negative donors to prevent the risk of transmission. The diagnosis and treatment of infection should be prompt and aggressive. However, in the absence of prophylaxis, SCID is inevitably fatal within a few months, and patients do not survive beyond a few years in the absence of transplantation.185 Indeed, hematopoietic stem cell transplantation (HSCT) is the mainstay of SCID treatments, and optimal results (> 90% survival) are obtained if the donor is an HLA-matched sibling.196,197 If no such donor is available, HSCT can be performed with a matched unrelated donor or with a haploidentical parent as the donor. However, if a haploidentical parent acts as the donor, mature T-lymphocytes must be removed from the graft to prevent graft-versus-host disease. Excellent results (> 95% survival) have been reported for SCID patients treated by HSCT before the age of 3.5 months,197 highlighting the importance of neonatal screening for SCID. HSCT results are less satisfactory for combined immunodeficiency with the residual presence of autologous T-lymphocytes, because there is a risk of graft rejection and of treatmentrelated toxicity, due to the need for chemotherapy for ablation of the immune system of the host.196 In particular, the outcome of HSCT is far from good in patients with major histocompatibility complex (MHC) class II deficiency, because the transplantation of hematopoietic stem cells does not correct the lack of MHC class II antigen expression on thymic epithelial cells.198 Enzyme replacement therapy may lead to detoxification and immune reconstitution in infants with ADA deficiency.199,200 Finally, gene therapy has been successfully used in infants with SCID due to ADA deficiency201 or to a γ c defect.85,202,203 However, 5 of 20 infants with X-linked SCID treated by gene therapy developed leukemic proliferation due to insertional mutagenesis (ie, insertion of the transgene near an oncogene, mostly LMO2, leading to transcriptional activation of the oncogene).204,205

9/18/12 5:01 AM

CHAPTER 48

Thus, studies of patients with SCID have laid the foundations for an understanding of the molecular and cellular mechanisms governing lymphoid development in humans. Identification of the molecular basis of SCID in humans has preceded the generation of gene-targeted mice on a number of occasions. Furthermore, important differences have emerged between the immunologic phenotypes of humans and mice, with mutations in orthologous genes associated with SCID. Finally, the early identification of SCID patients with molecular and immunologic tools has important clinical implications, optimizing survival after HSCT. Despite these advances, the gene responsible for disease remains unidentified in most cases of combined immunodeficiency and a small minority (< 5%) of infants with SCID. The deep sequencing of human exomes and genomes should lead to the identification of still more genes causing T-cell deficiencies in humans.

INBORN ERRORS OF B-CELL DEVELOPMENT OR FUNCTION Antibody deficiencies constitute the largest group of currently recognized PIDs (Table 48.2). 206–208 This is partly because these disorders were among the fi rst to be recognized to cause inborn errors in immunity and because they are among the easiest to detect with routine laboratory studies. Antibody deficiencies, regardless of their etiology, are associated with recurrent or persistent infections with encapsulated bacteria, particularly Streptococcus pneumoniae and Haemophilus influenzae. Patients develop infections typical of these organisms, including otitis, sinusitis, and pneumonia, but they are also likely to experience more severe, invasive infections, such as sepsis, meningitis, joint infections, and cellulitis. Other infections and clinical fi ndings are more specific to particular antibody deficiencies.

X-linked Agammaglobulinemia X-linked agammaglobulinemia (XLA), sometimes called Bruton agammaglobulinemia or congenital agammaglobulinemia, was fi rst described in 1952, when Colonel Bruton reported the case of an 8-year-old boy with recurrent pneumococcal sepsis and no globulin fraction on serum electrophoresis.209 The patient’s clinical course was improved by treatment with exogenous gammaglobulin. Once techniques had been developed for the evaluation of B cells in the peripheral blood, it became clear that patients with XLA had very few or no B cells in the bloodstream.210–213 This is the most consistent feature in patients with XLA. Although 10% to 15% of patients have higher than expected serum Ig concentrations, all patients have < 2% CD19 + lymphocytes in the peripheral blood (normal range 5% to 20% CD19 + cells). Patients are treated by gammaglobulin replacement and the aggressive use of antibiotics. In addition to infections caused S. pneumoniae and H. influenzae, patients with XLA are more vulnerable to enteroviral infections, particularly enteroviral encephalitis and

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vaccine-associated polio.214–218 Mycoplasma and ureaplasma infections are also more common and may cause arthritis, prostatitis, and meningitis, as well as pneumonia.219,220 The incidence of Giardia infection is also high in patients with XLA; some of these patients have protein-losing enteropathy and low serum IgG as the first sign of infection.221 Although significant complications and early death are still sometimes reported for patients with XLA,216 many patients now survive into adulthood.222 In 1993, two groups, using two separate approaches, showed that XLA was due to mutations in a previously unrecognized cytoplasmic tyrosine kinase, now called Bruton tyrosine kinase (BTK).223,224 One group identified the gene on the basis of linkage analysis223 ; the other isolated a cDNA encoding a tyrosine kinase from B-cell precursors.224 This gene mapped to the XLA critical region on the long arm of the X chromosome, making it an excellent candidate gene.224 BTK is an enzyme specific to hematopoietic cells that is expressed throughout B-cell differentiation.225 It is found in the platelets and myeloid cells, but not in T cells or plasma cells.226 However, the clinical effects of BTK deficiency appear to be limited to the B-cell lineage. Severe neutropenia is seen in some patients with XLA, particularly those who are very young.218,227 However, very low neutrophil counts are also seen in patients with defects specific to the B-cell lineage, such as μ heavy chain deficiency.228 This neutropenia may be due to bone marrow suppression by viruses in the absence of natural antibody. BTK has an amino-terminal pleckstrin homology domain, a proline-rich region, and an SH3 domain, followed by an SH2 and a carboxy-terminal kinase domain.224 It is activated and phosphorylated within minutes of pre-BCR or BCR activation.229,230 Activated BTK and PLCγ 2 then bind to the scaffold protein BLNK, allowing BTK to phosphorylate PLCγ 2, resulting in biphasic calcium flux.231 Over 600 different mutations have been identified in the BTK gene,232,233 most of which result in an absence of detectable BTK in monocytes and platelets.234–236 Amino acid substitutions, particularly those that do not affect the stability of the protein, tend to result in a milder phenotype characterized by an older age at diagnosis, higher serum Ig concentrations, and the presence of B cells in small, but detectable numbers.237–239 However, the genotype/phenotype correlation is not strong. Mutations of the BTK gene account for 85% of cases of early onset of infection and isolated defects in B-cell development.240 A similar clinical phenotype is seen in patients with mutations in genes encoding components of the pre-BCR and BCR, including the μ heavy chain,228,241–243 the surrogate light chain protein λ5,244 the signal transduction molecules Igα and Igβ (also called CD79a and CD79b),242,245,246 and the downstream scaffold protein BLNK.243,247 Bone marrow studies have shown B-cell development to be blocked at the pro-B cell to pre-B cell transition, the stage at which the pre-BCR is first expressed, in patients with mutations affecting BTK, the μ heavy chain, λ5, Igα , Igβ, or BLNK. BTK gene mutations cause a leaky defect in B-cell differentiation. Most patients with XLA have a small number of both pre-B cells in the bone marrow and immature

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TABLE

SECTION VIII

48.2

Disease

IMMUNOLOGIC MECHANISMS IN DISEASE

Antibody Deficiencies

Gene Defect

B cells T cells Serum Igs

Agammaglobulinemia with absent or very low B cells ↓↓↓ X-linked BTK N agammaglobulinemia μ heavy chain deficiency IGHM

↓↓↓↓

N

λ5 deficiency

IGLL1

↓↓↓↓

N

Igα deficiency

CD79A

↓↓↓↓

N

Igβ deficiency

CD79B

↓↓↓↓

N

BLNK deficiency

BLNK

↓↓↓↓

N

Inheritance

Pathogenesis

All isotypes decreased

XL

Defective signaling through the BCR

All isotypes decreased All isotypes decreased All isotypes decreased All isotypes decreased All isotypes decreased

AR

Defective signaling through the BCR Defective signaling through the BCR Defective signaling through the BCR Defective signaling through the BCR Defective signaling through the BCR

AR AR AR AR

Hypogammaglobulinemia with normal or low B cells CVID Multifactorial N or ↓ N or ↓ ↓ IgG and IgA; Usually variable IgM may sproradic be normal IgA deficiency

Multifactorial N

N

↓ IgA; normal IgG and IgM

ICOS deficiency

ICOS

N

N

CD19 deficiency

CD19

N

N

CD81 deficiency

CD81

N

N

↓ IgG and IgA; AR IgM may be normal ↓ IgG; ↓ or AR normal gA and IgM ↓ IgG; ↓ or AR normal gA and IgM

CD20 deficiency

MS4A1

N

N

↓ IgG; normal IgA and IgM

AR

TACI

TNFRSF13B

N

N

variable

AD or AR

BAFF-R

TNFRSF13C

↓

N

AR ↓ IgG and IgM; normal IgA

Class switch recombination defects X-linked hyper-IgM CD40LG syndrome

N

N

↓ IgG and IgA; XL IgM may be normal or ↑↑↑

CD40 deficiency

N

N

↓ IgG and IgA; AR IgM may be normal or ↑↑↑

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CD40

Usually sporadic

variable

Associated Features or Comments Detectable serum Igs in some patients

Autoimmune cytopenias; granulomatous disease; lymphoma Sometimes assoicated with IgG subclass deficiency

Defective T-cell activation Poor amplification of May have glomeruBCR signal lonephritis Poor amplification of May have gloBCR signal merulonephritis; coreceptor with CD19 Defective B-cell Poor antibody activation response to T cell–independent antigens Susceptibility gene Defective B-cell activation and not diseasesurvival causing; associated with autoimmunity Defective B-cell activation and survival Neutropenia, Impaired CD40LCryptosporidium mediated infection, biliary costimulation of tract disease, B and dendritic neuroectodercells by activated mal tumors CD4+ cells Neutropenia, Impaired CD40LCryptosporidium mediated infection, biliary costimulation of tract disease, B and dendritic neuroectodercells by activated mal tumors CD4+ cells

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CHAPTER 48

TABLE

48.2

INBORN ERRORS OF IMMUNITY

|

1245

Antibody Deficiencies (Cont.)

AID deficiency

AICDA

N

N

↓ IgG and IgA; AR or AD IgM ↑↑↑

UNG deficiency

UNG

N

N

↓ IgG and IgA; AR IgM ↑↑↑

Defective initiation of somatic hypermutation and class switch recombination Defective somatic hypermutation and class switch recombination

Lymphadenopathy, increased incidence of autoimmune disease Lymphoid hyperplasia

AD, autosomal deficient; AID, activation-induced cytidine deaminase; AR, autosomal recessive; BCR, B-cell receptor; CD, cluster of differentiation; CVID, common variable immunodeficiency; Ig, immunoglobulin; N, normal; UNG, uracil-DNA glycosylase; XL, X-linked.

B cells in the bloodstream.248–250 As in healthy controls, the number of B cells produced decreases with age,251 and most adults with XLA have less than 0.02% B cells in the blood. Mutations in the other genes cause a more severe block in B-cell development. Null mutations in the μ heavy chain, Igα , and Igβ genes result in a complete absence of CD19 + lymphocytes from the blood (< 0.01%). A patient with a hypomorphic mutation in Igβ and young children with mutations in λ5 or BLNK have been reported to have a small number of B cells (< 0.1% CD19 + cells). By contrast, mice with BTK, λ5, or BLNK defects have been reported to have 50%, 20%, or 10%, respectively, the normal number of B cells in the blood.

Hyperimmunoglobulin M Syndromes/Class Switch Recombination Defects Hyper IgM syndrome is characterized by normal or high serum IgM concentrations, with IgG and IgA either present at very low concentrations or undetectable.216,252,253 However, with the identification of the genetic etiologies of the most common forms of hyper-IgM syndrome, it has become clear that not all patients have high IgM concentrations. This has led to the suggestion of “class switch recombination defects” as a more appropriate designation for this group of disorders.253 We will use these terms interchangeably here. Approximately 65% of patients with hyper-IgM syndrome have the XR form of the disease, which is caused by mutations in the gene encoding the tumor necrosis factor (TNF) family member CD40 ligand (CD40L).254–256 These patients tend to have more severe illness than patients with XLA.216,252 The median age at diagnosis is 12 months, and patients generally have little or no detectable IgA and IgG in the serum, frequently accompanied by neutropenia and/ or opportunistic infections. The numbers of T cells and B cells are usually within normal limits. Infections with encapsulated bacteria, Pneumocystis, cytomegalovirus, parvovirus, Cryptosporidium, and Histoplasma are problematic in patients with CD40L deficiency. Sclerosing cholangitis secondary to Cryptosporidium infection252 and neuroendocrine or hepatobiliary carcinomas257,258 have been reported in a significant number of patients. Treatment consists of gammaglobulin replacement, the aggressive use of antibiotics,

Paul_CH48_final.indd 1245

granulocyte-colony stimulating factor (G-CSF) in cases of persistent neutropenia and, in some patients, HSCT.259 CD40L (also called CD154, gp39, TRAP, and TNFSF5) is a type II transmembrane protein transiently expressed on the surface of activated T cells260–262 and platelets. The binding of CD40L to its cognate receptor on B cells, CD40, induces B-cell activation, short- and long-term B-cell proliferation, and in the presence of cytokines, isotype switching260,262–264 through the induction of activation-induced cytosine deaminase (AID) production. By deaminating cytosine residues in VH and switch regions, AID performs the first step in both class switch recombination and somatic hypermutation.265,266 However, CD40 is also expressed on monocytes,267 dendritic cells,268 activated platelets,269 epithelial cells,270 and endothelial cells.271 The stimulation of these target cells via CD40L elicits an inflammatory response, with release of cytokines, including IL-12 in particular.267,272 The failure to elicit this inflammatory response accounts for the viral, fungal, and parasitic diseases seen in patients with CD40L deficiency. Several other genetic disorders resulting in hyper-IgM syndrome have been reported. A small number of patients with AR defects in CD40 have been described.273 These patients have a clinical phenotype identical to that seen in patients with mutations in CD40L. Approximately 10% to 15% of patients with class switch recombination defects have mutations in AICDA, which encodes the B cell–specific protein AID described previously.266,274,275 Defects in UNG, an enzyme responsible for eliminating the uracil molecules generated by AID activity, also account for a small number of cases.276 Patients with mutations affecting AID or UNG do not have the viral, parasitic, and fungal infections or neutropenia seen in patients with CD40L deficiency, but they are more likely to have lymphadenopathy and autoimmune disease.277

Common Variable Immunodeficiency Shortly after Bruton described the first case of agammaglobulinemia in a young boy, other authors reported adult patients, of both sexes, with severe hypogammaglobulinemia.278–280 Many of these patients appeared to have acquired, rather than congenital immunodeficiency, and both clinical and

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IMMUNOLOGIC MECHANISMS IN DISEASE

laboratory findings were highly variable.281,282 This disorder therefore came to be called acquired hypogammaglobulinemia or common variable immunodeficiency (CVID). Hypogammaglobulinemia can be seen in other immunodeficiencies and may result from infections, drug reactions, protein-losing conditions, or cancers, and it is therefore important to rule out these other possibilities before diagnosing CVID.283 Patients of any age may be diagnosed as having CVID, but onset occurs most frequently between the ages of 10 and 40 years.284–289 Respiratory tract infections, diarrhea, and autoimmune disorders are the most common findings on presentation. Laboratory analyses show low serum IgG and IgA concentrations, but IgM concentration may be low or within the normal range. B cells are easily detected in the blood of most but not all patients.290 However, most patients lack CD27+ memory B cells.291,292 About 10% of patients have a family history of immunodeficiency or autoimmune disease.286 Patients with CVID or IgA deficiency are more likely to have certain uncommon MHC haplotypes.293–295 The precise gene within this locus responsible for susceptibility remains unclear.296 Recent genome-wide association studies have suggested that some copy number variants are more common in CVID.297 In the last 10 years, several genetic disorders resulting in clinical signs consistent with CVID have been reported. A small number of CVID patients with homozygous mutations in ICOS have been identified.298,299 Heterozygous and homozygous mutations affecting TAC1, which belongs to the TNF receptor family (also known as TNFRSF13B), have been reported in 10% of patients with CVID and 1% of healthy controls.300–304 CVID patients with TACI alterations are more likely to have autoimmunity and splenomegaly than other patients with CVID.300,304 Homozygous mutations of the gene encoding another TNF receptor family member, BAFF-R, have been described in a brother and sister in whom immunodeficiency was first recognized after the age of 50 years.290 Homozygous mutations of the genes encoding CD19 or another component of the CD19 complex, CD81, have been reported in fewer than 10 patients.305–308 A child with a homozygous mutation of the gene encoding CD20 and a low serum IgG concentration has been reported.309 The clinical and laboratory findings for all the individuals with these mutations are highly variable, and it is not clear that any of these genetic alterations are sufficient to cause clinical symptoms when present on their own. In most patients, a combination of genetic and environmental factors is likely to play a role.

INBORN ERRORS OF PHAGOCYTE DEVELOPMENT OR FUNCTION A number of inborn errors affect the polymorphonuclear or mononuclear phagocytes of the myeloid lineage (Table 48.3). Granulocyte disorders mostly affect neutrophils, whereas mononuclear phagocyte disorders affect monocytes, macrophages, or dendritic cells. Some disorders affect phagocytes and other leukocytes; they may also sometimes affect nonhematopoietic cells. Some of these disorders will be covered

Paul_CH48_final.indd 1246

elsewhere in this chapter. The disorders mostly or exclusively affecting phagocytes have traditionally been grouped together under the umbrella of “phagocyte disorders.” We will not review all these disorders here, as this group is rapidly expanding. Instead, we will focus on two of these disorders that have played an important role in the history of this field, in which considerable progress has been made in recent decades. These two disorders—severe congenital neutropenia (SCN) and chronic granulomatous disease (CGD), first described by Rolf Kostmann, and CGD, first described by Robert Good— elegantly illustrate the quantitative and qualitative defects of phagocytes. Studies of these disorders have also provided substantial immunologic insight into granulocyte function and the respiratory burst of phagocytes. Many other equally fascinating quantitative defects, such as AR IRF8 deficiency, in which circulating monocytes and dendritic cells do not develop, have been described but will not be discussed here.310 Similarly, we will not consider qualitative disorders here, such as specific granule deficiency, which is characterized by a lack of specific granules, making it impossible to distinguish between neutrophil, basophil, and eosinophil granulocytes, and is caused by AR C/EBP-ε deficiency.311 A list of phagocyte disorders is provided in Table 48.3, under the caveat that the classification of these disorders is imperfect. SCN is characterized by absolute neutrophil counts of < 500/μl from early infancy, often falling below 200/ μl, with normal counts of other leukocytes.312–315 Patients with SCN have normal counts of basophil and eosinophil granulocytes. Myeloid maturation in the bone marrow is arrested at the promyelocyte or myelocyte stage of development. Differential diagnoses include complex and often syndromic PIDs with neutropenia, such as warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis syndrome, cartilage hair hypoplasia, CHS, dyskeratosis congenita, Fanconi anemia, reticular dysgenesis, and SchwachmanDiamond syndrome. Several other inborn errors of immunity do not affect the myeloid lineage directly but often result in transient or chronic neutropenia, due to infections or the use of particular drugs. Cyclic neutropenia (CN) is characterized by regular oscillations of the number of peripheral neutrophils, with a nadir at about 200/ μl and a period of about 21 days. SCN is also known as Kostmann syndrome, and CN is sometimes referred to as cyclic hematopoiesis. Both disorders were long absent from PID classifications, principally because patients with diseases of myeloid cells were seen by hematologists, whereas patients with lymphoid diseases were seen by immunologists. CN is marked by fever, oral ulcers, and bacterial infections during the nadir. The prognosis on G-CSF treatment is excellent316 ; these patients do not develop myelodysplasia or acute myeloid leukemia (AML).315 The clinical features of SCN are more severe, with mucocutaneous and deep-seated bacterial and fungal infections. The bacterial infections are caused by various Gramnegative and Gram-positive species, including staphylococci in particular. The fungi responsible for infections are equally diverse and include Candida albicans. Without treatment, the outcome in infancy or early childhood is poor. Before the advent of G-CSF treatment, half the patients died from sepsis in the first year of life, the other half dying during

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48.3 Affected Cells

Cognitive and neurologic defects in some patients Structural heart defects, urogenital abnormalities, and venous angiectasias of trunks and limbs

Myeloid differentiation

Myeloid differentiation, O2- production Adherence, chemotaxis, endocytosis, T/NK cytotoxicity

N N+M+ L + NK

k. Poikiloderma with neutropenia 2. Defects of motility a. LAD type 1

Myeloid differentiation

N

j. Cohen syndrome

N+L Mel

Myeloid differentiation

Endosome biogenesis

N+M

g. X-linked neutropenia*/ myelodysplasia h. P14 deficiency*

N

Mitosis

N

f. Cyclic neutropenia

i. Barth syndrome

Myeloid differentiation, chemotaxis, O2production ?

