Nod Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer, And Other Diseases (Molecular Biology Intelligence Unit) [1 ed.] 157059466X, 9781570594663, 9780585408798

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MEDICAL I N T E L L I G E N C E U N I T

2

Edward Leiter • Mark Atkinson

NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer and Other Diseases

R.G. LANDES C O M P A N Y

MEDICAL INTELLIGENCE UNIT 2

NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer and Other Diseases Edward Leiter The Jackson Laboratory Bar Harbor, Maine, U.S.A.

Mark Atkinson University of Florida Gainesville, Florida, U.S.A.

R.G. LANDES COMPANY AUSTIN , TEXAS U.S.A.

MEDICAL INTELLIGENCE U NIT 2 NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer and Other Diseases R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1998 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-466-X

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

NOD mice and related strains: research applications in diabetes, AIDS, cancer, and other diseases / edited by Edward Leiter, Mark Atkinson. p. cm. -- (Medical intelligence unit) ISBN 1-57059-466-X (alk. paper) 1. Diabetes--Animal models. 2. Mice as laboratory animals. I. Leiter, Edward. II. Atkinson, Mark, 1961- . III. Series. [DNLM: 1. Mice, Inbred NOD. 2. Diabetes Mellitus, Insulin-Dependent. 3. Disease Models, Animal. QY 60.R6 N761 1997] RC660.N63 1997 619'.93--dc21 97-31480 CIP

MEDICAL INTELLIGENCE UNIT 2 PUBLISHER’S N OTE

NOD Mice andproduces Related Strains: R. G. Landes Company books in six Intelligence Unit ser ies: M edical, M olecular Biology, N euroscience, TissueE ngineering, Biotechnology and in Environm ental. The Research Applications Diabetes, authors of our books are acknowledged leaders in their fields. Topics are unique; almostand withoutOther exception, Diseases no similar books AIDS, Cancer exist on these topics. Our goal is to publish books in im portant and rapidly changing areas of bioscience for sophisticated researchers and clin ician s. To achieve this goal, we have accelerated our publishing program conform to the fast pace at which ThetoJackson Laboratory inform ation grows in bioscience. of our books are Bar Harbor, Maine,Most U.S.A. published within 90 to 120 days of receipt of the manuscript. We would like to thank our readers for their continuing interest and welcome any comments or suggestions they may have for University of Florida future books.

Edward Leiter

Mark Atkinson

Gainesville, Florida, U.S.A. Judith Kemper Production Manager R.G. Landes Company

R.G. LANDES COMPANY AUSTIN , TEXAS U.S.A.

CONTENTS 1. NOD Mice and Related Strains: Origins, Husbandry and Biology Introduction ........................................................... 1

Edward H. Leiter Development of the NOD and Related Strains ......................... 2 NOD Distribution and the Politics of International Competition ................................................. 4 NOD Strain Characteristics ........................................................ 5 Spontaneous Development of Insulin Dependent Diabetes Mellitus ..................................................................... 6 Differences in Characteristics of NOD Substrains .................... 7 Clinical and Pathophysiologic Features of IDDM in NOD Mice ........................................................................... 8 T- Lymphoaccumulation and the Possible Origins of Insulitis ................................................................. 10 Other NOD Strain-Characteristic Immunodeficiencies ......... 12 Leukocytic Infiltrates in Non-Pancreatic Glandular Tissues .................................................................. 15 Miscellaneous NOD Strain Characteristics ............................. 17 Reproductive and Developmental Biology .............................. 18 Characteristics of NOD-Related Strains .................................. 19 Appropriate Controls for NOD Mice ...................................... 26 2. Genetics and Immunogenetics of NOD Mice and Related Strains .................................................................... 37

Edward H. Leiter Introduction .............................................................................. 37 NOD and the Genetics of Autoimmunity ............................... 39 The H2g7 Haplotype: A Scaffolding for Initiation of Insulitis and Diabetes ........................................................ 43 Rare Allelles Do Not Necessarily Equate with Diabetogenic Allelles: the TAP Gene Imbroglio ......................................... 47 Genetic Analysis of Insulitis: IDDM Susceptibility Inherited as a Threshold Liability ......................................................... 49 Genetic Segregation Analysis as a First Step in Identification of Non-MHC Idd LOCI ........................................................ 50 Speed Cogenics .......................................................................... 52 Congenic and Subcogenic Stocks Indicate the Presence of Multiple Non-MHC Susceptibility LOCI on Numerous Chromosomes ............................................... 53 Connecting Idd Loci with Immunophenotypes ...................... 56 Do NOD Islets Express Strain-Specific Idd Genes? ................. 58 Lessons From Genetics of IDDM in NOD Mice for the Genetic Prediction of IDDM in Humans............................. 59