N+M

e. Glycogen storage disease type 1b

Delayed cord separation, skin ulcers Periodontitis Leukocytosis

Retinopathy, developmental delay, facial dysmorphisms Poikiloderma, MDS

Neutropenia Hypogammaglobulinemia ↓CD8 cytotoxicity Partial albinism Growth failure Cardiomyopathy, growth retardation

Fasting hypoglycemia, lactic acidosis, hyperlipidemia, hepatomegaly Oscillations of other leukocytes and platelets Monocytopenia

B/T lymphopenia

Myeloid differentiation

Myeloid differentiation, chemotaxis, O2production

Subgroup with myelodysplasia

Associated Features

Myeloid differentiation

Affected Function

Inborn Errors of Phagocytes

1. Defects of neutrophil differentiation a. Severe congenital N neutropenia 1 (ELANE deficiency) b. SCN2* (GFI 1 N deficiency) c. SCN3 (Kostmann N disease) d. SCN4 (G6PC3 N+F deficiency)

Disease

TABLE

G6PC3: abolished enzymatic activity of glucose-6phosphatase, aberrant glycosylation, and enhanced apoptosis of N and F G6PT1: Glucose-6-phosphate transporter 1 ELANE: misfolded protein response WAS: Regulator of actin cytoskeleton (loss of autoinhibition) ROBLD3: Endosomal adaptor protein 14

AR

AD

AR

INTGB2: Adhesion protein (CD18)

C16orf57 gene: Pg unknown

AR

AR

Tafazzin (TAZ) gene: Abnormal lipid structure of mitochondrial membrane COH1 gene: Pg unknown

XL

AR

XL

AR

AR

GFI1: loss of repression of ELANE HAX1: control of apoptosis

ELANE: misfolded protein response

Genetic Defect/Presumed Pathogenesis

AD

AD

Inheritance

INBORN ERRORS OF IMMUNITY

(continued)

116920

604173

216550

302060

610389

300299

162800

232220

612541

610738

613107

202700

OMIM Number

CHAPTER 48

| 1247

9/18/12 5:01 AM

Paul_CH48_final.indd 1248

Adherence, chemotaxis

N+M+ L + NK N N+M N

c. LAD type 3

d. Rac 2 deficiency*

e. β-actin deficiency*

f. Localized juvenile periodontitis g. Papillon-Lefèvre syndrome

c. IFNγ receptor 1 deficiency

4. MSMD a. IL-12 and IL-23 receptor β1 chain deficiency b. IL-12p40 deficiency

b–e. Autosomal CGDs

IFNγ secretion IFNγ secretion IFNγ binding and signaling

M M+L

Killing (faulty O2production)

Killing (faulty O2production)

Chemotaxis

Chemotaxis

Formylpeptide-induced chemotaxis Chemotaxis

L + NK

N+M

h. Specific granule N deficiency* i. ShwachmanN Diamond syndrome 3. Defects of respiratory burst a. XL CGD N+M

N+M

Rolling, chemotaxis

N+M

b. LAD type 2*

Susceptibility to Mycobacteria and Salmonella Susceptibility to Mycobacteria and Salmonella Susceptibility to Mycobacteria and Salmonella

McLeod phenotype in patients with deletions extending into the contiguous Kell locus

Periodontitis, palmoplantar hyperkeratosis in some patients Neutrophils with bilobed nuclei Pancytopenia, exocrine pancreatic insufficiency, chondrodysplasia

Mental retardation, short stature Periodontitis only

Poor wound healing, leukocytosis

Mild LAD type 1 features plus hh-blood group plus mental and growth retardation LAD type 1 plus bleeding tendency

Associated Features

AR, AD

AR

AR

AR

XL

AR

AR

AR

AR

AD

AD

AR

AR

Inheritance

IFNGR1: IFNγR ligand binding chain

IL12B : subunit of IL-12/IL-23

IL12RB1: IL-12 and IL-23 receptor β1 chain

CYBA: Electron transport protein (p22phox) NCF1: Adapter protein (p47phox) NCF2: Activating protein (p67phox) NCF4: Activating protein (p40 phox)

CYBB: Electron transport protein (gp91phox)

C/EBPE: Myeloid transcription factor SBDS: Defective ribosome synthesis

CTSC: Cathepsin C activation of serine proteases

FPR1: Chemokine receptor

ACTB: Cytoplasmic Actin

KINDLIN3: Rap1-activation of β1-3 integrins RAC2: Regulation of actin cytoskeleton

FUCT1: GDP-Fucose transporter

Genetic Defect/Presumed Pathogenesis

107470

161561

601604

233690 233700 233710 601488

306400

260400

245480

245000

136537

102630

602049

612840

266265

OMIM Number

SECTION VIII

Adherence, chemotaxis O2 - production Motility

Affected Function

Inborn Errors of Phagocytes (Cont.) Affected Cells

48.3

|

Disease

TABLE

1248 IMMUNOLOGIC MECHANISMS IN DISEASE

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Paul_CH48_final.indd 1249

Mf only

f. Macrophage gp91 phox deficiency* g. IRF8 deficiency (AD form)*

Monocytes peripheral DC + NK + B

Alveolar macrophages

b. GATA2 deficiency (mono MAC syndrome)

c. Pulmonary alveolar proteinosis*

GM-CSF signaling

Multilineage cytopenias

Cytopenias

Susceptibility to Mycobacteria, Candida, myeloproliferation Susceptibility to Mycocbacteria, papillomaviruses, histoplasmosis, alveolar proteinosis, MDS/AML/ CMML Alveolar proteinosis

Susceptibility to Mycobacteria and Salmonella Susceptibility to Mycobacteria, Salmonella Isolated susceptibility to mycobacteria Susceptibility to Mycobacteria

CSF2RA

GATA-2: loss of stem cells

AD

Biallelic mutations in pseudoautosomal gene

IRF8: IL-12 production

CYBB: Electron transport protein (gp 91 phox) IRF8: IL12 production by CD1c+ MDC

STAT1

IFNGR2: IFNγR accessory chain

AR

AD

XL

AD

AR

306250

137295

601565

306400

600555

147569

ACTB, Actin beta; AD, autosomal dominant inheritance; AML, acute myeloid leukemia; AR, autosomal recessive inheritance; B, B lymphocytes; CD, cluster of differentiation; CEBPE, CCAAT/Enhancer-binding protein epsilon; CGD, chronic granulomatous disease; CMML, chronic myelomonocytic leukemia; CTSC, cathepsin C; CYBA, cytochrome b alpha subunit; CYBB, cytochrome b beta subunit; DC, dendritic cell; ELANE elastase neutrophil-expressed; F, fibroblasts; FPR1, formylpeptide receptor 1; FUCT1, fucose transporter 1; GATA2, GATA binding protein 2; GFI1, growth factor independent 1; GM-CSF, granulocyte macrophage-colony stimulating factor; HAX1, HLCS1-associated protein X1; IFN, interferon; IFNGR1, interferon-gamma receptor subunit 1; IFNGR2, interferon-gamma receptor subunit2; IL-12B, interleukin-12 beta subunit; IL-12RB1, interleukin-12 receptor beta 1; IFR8, interferon regulatory factor 8; ITGB2, integrin beta-2; L, lymphocytes; LAD, leukocyte adhesion deficiency; M, monocytes-macrophages; MDC, myeloid dendritic cell; MDS, myelodysplasia; Mel, melanocytes; Mf, macrophages; MSMD, Mendelian susceptibility to mycobacterial disease; N, neutrophils; NCF1, neutrophil cytosolic factor 1; NCF2, neutrophil cytosolic factor 2; NCF4, neutrophil cytosolic factor 4; NK, natural killer; OMIM, Online Mendelian Inheritance in Man; ROBLD3: roadblock domain containing 3; SBDS, Shwachman-Bodian-Diamond syndrome; STAT, signal transducer and activator of transcription; XL, X-linked inheritance. *Ten or fewer unrelated cases reported in the literature.

Monocytes peripheral DC

5. Other defects a. IRF8 deficiency (AR form)*

CD1c+ MDC

IFNγ signaling

M+L

e. STAT1 deficiency (AD form)* Killing (faulty O2production) Differentiation of CD1c+ MDC subgroup

IFNγ signaling

M+L

d. IFNγ receptor 2 deficiency

CHAPTER 48 INBORN ERRORS OF IMMUNITY

| 1249

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IMMUNOLOGIC MECHANISMS IN DISEASE

early childhood. Myelodysplastic syndromes (MDSs) and AML were rare. Antibacterial and antifungal prophylaxis cannot provide long-term protection. HSCT has been successful in some patients. Spectacular progress was achieved in 1989, with the discovery that recombinant G-CSF could restore normal counts of circulating granulocytes, in at least some patients.87 G-CSF treatment should be tailored to the individual patient. Most SCN patients and almost all CN patients respond to G-CSF treatment, although SCN patients require higher doses. It was subsequently shown that a substantial fraction of SCN patients treated with G-CSF developed MDS or AML, these conditions now constituting a more common cause of death in these patients than sepsis.317,318 No such effect was observed in CN patients. The cumulative incidence of MDS/AML is about 20% after 10 years on G-CSF treatment. Somatic mutations of the G-CSF receptor and monosomy 7 often precede MDS/AML. It is unclear whether G-CSF treatment itself favors these complications or whether treatment reveals natural complications of the underlying disorders. Patients who do not respond to G-CSF, or are undergoing malignant transformation, should be treated with HSCT.319–321 Multiple genetic etiologies of SCN have been identified. The first was responsible for autosomal deficient (AD) SCN, whereas more recent genome-wide linkage approaches have discovered causes of AR SCN. Heterozygous mutations of ELA2 are responsible for about 60% of SCN cases and most cases of CN.322–324 Penetrance appears to be complete. In most, but not all cases, the mutations cause either SCN or CN, although both phenotypes have been observed in a few rare kindreds. The mechanism of disease remained unclear for many years, as ELA2 encodes a cytoplasmic neutrophil elastase, a serine protease synthesized at the promyelocyte stage and stored in the primary granules of neutrophils. ELA2-deficient mice are not neutropenic but they do have impaired host defenses.325 By contrast, humans with heterozygous ELA2 mutations display impaired neutrophil granulocyte differentiation, due to high levels of intramedullary apoptosis. These mutations were recently shown to activate unfolded protein responses (UPRs) due to the detection of misfolded ELA2 proteins in the secretory pathway, resulting in cellular apoptosis.326,327 Thus, dominant-negative ELA2 mutations seem to cause SCN or CN not because ELA2 is involved in granulocyte differentiation, but simply because the resulting misfolded proteins, which are selectively expressed in granulocytes, activate the apoptosis-inducing UPR pathway in a “normal” manner. The degree of UPR activation seems to depend on the ELA2 mutation, possibly determining the type of phenotypic expression: CN (mild mutations) or SCN (more severe mutations). Rare cases of SCN are due to heterozygous dominant-negative mutations of the gene encoding GFI1,328 which encodes a transcriptional repressor of myeloid genes. GFI1 knockout mice are severely neutropenic and display an accumlulation of immature monocytes in the blood.329,330 A mouse model of the human mutation also displays specific neutropenia.331 However, the molecular mechanisms by which GFI1 mutations cause AD SCN remain unclear.314 Interestingly, GOF

Paul_CH48_final.indd 1250

mutations in the WASP gene have been shown to cause X-linked dominant SCN.81 The underlying mechanism involves unregulated actin polymerization, leading to defects of mitosis and cytokinesis that result in the apoptosis of granulocyte precursors.332 Some of the patients with these mutations have platelet and lymphocyte abnormalities. The original kindred described by Kostmann displayed AR inheritance.11,14,333 The underlying genetic defect was not identified until 2007, when causal mutations were discovered in the HAX1 gene, which encodes a mitochondrial protein.334 This is the most common genetic etiology of AR SCN. The mutations concerned are LOF and cause the mitochondrial membrane potential to dissipate, leading to the release of proapoptotic molecules into the cytoplasm, potentially accounting for SCN pathogenesis.335 However, the mechanism underlying SCN in HAX1-deficient patients remains unclear.314 Apoptosis rates are high in the neurons and lymphocytes of HAX1-deficient mice.336 HAX1-deficient patients from the kindred originally studied by Kostmann also displayed some neurologic signs.333 Moreover, HAX1 mutations affecting only isoform A were found to underlie SCN, whereas mutations affecting isoforms A and B (lacking part of exon 2 and expressed in neurons) were found to cause both SCN and various forms of neurologic impairment.337,338 Another genetic etiology of AR SCN has been identified in a few kindreds with mutations of the G6PC3 gene, encoding a member of the endoplasmic reticulum-resident glucose-6phosphatase family.339 As in patients with ELA2 mutations, these patients display signs of endoplasmic reticulum stress and UPR pathway activation, possibly due to impairment of the glycosylation of proteins transported in the secretory pathway. The knockout mice also display neutropenia.340 As this enzyme is ubiquitous, the other clinical features recorded in patients, including developmental defects of the heart or urogenital tract, are not particularly surprising. Overall, what have we learned from these experiments of nature? A number of genes cause CN and SCN, and there are probably multiple mechanisms of disease, converging on the apoptosis of myeloid precursors of neutrophil granulocytes. These studies have identified key players in neutrophil differentiation, in some cases even before the description of their impact in mice (HAX1), which differs for at least the two most common genetic etiologies of SCN (HAX1- and ELA2-deficient mice do not display neutropenia). These studies also identified the first gene for which LOF and GOF mutations underlie two different immunologic disorders: XR WAS and X-linked dominant SCN. These investigations are not yet complete, not only because the pathogenesis of SCN remains unclear for several genetic defects (HAX1, G6PC3), but also because no genetic etiology has yet been discovered for some patients with SCN, suggesting that new morbid genes remain to be discovered. CGD has also played an important historical role, as it was the first functional defect of phagocytes to be identified. It is also the most common and best characterized functional defect of phagocytes. It was first described clinically in individual patients in the late 1950s,341–344 and the first series of affected boys was studied in 1965.344 The phagocyte

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CHAPTER 48

phenotype was demonstrated in 1966, in a study showing the impaired killing of staphylococci by granulocytes,17,345 in which the terms “CGD” and “inborn error” were first used. Granulocytes from CGD patients were found to kill streptococci normally, these bacteria posing no particular threat to these patients.346 Shortly thereafter, in 1967, a lack of respiratory burst in the granulocytes of patients was reported.347,348 The concomitant identification of a respiratory burst phenotype in the mothers of affected boys confi rmed that most known cases resulted from XR inheritance.349 The nitroblue tetrazolium reduction assay was soon developed,350 and an AR form of CGD was discovered.350,351 A deficiency of phagocyte nicotinamide adenine dinucleotide phosphate activity was then demonstrated.352 The first review on CGD, an insightful and comprehensive paper, was published by Good et al. in 1968.353 These early studies led to the demonstration that patients lacked a particular enzyme, phagocytic nicotinamide adenine dinucleotide phosphate oxidase (abbreviated to Phox). This discovery preceded the identification of the underlying genetic lesions, as reviewed in detail elsewhere.354–359 Phox has five components, including membrane-bound gp91 and p22, which form the flavocytochrome b558. The other three components are cytosolic, and the complex forms after phagocyte activation, as occurs during phagocytosis. This complex is more potent in granulocytes than in other phagocytes. Upon formation of the Phox complex, reactive oxygen species, including superoxide and hydrogen peroxide (the levels of which can be assessed in various assays, including the nitroblue tetrazolium reduction assay) are released into the phagosomes, where they directly and indirectly contribute to the killing of ingested microbes. The patients have an abolished (CGD) or impaired (variant CGD) granulocyte respiratory burst. Mutations in any of the five components of Phox can be found in patients with XR or AR CGD. Mutations of the X-linked CYBB (encoding gp91, two-thirds of patients), CYBA (encoding p22), NCF1 (encoding p47, the most common AR form), and NCF2 (encoding p67) genes were identified in the 1980s, and it was not until 2010 that a patient with p40 deficiency was discovered.360 The CYBB gene, encoding one of the two membrane-bound components, was the first human morbid gene to be identified, based on its chromosomal location.361 Also interesting from a genetic standpoint, most, if not all mutations of NCF1 result from a rare gene conversion event between the gene and a nearby homologous pseudogene.362 CGD affects about 1/100,000 individuals. Typically, it is symptomatic in early childhood, whereas variant CGD may manifest later in life.363–366 Affected children display bacterial and fungal infections, but with different frequencies of the causal microbes. Most, but not all pathogens causing infections in CGD patients produce catalase, which was long thought to be an essential virulence factor in this setting.367 Some bacteria, such as Staphylococcus and Serratia, are major threats, whereas closely related bacteria, such as Streptococcus and Klebsiella, which pose a threat to many other PID patients, are almost completely innocuous in CGD patients. Aspergillus species pose a major threat to

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CGD patients, whereas other fungi, such as Candida, are rarely pathogenic in these patients. The defect in host defense is profound, as illustrated by the occasional detection of very weakly virulent microbes that appear to pose a threat only to CGD patients.368 Patients with AR CGD have a residual respiratory burst, albeit weaker than that of variant CGD patients, and this may account for their somewhat milder clinical phenotype and better prognosis.369 Interestingly, an impaired respiratory burst has been demonstrated in the macrophages and B cells, but not in the monocytes and granulocytes, of two kindreds with unique missense mutations in CYBB.370 These two kindreds display a pure Mendelian susceptibility to mycobacterial disease (MSMD) phenotype, consistent with the susceptibility of CGD patients to Bacillus Calmette-Guérin and M. tuberculosis.371 The results obtained for these two kindreds suggest that the respiratory burst in macrophages is essential for protective immunity against tuberculous mycobacteria. They also suggest that the respiratory burst in other phagocytes contributes to other manifestations of CGD, whether infectious or otherwise. Indeed, CGD patients also have inflammatory lesions, consisting of granulomas, that can affect almost all tissues and organs. The treatment of these lesions with steroids may precipitate the development of new infections. The prevention of infection in CGD patients has gradually been improved, initially by the use of oral trimetoprim-metronidazole, then by the subcutaneous administration of IFNγ,372 and finally by the addition of oral itraconazole to the treatment regimen.373 Most patients now survive until adulthood, provided that inflammatory and infectious complications are treated aggressively. The course of the disease is heterogeneous in CGD patients. Some patients do very well and survive without infection throughout adulthood, thanks to antibacterial and antifungal prophylaxis. Others experience invasive fungal disease and chronic inflammation, which may require transfusions of leukocytes and steroids, respectively, or, in some cases, HSCT. The success of HSCT has raised questions as to whether this procedure should be proposed to a larger number of patients early in life.359 Gene therapy trials were initiated but were stopped early due to insertional mutagenesis precipitating myeloid transformation in some cases.366,374 In any case, the investigation of CGD as an experiment of nature has resulted in the dissection of a fundamental mechanism by which phagocytes control microbes. The respiratory burst has been dissected in human phagocytes, largely through studies of patients with CGD. Mice lacking one or other of the components of Phox were engineered in the 1990s and their biochemical and infectious phenotypes matched those of human patients. There is little doubt that other functional defects of phagocytes will be discovered in the near future. Indeed, CGD patients are normally resistant to a large number of microbes, implying that other microbicidal pathways also operate in phagocytes.

SYNDROMIC IMMUNODEFICIENCIES There are several PIDs in which physical or laboratory findings unrelated to the greater susceptibility to infection are

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IMMUNOLOGIC MECHANISMS IN DISEASE

a prominent part of the clinical picture (Table 48.4).Some of these disorders are caused by mutations in genes that are broadly expressed and others are caused by defects affecting hematopoietic cells alone but involving processes not specific to immune defense. Interestingly, defects in the same signal transduction pathway or biologic process may cause different clinical manifestations.

Wiskott-Aldrich Syndrome and Defects in Cytoskeletal Organization The first cases of WAS were reported in 1937 by Wiskott, a German physician who described three brothers with bloody diarrhea, draining otitis, and severe eczema.21 In 1954, Aldrich et al. described a family in which 17 affected males had symptoms similar to those of the patients reported by Wiskott, with an XR pattern of inheritance.20 All the patients reported by both Wiskott and Aldrich died before the age of 3 years. Over time, it became clear that a low platelet number (thrombocytopenia) and unusually small platelets were the most frequent findings in WAS.375,376 The immune deficiency consists of recurrent infections, low serum IgM and high serum IgA and IgE concentrations, and impaired antibody production in response to carbohydrate antigens, particularly pneumococcal polysaccharides. Eczema is the least consistent component of the disease. Autoimmune disease

TABLE

48.4

and malignancies, such as EBV-related B-cell lymphoma in particular, are also more common in patients with WAS.377,378 In 1994, the gene responsible for WAS, a previously unknown gene, was identified by positional cloning. This gene encodes a hematopoietic cell–specific cytoplasmic protein, WASP, that acts as a scaffold, promoting rearrangement of the actin cytoskeleton in response to cell activation and assembly of the proteins involved in signal transduction.375,379,380 The carboxy-terminal VCA domain (verprolin homology, cofi lin homology, acidic region) is the most strongly conserved domain in protein family members. Other recognized domains of WASP include the amino-terminal WH1 domain, which is followed by the GTPase-binding domain (GBD) and a proline-rich region proximal to the VCA domain. In resting cells, WASP is in an autoinhibited conformation in which the VCA domain binds to a hydrophobic pocket within the GBD.381,382 When a GTPase, generally CDC42, binds to the GDB domain, the VCA domain is released and WASP forms a dimer in which a pair of VCA domains bind the actin-related proteins 2 and 3 complex.380 The actin-related proteins 2/3 complex recruits monomeric actin and stimulates the assembly of branched actin fi laments.383 WASP forms part of the immune synapse in activated T cells, B cells, and dendritic cells.384–388 It participates in the recruitment of downstream signaling molecules and cell

Syndromic Immunodeficiencies Gene Defect

B Cells

T Cells

Serum Igs

Inheritance

Pathogenesis

Wiskott-Aldrich syndrome

WASP

Progressive ↓

N

↓ IgM, ↑ IgA and IgE, IgG variable

XL

Defective cytoskeletal response to activation

AT

ATM

May be ↓

May be ↓

↓ IgA and IgE; IgG and IgM variable

AR

AT-like disease

MRE11

May be ↓

May be ↓

Variable

AR

Nijmegen breakage syndrome

NBS1

May be ↓

May be ↓

Variable

AR

Nijmegen breakage-like disease

RAD50

↓↓↓↓

N

All isotypes decreased

AR

Hyper-IgE syndrome

STAT3

N

N

Elevated IgE

AD

Defective phosphorylation of targets required for DNA repair Defective repair of DNA double-strand breaks Defective repair of DNA double-strand breaks Defective repair of DNA double-strand breaks Impaired response to cytokine and activation signals

Disease

Associated Features or Comments Thrombocytopenia with small platelets; eczema; autoimmunity; lymphoma Ataxia; telangiectasia; malignancy; progeria

Microcephaly; bird-like facies; growth failure Microcephaly; bird-like facies; growth failure Eczema; staphylococcal abscesses; delayed shedding of primary teeth

AD, autosomal dominant; AR, autosomal recessive; AT, ataxia telangiectasia; DNA, deoxyribonuclec aicd; Ig, immunoglobulin; N, normal.