3. The Identity and Ontogenic Origins of Autoreactive TLymphocytes in NOD Mice ................................................... 71

David V. Serreze Introduction .............................................................................. 71 Overview of T cell Ontogeny and Selection ............................. 71 The Diabetogenic Role of CD4+ Versus CD8+ T cells in NOD Mice ......................................................................... 75 TCR Gene Rearrangements Associated with β cell Autoreactivity in NOD Mice................................................. 78 Pancreatic β cell Autoantigens Targeted by Diabetogenic T Lymphocytes in NOD Mice ............................................... 83 Mechanistic Basis for the Development of Autoreactive T cells in NOD Mice .............................................................. 86 Conclusions ............................................................................... 90 4. The Immunopathogenic Roles of Antigen Presenting Cells in the NOD Mouse ......................................................... 101

Michael Clare-Salzler Introduction ............................................................................ 101 The Contribution of Hematopoietically Derived APC to the Development of Diabetes Susceptibility in the NOD Mouse .............................................................. 102 The NOD H2g7 .............................................................................................................................. 102 APC Co-Stimulatory Molecules, CD80 and CD86 ............... 104 APC Subpopulations ............................................................... 106 B Lymphocytes ........................................................................ 107 Macrophages............................................................................ 107 Dendritic Cells ......................................................................... 112 Conclusions ............................................................................. 113 5. The Natural History of Islet-Specific Autoimmunity in NOD Mice ........................................................................... 121

Jean-François Bach The Multi-Faceted Islet Specific Response ............................ 121 T Cell Repertoire ..................................................................... 126 The Nature of the β Cell Lesion .............................................. 128 Hypotheses on the Triggering of the β Cell Specific Response ................................................................. 130 Transient Early Protection from Diabetes by CD4 Immunoregulatory T Cells .................................... 130 The Search for Early Thymus and Bone Marrow Anomalies: An NK1+ T Cell Defect ........................................................ 132 Conclusions ............................................................................. 135

6. NOD Mice as a Model for Therapeutic Interventions in Human Insulin Dependent Diabetes Mellitus .................. 145

Mark A. Atkinson The Prevention of IDDM in Humans; Purpose and Historical Perspective ................................................... 145 The NOD Mouse as a Model for Prevention of IDDM in Humans ........................................................... 147 Genetic and Environmental Factors: Effect on Interpretation of Outcome Measures ........................... 148 Therapeutic Interventions in NOD Mice .............................. 150 Immunosuppression ............................................................... 152 Tolerance ................................................................................. 153 Immunostimulation ................................................................ 156 Reducing β Cell Metabolic Activity ........................................ 157 Dietary/Hormonal Manipulation .......................................... 158 Anti-inflammatory Agents ...................................................... 159 Human Trials for IDDM Prevention ..................................... 160 Conclusions ............................................................................. 162 7. The Use of NOD/LtSz-scid/scid Mice in Biomedical Research ........................................................... 173

Dale L. Greiner, Leonard D. Shultz Phenotypic Characteristics of scid Mice ................................. 173 Defective DNA Repair ............................................................. 174 Robust Innate Immunity ........................................................ 175 Care and Management of scid Mouse Colonies .................... 175 Utility of C.B-17- scid Mice as Research Tools ....................... 176 Characteristics of NOD/Lt/Sz- scid Mice ................................ 178 NOD/LtSz- scid Mice in Diabetes Research ............................ 181 Induction of Chemical Diabetes in NOD/LtSz- scid Mice ..... 184 NOD/LtSz- scid Mice as Islet Graft Recipients ....................... 185 Human Cell Engraftment in NOD/LtSz- scid Mice ............... 186 Engraftment of Hemopoietic Stem Cells ............................... 187 Epstein Barr Virus (EBV)-Related Tumors ........................... 189 Human Immunodificiency Virus-1 (HIV-1) Infection of Hu-PBL-NOD- scid Mice ................................................ 190 NOD/LtSz- scid Mice as Models for Human Tumor Grafts ....................................................................... 191 Use of NOD/LtSz- scid Mice in Parasitic Research ................ 191 New Models of NOD/LtSz-Immunodeficient Mice .............. 193 Conclusions ............................................................................. 194 Index ......................................................................................... 205