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motility. In the absence of WASP, T-cell proliferation, differentiation, and survival are impaired, and the number of T cells tends to decrease with age.389 WASP is also important for maintenance of the function and normal numbers of circulating invariant NKT cells and regulatory T cells.390–393 Lymphocytes with normal WASP expression have a strong selective advantage in proliferation and survival over WASP-negative cells. This has been demonstrated in heterozygous carriers of a WASP mutation (the mothers of affected boys),394,395 in chimeric mice treated with a mixture of wildtype and WASP-deficient bone marrow,396 and in patients with WAS with a second mutation of the WASP gene.397–400 In as many as 11% of patients with WAS, there is evidence of either a reversion of the primary mutation to the wild-type sequence, or of a second mutation compensating for the disease-causing mutation in at least some cells.400,401 Gene reversion is seen most frequently in T cells, but it has been detected in NK cells and B cells. The first example of somatic gene reversion improving the clinical course of an inherited disorder was documented in 1994 for another PID, ADA deficiency.402,403 Such reversion now appears to be less rare than initially thought, at least in WAS patients. A wide diversity of WASP mutations has been observed in affected patients, with some genotype/phenotype correlation.404 However, this correlation is not strong enough to predict clinical outcome. Amino acid substitutions and some splice variants are more likely to be associated with mild disease, particularly if some WASP protein is detectable on western blots. A subset of patients with mutations in WASP have thrombocytopenia or even intermittent thrombocytopenia as the only manifestation of their disease.405,406 These patients usually display alterations to the WHI domain. The WHI domain binds a protein called the WASP-interacting protein (WIP), which stabilizes WASP and contributes to actin polymerization.407,408 A nonsense mutation in WIP, introducing a premature stop codon, has been identified in a single patient with no WASP protein in monocytes and a clinical phenotype very similar to that of patients with WAS.409 Activating mutations in WASP have been reported in a small number of patients with severe congenital neutropenia but not thrombocytopenia.81,410,411 Additional clinical manifestations vary in the 17 reported patients: some displayed low levels of proliferation in response to CD3 cross-linking, some had small numbers of monocytes, and one patient had large platelets. Several had low CD4 + T-cell counts. The activating mutations result in amino acid substitutions in the GBD of WASP, preventing the autoinhibitory binding of the VCA domain to the GBD and resulting in the disorganization of actin polymerization.332 These mutations led to the first description of two unrelated phenotypes caused by GOF and LOF mutations in a gene responsible for immunodeficiency. Another two immunodeficiencies resulting in abnormal regulation of the actin cytoskeleton have recently been described. DOCK8 is a GTP exchange factor that binds to CDC42. Homozygous or compound heterozygous mutations in the gene encoding this protein result in a syndrome with some similarities to both hyper-IgE syndrome (HIES)

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and WAS.412,413 Patients with DOCK8 deficiency have low serum IgM and markedly high IgE concentrations, like patients with WAS. However, one of the most striking characteristics of their immunodeficiency is severe viral infections of the skin caused by herpes simplex virus (HSV), varicella virus, molluscum contagiosum virus, and papillomaviruses. The incidence of cancer is also higher than normal in these patients. Thrombocytopenia is not reported in this syndrome. Heterozygous dominant-negative mutations of Rac2, which encodes a GTPase similar to CDC42, have been reported in three patients with an early onset of severe bacterial infections, high neutrophil counts, impaired respiratory burst, and poor chemotaxis.414–416 One of these patients was identified during newborn screening for SCID based on low levels of T cell.416

Ataxia Telangiectasia and Defects in the Deoxyribonucleic Double-Strand Break Response An unusual combination of ocular telangiectasia and cerebellar ataxia was first described in French medical journals in 1926 by Syllaba and Henner19 and then in 1941 by Louis-Bar.18 In the mid-1950s, Broder and Sedgwick named this disorder ataxia telangiectasia (AT) and noted the high incidence of pulmonary infections in affected patients.24 AT is a complex, highly variable, progressive disorder characterized by the onset of an unsteady gait after the first year of life, telangiectasia first noted when the child is 2 to 8 years old, unusual eye movements, drooling, recurrent respiratory infections, premature aging, infertility, an affable personality, and a high risk of cancer.25 Patients are usually wheelchair-bound by adolescence and often die from cancer or pulmonary disease before the age of 20 years.417 Lymphocytes from patients with AT are particularly susceptible to radiation damage and display frequent translocations involving chromosomes 7 and 14, involving the sites encoding the antigen receptor genes.418–420 AT is caused by homozygous or compound heterozygous mutations of ATM (ataxia telangiectasia mutated),421,422 a large gene consisting of 66 exons. Most of the mutations in patients with classical AT are premature stop codons or frameshift mutations resulting in a null phenotype.423 The protein encoded by ATM consists of 3056 amino acids, the 350 most carboxy-terminal residues displaying sequence similarities to the catalytic domain of phosphatidylinositol 3-kinases. However, ATM functions as a serine/threonine kinase. The amino-terminal end of ATM binds to several substrates, including BRACA1 and p53.424 ATM is predominantly a nuclear protein that responds rapidly to doublestrand DNA breaks and oxidative stress.425,426 In response to double-strand DNA breaks, a complex consisting of Mre11, Rad50, and Nbs1 (MRN complex) binds to DNA ends and recruits ATM.427 ATM is then able to phosphorylate Rad50, which acts as a scaffold for downstream targets. ATM also phosphorylates a wide array of target proteins involved in DNA double-strand break repair, cell cycle control, and stress responses.425 ATM is also activated, in a pathway independent of the MRN complex, by high levels of reactive oxygen species production.426

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IMMUNOLOGIC MECHANISMS IN DISEASE

Mutations of the genes encoding each of the components of the MRN complex are associated with clinical disorders with some, but not all, of the features of AT. Homozygous mutations in Nbs1 (also called nibrin) cause Nijmegen breakage syndrome,427–429 a disorder associated with profound microcephaly, chromosomal breakage, and a high risk of cancer, but not ataxia or telangiectasia.430 A syndrome that has been referred to as AT-like disorder is caused by hypomorphic mutations of the Mre11 gene.431–433 Affected patients have ataxia and some have microcephaly, but no telangiectasia is observed. A single patient has been reported with microcephaly and hypomorphic mutations of the Rad50 gene.434 All three disorders are associated with abnormally high sensitivity to ionizing radiation.

Hyperimmunoglobulin E Syndrome Autosomal dominant HIES, also called Job syndrome, is a multisystem disorder characterized by eczema-like skin rashes, recurrent skin and pulmonary abscesses, pneumonia, hyperextensible joints, a high incidence of bone fractures and scoliosis, late shedding of primary teeth, and markedly high serum concentrations of IgE with eosinophilia.435–438 Heterozygous dominant-negative mutations of the gene encoding the (signal transducer and activator of transcription) STAT3 are responsible for this disorder.439,440 Like other members of the STAT family, STAT3 is activated by various cytokines and growth factors using the JAK-STAT pathway. The receptors containing the widely expressed ligand binding chain gp130, including receptors for IL-6, IL-11, IL-27, OSM, CNTF, and cardiotrophin-1 all activate STAT3. These receptors activate STAT3, which forms dimers that enter the nucleus and enhance the transcription of genes encoding mediators of inflammation and immunity.441 Paradoxically, STAT3 may have both pro- and anti-inflammatory effects, depending on the cell type and activating signal.442 In mice, homozygous null mutations of STAT3 are lethal early in embryogenesis.443 In patients with autosomal dominant HIES, the mutations of STAT3 cluster in the regions of the gene encoding the SH2 and DNA-binding domains.444 Homozygous mutations of the gene encoding the JAK Tyk2 may also result in an immunodeficiency characterized by rashes, unusual susceptibility to viral and fungal infections, and high serum IgE concentration.445 However, the phenotype of patients with Tyk2 mutations is variable, and one patient was found to be particularly susceptible to mycobacteria and viruses but to have none of the cardinal features of HIES: high serum IgE concentration, rashes, and staphylococcal disease.446 As noted previously, mutations in DOCK8 also result in high serum IgE concentration and susceptibility to infections.

INBORN ERRORS OF IMMUNITY TO A NARROW RANGE OF INFECTIONS PIDs were originally defi ned by the identification of immunologic phenotypes. As the first immunologic abnormalities detected were profound, such as neutropenia and agammaglobulinemia, PIDs were, unsurprisingly, initially

Paul_CH48_final.indd 1254

associated with multiple, recurrent, and often opportunistic infections. The first exception to this rule was provided by the description of XR lymphoproliferative disease (XLP), which manifests as various EBV-driven clinical phenotypes in previously healthy individuals (Table 48.5). The clinical features of this disease, including hemophagocytosis, lymphoma, and agammaglobulinemia, were described between 1974 and 1976, and all were attributed to an XR inheritance of susceptibility to EBV in a large kindred.447–450 The variety of phenotypes triggered by EBV was confirmed by studies of additional kindreds.451 The morbid gene, SAP, was identified in 1998 and shown to be specifically expressed in T cells.452,453 A related XR disorder associated principally with hemophagocytosis was found to be due to mutations in XIAP.72,454,455 The pathogenesis of XLP was fully deciphered only recently, with the discovery that SAP-deficient cytotoxic T cells from healthy female carriers could not respond to EBV-infected B cells.456 Moreover, somatic reversion of SAP mutations in cytotoxic CD8 + T cells was documented in XLP patients with the mildest forms of disease.456a Other well-known examples of narrow vulnerability to infection are provided by AR deficiencies of any component of the complement membrane attack complex (C5 to C9), and XR deficiencies of properdin and factor H. Patients with these conditions display selective susceptibility to recurrent, invasive Neisseria infections. The fi rst AR deficiencies of C6, C7, and C8 were reported in 1974 to 1976,457–461 whereas the first XR properdin deficiency was not reported until 1982,462 and AR deficiencies of factors I463 and D464 were reported even later. Properdin and factors I and D act by stabilizing the alternative pathway. Remarkably, alternative and terminal complement defects are almost exclusively associated with gonorrheal and meningococcal diseases.71,465,466 A third example, although it should really be considered the first in terms of chronology, is provided by epidermodysplasia verruciformis (EV), a rare, lifelong disorder characterized by an abnormal and selective susceptibility to disseminated and persistent warts following the infection of keratinocytes with weakly virulent skin-tropic human β papillomaviruses.73 EV is also associated with an increase in the risk of nonmelanoma skin carcinomas, with no other clinical signs in most patients.467 The viral etiology of EV was demonstrated between 1946 and 1959, and specific human papillomavirus genotypes were implicated in this disease in 1978.23,468 EV-causing human papillomaviruses are now known to cause asymptomatic infections in the general population. Cockayne first suggested in 1933 that EV could be inherited as an AR condition.22 EV may therefore be seen as one of the fi rst PIDs described, although the lack of a detectable immunologic phenotype long prevented this disease from being considered as such. The fi rst genetic etiologies of EV were identified in 2002, with the identification of mutations in EVER1 (TMC6) and EVER2 (TMC8).28 Both EVER1 and EVER2 are strongly expressed in circulating lymphocytes, including CD4 + and CD8 + T cells, B cells, and NK cells. These two genes are also expressed in keratinocytes, in which the EVER proteins form a complex with the zinc transporter ZnT1. Inactivating

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TIR-MyD88 signaling pathway Increased response of the CXCR4 chemokine receptor to its ligand CXCL12 (SDF-1)

TLR3-dependent IFNα, -β, and -l induction UNC-93B-dependent

Lymphocytes + monocytes Granulocytes + lymphocytes

3. MyD88 deficiency

4. Warts, hypogammaglobulinemia, infections, myelokathexis syndrome 5. Epidermodysplasia verruciformis 6. HSE* a. TLR3 deficiency*

Trypanosomiasis

CMC CMC

CMC

Invasive candidiasis and peripheral dermatophytosis

AD

AD AD

AR

AR

AD

AR

AD

AR

AD

AR

Mutation in APOL-I

Mutation in IL17F Mutations in STAT1

Mutation in IL17RA

Mutations of CARD9

Mutation of TRAF3

Mutations of UNC93B1

Mutations of TLR3

Mutations of EVER1, EVER2

Gain-of-function mutation of IKBA, resulting in impaired activation of NF-kB Mutation of IRAK4, a component of TLR- and IL-1Rsignaling pathway Mutation of MYD88, a component of the TLR and IL-1R signaling pathway GOF mutations of CXCR4, the receptor for CXCL12

Mutations of NEMO (IKBKG), a modulator of NF-kB activation

Genetic Defect/Presumed Pathogenesis

606496 Not in OMIM yet 603743

605461

212050

610551

613002

226400

193670

612260

607676

612132

300291, 300584, 300301

OMIM Number

|

AD, autosomal dominant inheritance; AR, autosomal recessive inheritance; CMC, chronic mucocutaneous candidiasis; EDA-ID, anhidrotic ectodermal dysplasia with immunodeficiency; GOF, gain-of-function; HPV, human papilloma virus; HSE, HSV-1 encephalitis; IFN, interferon; IL, interleukin; OMIM, Online Mendelian Inheritance in Man; STAT, signal transducer and activator of transcription; TIR, toll and interleukin-1 receptor; TLR, Toll-like receptor; XL, X-linked inheritance. *Ten or fewer unrelated cases reported in the literature.

IL-17F–containing dimers GOF STAT1 mutations that impair the development of IL-17–producing T cells APOL-I

IL-17RA signaling pathway

IFNα, -β, and -l induction CARD9 signaling pathway

HSE

HSE

Bacterial infections (pyogens)

AR

AD

XL

Inheritance

INBORN ERRORS OF IMMUNITY

9. Trypanosomiasis*

b. IL-17F deficiency* c. STAT1 GOF

Epithelial cells, fibroblasts, mononuclear phagocytes T cells T cells

Mononuclear phagocytes

CNS resident cells and fibroblasts

c. TRAF3 deficiency

7. Predisposition to fungal diseases* 8. CMC a. IL-17RA deficiency*

CNS resident cells and fibroblasts

b. UNC93B1 deficiency

CNS resident cells and fibroblasts

IFNα, -β, and -l induction TRAF3-dependent

HSE

TIR-IRAK signaling pathway

Lymphocytes + monocytes

2. IRAK4 deficiency

Keratinocytes + leukocytes

Hypogammaglobulinemia, reduced B-cell numbers, severe reduction of neutrophil count, warts/ HPV infection HPV (group B1) infections and cancer of the skin

NF-κB signaling pathway

Lymphocytes + monocytes

b. EDA-ID, autosomal-dominant*

Anhidrotic ectodermal dysplasia + specific antibody deficiency (lack of Ab response to polysaccharides) + various infections (mycobacteria and pyogens) Anhidrotic ectodermal dysplasia + T-cell defect + various infections Bacterial infections (pyogens)

NF-κB signaling pathway

Lymphocytes + monocytes

Associated Features

1. Anhidrotic EDA-ID a. EDA-ID, XL (NEMO deficiency)

Functional Defect

Inborn Errors of Immunity Against a Single Infection Affected Cell

48.5

Disease

TABLE

CHAPTER 48

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mutations in EVER1 or EVER2 affect intracellular zinc distribution and the activity of transcription factors stimulated by zinc.469 The remaining 25% or so of patients with EV and no demonstrated mutations in EVER1 or EVER2 include patients from families displaying AR,470 XR,471 and AD inheritance.472 A third genetic etiology, with mutations in RHOH underlying a specific T-cell deficiency, was recently discovered, suggesting that the pathogenesis of EV in EVER-deficient patients might involve T cells.472a These pioneering studies together set the stage for the discovery of new inborn errors of immunity underlying life-threatening infections in previously and otherwise healthy children. The idea that PIDs may underlie infectious diseases in patients normally resistant to most other microbes gained ground from the mid-1990s onwards, with the identification of AR IFN-γR1 deficiency as the fi rst genetic basis of MSMD (Fig. 48.2).473,474 Mutations in two X-linked (NEMO, CYBB) and six autosomal (IFNGR1, IFNGR2, IL12B, IL12RB1, STAT1, IRF8) genes have since been discovered.74,310,370,475 MSMD was first described in the 1950s, as unexplained infections caused by the Bacillus CalmetteGuérin vaccine.476–478 The high levels of locus and allelic heterogeneity have resulted in the defi nition of 15 different disorders, accounting for only about half the known cases.

These defects are physiologically related, as they all result in an impairment of IFNγ immunity. Mutations affecting IFNGR1, IFNGR2, and STAT1 impair cellular responses to IFNγ. Mutations affecting IL12B and IL12RB1 impair the IL-12–dependent induction of IFNγ, accounting for MSMD, and the IL-23–dependent induction of IL-17, accounting for the mild CMC documented in some of these patients.479,480 MSMD-causing mutations in NEMO impair the T cell– and CD40L-dependent induction of IL-12 by dendritic cells.481 MSMD-causing mutations in CYBB impair the respiratory burst in macrophages.370 In both these cases, subtle mutations in genes for which null alleles are known to cause more complex PIDs—anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) and CGD, respectively—have highlighted the effects of specific alleles on a specific signaling pathway (NEMO) or a specific cell (CYBB).482 Heterozygous mutations in the IRF8 gene prevent the development of IL-12–producing CD1c + CD11c + dendritic cells.310 Patients with MSMD display clinical disease caused by weakly virulent mycobacteria, such as Bacillus Calmette-Guérin vaccines and environmental mycobacteria. They also often suffer from nontyphoidal, extraintestinal salmonellosis. They are highly prone to tuberculosis upon exposure to M. tuberculosis. AR IL-12Rβ1

Mycobacteria IL-12Rβ1

p19 IL-23

TYK2

IL-23R

JAK2

p40 IL-12Rβ1

IL-12 gp91phox

p35 IL-12Rβ2

TYK2 JAK2

CD40

NEMO IRF1

CD40L

IRF8 JAK1 JAK2

IFN-γR1 IFN-γR2

IFN-γ

STAT-1 (GAF)

1 JAK 2 JAK IFN-γ

Dendritic cells/Phagocytes

IFN-γR

T Lymphocytes/NK cells

FIG 48.2. Inborn Errors in the Interleukin (IL)-12/23-Interferon (IFN)γ Pathway Underlie Mendelian Susceptibility to Mycobacterial Diseases (MSMD). Schematic diagram of cytokine production and cooperation between phagocytes/dendritic myeloid cells and natural killer/T lymphocytes. The IL-12/IFNγ circuit, the CD40/CD40L pathway, and the oxidative burst (mediated in part by CYBBencoded gp91, a component of the nicotinamide adenine dinucleotide phosphate phagocyte oxydase) are crucial for protective immunity against mycobacterial infection in humans. Mutations in IFNGR1 or IFNGR2, encoding the ligand-binding and associated chains of the IFNγR, impair cellular responses to IFNγ. Likewise, heterozygous dominant-negative mutations in STAT1 impair IFNγ but not IFNα/β responses. Mutations in IL-12p40 or IL-12Rb1 impair IL-12–dependent induction of IFNγ. Mutations in CYBB that selectively impair the respiratory burst in monocyte-derived macrophages are associated with MSMD. Heterozygous dominant-negative mutations in IRF8 impair the development of IL-12–producing CD1cCD11c dendritic cells. Proteins for which mutations in the corresponding genes have been identified and associated with MSMD are shown in red. The allelic heterogeneity is described in Table 48.1.

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CHAPTER 48

deficiency was the first genetic etiology of the severe forms of pediatric tuberculosis to be identified.483–486 Such identification of the defects underlying MSMD has been very beneficial to patients. Poor producers of IFNγ, due to IL-12 deficiency, for example, benefit from preventive or curative treatment with recombinant IFNγ. Conversely, patients whose cells do not respond to IFNγ, such as those with complete IFNγR1 deficiency, should undergo HSCT. These studies have also had important immunologic implications. These experiments of nature have shown that IFNγ is essential for immunity against mycobacteria and a few other intracellular bacteria, fungi, and parasites that infect macrophages. Surprisingly, however, these patients are not prone to infections with other intracellular agents, including most viruses, in particular. The rare viral illnesses occurring in these patients may have been favored by mycobacterially induced immunosuppression. IFNγ plays a more important role as a macrophage-activating factor than as an antiviral molecule.487 The “Th1” arm of immunity, if such a lineage exists in humans, is not as broadly potent in host defense as reported for the mouse model. The dissection of MSMD has, therefore, revealed the narrow role of the “Th1” branch of T helper cell immunity. These studies have also suggested that mycobacterial diseases in other medical settings may result from the impairment of IFNγ immunity. Finally, these studies lend weight to the idea that otherwise healthy children with other infectious diseases may suffer from single-gene inborn errors of immunity.70 No germline mutation affecting the “Th2” arm of immunity has yet been identified, but mutations impairing the recently described “Th17” arm of immunity have been identified during investigations of CMC disease (CMCD), which was clinically described in the late 1960s and shown to display AR or AD inheritance in the early 1970s.488–490 CMC is characterized by persistent or recurrent infections of the nails, skin, and oral and genital mucosae with the fungus C. albicans. It is common in patients with various inherited or acquired T-cell deficits that are also associated with other infections.491 In patients with AD HIES caused by dominant-negative STAT3 mutations, the two principal infectious threats are CMC and staphylococcal diseases.65,492 These patients have a small proportion of IL-17–producing T cells.479,493,494 However, this does not formally demonstrate a role for impaired IL-17 immunity in the CMC observed in these patients, as this immunologic phenotype may be silent or even be responsible for other phenotypes, infectious or otherwise. Interestingly, however, patients with autoimmune polyendocrinopathy type I suffer from a single infectious disease, CMC, which is accompanied by numerous autoimmune signs.495 They have high titers of neutralizing auto-Abs against IL-17 cytokines.496,497 Again, this observation alone does not prove that impaired IL-17 immunity underlies CMC in such patients, as the autoAbs may be clinically silent or may attenuate autoimmune signs. However, these clinical observations, together with the results of studies in mice, suggest that impaired IL-17 immunity may generally underlie CMC.491 These studies paved the way for the discovery of patients with isolated

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CMC (CMCD) due to inborn errors of IL-17F or IL-17RA immunity.76 These patients were otherwise healthy and normally resistant to other infections, with the exception of a few cutaneous staphylococcal infections. One patient displayed AR complete IL-17RA deficiency, whereas patients from another kindred displayed AD partial IL-17F deficiency. These fi ndings provided the fi rst genetic etiologies of CMCD and suggested that IL-17A and IL-17F were essential for mucocutaneous immunity against C. albicans, but otherwise largely redundant in host defense, at odds with fi ndings for the mouse model. Moreover, the use of a genome-wide approach led to the discovery of GOF mutations of STAT1 in other patients with CMCD, some of whom also displayed features of autoimmunity.77,498 Previously described STAT1 mutations were LOF (null or hypomorphic) and associated with AD or AR predisposition to viral and/or mycobacterial diseases.499–501 By contrast, the CMCD-causing STAT1 mutations are GOF. They prevent the nuclear dephosphorylation of activated STAT1containing complexes, thereby increasing transcriptional activity in response to IFNγ, IFNα / β, IFNλ , and IL-27, the biologic functions of which are highly dependent on STAT1. Cytokines that activate STAT3 predominantly and STAT1 to a lesser extent, including IL-6, IL-21, and IL-23, also trigger enhanced STAT1-dependent responses in these patients. Patients with GOF mutations in STAT1 have small proportions of IL-17 T cells, because IFNγ, IFNα / β, IFNλ , and IL-27 are inhibitors of IL-17 T-cell differentiation via STAT1, or because IL-6, IL-21, and IL-23 are inducers of IL-17 T cells via STAT3 but not via STAT1, or both.77 Impaired IL-17 immunity therefore underlies CMCD in these patients. Surprisingly, some CMC patients with STAT1 mutations were recently reported to suffer from reactivations of viral disease.501a This situation is reminiscent of the observation that patients with HIES that are heterozygous for STAT3 develop shingles due to impaired T-cell memory.502 Overall, STAT1 LOF alleles underlie viral diseases that occur during primary infection due to the impairment of antiviral IFN activity. By contrast, GOF STAT1 mutations may confer a predisposition to the reactivation viral diseases (including some caused by the same herpes viruses) due to insufficient T-cell memory. In any case, studies of CMCD revealed that the “Th17” arm of immunity is apparently redundant for host defense against most microbes in humans, at odds with fi ndings for the mouse model. Studies of MSMD and CMC have indicated that helper T-cell immunity is unlikely to be restricted to the “Th1” and “Th2” arms, even with the addition of the third partner “Th17.” Indeed, mutations of IFNγ and IL-17 immunity underlie predispositions to only a few of the millions of microbes in the environment, including hundreds of known pathogens. Another step forward came with the investigation of children with invasive pneumococcal disease (IPD). Patients with inborn errors of IL-17 or IFNγ immunity typically display recurrent or persistent infectious diseases, consistent with the production of these cytokines principally by T cells. Only patients with IL-12 and IL-12R deficiencies

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seem to display a single episode of mycobacterial disease, implying that these cytokines are necessary for immunity against primary infection with mycobacteria but not for immunity to latent or secondary infection.78 IPD was long known to be favored by inborn errors of pneumococcal opsonization, such as defects of the classical complement pathway or of antibody responses to capsular glycans, and asplenia.503 Patients with any of these PIDs are actually susceptible to a wide range of encapsulated bacteria. Interest in the investigation of IPD was stimulated in 2001 by the genetic dissection of a rare PID, known as EDA-ID.504,505 IPD is, by far, the most common infection in these patients, but susceptibility to other pyogenic bacteria, mycobacteria, and some fungi and viruses has been recorded. Inflammation is weak or delayed in the course of infection. The only consistent immunologic abnormality is a lack of antibodies directed against glycans. This fi nding is consistent with the observation that IPD is often recurrent in these patients, with recurrences in some cases even being caused by the same serotype. Inborn errors of NF-κ B were identified as responsible for the disease: patients with XR-EDA-ID carry hypomorphic mutations in NEMO,506 whereas patients with AD-EDA-ID carry hypermorphic mutations in IKBA.507 The key role of NF-κ B in developmental processes accounts for the developmental features of the disease in affected children. Impaired immunity has a broad basis, consistent with the broad role of the multiple TNF, IL-1, and TLR receptors downstream from NF-κ B. The NEMO mutations underlying EDA-ID are hypomorphic; null alleles cause the death in utero of male embryos and incontinentia pigmenti in women.508 These hypomorphic mutations are associated with immunologic and infectious clinical phenotypes, some underlying IPD509 and others underlying mycobacterial diseases481 in otherwise healthy patients. NEMO mutations probably defi ne the broadest phenotypic diversity in the field of PIDs, ranging from death in utero or lifethreatening neonatal disease to a mild and transient B-cell deficiency in adults. The mechanisms underlying IPD and other infections in patients with NEMO or IKBA mutations are progressively being deciphered through the identification of patients with germline mutations in genes encoding products acting upstream or downstream from NEMO. Children with isolated IPD provide the best example, as some have been shown to suffer from IRAK-4 or MyD88 deficiency.510,511 These children have no developmental phenotype and are normally resistant to viruses, fungi, and mycobacteria. They suffer from IPD and, more rarely, from invasive staphylococcal diseases. Gram-negative infections are rare, with the exception of those caused by Pseudomonas and, more rarely, Shigella.512–514 The patients display weak, delayed biologic and clinical signs of inflammation during infection. The patients’ cells do not respond to agonists of most TLRs (other than TLR3 agonists) and IL-1Rs (including IL-1, IL-18, and IL-33). Surprisingly, these patients are susceptible to a few pyogenic bacteria, but normally resistant to other bacteria and parasites, fungi, and viruses. The clinical status of patients improves spontaneously from adolescence onwards, with no deaths or invasive infections observed in the absence of prophylaxis. This implies that

Paul_CH48_final.indd 1258

TLRs and IL-1Rs are not required for protective T- and B-cell immunity to these infectious agents. This experiment of nature has not only confi rmed that IPD may have a genetic basis, as previously shown in patients with inborn errors of opsonization, but has also revealed that TLR and IL-1R immunity is largely redundant in host defense. This is at odds with theoretical models attributing a broad role in host defense to TLRs, as microbial sensors or pathogenassociated molecular pattern receptors, and with experiments conducted in MyD88-deficient mice, which were found to be susceptible to more than 35 of the pathogens tested.75 However, this fi nding is entirely consistent with evolutionary genetic studies, which have shown surfaceexpressed human TLRs to be subject to weaker selection pressure than intracellular TLRs.515 Impaired TLR immunity may even play no more than a modest role in the development of infections in MyD88- and IRAK-4–deficient patients. It is not inconceivable that impaired IL-1 immunity alone accounts for the development of the few pyogenic bacterial infections observed in these patients. TLR3, the only TLR that does not signal via MyD88 and IRAK-4 but instead uses TRIF as its sole adaptor, was serendipitously found to be essential for protective immunity against HSV-1 in the central nervous system (CNS), in the course of primary infection, in at least some children (Fig. 48.3).75 The other intracellular TLRs—TLR7, TLR8, and TLR9—which are stimulated by nucleic acids and generally thought to play an important role in antiviral defense, were found to be redundant in humans against most viruses.512 However, the strong purifying selection operating on these genes suggests that past pathogens or other physiologic processes have exerted selection pressure on these four receptors.515 The role of TLR3 in host defense was deciphered by investigations of children with HSV-1 encephalitis (HSE).516 This disease is the most common sporadic viral encephalitis in western countries. In this terrible disease, the virus is restricted to the CNS. It is absent from the bloodstream and does not spread to other organs. HSE is neurotropic in terms of both the route it follows and its destination: it reaches the CNS via cranial nerves. Patients with the most severe myeloid and lymphoid PIDs, including children with no T cells, display no particular susceptibility to HSE. The disease is sporadic in the vast majority of cases, with only four multiplex kindreds reported in 60 years, but there is a high frequency of parental consanguinity (14% in the French survey) in these cases, suggesting that HSE may be due to single-gene inborn errors of immunity displaying incomplete clinical penetrance.516 The first genetic etiology of HSE was identified as AR UNC-93B deficiency, resulting in an impairment of cellular responses to the four intracellular TLRs, including TLR3.517 Involvement of the TLR3 pathway was then suspected, because IRAK-4– and MyD88deficient patients, whose cells do not respond to TLR7-9, are not prone to HSE. TLR3 was formally implicated in the disease when AD and AR TLR3 deficiencies were discovered in other patients with HSE.518,519 The subsequent identification of children with AR or AD TRIF deficiency confi rmed the role of TLR3-TRIF and further suggested that childhood HSE might result from a collection of highly diverse

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INBORN ERRORS OF IMMUNITY

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1259

HSV-1 IFN-λR

TLR3

IL10RB TRIF TBK1 IKK-ε

IRF-3

UNC-93B TRAF3

TYK-2 JAK-1

NEMO IKK complex

IFN-α/βR

NF-κB

TYK-2 JAK-1 STAT-1

STAT-2

IFN-α/β and-λ IRF9

Neurons/Oligodendrocytes FIG 48.3. Inborn Errors of Toll-Like Receptor (TLR)3-Dependent, Interferon (IFN)α/β and -l Immunity Underlie Childhood Herpes Simplex Virus (HSV) 1 Encephalitis (HSE). Schematic representation of the production of and response to IFNα/β and IFNλ in anti-HSV-1 immunity in the central nervous system, based on the genetic dissection of children with HSE. Like most viruses, HSV-1 produces double-stranded ribonucleic acid (dsRNA) intermediates during its replication. TLR3 is an endosomal transmembrane receptor for dsRNA. The recognition of dsRNA by TLR3 induces activation of the IRF-3 and NF-kB pathways via TRIF, leading to IFNα/β and/or IFNλ production. TLR3, UNC-93B, TRIF, TRAF3, TBK1, and NEMO deficiencies are all associated with impaired IFNα/β and/or IFNλ production and predisposition to HSE in the course of primary infection by HSV-1. The binding of IFNα/β and IFNλ to their receptors induce the phosphorylation of JAK1 and TYK-2, activating the signal transduction proteins signal transducer and activator of transcription (STAT)-1, STAT-2, and IRF9. This complex is translocated as a heterotrimer to the nucleus, where it acts as a transcriptional activator, binding to specific deoxyribonucleic acid response elements in the promoter region of IFN-inducible genes. STAT-1 and TYK2 deficiencies are associated with impaired IFNα/β responses and, for STAT1, impaired IFNλ responses and predisposition to HSE. Proteins for which genetic mutations have been identified and associated with susceptibility to isolated HSE are shown in blue. Proteins for which genetic mutations have been identified and associated with susceptibility to mycobacterial, bacterial, and viral diseases, including HSE, are shown in green. Proteins for which genetic mutations have been identified but not associated with susceptibility to infectious diseases are shown in red. This figure will be revised as new results are obtained with the genetic and immunologic dissection of children with HSE and other viral diseases.

but immunologically related single-gene lesions.520 HSEcausing heterozygous mutations of TRAF3 further highlighted the potential role of subtle mutations in pleiotropic genes in narrow clinical phenotypes. In AD TRAF3 deficiency, the mutation is dominant-negative and impaired TLR3 responses account for HSE, whereas the other cellular phenotypes, such as impaired responses to members of the TNF receptor superfamily, are clinically silent.521 There is a broad immunologic phenotype but a narrow infectious phenotype, because the thresholds for clinical consequences differ between cellular phenotypes. Patients with NEMO mutations are broadly susceptible to viral infections, including HSE, reflecting the impairment of antiviral IFN production in response to the stimulation of multiple receptors, including TLR3, in their cells.522 The target genes involved in HSE, downstream from TLR3, were identified as antiviral IFN genes in 2003, when patients with LOF mutations of STAT1 were found to be prone to multiple viral diseases, including HSE.500,523,524 All these genetic etiologies display complete penetrance at the cellular level (in fibro-

Paul_CH48_final.indd 1259

blasts) but incomplete clinical penetrance, accounting for the sporadic nature of HSE. Interestingly, these defects also predispose subjects to childhood HSE, in the course of primary infection, but do not seem to impair immunity against latent HSV-1 infection, in the CNS and elsewhere. The molecular dissection of HSE also led to its cellular dissection. The TLR3 pathway was recently shown to be largely redundant for poly(I:C) responses in keratinocytes and leukocytes, whereas it is essential in CNS-resident cells, including astrocytes, neurons, and oligodendrocytes.524a However, anti-HSV1 immunity has been shown to be critically dependent on the TLR3-dependent production of IFNα / β in neurons and oligodendrocytes only. These fi ndings strongly suggest that HSE results from a CNS-intrinsic defect of antiviral immunity. HSE probably results from a collection of single-gene inborn errors of TLR3 intrinsic immunity operating in CNS-resident cells, including neurons and oligodendrocytes in particular. Overall, the genetic dissection of HSE has shed new light on host defenses, revealing that the TLR3 pathway is responsible for ensuring CNS-intrinsic

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protective immunity against HSV-1 during primary, but not latent infection.

INBORN ERRORS OF TOLERANCE The second most common phenotype associated with PIDs is autoimmunity (Table 48.6). The random process of assembling V, D, and J elements of TCR and Ig genes through the process of VDJ recombination inevitably generates TCRs and BCRs recognizing self-antigens. Tonal signaling in response to self-antigens is essential to promote the progression of T- and B-cell development in the thymus and bone marrow, respectively. However, the prevention of autoimmunity requires T- and B-lymphocytes with high affinity for self-antigens to be deleted or kept in check. The mechanism underlying immune tolerance in the T-lymphocyte compartment include deletion of self-reactive clones,525,526 conversion of autoreactive T-lymphocytes to self-antigen– specific regulatory T (Treg) cells, and the apoptosis of self-reactive T cells. Autoreactive B-lymphocytes are eliminated from the bone marrow through receptor editing (whereby the reexpression of RAG genes promotes sequential rearrangements of the immunoglobulin genes,

TABLE

48.6

thus modifying antigen specificity).527 Furthermore, B cell– activating factor levels regulate the survival of autoreactive B-lymphocytes in the periphery.528 These processes define central and peripheral mechanisms of tolerance, depending on the developmental stage at which they occur. As discussed previously, autoimmune manifestations have frequently been reported in patients with combined immunodeficiency and residual T- and B-cell development, but not in infants with SCID. In addition, some rare monogenic disorders associated mostly, if not exclusively, with autoimmunity highlight the critical role played by tolerogenic mechanisms in immune homeostasis and function (see Table 48.6).92 Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, also known as autoimmune polyglandular syndrome type 1) is an AR disorder characterized by multiple autoimmune endocrine manifestations (hypoparathyroidism, Addison disease), CMC, and nail dystrophy. The syndrome was probably first described in the 1920s to 1940s.30 In 1981, Neufeld et al. collated information for 295 patients with autoimmune Addison disease as a component of polyglandular autoimmune syndrome, and established that APECED/autoimmune polyglandular syndrome

Inborn Errors of Tolerance

Disease

Gene Defect

Inheritance

Immunologic Phenotype

APECED

AIRE

AR

None

IPEX

FOXP3

XL

↓↓↓ FOXP3+ Treg cells

ALPS ALPS-FAS

FAS (TNFRSF6)

AD, rarely AR

ALPS-FASLG

FASLG (TNFSF6)

AD, AR

ALPS-CASP10

CASP10

AD

Caspase-8 deficiency N-RAS defect

CASP8

AD

NRAS

Sporadic

K-RAS defect

KRAS

Sporadic

DN T cells are ↑ or N

FADD deficiency

FADD

AR

↑ DN T cells

CD25 deficiency

IL2RA

AR

T cells ↓ or N

ITCH deficiency

ITCH

AR

None known

Pathogenesis

Associated Features

Defect of central tolerance Defect of Treg

Autoimmune endocrinopathies, candidiasis Autoimmune enteropathy, IDDM, skin rash

↑ TCRαβ+ CD4− CD8− cells (DN T)

Impaired apoptosis via FAS

↑ TCRαβ+ CD4− CD8− cells (DN T) ↑ TCRαβ+ CD4− CD8− cells (DN T) Slight ↑ of DN T cells DN T cells are ↑ or N

Impaired apoptosis via FAS Impaired apoptosis (intrinsic pathway) Impaired apoptosis (intrinsic pathway) Activating N-RAS mutations that impair apoptosis Activating N-RAS mutations that impair apoptosis Impaired apoptosis

Splenomegaly, adenopathies, autoimmunity, increased risk of lymphoma Splenomegaly, adenopathies, autoimmunity, SLE Splenomegaly, adenopathies, autoimmunity Recurrent infections, splenomegaly, adenopathies Splenomegaly, adenopathies, lymphoma

Impaired homeostasis of T cells Not known

Splenomegaly, adenopathies, lymphoma Recurrent infections, liver dysfunction, encephalopathy Recurrent infections, autoimmunity (IPEX-like) Autoimmunity, developmental delay, microcephaly, lung disease

AD, autosomal deficient; ALPS, autoimmune lymphoproliferative syndrome; APECED, autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy; AR, autosomal recessive; CD, cluster of differentiation; DN, double negative; FADD, Fas-associated protein with death domain; IDDM, insulin-dependent diabetes mellitus; IPEX, immune dysregulation polyendocrinopathy enteropathy X-linked; N, normal; SLE, systemic lupus erythematosus; Treg, regulatory T; XL, X-linked.

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CHAPTER 48

type 1 consisted of a combination of at least two of the following three featurs: Addison disease, hypoparathyroidism, and CMC.529 Other autoimmune manifestations are also common (hepatitis, ovarian failure).530 Although it was obvious that identification of the gene underlying APECED could shed light on the mechanisms governing immune tolerance, it was not until 1997 that two groups independently and simultaneously established that the disease was due to mutations of the autoimmune regulator (AIRE) gene.531,532 This discovery constituted a major breakthrough in the understanding of central T-cell tolerance and led to fundamental studies in mice. AIRE is a transcription factor expressed by terminally differentiated medullary thymic epithelial cells, in which it regulates the expression of tissue-specific antigens.533 AIRE-dependent tissue-specific antigens associated with HLA molecules are presented to developing T cells. Through this mechanism, self-reactive T-lymphocytes are deleted from the thymus.534 Consistently, mutations of AIRE cause autoimmunity that is not restricted to endocrine glands and other tissues, but also includes the production of autoantibodies against various cytokines, including IL-17A, IL-17F, and IL-22, which play a key role in defense against Candida spp.535,536 This accounts for the occurrence of CMC in patients with APECED. The autoimmune phenotype of autoimmune polyendocrinopathy type I patients might well be both broader and more profound in the absence of neutralization of these cytokines, which have been shown to play an important role in autoimmunity in the mouse model. Conversely, it is remarkable that the most severe autoimmune disorder known occurs despite the neutralization of these cytokines, indicating the IL-17–independent nature of at least a few autoimmune diseases in humans. In 1959, Russell et al. described a naturally occurring mutant mouse strain (the “scurfy” mouse) with an XR phenotype characterized by enteropathy, runting, dermatitis, and early lethality.537 In 1982, Powell et al. described a large family in which 19 male subjects presented early-onset enteropathy, dermatitis, and endocrine abnormalities. Most of the affected male subjects died before the age of 3 years.538 Other kindreds with a similar phenotype were subsequently described and the disease was named immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome. Linkage analysis mapped the scurfy locus in mice and the IPEX locus in humans to the same syntenic region of the X chromosome.539,540 The demonstration that disruption of the Foxp3 gene is responsible for the scurfy phenotype in mice541 led to a candidate gene approach culminating in the identification of FOXP3 gene mutations in patients with IPEX.542 FOXP3 is a transcription factor required for the development and function of Treg cells.543,544 In patients with IPEX, activated autoreactive T-lymphocytes infiltrate target tissues and secrete cytokines, causing tissue damage. The patients lack CD4 + CD25hi FOXP3 + (Treg) cells. Treg cells may be generated in the thymus (natural Treg) or in the periphery (induced Treg), and these cells have suppressive activity.545 Patients with IPEX require immune suppression, but the only definitive treatment currently available is allogeneic HSCT.546

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Inborn Errors of Interleukin-2 Responses Treg cells express the high-affinity IL-2R. IL-2 plays an important role in both T-lymphocyte proliferation and immune homeostasis, by increasing FOXP3 expression and enhancing the suppressive activity of Treg cells.547 Mutations of the IL2RA (CD25) gene, which encodes the α chain of the IL-2 receptor, are inherited as an AR trait and cause IPEXlike signs of immune dysregulation and lymphoproliferation (lymphadenopathy, hepatosplenomegaly), associated with susceptibility to severe infections.548,549 The presence of circulating FOXP3 + Treg cells in CD25-deficient patients549 indicates that CD25 is dispensable for the generation of natural Treg cells, for which a low IL-2R signaling threshold may suffice.550 However, signaling through the high-affi nity IL-2R is important for the generation of Foxp3 + -induced Treg lymphocytes from naive peripheral T-lymphocytes.551 Moreover, CD25 deficiency leads to an impairment of the survival and fitness of mature natural Tregs in mice.547,552 The binding of IL-2 to IL-2R activates the JAK-STAT signaling pathway, resulting in STAT5B phosphorylation, dimerization, and nuclear translocation, with the expression of IL-2–dependent genes, including FOXP3, Bcl-2, and CD25.553 Furthermore, STAT5B is also activated in response to signaling through the growth hormone receptor, inducing the expression of insulin growth factor-1, a critical growth mediator.553 In 2003, Kofoed et al. described a patient with short stature and growth hormone insensitivity, severe infections due to viruses and P. jiroveci, and lymphoid interstitial pneumonitis with a homozygous missense mutation of STAT5B.554 Additional patients with a similar phenotype combining short stature, immunodeficiency, and autoimmunity have been reported,555 thus substantiating the role of STAT5B-mediated signaling in immune function and homeostasis.

Inborn Errors of Apoptosis A syndrome consisting of generalized lymphadenopathy, autoimmune cytopenia, and hypergammaglobulinemia was described in 1967 by Canale and Smith.556 In 1992, Sneller et al. reported other patients with this constellation of symptoms and showed that these patients had larger than normal numbers of circulating T cells expressing the αβ form of TCR, but lacking the expression of CD4 and CD8 on their surface.557 These cells have since been named DN T cells, and the disease (originally named Canale-Smith syndrome) has been renamed autoimmune lymphoproliferative syndrome (ALPS). Sneller et al.557 recognized the similarity of the ALPS phenotype to a mouse model of genetically determined lymphoproliferation (lpr/lpr mice) shown to result from mutations of the Fas gene.558 The apoptosis of autoreactive lymphocytes plays an important role in the preservation of immune homeostasis. Interactions between FAS ligand, produced by activated lymphocytes, and FAS (CD95) trigger an intracellular signaling pathway that ultimately results in the activation of caspases and cell death.559 FAS mutations are the leading cause of ALPS in humans.560–562 These patients have a higher

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than normal risk of developing cancers (especially B-cell lymphomas), which are observed in 10% of patients with FAS mutations.563 ALPS is mostly inherited as an AD trait and is due to dominant-negative mutations inhibiting FAS-mediated signaling and caspase activation.560 ALPS may more rarely be AR and due to biallelic FAS mutations completely abolishing the production or function of FAS. Furthermore, somatic mutations in the FAS gene may also cause ALPS.564 Finally, combination of a germline FAS mutation affecting one allele and somatic mutations affecting the other allele have recently been reported in some patients with progressive development of the disease phenotype.565 Other rare causes of ALPS include mutations of the gene encoding the FAS-associated death domain protein,566 FAS ligand,567,568 caspase-8,569 and caspase-10.570 The apoptosis of chronically activated T-lymphocytes can also be induced through a FAS-independent mechanism involving the release of cytochrome c and the activation of caspase-9.571 This “intrinsic” (or mitochondrial) pathway of apopotosis is elicited in response to cell damage and cytokine (eg, IL-2) deprivation. GOF mutations of N-RAS may disrupt this pathway and cause ALPS.572 Somatic mutations of K-RAS may also cause ALPS,573,574 but this variant does not result in an increase in the number of DN T-lymphocytes. ALPS is diagnosed on the basis of clinical features, the demonstration of a larger than normal number of DN T cells, and defective apoptosis in response to FAS stimulation or activation of the intrinsic pathway.562,575 Treatment is based on the use of immunosuppressive drugs and surveillance for lymphoma.561,563 The study of patients with ALPS has provided an important confirmation that mechanisms for controlling T-cell survival are important in immune homeostasis. However, it has also raised interesting questions that remain unanswered: why do patients with ALPS suffer principally from autoimmune cytopenia, whereas AIRE and FOXP3 defects are mostly associated with organ-specific autoimmunity? Why is the phenotype of lpr mice (increased in the risk of kidney disease) different from that of FAS-mutated patients? Deep sequencing of the exomes and genomes of patients with unexplained autoimmunity will undoubtedly shed new light on the mechanisms governing central and peripheral tolerance in B cells and T cells.