EDITORS Dr. Edward Leiter Dr. Leiter is a Senior Staff Scientist at The Jackson Laboratory in Bar Harbor, Maine. He earned a B.S. in Biology from Princeton University, and both M.S. and Ph.D. degrees in Biology/Cell Biology from Emory University. His research interests include the genetics and pathogenesis of insulin dependent and non-insulin dependent diabetes, and β cell metabolism. Chapters 1, 2 Dr. Mark Atkinson Dr. Atkinson is an Associate Professor of Pathology and Director for The Center for Immunology and Transplantation at The University of Florida College of Medicine in Gainesville, Florida. He earned a B.A. in Microbiology at The University of Michigan and a Ph.D. in Pathology from The University of Florida College of Medicine. His research interests are primarily focused on insulin dependent diabetes and include studies of cellular immunity, environmental factors associated with variances in the incidence of the disease, and designing interventions for disease prevention. Chapter 6

CONTRIBUTORS Dr. Jean-Francois Bach Dr. Bach is a Professor and Director of the Autoimmunity Section at the Hospital Neker in Paris, France. His research interests include many aspects of immunology (especially those relating to lymphocyte development and function), autoimmunity, genetic susceptibility, and interventions aimed at preventing insulin dependent diabetes. Chapter 5 Dr. Michael Clare-Salzler Dr. Clare-Salzer is an Associate Professor of Pathology and Medicin e at The Un iversity of Florida College of Medicin e in Gainesville, Florida. He earned a B.S. in Chemistry from The University of Notre Dame and an M.D. from The State University of New York (SUNY) School of Medicine. His research interests include various aspects of the pathogenesis of insulin dependent diabetes, macrophage function, and sepsis. Chapter 4

Dr. Dale Greiner Dr. Greiner is a Professor of Medicine in the Diabetes Division at The University of Massachusetts Medical School in Worcester, Massachusetts. In addition, he holds a joint appointment with The Department of Pathology at The University of Connecticut Health Center. He earned a B.A. in Biology and a Ph.D. in Microbiology and Immunology from The University of Iowa. His research interests include lymphocyte development and function, autoimmunity, and AIDS. Chapter 7 Dr. David Serreze Dr. Serreze is an Associate Staff Scientist at The Jackson Laboratory in Bar Harbor, Maine. While at The University of Maine, Dr. Serreze earned a B.S. in Biology, an M.S. in Microbiology, and a Ph.D. in Microbiology. His research interests include immunoregulation, function of class I major histocompatibility molecules, and the genetics of insulin dependent diabetes. Chapter 3 Dr. Leonard Shultz Dr. Shultz is a Senior Staff Scientist at The Jackson Laboratory in Bar Harbor, Maine. In addition, he holds joint appointments as a Graduate Faculty Member in Zoology at The University of Maine and a Research Professor at The University of Massachusetts Medical School. He earned a B.A. in Biology from Northeastern University and a Ph.D. in Pathogenic Bacteriology at The University of Massachusetts Medical School. His research interests include the development and regulation of the immune system, AIDS, and tumor immunology. Chapter 7