INBORN ERRORS OF LYMPHOCYTE CYTOTOXICITY UNDERLYING HEMOPHAGOCYTOSIS In 1952, Farquhar and Claireaux described the cases of a brother and sister who presented with high fever, progressive pancytopenia, hepatosplenomegaly, and bruising.576 Unfortunately, a rapid progression to death was observed in both cases. Postmortem examination revealed a prominent infi ltrate of lymphocytes, plasma cells, and histiocytes in the liver, spleen, and bone marrow. Other similar cases were subsequently reported. In some cases, the disease occurred in multiple family members and was thought to be intrinsic, whereas the sporadic presentation in other patients was

Paul_CH48_final.indd 1262

thought to be a consequence of infections or cancers. This led to a distinction being made between primary and secondary forms of hemophagocytic lymphohistocytosis, although it is now clear that many sporadic cases are actually genetic in origin and that episodes of familial cases can be triggered by infection (Table 48.7; Fig. 48.4). Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically determined condition characterized by impaired lymphocyte-mediated cytotoxicity.577–581 In patients with FHL, an inability to clear viral infections results in the persistent activation of CD8 + and NK lymphocytes, high levels of IFNγ production, macrophage activation, and the uncontrolled release of proinflammatory cytokines (TNFα , IL-6).582 Clinical features of FHL include episodes of high fever, pancytopenia, liver and spleen enlargement, and neurologic signs. The study of patients with hemophagocytic lymphohistiocytosis has made an important contribution to definition of the role of lymphocyte cytotoxicity in immune homeostasis. The elimination of virus-infected cells is dependent on CD8 + cytotoxic T-lymphocytes (CTLs) and NK lymphocytes, which release cytotoxic proteins (granzyme B, granulolysin) into target cells through cell membrane pores formed by perforin multimers, causing the activation of caspases and apoptosis.583 The cytotoxic proteins are contained in endosomal lytic granules. Following the interaction of CTL or NK cells with the target cell, rearrangement of the cytoskeleton guides the transport of the lytic granules toward the immunologic synapse.584 During this process, the small GTPase Rab27a promotes docking of the lytic granules, Munc 13-4 induces priming of the cytolytic granules, and syntaxin-11 and Munc 18-2 (also known as syntaxin-binding protein) promote the fusion of the lytic granules with the cell membrane. The lytic proteins are then released by exocytosis through pores formed by perforin (see Fig. 48.3).583 Mutations in the genes encoding Rab27a, Munc13-4, syntaxin-11, Munc 18-2, and perforin cause AR FHL. In addition, patients with Rab27a deficiency (also known as Griscelli syndrome type 2) show partial albinism, because the Rab27a protein is also important for melanin transport in melanocytes, as seen in patients with CHS (see following discussion). An important step in the diagnosis of FHL is the demonstration of defective cell-mediated cytotoxicity.585,586 NK cells present in peripheral blood mononuclear cell preparations from healthy controls kill 51Cr-labeled K562 erythroleukemic cells; a profound defect in this killing is observed in FHL patients. Moreover, during cytotoxic processes, degranulation is accompanied by the surface expression of proteins (such as CD107a) that are normally sequestered in endosomal granules. FHL-associated defects in the docking, priming, and fusion of cytolytic granules impair the expression of CD107a in response to the in vitro activation of CTLs and NK lymphocytes.587 Finally, flow cytometry can be used to detect defects of perforin expression.585,586 FHL is fatal in the absence of treatment, which is based on the prompt recognition and specific therapy of underlying

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48.7 Circulating T Cells

Circulating B Cells

Normal

Normal

c. Syntaxin 11 deficiency, FHL4

d. STXBP2 (Munc 18-2) deficiency, FHL5

Normal

Normal or reduced

Normal or reduced

Normal

Normal

Normal

Normal or low

Normal or low

Normal or low

Normal

Normal

Normal

Normal

Normal

Clinical and immunologic abnormalities triggered by EBV infection, including hepatitis, hemophagocytic syndrome, aplastic anaemia, and lymphoma Dysgammaglobulinemia or hypogammaglobulinemia, low to absent NKT cells Clinical and immunologic abnormalities triggered by EBV infection, including splenomegaly, hepatitis, hemophagocytic syndrome, colitis

Severe inflammation, persistent fever, splenomegaly; hemophagocytosis, decreased to absent NK activity Severe inflammation, fever, splenomegaly, hemophagocytosis possible bowel disease; decreased NK and CTL activities with partial restoration after IL-2 stimulation

Severe inflammation, persistent fever, cytopenias, splenomegaly; hemophagocytosis, decreased to absent NK and CTL activities Severe inflammation, persistent fever, splenomegaly, hemophagocytosis, decreased NK and CTL activities

Partial albinism, recurrent infections, late-onset primary encephalopathy, increased lymphoma risk;n eutropenia, giant lysosomes, low NK and CTL activities, elevation of acute-phase markers Partial albinism, elevation of acute-phase markers, encephalopathy in some patients; low NK and CTL activities Partial albinism, increased bleeding; neutropenia, low NK and CTL activity

Associated Features

XL

XL

AR

AR

AR

AR

AR

AR

AR

Inheritance

Mutations in XIAP encoding an inhibitor of apoptosis

Mutations in SH2D1A encoding an adaptor protein regulating intracellular signals

Mutations in UNC13 (as named in OMIM) required to prime vesicles for fusion; note that also in OMIM the “official” name is UNC13D deficiency with the alternative title of MUNC13D deficiency Mutations in STX11, required for fusion of secretory vesicles with the cell membrane and release of contents Mutations in STXBP2, required for fusion of secretory vesicles with the cell membrane and release of contents

Mutations in PRF1; perforin, a major cytolytic protein

Mutations in RAB27A encoding a GTPase that promotes docking of secretory vesicles to the cell membrane Mutations in the AP3B1 gene, encoding for the b subunit of the AP-3 complex

Mutations in LYST, impaired lysosomal trafficking

Genetic Defect/Presumed Pathogenesis

300635

308240

613101

603552

608898

603553

608233

607624

214500

OMIM Number

INBORN ERRORS OF IMMUNITY

|

AR, autosomal recessive; CTL, cytotoxic T-lymphocyte; EBV, Epstein-Barr virus; FHL, familial hemophagocytic lymphohistiocytosis; Ig, immunoglobulin; IL, interleulin; NK, natural killer; OMIM, Online Mendelian Inheritance in Man; XL, X-linker; XLP, XR lymphoproliferative disease.

b. XIAP deficiency, XLP2

3. Lymphoproliferative syndromes a. SH2D1A defiNormal ciency, XLP1

Normal

Normal

Normal

b. UNC13D (Munc13-4) deficiency, FHL3

Normal

Normal

c. Hermansky-Pudlak syndrome, type 2 2. FHL syndromes a. Perforin deficiency, FHL2

Normal

Normal

b. Griscelli syndrome, type2

Normal

Serum Ig

Inborn Errors of Lymphocyte Cytotoxicity

1. Immunodeficiency with hypopigmentation Normal Normal a. Chediak-Higashi syndrome

Disease

TABLE

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IMMUNOLOGIC MECHANISMS IN DISEASE

cytolytic granule polarization Munc13-4 Rab27a

Cytotoxic T lymphocyte

docking Munc18-2 Syntaxin 11

priming fusion perforin

Target cell

granzyme

FIG 48.4. Schematic Representation of Cytolytic Granules Trafficking and Release. Upon recognition of target cells, cytotoxic T-lymphocytes mobilize cytolytic granules toward the point of contact with the target cell. The Rab27a protein mediates docking of the cytolytic granule; the syntaxin-11/Munc18-2 complex also participates at this process. Then, Munc13-4 favors priming of the granule, allowing a shift of syntaxin-11 from a closed to an open conformation. Eventually, the membrane of the cytolytic granule fuses with the cell membrane and granzyme, contained in the cytolytic granules is delivered to the target cell through pores formed by perforin.

infections associated with chemotherapy and immunosuppressive regimens.588 However, although such treatment may induce remission, relapses are the rule. The only curative approach to FHL is thus HSCT. Optimal results have recently been obtained with reduced-intensity conditioning regimens.589 CHS associates features of FHL with partial albinism, peripheral neuropathy, and the presence of giant lysosomes, which are easy to identify in leukocytes.590 In this disease, the defect lies in the sorting of proteins to secretory endosomes.591,592 This defect affects not only cytotoxic lymphocytes (accounting for FHL-like clinical features), but also melanocytes, which are unable to transfer melanin to keratinocytes and other epithelial cells, thus accounting for albinism.

X-Linked Lymphoproliferative Disease In 1975, Purtilo et al. described a large family in which 6 of 18 male subjects died from a lymphoproliferative disease;

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infectious mononucleosis due to EBV infection preceded clinical signs of the disease and death in at least three of these cases. At the postmortem examination, lymphocytic and histiocytic infi ltration was apparent in multiple organs.448 This constituted the first report of XLP in humans. In normal individuals, EBV causes a self-limiting disease: infectious mononucleosis. The virus establishes a latent infection in B-lymphocytes, salivary glands, and some epithelial cells, but is kept under control by CD8 + CTL and NK lymphocytes.593 By contrast, EBV infection may lead to life-threatening complications in patients with genetic defects in affecting the mechanism controlling EBV infection. Male subjects with XLP are uniquely susceptible to EBV,594 although EBV is also a common trigger of the acute disease phenotype in patients with FHL. Most male subjects with XLP carry mutations in the SH2D1A gene encoding a small adaptor molecule: SLAM-associated protein (SAP).595–598 Various molecules of the SLAM family are expressed on the surface of CTL and NK cells. Through SAP, they trigger activatory signaling pathways that ultimately lead to the killing of EBV-infected cells.597 In the absence of SAP, these activatory pathways are inhibited, and EBV infection remains uncontrolled.599 The persistence of the virus within B-lymphocytes triggers the continuous activation of virus-specific CD8 + CTLs, which display hyperproliferation and release large amounts of IFNγ. This ultimately leads to a macrophage activation syndrome, as described for patients with FHL. Alternatively, EBV may cause neoplastic degeneration of the infected B-lymphocytes (lymphoma). SAP is also important for the function of follicular helper T cells (TFH), which interact with B-lymphocytes in secondary lymphoid organs and secrete IL-21, promoting B-cell activation, germinal center reaction, memory B-cell development, and plasmablast differentiation.600 Consistent with these findings, male subjects with XLP and SAP defects often develop hypogammaglobulinemia with a lack of memory B-lymphocytes. Finally, SAP is also important for the development of NKT lymphocytes, which are typically absent in SAP-deficient patients.601 The cellular pathogenesis of XLP in SAP-deficient patients was recently clarified by two studies. First, CTL from female carriers expressing the mutant SAP were shown not to recognize and kill EBV-infected target cells, whereas CTL from the same individuals expressing the wild-type SAP did.456 Second, somatic reversions of the germline SAP mutations in CTL from affected male subjects were associated with the control of EBV infection.456a These data therefore implicate CTL, as opposed to other lymphocytes that also normally express SAP, in the pathogenesis of XLP. A minority of patients with XLP carry defects in another gene (BIRC4) encoding the X-linked inhibitor of apoptosis.602 Consistently lymphocytes from patients with BIRC4 mutations are particularly susceptible to apoptotic stimuli. However, unlike SAP deficiency, X-linked inhibitor of apoptosis defects are not associated with lymphoma and are more often present with hemophagocytic lymphohistiocytosis, even in the absence of EBV infection.455 XLP is a severe disease. Most patients die from fulminant hepatitis or B-cell lymphoma, and survivors often display

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CHAPTER 48

hypogammaglobulinemia.594 XLP is diagnosed on the basis of an analysis of SAP expression603,604 and is ultimately confirmed by mutation analysis. HSCT is the only curative treatment available and should probably be attempted before primary EBV infection, if possible, in genetically affected individuals.605 It should be stressed that we owe our full appreciation of the consequences of SAP deficiency to the study of XLP patients, because mice are not susceptible to EBV infection. XLP demonstrates the importance of studying human disease in natura. Sap− /− mice have been generated by gene targeting,606 but they cannot fully replicate the human disease, because EBV does not infect murine cells. Other viruses, such as lymphocytic choriomeningitis virus in particular, have been used to model XLP in mice. This has made it possible to show that CD4 + and CD8 + lymphocytes proliferate in an unrestrained manner in Sap− /− mice infected with lymphocytic choriomeningitis virus and that this proliferation is associated with a marked increase in the production of IFNγ.606 These are important observations, but they fall short of the broad and dramatic phenotype of XLP patients following EBV infection.

CONCLUSION We have attempted to cover the diversity of inborn errors of immunity by highlighting disorders affecting different branches of the host response and resulting in various phenotypes, infectious and otherwise. The field is so large and diverse that we could easily have selected other topics. For example, we did not review the inherited disorders of complement, despite their recently discovered surprising association with hemolytic uremic syndrome and related disorders.607 It is also clear that the rapidly expanding and fascinating group of autoinflammatory disorders merits more attention.66 Patients with these diseases do not display autoimmunity, in the classical sense of the term, because they have no detectable autoreactive T cells and B cells. Most patients suffer from enhanced IL-1– or TNF-mediated inflammation. Similarly, we did not discuss the inborn errors of enhanced IFNα / β production, resulting in AicardiGoutière syndrome and systemic lupus erythematosus.608 This chapter focuses on pediatric PIDs, discussing only briefly the most common form of PID in adults, common variable immunodeficiency, which has taken much longer to dissect than anticipated, possibly because patients with this disease have more than one genetic lesion.82 The possibly polygenic basis of some immunologic diseases is not reviewed here, and we have not even touched on autoimmune diseases that are almost Mendelian, such as type I diabetes, which is closely associated with HLA-II alleles, such as HLA-DQ8, in particular.609 Finally, we do not consider here the ground-breaking discoveries of Mendelian resistance to certain infectious diseases.610 Patients with AR DARC, CCR5, or FUT2 deficiencies are intrinsically resistant to Plasmodium vivax, human immunodeficiency virus, and norovirus, respectively. Individuals carrying at least one

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functional allele at any of these loci therefore display AD susceptibility to the corresponding pathogen. In these cases, the PIDs are represented by the wild-type alleles, which are currently more common, but the selective pressure exerted by the pathogens may gradually inverse the allele frequencies.611 These are only some of the topics not covered here due to space constraints. We have undoubtedly omitted many other fascinating lines of research in the field of PIDs and we apologize to our colleagues for these omissions. The field of PIDs is one of the most rapidly expanding fields of research in immunology. New phenotypes are continually being explored. As host defenses are mediated not only by leukocytes, but by almost all cells and tissues, a myriad of diseases, infectious and otherwise, may result from genetic lesions affecting host defense genes. We anticipate that thousands of PIDs will be deciphered in the future. In any case, we have attempted to highlight the main clinical and immunologic implications of these studies. Over the last 60 years, more than 200 disorders have been clinically described, immunologically deciphered, and genetically dissected. New therapeutic approaches have been pioneered in patients with PIDs, including IgG substitution, enzyme replacement, recombinant cytokine, HSCT, and gene therapy. This field has, arguably, been one of the most successful in pediatrics and clinical immunology. The immunologic lessons learned from these experiments of nature are also of considerable interest. Some ground-breaking immunologic discoveries have resulted directly from the dissection of PIDs, such as the discovery that AIRE was responsible for autoimmune polyendocrinopathy type I.612 Many other genes first discovered in patients with PIDs, only some of which have been discussed here, have paved the way to new avenues of immunologic investigation. PIDs have also provided considerable insight in situations in which the morbid genes were first described in the mouse model. Indeed, whether identified by genome-wide or candidate gene approaches, these genes have, in some cases, been assigned a new function, or at least had their known function redefined, in the human model. There are grounds for optimism, as the increasingly careful and widespread clinical care of 7 billion patients will provide an extraordinary wealth of phenotypic description. Moreover, spectacular technologic progress in genetics is making it possible to study the genome of these patients rapidly, searching for morbid lesions at high speed. Finally, tremendous progress in basic immunology, in the mouse model in particular, has provided investigators with the concepts and tools they require to connect PID genotypes and phenotypes experimentally. The identification of causal relationships between gene lesions and clinical phenotypes is based on the molecular and cellular dissection of immunologic pathogenesis. These studies will be of benefit to patients, while providing new insight into the function of host defense genes. We like to think that PIDs is a new frontier in basic immunology and that some of the most extraordinary, paradigm-shifting, immunologic discoveries in the near future will be generated by the investigation of human patients.

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9/18/12 5:01 AM

INDEX

Page numbers followed by an f denote figures; page numbers followed by a t denote tables.

A Abbreviations, found in text, 71–72t Accelerated trejection (acute humoral rejection), 1164–1165 Accessory molecules, in NK-cell activation, 419 Acquired immunity, intracellular bacteria and, 987–991 Activation-induced deaminase, 169–171 Activation receptors, NK-cell, 411– 418, 428 Actively acquired tolerance, 765–766 Acute infection syndrome, HIV, 1019–1021 Acute neutrophil-mediated inflammatory disorders, chemokines and, 706 Acute rejection, graft-versus-host disease and, 1165–1171 Adapter-mediated signaling, 309–311, 309–310f Adapter proteins, b-cell receptor signaling and, 251–252 Adaptive immune system innate immunity relations, 367– 368, 368f origins of, 123–126 protein domains in, 372f viral infections and, 970–972 Adaptive immunity chemokine regulation, 700–702 dendritic cell link to innate immunity, 391–393 gnathostomes, 102–106 lymphoid tissues, 106–112 origins of, 123–126 vertebrates, 90–116 Adaptive immunosurveillance, 1223–1224 Adenoids, 61 Adhesion molecules, spleen and, 59–60 Adipocyte activation, complement and, 890f Adoptive T-cell therapy, in immunotherapy, 1233–1234 Adrenergic neurons, 859–860 Aeroallergens, 1115

Afferent lymphatics, 383 Afferent response, Fc receptors in, 593–594 Afferent trafficking to secondary lymphoid tissues, 700 Affinity antigen-antibody interactions, 183–194 average, 189–190, 190f B-cell receptor, 250–251 heterogeneity of, 188–189 intrinsic, 191–192 maturation, 139, 270–271 somatic hypermutation, 139 Aging immune system and, 962 tumor immunity and, 1222 Agnathan variable lymphocyte receptor (VLRA), 99–102, 100–101 Allelic exclusion, 287, 288t Allergens, 1115–1116 classification, 1115 Allergic airway disease, chemokines and, 706–707 Allergic disorders, 1113–1153 cytokine regulation of Th2 cells, 1129 environmental antigens, 1079f, 1121f epigenetic influences, 1123–1124 historical perspective, 1113–1115 immune responses, 1114t impaired T regulatory cells, 1119–1120 key terms, 1114t natural killer T cells in, 1118 phases of, 1151f Th17 cells in, 1118–1119 type 2 polarized immune responses, 1116–1124 determination of susceptibility, 1120–1121 factors regulating responses, 1124–1134 Allergic encephalitis, 1091f Allergic rhinitis, 1152 Allergy, 31 Allograft rejection, regulatory T cells, 826–828, 827f

Allorecognition, 116–119 Allotypes, 131–132 Altered peptide ligands, 575, 576f Alternative pathway (AP), 869–870, 870f C3 convertase, 873 component deficiencies, 880 Alum, 1042 Amino acids, individual, 575 Amino acid sequences, MHC molecules, 506 Amphibians adaptive immunity, 104–105 lymphoid tissues, 108–109 Anaphylatoxins, 875–877 control of, 873–874 Anaphylaxis, 1151–1152 Anergic T cells, 785–786 Anergy/unresponsiveness/neonatal tolerance, tumor immunity and, 1222 Anti-Baff, 679 Antibodies (Abs). see also specific antibody antigenic determinants recognized by, 539–554 bispecific and bifunctional, 211 concentration, 184, 184f cross-reactivity, 199–202, 200f energetics of binding, 185f engineered, 1231–1233 historical perspective, 132 human or humanized, 212 specificity, 199–202 tumor immunity and, 1222 Antibody diversity structural diversity of, 34 theories on, 31–32, 32f Anticomplement therapies, 885–887, 885f, 886t Antigen. see also specific antigen historical perspective, 132 separation of bound and free, 194–195 structure, immunogenicity and, 539–582 Antigen-antibody complexes, reactions, kinetics of, 186–187

1267

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INDEX

Antigen-antibody interactions, 139–140 interaction with multivalent ligands, 192–193 molecular flexibility, 139 monoclonal antibodies and, 183–214 other methods, 202–208 role of water, 139 solution with monovalent ligand, 187–192, 187f thermodynamics and, 139–140 thermodynamics and kinetics, 183–187 Antigen-binding site, 138–139, 139f Antigenic determinants protein and polypeptide, 545–554, 546–547f recognized by T cells, 554–580, 556f, 556t Antigenic structures, 554–556 Antigen presentation alternate pathways, 538 anatomical differences, 345 in atopic individuals, 1130–1133 leupeptin inhibition of, 559f steps in, 558f Antigen presentation pathways, 524 Antigen-presenting cells (APCs), 1091f Antigen processing, 524–538 cytoplasmic pathway leading to MHC, 563f viral immune evasion, 528–529, 530f Antigen-processing compartments, removal of, 534–535, 533–534f Antigen (signal 1), activation of T cells, 746 Anti-infective immune response, balance with host pathology, 929 Anti-inflammatory cytokines, interleukin-33 as, 648–649 Antimicrobial peptides, 86, 833–834 innate immunity and, 368–369 Anti-RANK ligand, 679 Antiretroviral therapy, 1018f Antiviral agents, development of, 1017–1018 Antiviral immune responses, 944–952 Aphylaxis (anaphylaxis), 30 Apoptosis, 982 in autoimmune process, 1079–1080 inborn errors, 1261–1262 inhibition by virus, 966, 966f mediated by caspase complexes, 727–729, 728f structural biology of, 737f TNF, 665, 667f

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Apoptotic protease-activating factor proteins, 738 Appendix, 64 Artemis, 163 Asthma and allergy, 1153 helper T cells in, 717 Ataxia telangiectasia, 1253–1254 Atherosclerosis, chemokines and, 705–706 Atopic dermatitis, 1153 Atopic disorders, general features, 1113–1115 Autochthonous tumors, 1215–1217, 1215f Autoimmune bullous dermatoses, 1101–1102, 1101t Autoimmune diseases, 1069–1112 central nervous system and ocular, 1089–1094 dendritic cells in, 394 endocrine, 1082 extracellular bacteria and, 1015 Fc receptors and, 597–598 human, abnormal regulatory T cell function, 831–832, 832t organ-specific and systemic, 1072–1073 prevalence of, 1071t regulatory T cell models, 823–826 therapeutic considerations, 1080–1081 Autoimmune gastritis, 1095 Autoimmune hepatitis, 1095–1096 Autoimmunity, 29–30, 1069–1112 complement in, 884 helper T cells in, 717 impaired cell death in, 725–726 initiation, 1076–1078, 1079f kinetics of immune response, 1080, 1081f pathogenicity of, 1075f regulatory circuits in process, 1078–1080 TNF superfamily and, 670 viral infections and, 962–963 Autoinflammatory diseases, 1072–1073 Autophagy, 46, 982–983 IL-1α and, 646–647 Autoreactive T cells, mature, fate of, 784–785, 785f

B Bacille Calmette-Guérin, vaccines replacing, 1062 Bacteria. see also Extracellular bacteria; Intracellular bacteria gram-positive and gram-negative, 1001–1005, 1003f

Bacterial complement evasion strategies, 889f Bacterial vaccines, 1054–1059 BAFF systems Anti-Baff, 679 CD40L and, 672–674 BALT. see Bronchus-associated lymphoid tissue Bam32, protein kinase activation and cytoskeletal rearrangements, 254–255 Basophils, 480–486, 480t, 484f allergic response, 1139–1144 mediators, 483t in viral infections, 947–948 B1-B cells, 235 BCAP, protein tyrosine kinase and P13K, 254 B-cell antigen receptors, discovery of, 37–38 B cell epitopes, 580–581 B cell homeostasis, 724–725 B cell precursors, chemokines in migration of, 226 B cell receptors (BCR) antigen-induced clustering of, 249–250, 249f editing in bone marrow, 791 second messengers, 255–256 signaling, 234, 246–260, 247f affinity and isotype in, 250–251 B-cell coreceptors and, 255t clustering and, 251 coreceptor regulation, 256–259, 257f in human disease, 259–260 signaling cascades, 251–256, 252t, 253f dynamics of, 256 structure, 248–249, 249f B cell responses, complement as regulator of, 887–888, 888f B cells abnormalities of development, 241–242 antigen persistence in vivo, 277 antigen presentation, 262–263 antigen-specific activation, 263 cell cycle arrest and apoptosis, 269–270 controlling antibody class, 266 dendritic cell maturation, 263 development alternative strategies for, 243–245 bone marrow, 240–241, 244f fetal, 240 humans versus mouse, 240–243

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INDEX

impact of micro-ribonucleic acids, 222 inborn errors, 1208t, 1243–1247 pathways, 244–245, 244f discovery of, 34–35 emergence of, 126 germinal center cycle, 267–272, 268f immature, light chain rearrangement, 230–231 malignancies, 242–243 mechanism of help, 39–40 migration and maintenance, 233–234 peripheral maturation stages, 231– 240, 232f response to HIV, 1026, 1026f self-reactive, 787–794, 788f, 789– 790t, 790f somatic hypermutation, 269 stem cells and, 216–218, 217f, 219f tolerance, 787–794, 1074–1075 tolerance and receptor editing, 237–239, 238t tumor immunity and, 1218, 1222 viral infections, 959–961 B cell vaccines, 932–933 Bcl-2 gene family homology structures, 739 programmed cell death and, 735– 736, 735f B/F plot, affinity, 190–191, 191f Bim, negative selection and autoimmunity, 772 Birds adaptive immunity, 105–106 lymphoid tissues, 109–110 B lineage cells development, transcription factors regulating, 218–222, 220f gene expression and immunoglobulin rearrangement, 226, 227f regulators of, 224t BLINK, protein tyrosine kinase and PLC-γ2, 252–253 Blocking factors, tumor immunity and, 1222 Blood group antigens, 542–546, 543f Blunt signal ends, 158 B lymphocytes development, 4–5 mice, 215–236 development and biology, 215–245 pre-germinal center development, 261–267, 262f suppression, Fc receptors and, 591 B lymphopoiesis, during ontogeny, 215–216, 216f

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2B4 molecule, 417 Bone marrow architecture: cellular/functional niches, 49 critical microenvironmental interaction, 223–226, 225f, 230f developmental stages, 222–231, 223f human versus mouse, 240–241, 240f early B-cell progenitors in, 216–218, 217f, 219f functions, 48–49 macrophages in, 451–452 phenotypic definition, 223, 224f Bony fish adaptive immunity, 103–104 lymphoid tissues, 107–108 Bronchus-associated lymphoid tissue (BALT), 62 β-selection, 337–338 Bystander suppression, 1076f

C C3 activation and inactivation, 855f deficiency, 880 C4, activation and inactivation, 855f Calcium, as a second messenger, 255–256 Calcium-nuclear factor, 311–312, 311f Cancer, 1200–1234 antigens, 1207–1209 chemically induced, 1225 chemokines and, 707 dendritic cells in, 394 experimental, 1203–1207, 1203f helper T cells in, 717 immune surveillance, 1223–1226, 1224t immunity effector mechanisms in, 1217–1220 factors limiting, 1220–1223 inflammation and, 1225–1229, 1226f preventing relapse and targeting variants, 1234 ultraviolet light-induced, 1225 virally induced, 1223 viruses and, 963 Cancer-specific antigens, 1209–1211, 1209f Cancer stroma, 1201–1203 Carbohydrate antigens, 540 Cardiac autoimmune diseases, 1104–1105

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1269

Cardiotrophin-1, 613–614, 613t Cartilagenous fish, lymphoid tissues, 106–107 Caspase complexes apoptosis initiation mediated by, 727–729, 728f regulation of apoptosis, 731–733, 732f structure, 740 Catalytic antibodies, 211 CD4, TCR subdivisions, 327 CD8, TCR subdivisions, 327 CD4/CD8, lineage choices, 346–350, 347f, 349f CD3 complex, 281–283, 282–283f, 304–305 CD1d-restricted NK T cells, 432–447, 433t CD40L, BAFF systems and, 672–674 CD2 molecule, 417 CD94/NKG2, 410 C1 domains, 125–126 CDR-H3, diversity and constraints, 137–138, 138–139f CD4 T cells differentiation and plasticity, 708–717 early depletion in HIV, 1020f encountering antigen, 709 fates, 709–711, 709f response to HIV, 1025–1026 subsets with regulatory activities, 714 thymus to periphery, 708 CD4+ T cells, 359–360, 364f CD8+ T cell development and, 754–755 CD8+ T cell priming and, 747–748, 748f 25+FOX3+ regulatory, 798–801, 799–801f viral infections, 952–955, 953f CD8+ T cells, 360–361, 365f cell-mediated cytotoxicity, 891–892 Qa-1 restricted, 823 response to HIV, 1023–1025, 1025f viral infections, 955–959, 956f Cell contact-dependent mechanism of suppression, 812–814 Cell-mediated cytotoxicity, 891–910 Cell-to-cell communication, 41 Cell-to-cell interaction networks, tolerance by, 767–768 Cell types, invertebrates, 73–77 Cellular homeostasis, multicellular organisms, 718–720 Cellular immunology, birth of, 26–27 Cellularists versus humoralists, 26

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INDEX

Cellular mechanisms, parasitic infection, 912–913, 914f Cernunnos/XRCC4-like factor, 163 Chemically induced cancers, 1225 Chemokine receptor antagonists, 704f Chemokine receptors general evolution, 122–123 on hematopoietic cells, 689t phenotypes of knockout mice, 697t structure, 690f structure and classification, 682– 683, 684f transmembrane, 688t Chemokines, 41, 119–123, 681–707 atypical system components, 684–690 biologic response modifiers, 707 disease and, 702–707 early inflammatory response to virus, 947 general evolution, 122–123 genes and evolution, 690–691, 691t human disease and, 703t immunologic classification, 691–692 inhibition by virus, 966 issue of redundancy, 693 leukocyte responses to, 693 lymphoid organ development, 56–57 in lymphoid organ development, 48t molecular organization, 681–693, 682–683 myelosuppressive, 698 neutrophil-derived, 475–476 nomenclature and immunologic function, 685–687t, 687f presentation mechanisms, 693 proinflammatory, 119–120 regulation of action, 695–696, 695f regulation of hematopoiesis, 696– 698, 696f regulation of immune response, 698–702 resistance to intracellular bacteria, 992 signaling pathways, 693–695, 694f sources of CC chemokines, 692t sources of CXC, CX3C and C chemokines, 691t spleen and, 59–60 therapeutic applications, 707 thymus and, 51–52 Chondrichthyan germline-joined genes, 98–99

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CH region, genomic organization of, 154, 155f Chromatin marks, 176 Chromosomal translocations and diseases, 288t, 288–289 Chronic mucocutaneous candidiasis, 637–638 Chronic rejection, graft-versus-host disease and, 1171–1173, 1172f Ciliary neurotrophic factor, 613–614, 613t CIS/SOCS/JAB/SSI family, inhibitory adaptor proteins, 636–637 CK elements, 156 Classical pathway (CP), 864–865, 868f component deficiencies, 880 Class switch recombination, 177–182, 180f Clonal deletion experimental and conceptual swing against, 766–767 thymic, 771–772 Clonal ignorance, self-reactive B cells, 794 Clonal selection theory, 33, 33f, 766 Clq, structure, 867f Clq receptors, 876–877 Clr/Cls/Mannan-Binding Lectinassociated serine protease-2 family, 878 Cnidaria, histocompatibility reactions, 117 Collectin family, 878 Colon lymphoglandular complexes, 64 Combination vaccine, 1039 Commensals bystander effect on peripheral responses, 847–848 control of effector and regulatory responses, 845f, 846–847 role in mucosal inflammation, 847 Common ancestor hypothesis, 67, 68f Common variable immunodeficiency, 1245–1246 Complement, 863–890 adipocyte activation, 890f in autoimmunity, 884 components and pathways, 864– 872, 865–866 definitions and history, 863–864 in degenerative disease, 885 in disease, 883–885 evasion and hijacking by pathogens, 889 in ischemia-reperfusion injuries, 883–884 lipid metabolism and, 889–890 novel roles of, 887–890

ontogeny, genetics, and protein families, 877–879 in renal disease, 885 in sepsis, 884 in transplant rejection, 884 Complement activation control, 872–873f, 872–875 family, 878 inhibition by virus, 968–969, 968f Complementary-determining region 3 (CDR3), 287–288, 518f Complement autoantibodies, 879–883, 882–883 Complement deficiencies, 879f Complement mutations, 879–883 Complement polymorphism, 879–883 Complement protein point mutations, 882 Complement protein polymorphisms, 882 Complement proteins deficiencies, 881 expression patterns, 878–879, 879f Complement receptors, 875–877 Complement system, 82–84, 83f Complotype, 882, 883f Conformational flexibility, antigenantibody interactions, 185–186 Congenic strains, identifying MHC, 1157, 1157t, 1158f Congenital immunodeficiency diseases, 1223 α/β constant domains, 97 Costimulation CD28, 318–320, 318f receptors other than CD28, 320– 321, 320f Costimulatory receptors (signal 2), activation of T cells, 746 Cowpox pustules, 26, 27f CP/CP C3 convertase, 872–873 C3 receptors anaphylactic fragments, 876 opsonic fragments, 875–876 C4 receptors anaphylactic fragments, 876 opsonic fragments, 875–876 Crossmatch, 1198 Cross-reactive idiotypes, 131 Cross-reactivity, 130 Cryptopatches, 63–64 C-type lectin receptors, 379–380 C-type lectins, 79 Cutaneous autoimmune diseases, 1101–1104, 1102f CXCR2, CXCR4, and CXCR7, 696– 697, 696f, 697t

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INDEX

Cytokine receptors diseases of, 637–638 pleiotropy and redundancy, 618–619 soluble, 619–620, 619–620t Cytokines, 119–123. see also Type 1 cytokines altered T-reg cell-promoting, 1133–1134 in autoimmune process, 1078 common ϒ-chain, role in memory T cell homeostasis, 753–754 downmodulation of signals, 635–636 early inflammatory response to virus, 947 γC family, 120 heterodimeric, 121 immunomodulatory effects, 1174–1175 in immunotherapy, 1233 inhibition by virus, 966 lymphoid organ development, 56–57 made by subsets of T cells, 715 modulation of, 638 neutrophil-derived, 475–476, 476t NK cells and, 395–396 pleiotropy and redundancy, 618–619 receptors share gp130, 613–614, 613t receptors share λ, 612, 612t regulation of Th2 cells in allergic disease, 1129, 1129f resistance to intracellular bacteria, 992 shared receptor molecules, 617 signaling molecules important for, 635 species specificity, 623 TH2, 120–121 treatment of irritable bowel disease, 824t Cytolytic granules, trafficking and release, 1264f Cytoplasmic domains, 148 Cytoskeletal organization, defects, 1252–1253 Cytoskeletal regulation, 316, 316f Cytosolic deoxyribonucleic acid sensors, 78 Cytotoxic gene expression, 892–895 Cytotoxic granules, 895–896, 895f killer cell protection from, 905–906 Cytotoxicity cell-mediated, 891–910 NK cells, 398–399

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D Daicyl glycerol, as a second messenger, 256 Death receptor pathways, 907–909, 908f Death receptors, signal transduction pathways of, 728f Defense mechanisms conservation of, 68–69 rapid evolution of, 67–68 Defensins, 86 Degenerative diseases complement in, 885 viral infections and, 962–963 De Natura Hominis (On the Nature of Man), 22 Dendritic cells, 381–394 anatomic distribution, 382–383, 383t antigen presentation, 389–391 antigen processing, 391 in clinical immunology, 394 control of tolerance, 393–394 conventional, cross-presentation by, 392–393 cultures, 386 development, 383–391 transcriptional regulations of, 386–387, 387t discovery of, 46, 381 follicular, antigen scanning on, 271–272, 271f growth factors for, 386 in HIV infection, 1026–1027 homeostasis, 388–389, 389f, 390t, 725 human, 387–388, 388f initiating adaptive T-cell immunity, 392 as initiating antigen-presenting cells, 357–358 initiation of T-cell responses, 381 innate and adaptive immunity, 391–393 interactions with innate lymphocytes, 392 maturation, 263, 390–391 migratory pre-dendritic cells, 384 monoclonal antibodies to, 381–382 monocyte relations, 383, 385f plasmacytoid, 386, 391 progenitors in bone marrow, 384 quality of T-cell response, 392 skin and other body surfaces, 383 subsets, 382 transition to adaptive immune response, 700 viral infections, 948–951, 949f

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1271

Deoxyribonucleic double-strand break response, 1253–1254 Dextran-binding myeloma proteins, 543–545 Diabetes, type 1, 1083–1089, 1084f, 1088f Differentiation 3 molecules, 281–282, 282f Dimers, 148 Diphtheria vaccine, 1054–1055 Disease, ancient theories, 22–23 DNA, cytosolic sensors of, 379 DNA damage response factors, 164 DNA ligase IV, 163 DNA-PK complex, 162 DNA polymerase X family members, 163 Double negative compartment, generation of T-cell precursors, 330 Downstream signaling pathways, 311–314 Down syndrome cell adhesion molecules, 79–80 Drug development, chemokines and chemokine receptors, 707 D segments, 99

E Effector function, NK cells, 398–399 Effector mechanisms cancer immunity, 1217–1220 host resistance, 920–926 Effector molecules, 85–86 Effector T cells, 357–362 activation by Fc receptors, 590–591 allergic response, 1139–1144 clonal expansion of, 747 contraction of population, 749–750 differentiation, 359–361 function in nonlymphoid organs, 361–362 generation of memory T cells from, 362–363 heterogeneity and memory cell potential, 750–751 inflammatory cytokines and, 746–747 maintenance and function of T-cell memory, 752–757 methods of detecting, 745 migration, 748–749 model of development, 743–745, 744f transcriptional control of, 751–752 Efferent response, Fc receptors in, 594–597, 594f Efferent trafficking, 701–702 Ehrlich, Paul, 28–29, 31

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INDEX

Elbow joints, Ig, 140, 140f ELIspot assay, 199 Endothelial glycoproteins, 1162 Engineered antibodies, 1231–1233 Environmental antigens in allergic disorders, 1079f, 1121f innate immune response, 1081f, 1124–1128 Enzyme-linked immunosorbent assay (ELISA), 197–198, 198f Eosinophil-derived mediators, 1144–1146 Eosinophils, 478–486, 478f biology, 1144–1151, 1145f as effector cells in allergic responses, 1147 mechanism of action, 1146–1147 mediators, 480t recruitment to sites of inflammation, 1146 in viral infections, 947–948 Epithelial barrier, innate immunity and, 368 Epitopes, 130 ERK, role in negative selection, 771–772 Error-prone polymerases, 174 Erythropoietin, 618 Evolution convergent, 69 defense mechanisms, 67–68 immune system, 67–129 Extracellular bacteria antigen-nonspecific host defense response, 1008–1011 B cell response, 1010–1013 deleterious host responses, 1014–1015 diseases associated with, 1003t immunity to, 1001–1015 invasion of the host, 1007–1008 local response (inflammation), 1009–1010 mucosal defense, 1008–1009 protective mechanisms of antibodies, 1013–1014 systemic response to invasion, 1010–1011 T cell response, 1014 virulence factors, 1005–1007 Extracellular soluble receptor, with effector cascade, 82–85

F Fab, 135–139 historical perspective, 132–133 structure, 135, 135f FAS ligand system, 674–675 Fb, structure, 135, 135f

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Fc historical perspective, 132–133 structure and function, 141–142, 143t FcεRII (CD23), 1139 FcεRI-mediated signal transduction, 1138 FcεRI surface expression, 1138 FcγRIII (CD16), 413 Fc receptors chromosomal organization, 586f disease associations, 597–599 human and mouse comparison, 587f, 590f immunoglobulin interactions, 146–147, 147f ribbon diagram, 589f role in immune regulation and inflammation, 583–600 signaling, 591–593, 592f structure and expression, 584–589, 585f three-dimensional structure, 588–589 in vitro activity, 589–591 in vivo functions, 593–597 Fetal liver, 47–48, 48f macrophages in, 451–452 Fibrinogen-related proteins, 85 Follicular B cells, 233 Food allergens, 1115 Food allergy, 1152–1153 Foxp3+ regulatory T cells, 713–714 biologic properties, 801–808 development extrathymically, 804–805 development in the thymus, 801–802 gene expression by, 806–807, 807t helios, 806f molecular analysis of suppression and anergy, 809–810 production of suppressor cytokines, 811–812 suppressor mechanisms, 808–818, 808f, 809t T-cell receptor repertoire analysis, 804 thymically derived, 841–842, 841f TNF-β, 805f, 811t, 812f Foxp3- regulatory T cells, 820–823 Fracastoro’s seeds, 23–24 Free energy, antigen-antibody interactions, 184–185 Fv, structure, 135, 135f

G Gamma activated site motifs, 634 Gamma globulins, historical perspective, 132

Gastrointestinal autoimmune diseases, 1094–1095 GATA3, CD4-specific driver, 347–348 γδ-selection, 339–340 Gene libraries, use to derive monoclonal antibodies, 209–210 185/333 Genes, 86 Genetic diseases, perforin or granule exocytosis, 897–898 Germinal center (GC) B cells, 234 cellular dynamics, 267–268, 268f dark zone, 270f, 273f differentiation, 241 follicular helper T cells, 272, 273f Germline precursors, 158 Germ theory of disease antecedents of, 22–24 conceptual basis of, 24–25 development of vaccines and, 24–26 experimental evidence for, 25 summation, 26 Gi-dependent effectors, 693–694, 694f Gi-independent effectors, 695 β1-3 Glucan receptors, 80–81, 80f Glycoconjugate-based vaccines, 1038–1039 Glycosylation, 132 Graft-versus-host disease chronic rejection and, 1171–1173 physiologic interactions that modulate, 1173–1176 regulatory T cells, 826–828, 827f T cells and, 1165–1171 Granule exocytosis, 896–897, 896–897f genetic diseases caused by, 897–898 Granule-mediated cell death, 895–907 Granulocyte macrophage-colony stimulating factor, 612–613 Granulocyte macrophage-colony stimulating factor receptor, 615–616 Granulocytes, 468–486 tumor immunity and, 1219–1220 in viral infections, 947–948 Granuloma, formation, intracellular bacteria and, 995–998, 996f Granulysin, 905 Granzyme A, 901–903, 902f Granzyme B, 903–904, 903f Granzyme K, 904 Granzyme M, 905 Granzymes, 398, 892–893, 893f extracellular roles of, 907 extracellular signals regulating expression, 893–894

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INDEX

features of the distinct cell death pathways induced by, 901t programmed cell death pathways, 899–901 transcriptional regulation of, 894–895 viral inhibitors, 906–907 Granzymes C and H, 904 Graves disease, 1082–1083 Greek theories of disease, 22 Growth factor receptors, 326 Growth hormone receptor, 604f Gut-associated lymphoid tissue, 62–63, 833 development, 63 Preyer’s patches, 62, 63f trafficking in and out, 62

H Haemophilus influenza type b vaccine, 1056 Hairpin coding ends, 158 Halothane hepatitis, 1100 Haptens, 539–540, 540t Hashimoto thyroiditis, 1082–1083 Heavy chain structure and function, 141–148 switch regions and functions, 176– 182, 180f Heavy chain constant regions, 154 Helper T cells clonal selection, 264 differentiation and plasticity, 708–717 epigenetic regulation, 714–717, 716f epitopes, 580–581 follicular, 264, 265f, 713 antigen-specific memory, 276–277 B-cell contact, 264–266 germinal center, 272, 273f immune-mediated disease and cancer, 717 lineage commitment versus plasticity and flexibility, 715–716 Hemagglutination, 206–207 Hemagglutination inhibition, 206–207 Hematopoiesis invertebrates, 73–77 vertebrates, 76 Hematopoietic antigen-presenting cells, 353 Hematopoietic cells origins, 47–48 positioning in peripheral tissue, 698 Hematopoietic chimerism, 1183f

Paul_Index_final.indd 1273

Hemocytes, responses of, 76–77 Hepatic autoimmune diseases, 1095– 1101, 1096t, 1098f Hepatitis A and B vaccine, 1051–1052 Heterodimers, 129–130, 130–131, 604–605 Hexahelical bundle, 739–740 Hexamers, 148 H-2 haplotypes, 491t mutations, 504 structure, 509f High endothelial venules, lymph nodes and, 55–56 Higher-order receptor oligomers, 604–605 Higher order structure, 148 Hinges, Ig, 140–141, 140f, 140t The Hippocratic Corpus, 22 Histocompatibility, reactions, invertebrates, 116–119 Histocompatibility testing, family testing, 499–501, 500f A History of immunology (Silverstein), 35 History of Transplantation Immunology (Brent), 35 Homodimers, 604–605 Horror autotoxicus, 765 Host defense antigen-specific response, 1010–1011 innate recognition and, 911–921 Host resistance, effector mechanisms of, 920–926 Hotspot focusing, 176 Human cytomegalovirus vaccine, 1060–1061 Human immunodeficiency virus (HIV) chemokines and, 702–704, 703f discovery of, 43–44 early immune responses, 1020f helper T cells in, 717 immune response to, 1022–1028 immunogenetics, 1028–1029, 1028f immunology of, 1016–1070 origin, transmission and progression, 1016–1022, 1017–1018 prevention, 1029–1031, 1030f vaccine, 1059–1061, 1060t Human killer immunoglobulin-like receptors, 407–408, 408f Human leukocyte antigen (HLA) color ribbon representation, 511–512f disease associations, 501t donor kidney graft survival, 498f

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1273

drug hypersensitivity reactions, 503–504, 503t gene nomenclature, 489f, 490–491t in hematopoietic cell transplantation, 1198 HIV control and, 1023f location of pockets, 512f ribbon diagram, 512f rates for binding, 514t Human papillomavirus vaccine, 1054 Humoral immunity ascendance of, 28 beneficiaries of early focus on, 29–31 fundamental strategy of, 141 longevity of, 762–763, 762t Humoral mechanisms, parasitic infection, 911–912, 914f Hybridomas derivation of, 209, 209f species other than mice, 209 Hybrid resistance, 1163f Hyperacute rejection, 1163–1164, 1163f Hyperimmunoglobulin E syndrome, 1254 Hyperimmunoglobulin M syndrome, 1245 Hypersensitivity, discovery of, 30–31 Hypothalamic-pituitary-adrenal axis, 860–861

I Idiotypes, 130–131, 131f Immune cells, adrenergic neurons modulate responses, 859–860 Immune effector mechanism, programmed cell death as, 727 Immune function, macrophage relations to, 450–466, 450f Immune homeostasis, regulatory T cell models, 826 Immune invasion, mechanisms of, 926–929 Immune recognition, evasion of, 926–927 Immune regulation Fc receptor role in, 583–600 mucosal, 840–843 programmed cell death and, 719–727 Immune response antigen-specific, 952–961 parasites, 910–936 Immune subversion, 42–43 Immune suppression, evasion of, 927

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1274

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INDEX

Immune system aging, 962 evolution of, 67–129 homeostasis impaired neurophysiologic control, 861–862 neural signals and, 857 innervation of, 852–853, 852f neuron response to molecules of, 855–856 specialization within, 34–35, 37–39 Immunity early concepts, 23 inborn errors, 1235–1265 narrow range of infections, 1256f, 1254–1260, 1255t, 1259f major gene families in, 70–73, 70f viruses, 937–972 Immunization changes in lymph nodes and, 57 regulatory T cells and, 817f Immunoblot (Western Blot), 207, 207f Immunochemistry, dawn of, 31–34 Immunodiffusion, Ouchterlony method and, 204–205 Immunoelectrophoresis, 205, 206f Immunogenicity antigen structure and, 539–582 autochthonus cancers, 1215–1217 Immunoglobulin A (IgA), 94–95, 145, 145f generation and secretion, 834 host microbe interaction, 835 T-dependent and -independent generation, 835 Immunoglobulin D (IgD), 93–94, 93f, 144, 792–793 Immunoglobulin E (IgE), 94–95, 146, 1137 regulation of synthesis, 1134–1139, 1135–1136f Immunoglobulin gene loci, 152–157, 153t Immunoglobulin gene organization, 98–99 Immunoglobulin G (IgG), isotypes related to, 94–95 Immunoglobulin heavy chain isotypes, 90–96, 91f Immunoglobulin H (IgH), germline variable, 153–154 Immunoglobulin light chains, 95–96 Immunoglobulin M1gi (IgM), 92 Immunoglobulin M (IgM), 90–91, 142–144 B cells, 792

Paul_Index_final.indd 1274

Immunoglobulin new antigen receptor, 92–93, 93f Immunoglobulin R (IgR), 93–94, 93f Immunoglobulins (Ig), 129–149 chemical nature of molecules, 31 classes and allotypes, 131–132 distinct classes of, 38 domains, 133–135, 133f “elbow joints” and “hinges,” 140–141 gene alterations in germinal centers, 168–182, 168f gene assembly, 150–152 glycosylation, 132 as heterodimers, 129–130, 130–131 historical perspective, 132 isotypes and idiotypes, 130–131, 131f membrane and secretory, 130 membrane versus secreted, 154–155, 155f molecular genetics, 150–182 nomenclature, definitions, 133t paratopes and epitopes, 130 structure of, 31–34 transport, 148–149 vertebrates, 90 Immunoglobulin superfamily, 68f, 73, 133–134, 133f natural killer receptors, 89–90 Immunoglobulin W (IgW), 93–94, 93f Immunoglobulin Z/T (IgZ/T), 93f, 94 Immunologic memory, 741–764 lifelong, 741–743, 743f Immunologic synapse, 314–315, 314f Immunologic tolerance, 35–37, 765–794 history, 765–768 Immunology, as a discipline, 26–29 Immunopathogenesis, malaria, 930f Immunopathologic mechanisms, 928–931 Immunoprevention, 1229–1230 Immunoreceptor tyrosine-based activation motif (ITAM), 306–308, 307f, 322 Fc receptor signaling, 591–592 Immunoreceptor tyrosine-based inhibitory motif (ITIM), Fc receptor signaling, 592–593 Immunoregulation, concept of, 42 Immunosuppressive medications, 1176–1177, 1177t, 1199 Immunotherapy, 1230–1234, 1230f programmed cell death as, 727 Inbred strains, 1154–1155, 1155f

Infection, induction of lifelong memory through, 741–743, 743f Infectious agents, immunity to, regulatory T cells, 830–831 Infectious tolerance, 1076f Inflammasomes, 376–377, 994 Inflammation acute, regression after, 1227 bacterial-triggered, 991–995 cancer and, 1225–1229, 1226f chronic, 1228 Fc receptors and, 598–599, 599f mucosal, role of commensals in, 847 Inflammatory bowel disease, 848 Inflammatory cytokines (signal 3), effector T cells and, 746–747 Inflammatory mediators, 855f, 854t Inflammatory reflex, 857–859, 858– 859f, 861t Inflammatory response cytokines and chemokines, 947 tumor growth, 1228–1229 Influenza vaccine, 1049–1051 Inhibitory adaptor proteins, CIS/ SOCS/JAB/SSI family, 636–637 Innate immune responses, 77–90 Innate immune system, 367–380 adaptive immunity relations, 367– 368, 368f cells of, 947–948 protein domains in, 372f response once barriers are breached, 369–370 role in transplant rejection, ??t, 1175–1176 Innate immunity antiviral responses, 944–952 chemokine regulation, 698–700 conservation across species, 375t cytosolic components-25, 45–46 dendritic cell link to adaptive immunity, 391–393 direct-acting antimicrobial factors, 368–369 frontline of, 368–369 humor circuit regulation of, 860–861 intracellular bacteria and, 986–987 pattern recognition, 370–380 rediscovery of, 43–45 sensors, 359f Innate immunosurveillance, 1225 Innate lymphoid cells, 843–845, 844f Innate recognition, host defense and, 911–921

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INDEX

Innate surface recognition molecules, signaling through, 81–82 Innate-type lymphocytes, agonist selection of, 352–353 Insect venom allergens, 1115–1116 Instruction theories, 31–32 Integral membrane proteins, 79–80 Interferon-γ-receptor, 603f Interferon receptors, signaling through, 623 Interferon regulatory factor family proteins, 635 Interferons, 121, 620–623 nomenclature, 601–602 Interleukin-1, 119 Interleukin-2, 120, 606–618, 607t, 611f inborn errors, 1261 Interleukin-3, 612–613 Interleukin-4, 120–121, 606–618, 607t, 611f Interleukin-5, 120–121, 612–613 Interleukin-6, 119, 613–614, 613f, 615t Interleukin-7, 606–618, 607t, 611f, 753–754 Interleukin-8, 119 Interleukin-9, 120–121, 606–618, 607t, 611f Interleukin-10, 121–122, 621–623 Interleukin-11, 613–614, 613t Interleukin-13, 120–121 in allergic response, 1149f Interleukin-15, 395–396, 606–618, 607t, 611f, 753–754 Interleukin-17, 122 Interleukin-19, 621–623 Interleukin-20, 621–623 Interleukin-21, 606–618, 607t, 611f Interleukin-22, 621–623 Interleukin-23, mucosal inflammation and, 848–849, 849f Interleukin-24, 621–623 Interleukin-25, in allergic inflammation, 1129 Interleukin-26, 621–623 Interleukin-27, 613–614, 613t Interleukin-29, 621–623 Interleukin-33 as an anti-inflammatory cytokine, 648–649 in atopy, 1129–1130 IL-1 subfamily, 647 processing of precursor, 648 Th2 responses and, 647–648 as a transcription factor, 649 Interleukin-1α, 642–643 autophagy and, 646–647 biologic functions, 643 membrane-associated, 642–643

Paul_Index_final.indd 1275

mice deficient in, 643 processing and secretion of, 643 Interleukin-28A, 621–623 Interleukin-1β, 643–647 gain of function mutation in cryopyrin, 645 inducible cytokine, 644 mice deficient in, 646 non-caspase-1 processing of, 645 polymorphisms in P2X7, 645 processing and secretion, 644–645 reactive oxygen species and, 646 Interleukin-28B, 621–623 Interleukin-1 family, 639–659, 640t IL-33 as member of, 647 IL-1β, 643–644 influence on TH17 responses, 641–642 innate responses, 639–640 organization into 3 subfamilies, 640–641, 640–641 Interleukin-31R, 615–616 Interleukin-12Rβ1, 615–616 Interleukin-12Rβ2, 615–616 Interleukin-1 receptor family, 647t Interleukin-2 receptors, 617–618, 618t Interleukin-18 subfamily, 649–653, 650f Interleukin-36 subfamily, 655–658, 656f in human disease, 657–658 IL-38, 658 IL-36 α, β, and γ, 657 in metabolic regulation, 657 in psoriasis, 657 Interleukin-37 subfamily, 653–655, 655f Intestinal protozoa, vaccination against, 932 Intracellular bacteria antimicrobial peptides, 985 B cells, 990 CD4 T cells, 989 CD8 T cells, 989 cell biology, 976–986 cell death patterns, 982–983 cell-to-cell spreading, 981–982 cytokines and chemokines, 992 egression into cytoplasm, 981–982 genetic control of resistance, 998–1000 granuloma formation and, 995–998 guanine triphosphatases and, 985–986 immunity to, 973–1000 infections, features of, 973–976, 974t inflammasome, 994

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1275

inflammation triggered by, 991–995 intracellular iron and, 983–984 kinetics of infection with, 991–992, 991f leukocyte extravasation, 993–994 leukocyte recruitment, 992–993 macrophage heterogeneity and polarization, 994–995 major infections caused by, 975t memory T cells, 990–991 mononuclear phagocytes, 986 peripheral T cell responses, 987– 988, 988f phagocytes and, 977f phagocytosis and phagosome dynamics, 980 polymorphonuclear granulocytes, 986–987 regulatory T cells, 990 toxic effector molecules and, 984– 985, 984f unconventional T cells, 990 Intracellular recognition, 77–78 Intraepithelial lymphocytes, 835–838, 835t functions, 836 homeostasis of, 836 Intrinsic growth factor, T-cell death, 723 Invertebrate cell types, 73–75, 74f Invertebrates histocompatibility reactions in, 116–117 responses to viruses, 87 IRS proteins, 635 Ischemia-reperfusion injuries, complement in, 883–884 Iscomatrix, 1046 Isotypes, 130–131, 131f

J JAK3 mutations, 625 JAK2 mutations and translocations, 626 JAKs, 623–624, 623f, 624t, 626–628t determination of specificity, 629–634 importance in signaling, 607t, 624–625 JAK-STAT activation, 626–629, 629f other substrates for, 634–635 J chain, 96, 148 Jerne’s natural selection theory of antibody formation, 32–33 JH, diversity and, 153–154 JK elements, 156

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1276

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INDEX

K Kaposi sarcoma, chemokines and, 705 Kappa light chain genes, 156 Killer cells, 891–892 cellular resistance to granulemediated death, 906 protection from cytotoxic molecules, 905–906 Knockout mice, cytotoxicity, 898 Koch, Robert, 25–26

L Lactoferrin, 369 Lambda light chain genes, 156–157 Landsteiner, Karl, 29–30 Latex allergens, 1115 Lectin-like receptors, natural killer gene complex, 416–417 Lectin pathway (LP), 870–871 component deficiencies, 880 Leishmaniasis, vaccination against, 933–934 Leishmania vaccine, 1067 Leucine-rich repeats, 70–73, 70f Leukemia inhibitory factor, 613–614, 613t Leukocyte transmigration, 472–473, 473f Licensing hypothesis, natural killercell tolerance, 422–424, 423f Lipid ligands, natural killer T cells, 433–435, 434f Lipid metabolism, complement and, 889–890 Liposomes, 1044 Live-attenuated vaccines, 1036–1037 λ locus, 156–157, 157f λ-related surrogate light chains, 157 Luminal antigens, 837 Ly49, 402–407, 404–405f Lymph nodes changes after immunization, 57 conduit system, 55 development, 56 high endothelial venules and, 55–56 macrophages in, 454 microenvironments, migration within, 701 structure and organization, 53–54, 54f vasculature, 54–55 Lymphocytes activation models, 39, 40t adrenergic neurons modulate responses, 859–860

Paul_Index_final.indd 1276

cytotoxicity, inborn errors, 1262– 1265, 1263t discovery of costimulation, 39 Lymphoid follicles, 63–64 Lymphoid malignancy, programmed cell death and, 726 Lymphoid neogenesis, 65t Lymphoid organogenesis, 56–57 Lymphoid organs, 47–66 dendritic cells, 382–383 primary, 47–53, 48f resident macrophages in, 451–452, 452f, 452t secondary, 53–64 Lymphoid tissues, 47–66 bronchus-associated, 62 evolution in vertebrates, 106–112, 106f gut-associated, 62–63 mucosal-associated, 60–61 nasal-associated, 61–62 tertiary, 64–66, 64f Lymphokine-activated killer cells, 395–396 tumor immunity and, 1219 Lymphoproliferative disease, impaired cell death in, 725–726 Lymphotoxins autoimmunity and, 670 in host defense, 671t lymphocyte development and, 670 mice deficient in, 669t TNF family and, 667–670, 668f

M Macrophages enhanced recruitment of monocytes, 455–457, 456f gene expression and secretion, 464–465 landmarks in the study of, 448–450 marginal zone, 453–454, 454f modulation of phenotype, 465– 466, 465t phagocytic recognition and intracellular infection, 457– 464, 458–464f, 461t phagocytosis and, 448–467 properties and relation to immune function, 450–466, 450f red pulp, 455 tumor immunity and, 1219–1220 viral infections, 948 white pulp, 454 Macular degeneration, age-related, chemokines and, 706

Major antihelminth effector mechanisms, 923t Major histocompatibility complex-Ib molecules cluster of differentiation 1, 513–514, 514f H2-M3, 513 Major histocompatibility complex-II molecules antigen processing and peptide loading, 535–536 assembly and transport to endosomal pathway, 531–533, 532f delivery to cell surface, 536–537 high-resolution structures, 510–511, 511f interactions of peptides with binding groove, 572t invariant chain peptide, 513–514f motifs for peptides, 570t source of antigen for, 514 Major histocompatibility complex-I molecules high-resolution structures, 509– 510, 511–512 motifs for peptides, 570t Major histocompatibility complex-I v molecules, 515–516, 516f Major histocompatibility complex-like molecules peptides bound by, 506–509, 507–508t structure of, 505–522 Major histocompatibility complex (MHC) antigen processing and presentation, 524–531, 527f classical and neoclassical class I and II, 112–113 class I/II expression, 113 class I/II structure through evolution, 112, 114–115f control of expression, 504–505 discovery of, 36–37 features, 1159–1163, 1159t gene organization, 113–114 immunologic function, 489–493 inhibition by viral infection, 971f Mhc and disease, 501–503, 502t Mhc evolution, 496–497 Mhc genetics, 493–496, 496–497f Mhc polymorphism, 496 molecular interactions, 522 natural killer-cell tolerance and, 421–422 natural killer receptor complexes, 520–521, 520f

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INDEX

nomenclature, 487–489, 488t origins, 123 proteins and, 487–523 role in memory T cell homeostasis, 752–753 structure of, 505–522 target cell, natural killing and, 400–401, 400f transplantation and, 497–499 vertebrates, 112–116 Malaria chemokines and, 704–705 extracellular stages, vaccines, 932 immunopathogenesis of, 930f, 933f liver stages, vaccines, 934 vaccine, 1064–1066, 1066t MALTS. see Mucosal-associated lymphoid tissues Mammals adaptive immunity, 105–106 generation of diversity, 110 Mapping epitopes, 545–546 Marginal zone B cells, 237 Marginal zone macrophages, 453–454, 454f Mast cell-derived mediators, 1140–1142 Mast cells, 468–486, 480–486, 480t, 482f allergic response, 1139–1144, 1140f mediators, 483t in viral infections, 947–948 Measles vaccine, 1052–1053 Medulla, role in negative selection, 350–351 Melanization, 85 Membrane attack complex, 874–875, 874f Membrane immunoglobulin, 130 Membrane regulatory protein, deficiencies, 881–882 Memory B cells, 234–235 differentiation and phenotype, 761–764 evolution, 268–269 generation of, 757–761, 758f germinal centers, 758–759f, 758–760 interplay with plasma cell compartments, 764 longevity of, 763 longevity of humoral immunity, 762 quiescent memory, 762 response, 272–277, 274f Memory (long-lived) plasma cells differentiation and phenotype, 761–762

Paul_Index_final.indd 1277

generation of, 757–761, 758f interplay with memory B cells, 764 longevity of, 763–764 Memory response precursors, 274–275 Memory response to antigen recall, 275–276 Memory T cells generation from effector cells, 362–363 generation of, 743–757, 744f phase I, 745–749 phase II, 749–752 phase III, 752–757 methods of detecting, 745 model of development, 743–745, 744f phenotypic and functional properties, 755–757, 755t programmed cell death, 724 secondary response, 757 TNF superfamily and, 672 transcriptional control of, 751–752 Meningococcus vaccine, 1057–1059 Metchnikoff, Elie, 26–28 Metchnikoff’s legacy, innate immunity, 44–46 Miasma theory of disease, 22 Microbiota, 369 role in mucosal immunity, 845– 848, 845f Micro-ribonucleic acids, phenotypic stability and, 717 MINK, Jnk and Bim in negative selection, 772 Minor histocompatibility antigens, 1161–1162 Missing self-hypothesis, 1162–1163 Modified membrane regulators, as anticomplement drugs, 885–886 Molecules, found in text, 71–72t Monoclonal antibodies antigen-antibody interactions and, 183–214 applications, 210 cross-reactions of, 213 derived from single B cells, 210 gene libraries, 209–210 nucleotide aptamers as alternative to, 212–213 polyclonal versus, 214 specificity, 213 Monoclonal anticomplement antibodies, 887 Monoclonal T cells, 555–556 Monocyte-/macrophage-targeted chemokines, 699–700

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1277

Monocytes, adrenergic neurons modulate responses, 859–860 Monogamous bivalency, 192–193 Monophosphoryl lipid A and derivatives, 1043–1044 Monovalent ligand, antigen-antibody interactions with, 187–192, 187f, 208–214, 208f Mucosal antibodies, 834–835 Mucosal-associated lymphoid tissues (MALT), 60–61 immune response, 61 tonsils and adenoids, 61 Mucosal immune system, 833–849 compartmentalization, 833–834 role of microbiota in, 845–848, 845f viral infections, 961–962 vitamin A and, 838–840 Mucosal inflammation inflammatory bowel disease, 848–849 role of commensals, 847–848 Mucosal mononuclear phagocytes, 836–837, 837f origin, 837 Mucus, 834 Multigene families, 69 Multiple sclerosis, 1089–1092 Multispecificity, 202 Multivalent ligands, antigen-antibody interactions with, 192–193 Mumps vaccine, 1052–1053 Mycobacterial infections, defects in ability to clear, 637–638 Myeloid-derived suppressor cells, tumor immunity and, 1223 Myelosuppressive chemokines, 698

N Naïve T cells, 355–357 abundance, 356–357 generation, 355 recirculation, 355–356 survival, 356 Nasal-associated lymphoid tissue (NALT), 61–62 development, 62 Natural cytotoxicity receptors, 417–418 Natural killer cell-targeted chemokines, 700 Natural killer lectins, phylogeny of, 88–89 Natural killer-like cells, 88 Natural killer (NK) cell receptors, 402–419 activation receptors, 411–418, 428 canonical, 432

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1278

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INDEX

Natural killer (NK) cell receptors (cont.) HMC-independent, 411 immunoglobulin superfamily, 89–90 mechanism of inhibition, 420–421 recognition of epithelial tissues, 418–419 specific for MHC-1 molecules, 402–404, 403–404t, 409–411 virus infections, 951–952, 951t Natural killer (NK) cells, 88, 395–431 dendritic cell interactions and, 430 development, 424–426 effector functions, 398–399 host defense against pathogens and, 427 in human disease, 431 maternal-fetal interactions and, 430 memory-like responses, 429 modulation by virus, 969–970, 969f molecular definition, 397–398 recognition of targets, 399–400 current principles, 401–402, 401f response to HIV, 1022–1023 role in immune responses, 426–428 selective surface markers, 396–397 signal transduction in, 419–421 versus T cells, 395 tolerance, 421–424 licensing hypothesis, 422–424, 423f tumor immunity and, 1219 tumor surveillance and, 426–427 viral infections, 951–952, 951t Natural killer (NK) T cells in allergic disease, 1118 antigenic ligands, 432–433, 433f autoimmunity, inflammation, and allergy, 445 cancer, 446 CD1d-restricted, 432–447, 433t cell-mediated cytotoxicity, 891–892 development and homeostasis, 436–440, 437f functions, 441–442 fungal and parasitic infections, 445 lipid ligands of, 433–435, 434f lipid recognition by, 435–436, 435f liver, intravascular patrolling, 441f microbial infections, 443–445, 444f organ-specific sublineages, 441 tissue distribution and recirculation, 440–441 tumor immunity and, 1219 vaccine adjuvant properties, 432f, 441 viral infections, 445, 951–952, 951t

Paul_Index_final.indd 1278

Natural killing activity, across metazoa, 87–88 Necrosis, 982 Negative selection, self-reactive T cells, 350–352 Neonatal Fc receptor, 515, 515f Nervous system monitoring of immune activity, 853–855 organization of, 850–851, 852f organ system homeostasis, 851–852, 852f systemic cytokine modulation, 854f Neurophysiologic reflex mechanisms, 850–862, 851f Neutrophil receptors, 472, 474t Neutrophils, 369–370, 468–478 angiogenesis and tumor growth, 477–478 in diseases, 478 effector functions, 475–478 generalities, 468–469, 469f granules, 471f in immunoregulation, 476–477 microbial mechanisms, 469–472, 470f resolution phase of inflammation, 475 role in acute inflammation, 472– 473, 473f in viral infections, 947–948 Neutrophil-targeted chemokines, 699 NF-AT, 634 NF-κB, 634 opposing effects of p53, 1227f Nicotinamide adenine dinucleotide phosphate (NADPH), 469f Nicotinamide adenine dinucleotide phosphate-oxidase, 45 NKG2D, 413–416 Nod-like receptors, 375–376 Noncytolytic cells, expression, 892–893 Nonlymphoid organs dendritic cells, 382–384 resident macrophages in, 451–452, 452f, 452t, 455 Notable nucleotide oligomerization domain, 375–376, 375t Notch signaling, 334 Novel neurotrophin-1/B cellstimulating factor-3/ cardiotrophin-like factor, 613–614, 613t Nucleated cells recovery mechanisms, 875f resistance and recovery mechanisms, 874–875, 874f

Nucleic acid-based vaccines, 1040 Nucleotide aptamers, 212–213 Nucleotide-binding domain leucinerich repeat, 77–78, 77f Nucleotide-binding oligomerization domain-containing proteinlike receptors, 848–849, 849f Nuocytes, in atopy, 1129–1130 Nur77, role in negative selection, 771–772

O OB-R, 615–616 Ocular autoimmune diseases, 1092– 1094, 1094f Oil-in-water emulsions, vaccines, 1042–1043, 1046 Oncostatin M, 613–614, 613t Ontogeny, T-cell development, 332–333 Opsonins, 875–877 Oral tolerance, 841 Ouchterlony method, immunodiffusion and, 204– 205, 205f

P p53, opposing effects of NF-κB, 1227f Parasites, 910–936 cellular mechanisms, 912–915, 914f downregulation of innate signaling pathways, 917–918 extracellular, 922–926 global impact and control measures, 911t humoral mechanisms intracellular, 920–922 pattern recognition receptors, 915–917 Parasite vaccine, 931–936 Paratopes, 130 Pasteur, Louis, 25–26 Pattern recognition receptors bacterial recognition, 979f innate immunity, 370–380 parasites, 915–917 Pax 5/B-cell-specific activator protein, 164 Penaedins, 86 Pentamers, 148 Peptide-based vaccines, 1040–1041 Peptide loading, in the endoplasmic reticulum, 527–529, 529f Peptide transport, in the endoplasmic reticulum, 526

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INDEX

Peptide trimming in the cytosol, 525–526 in the endoplasmic reticulum, 526–527 Peptodoglycan-recognizing protein, 80–81, 80f Perforin, 892–893 delivery of cytotoxic molecules into target cells, 898–899, 900f genetic diseases caused by, 897–898 transcriptional regulation of, 894–895 Peripheral T cells intracellular bacteria and, 987–988, 988f programmed cell death and homeostasis, 720–721, 721f tolerance, 773–787 Peripheral T lymphocyte responses and function, 355–366 Peripheral tolerance endogenous peptide antigens, 781–784 exogenous peptide antigens, 775–776 exogenous superantigens, 775 Pernicious anemia, 1095 Peroxidasin, 85 Pertussis vaccine, 1054–1055 Peyer patch, macrophages in, 454 P47 GTPases, 79 pH, antigen-antibody interactions, 185–186 Phagocytes development, inborn errors, 1212– 1214t, 1246–1252 intracellular bacteria and, 977–978, 977f Phagocytosis, 76–77, 980 macrophages and, 448–467 neutrophil-mediated, 473–475, 475f Pharmaceutical allergens, 1115 Phosphatidylinositol 3-kinase, 635 Phosphoinositides (PI), as signaling mediators, 256 Phospholipase A2, 369 Phox homology (PX) domain, 254 P13K, protein tyrosine kinase and, 254 Plasma cells extrafollicular, development, 266–267 pre-germinal center, 275 Plasmacytoid dendritic cells, 386, 391 Plasma regulators, as anticomplement drugs, 887 Platelet-derived chemokines, 698–699 Plekstrin homology (PH) domain, 254

Paul_Index_final.indd 1279

Pneumococcus vaccine, 1056–1057 Poliomyelitis vaccine, 1048–1049 Polyclonal T cells, 554–555 Polymerase λ, 164 Polymerase μ, 164 Polymorphism, 69 Polypeptide, historical perspective, 133 Polysaccharide-based vaccines, 1038 Polysaccharides, immunogenicity of, 545 Porifera, histocompatibility reactions, 116–117 Posttranscriptional regulation, 895 Pre-B-cell receptors, immunoglobulin heavy chain and, 226–229, 228–229f Preyer’s patches, 62, 63f Primary biliary cirrhosis, 1098–1099 Primary sclerosing cholangitis, 1099–1100 Programmed cell death, 718–740, 719f activated by granzymes, 899–901 cellular and molecular mechanisms, 727–731 central role of mitochondrion, 729–730 as immune effector mechanism, 727 immune regulation and, 719–727 as immune therapy, 727 lymphoid malignancy and, 726 lymphoproliferative disease and autoimmunity, 725–726 multicellular organisms, 718–720 precise regulation by families of molecules, 731–738 structural regulation of, 738–740 Programmed necrosis, 730–731 Promyelocytic leukemia zinc finger, 353 Prophenoloxidase cascade, 85 Protease inhibitors, 1018f Protein inhibitors of activated STAT, 637 Protein tyrosine kinase B-cell receptor signaling and, 251 cytoskeletal rearrangements and, 254–255 P13 K and, 254 PLC-γ2 and, 252–253 Protein vaccines, 1037 Proximal signaling, T cells, 308–311 Public idiotypes, 131 Pyroptosis, 983

Q Quantitative precipitin, 203–204, 203f

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1279

R Radioimmunoassay (RIA), 194–199 corrections for B, F, and T, 197, 196f nonequilibrium, 197 sensitivity limitations, 195–196 solid-phase methods, 195 solution methods, 194–195 RAG gene expression, 166 receptor editing and receptor revision, 168 RANK ligand, RANK, and osteoprotegerin system, 676 anti-RANK ligand, 679 Ras-mitogen-activated protein kinase, 312–313, 312f pathway, 635 Rearranging genes, organization of, 99 Rearranging machinery, 123–124 Rearranging receptors, origins, 123–126 Recombinant protein vaccines, 1037–1038 Recombination class switch, 177–182, 180f function of signal sequences, 151– 152, 154f variable assembly, 150–151, 152f V(D)J, 157–168, 158f, 159f Recombination products, 158 Red pulp macrophages, 454 Regression, prognostic significance, 1228 Regulatory T cells, 713–714, 795–832 in allergic disorders, 1119–1120 disease and, 823–832 double-negative, 823 downregulation of dendritic cells, 813f high affinity/avidity interactions, 770–771 induction at mucosal sites, 842–843 infectious tolerance, 823 parasite persistence and, 927–928 plasticity, 819–820 mucosal sites, 842 specialization, 818–819, 818f tolerance and autoimmunity, 784 transcriptional regulation of development, 803–804, 803f Tr1 cells, 821–823 tumor immunity and, 1222 Renal autoimmune diseases, 1100– 1101, 1101t Renal disease, complement in, 885 Reptiles adaptive immunity, 105 lymphoid tissues, 109

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1280

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INDEX

Rerum Rusticarum Libri Tres (On Agricultural Topics), 22 Retinoic acid, infection and immunity, 839–840, 839–840f Retinoic acid-inducible gene receptors, 377–378, 378f, 946f, 965f Reverse immunology, 1208 Rheumatoid arthritis, 1105–1107f, 1105–1108 Rig-I-Like Receptors (RLR), 78 RNA transcription, 175 Roman theories of disease, 22 Rotavirus vaccine, 1053–1054 Rubella vaccine, 1052–1053 Runx3, CD8-specific driver, 348

S Salmonella O antigens, immunochemistry of, 540– 542, 541–542, 541f Salt concentration, antigen-antibody interactions, 185–186 Saponins, 1046 Scatchard analysis, affinity, 187–188, 188–189f Scavenger receptors, 79 Scleroderma (systemic sclerosis), 1108–1109, 1108t Secretory immunoglobulin, 130 Selection theories, 31–32 Self-antigens, 1211–1215 Self/nonself discrimination, 36–37 Self-reactivity, T cells, 768–769 Sepsis, complement in, 884 Severe combined immunodeficiency (SCID) cytokine-related causes, 637 JAK3 mutations, 625 mice, 235–237, 236f molecular mechanisms, 1238–1241, 1241f Side chain theory (Ehrlich), 28–29, 29f Signaling lymphocytic activation molecule, 417 family, 321, 321f Signal transduction in NK cells, 419–421 protein modules in, 254 TNF receptors, 665 Sips plot, 190 Skin dendritic cells in, 383–384 innate immunity and, 368 SMADS, 634 Small molecule complement inhibitors, 887 Smallpox, 26, 27f, 1032–1034

Paul_Index_final.indd 1280

Solitary ascidians, histocompatibility reactions, 118 Somatic generation of diversity, 69–70 Somatic hypermutation, 139, 171–176, 173f B cells, 269 mismatch repair, 173–174 rearrangement or, 124–125 Species differences, T-cell development, 332–333, 333t Spleen architecture and cellular composition, 58–59, 58f chemokines and adhesion molecules, 59–60 development, 60 function, 57–58 plasticity and virus infection, 60 resident macrophages in, 453 Src homology 2 (SH2)domain, 254 SRC homology 3 (SH3) domain, 254 Staphylococcus aureus vaccine, 1064 STATS, 626–628t deoxyribonucleic acid binding and, 631–632, 632f docking, 630–631, 631f evolutionarily old, 633 nonphosphorylated, 632 optimal binding sites, 632 specificity of, 633 transcriptional activation by, 632–633 types of functions mediated by, 633–634 Stem cell factor, 618 Stromal cells, 1201–1203 Subunit vaccines, 1037–1039 Superantigens, 1162 binding to nonclassic V domain, 139 Suppressor T cells history, 795–798, 797–798 tolerance by, 767–768, 795–832 Surface plasmon resonance (SPR), 207–208 Syndromic immunodeficiencies, 1251– 1255, 1252t Systemic lupus erythematosus (SLE), 1109–1111, 1110f

T Taxonomy, 1072–1073 T-cell activation, complement as regulator of, 888 T-cell affinity/avidity, 769–770 T cell anergy, evidence for, 774–775

T-cell differentiation, chemokines and, 702 T-cell identity, 325–326 T-cell polarization, 42 T-cell precursors, double negative compartment, 330 T-cell priming, 745–746 αβ T-cell receptor-ligand recognition, 290–298 binding, 290–293, 291t, 293f topology and cross-reactivity, 293– 294, 294f T-cell receptors (TCR), 96–98, 279– 305. see also TCR αβ cells, positive versus negative selection, 342–345 αβ receptor rearrangement and selection, 327–329, 329t αβ receptor structure, 289–290, 289f, 308 αβ structure, 289, 289f α/β variable domains, 97–98 αβ versus γδ fates, 339–342 activation markers, 326 α/δ locus, 284–285 allelic exclusion, 287, 288f antigen receptor signaling, 358–359 autoreactive αβ cells, 329–330 β locus, 285–286, 285f β-selection, 337–338 CD3, 281–282, 282–283f, 307f CDR3, 287–288 chromosomal translocations and diseases, 288–289, 288t complex, 306 components, 325–326 coreceptors and signal modulators, 326 developmental stage markers, 326 discovery of, 37–38 distinctive paths for αβ cells, 330 diversity, 287 features and immunoglobulin diversity, 304–305, 305f γ/δ, 98 γδ cells multiple lineage of, 341–342 specification, 341 γ/δ cells, viral infections, 948 γδ ligands, 298–302, 300f, 301t γδ loci, 339, 340f γδ structure, 289, 289f genes, 284–290, 284f γ locus, 286 growth factor receptors, 326 new antigen receptor, 92–93, 93f peptide/MHC interactions, 769f polypeptides, 279–281, 280t

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INDEX

proliferation and costimulation, 359 transcription and rearrangement, 286–287 tyrosine kinase, 308f, 308–309 αβ T-cell receptors (TCR) CD4 and CD8 role, 296–297 MHC interactions, 294–295, 294f, 295t, 296f superantigens, 297–298, 299f γδ T-cell receptors (TCR) antigen recognition, 302–303 thymic ligand recognition, 302, 303f T cells breaking tolerance, 786–787, 787f calcium-nuclear factor, 311–312 cell adhesion regulation, 317–318, 317f costimulation, CD28, 318–320, 318f cytoskeletal regulation, 316, 316f development, 325–330, 328f affinity to self-antigens, 344, 344f anatomical organization of, 330–332, 331f effector response molecules, 326–327 inborn errors, 1204–1205t, 1238–1243 intrathymic, 327–330 migration pathways for, 330–331 variations, 332–333 in vitro context for, 333–334 discovery of, 34–35 early lineage choices and commitment, 334–336, 336f emergence of, 126 helper (see Helper T cells) ignorance toward self-antigens, 784 immunologic synapse, 314–315, 314f inhibitory adaptors, 322–323 inhibitory costimulation, 323 negative regulation of activation, 321–323 negative selection, 350–352 Ras-mitogen-activated protein kinase, 312–313, 312f rejected mediated by, 1165–1171, 1166f self-reactivity, 768–769 signaling and activation, 306–324 signal lymphocyte activation molecule family, 321, 321f tolerance, 1074

Paul_Index_final.indd 1281

toperogenic antigens in vivo, 776–779t triggering mechanism, 315–316 viral infections, 952–959 T-cell signaling paradigms, 40 T-cell subsets, 40 T-cell vaccines, 933–934 Temperature, antigen-antibody interactions, 185–186 Terminal deoxynucleotidyl transferase and N regions, 163–164 Terminal pathway (TP), 871–872, 871f component deficiencies, 880 control, 874 protein family, 877–878 Tertiary lymphoid tissues, 64–66, 64f chronic inflammation to lymphoid microenvironments, 64–65 functions, 65–66 plasticity and adaptability, 66 Tetanus vaccine, 1054–1055 Th1 and Th2 variants, 441, 443f mechanisms underlying response, 919–920 Th1 cells, 711 chronic responses, 929 mucosal inflammation and, 848– 849, 849f Th2 cells, 711–712 chronic responses, 930–931 cytokine-producing, as effector cells, 1147–1151 cytokine regulation, allergic inflammation, 1134 immune response, thymic stromal lymphopoietins, 1129, 1129f molecular mechanisms, alternations in, 1133 Th3 cells, 821 Th9 cells, 713 Th17 cells, 712–713 in allergic disorders, 1118–1119 barrier functions and mucosal immunity, 843–845 mucosal inflammation and, 848– 849, 849f Th22 cells, 713 Thermodynamics, antigen-antibody interactions and, 139–140 Thermodynamics of affinity, antigenantibody interactions, 183–184 Thioester group structure, 869f Thioester protein family, 877–878 Th-POK, CD4-specific driver, 347–348 Thrombocytopenia, heparin-induced, chemokines and, 706

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1281

Thrombopoietin, 618 Th subsets, 710t Thymic clonal deletion, 766, 771–772 Thymic deletion, programmed cell death, 720 Thymic dendritic cells, 393 Thymic domains, 330, 331f Thymic tolerance, 768–771 Thymus adhesion molecules and chemokines, 51–52 anatomy, 50, 50f, 331, 331f architecture, 50, 50f cellular composition and functions, 51 development, 52–53 evolution, 110–112 expression of self-antigens, 772–773 functions, 49–50 resident macrophages in, 453 Tissue-specific antigens, 1162 Tissue-specific lymphocyte homing, 702 TL1A-DR3 system, 676 T lymphocytes death, extrinsic or antigenstimulated, 721–723 tumor immunity and, 1218 T lymphopoiesis, 698 Tolerance B-cell, 787–794 central and peripheral, 1073–1074 dendritic cell control of, 393–394 donor-specific, 1177–1192 Fc receptors and, 597–598 immunologic, 765–794 inborn errors, 1260–1262, 1260t natural killer-cell, 421–424 licensing hypothesis, 422–424, 423f oral, 841 peripheral T cells, 773–787 Toll-like receptors, 80f, 81, 371–372, 374f agonist adjuvants, 1044–1045 complement interactions with, 888–889 parasite molecular patterns recognized by, 915t signaling, viral invasion of, 964f signaling pathways, 944–945, 945f Toll receptors, 80f, 81 immune deficiency pathways and, 82 Tonsils, 61 Toxoids, 1037 TRAIL receptor system, 675–676

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1282

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INDEX

Transcription factors B lineage development, 218–222, 220f lymphoid organ development, 56–57 Transforming growth factor-β, 121–122 Transfusion medicine, 29–30 Transitional B cells, 231–233, 232f Transmembrane domains, 148 Transplantation, 35–37 graft rejection, donor antigens for, 1159–1163 heart and lung, 1192–1193 hematopoietic cells, 1194–1195 immunologic issues in, 1198–1199 kidney, 1192 liver, 1192 pancreas and islets, 1194 rejection chemokines and, 706 complement in, 884 diagnosis, 1198–1199 mechanisms of, 1163–1173 physiologic interactions, 1173–1176 prevention, 1176–1192, 1181f sensitized candidates for, 1198 Transplantation immunology, 1154–1199 genetic principles (Laws of Transplantation), 1156, 1156t origin of, 1154–1158 Tr1 cells, 821–823 Treg selection, 351–352, 352f T regulatory cells, 723–724 Triggering mechanism, 315–316 Trigger mechanisms, bacterial invasion, 976–977 Tripartite motifs (TRIM), 78–79 Tuberculosis vaccine, 1061–1064, 1063t Tumor immunity, regulatory T cells, 828–830, 829 Tumor infi ltrates, prognostic significance, 1228 Tumor microenvironment, tumor immunity and, 1220–1221 Tumor models, 1204–1207 Tumor necrosis factor blockade, in inflammatory diseases, 678 in cancer therapy, 679–680 CD40L and BAFF systems, 672–674 death receptors and ligands, 674–675 human genetic diseases and, 677t inhibitors of, 677 ligand family, 659–660, 660t

Paul_Index_final.indd 1282

lymphotoxin family, 667–670 mediated gene induction, 668t mice deficient in, 669t pathways in infectious diseases, 673t programmed cell death, 738–740 RANK ligand, RANK and osteoprotegerin system, 676 receptor-ligand complex, 661–663, 662–663f receptor superfamily, 660–661, 660t, 661–662f, 733–734, 734f serious adverse effects, 678–679 signaling pathways and cellular responses, 663–667, 664f, 666f T cell cosignaling and, 670–672 therapeutics, 677–680, 677t TL1A-DR3 system, 676 TRAIL receptor system, 675–676 TWEAK-FN14 system, 674 virtual orthologs, 663, 663f, 663t Tumor necrosis factor-α, 119 mucosal inflammation and, 848– 849, 849f TWEAK-FN14 system, 674 Two-phase systems, affinities, 193–194 TYK2 mutations, human immunodeficiency and, 625 Type 1 cytokines, 626–628t nomenclature, 601–602 receptors families and their relations, 606–618, 607t, 611f receptors for, 604, 604t structural considerations, 602–604, 602f, 603t Type 2 cytokines, 620–623 Tyrosine kinase, 308f, 309, 635 regulation of, 308–309, 309f

U Ultraviolet light-induced cancers, 1225 Urochordates, histocompatibility reactions, 117–118

V Vaccination history of, 26 induction of lifelong memory through, 741–743, 743f prevent pathology, 935 therapeutic, 1231, 1232f Vaccine adjuvants, 1040–1047, 1042t clinical development phase, 1044– 1047, 1045t licensed, 1042–1044, 1042t

LT and LTK63, 1046–1047 novel combinations, 1046 Vaccines, 1032–1068 challenges for the future, 1067–1068 classification, 1036–1041 dendritic cell-targeted, 394 in development, 1059–1067 development of, 24–26, 1034t helminthes, 935 historical perspective, 1032–1035 HIV, 1029 licensed, 1048–1059 malaria, 1064–1066, 1066t mode of action, 1036f novel technologies for design, 1039–1041 parasite, 931–936 routes of administration, 1047–1048 T-cell, 933–934 tetanus, 1054–1055 varicella, 1052–1053 Variable domain chitin-binding proteins, 86 Varicella vaccine, 1052–1053 Variolation, 26, 1032–1034, 1033f V(D)J recombination, 157–168, 158f, 159f accessibility and transcription, 166–167 in B-cell development, 164–168, 165f V domain, 134–135, 134f constraints on sequence and structure, 136–137 generation by recombination, 135–136 segmental conservation and diversity, 136–137f Vertebrates adaptive immunity, 90–116 hematopoiesis and transcription factors in, 76 VH evolution, 96 VH regions, 98 VH segments, 153–154, 153f Viral antigens, 1215 Viral granzyme inhibitors, 906–907 Viral infection dendritic cells in, 394 spleen and, 60 Viral-like particles, 1038 Virally induced cancers, 1223 Viral receptors, 942t Viral vaccines, 1048–1054 Viral vector-based vaccines, 1039–1040 Virology, 938–944 Virosomes, 1044

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INDEX

Virulence determination, 917–919, 918f Virulence factors, bacterial, 1005–1007 Viruses autoimmunity and, 962–963 cell entry and replication, 941–944 characteristics, 940t classification, 938 degenerative diseases and, 962–963 early recognition, 944–946, 945f, 946f immunity to, 937–972 immunoevasion by, 963–972 persistence, 944 taxonomy of, 939t transmission, 938–941

Paul_Index_final.indd 1283

Vitamin A, mucosal immunity and, 838–840 Vitiligo, prognostic significance, 1228 V(J) domains, 125–126 Vκ locus, 156, 156f

W WHIM syndrome, chemokines and, 705 White pulp macrophages, 454 Whole-killed (inactivated) vaccines, 1037 Wiskott-Aldrich syndrome, 1252–1253 WSX-1/TCCR type I receptor, 638

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1283

X Xenotransplantation, 1195–1198, 1196f X-linked agammaglobulinemia (XKA), 1243–1245 X-linked lymphoproliferative disease, 1264–1265 X-linked severe combined immunodeficiency disease (XSCID), 611, 611t XRCC4, 163

Z Zipper mechanism, bacterial invasion, 976

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CHAPTER 2 REFERENCES

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CHAPTER 18 REFERENCES

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