PREFACE Serendipity - making a fortunate or unexpected discovery by accident. iscovery of a female mouse with autoimmune, insulin dependent diabetes mellitus (IDDM) in an incipient inbred line initially selected for normoglycemia was surely a serendipitous event. The subsequent selection for IDDM in the progeny of this female produced the current Nonobese Diabetic (NOD) strain. Prior to development of the NOD strain, the only animal model of autoimmune IDDM predictably developing the disease at high frequency was the BioBreeding (BB) rat. The advent of the NOD mouse provided researchers working in the field of autoimmunity with the needed perspective to assess findings in BB rats relative to what is known about pathogenic processes in humans. There are common etiopathogenic features observed in all three genera, as exemplified by involvement of susceptibility conferring major histocompatibility complex (MHC) haplotypes at the genetic level and of autoreactive T cells at the effector level. At the same time, there are sufficient distinctions between the rat and mouse models to remind the clinical investigator that rodents can only model various aspects of a human disease syndrome, while at the same time exhibiting speciesunique features. For example, resistance to development of severe ketoacidosis—despite severe hyperglycemia—is a genus-specific feature peculiar to the mouse model. Many generations of strict inbreeding have produced unusual phenotypes in both rodent models that distinguish them from each other, and probably from most humans destined to develop IDDM. The diabetes-prone BB rat is severely T-lymphocytopenic in peripheral lymphoid organs whereas the NOD mouse reflects the other extreme: T-lymphoaccumulation. Yet both BB rats and NOD mice develop a veritable “Pandora’s box” of organ-specific lymphocytic infiltrations, showing that immunoregyulatory pathways are compromised in both models, albeit in different ways. In-depth analysis of as many animal models as possible enhances an investigator’s appreciation of the etiologic complexity of IDDM in humans. Given the differences as well as the similarities between IDDM development in BB rats and NOD mice, a diabetes prophylactic treatment effective in both models becomes a potential treatment to prevent IDDM in humans. The Diabetes Prevention Trial using prophylactic insulin treatment discussed in chapter 6 is an illustration of how promising results obtained in several animal models can be translated into human clinical trials.

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As detailed in chapter 5, the NOD mouse has been especially instructive for exploration the relationship between insulitis developNOD Mice and of Related Strains: Research Applications ment and the in ultimate expression of clinical disease. Insulitis in NOD Diabetes, AIDS, Cancer and Other Diseases mice entails the selective destruction of β cells following infiltration of the pancreatic islets by leukocytes (principally macrophages, T cells, and B lymphocytes). Certain of the genetic defects permitting development of destructive insulitis have been traced to bone marrow derived antigen presenting cells. The fact that IDDM susceptibility “tracks” with NOD hemopoietic stem cells makes the NOD mouse an attractive model to apply gene therapy approaches. Defects in T cell repertoire selection, discussed in chapters 3 and 4, have been correlated with specific immunophenotypic defects. If the only contribution of the NOD mouse were to the advancement of our understanding of the genetic and pathophysiologic basis for autoimmune endocrinopathies such as insulitis, thyroiditis, lupus and sialoadenitis, it would be a major contribution indeed. However, as pointed out in chapter 1 of this volume, the contributions of the NOD mouse to immunologic research comprise but a small aspect of its value to science. A genetically well-characterized inbred strain that is also phenotypically well-studied provides researchers with an important tool for dissecting how genes interact with the environment to control not only autoimmune phenotypes, but also many ISBN: 1-57059-466-X others. As described in the first several chapters of this volume, the genome of the NOD mouse has been intensively studied, and as described in chapter 6, this strain’s physiologic, endocrinologic and immunologic responses to multiple aspects of the physical environment have been published. The astounding breeding performance of the NOD mouse, coupled with the approximately 50% polymorphism in genomic simple sequence repeats when compared to those of other inbred strains, should bring this strain to the attention of all mouse geneticists doing physical mapping projects. MHC-congenic stocks of NOD mice are available that are IDDM-resistant, but which retain the high reproductive potential of standard NOD mice. These mice are ideal for outcross with other inbred strains carrying single gene mutations that can be physically mapped NOD mice andpositionally related strains: research in diabetes, (and thence cloned) in applications genetic segregation analyses. The AIDS, cancer, and / edited by Edward Leiter, Mark Atkinson. NOD genome willother also diseases be useful for genetic control of multigenic disorcm. -- (Medical intelligence unit) ders,p.including deafness and inflammatory bowel disease. ISBN 1-57059-466-X (alk. paper) The deficiencies in immunoregulation discussed in chapters 3 and 1. Diabetes--Animal models.mouse 2. Micetoasthe laboratory animals. 4 should bring the NOD attention of cancer researchers. I. Leiter, Edward. Atkinson, Mark, 1961- . III. Possibly due to aII. dysfunctional population ofSeries. natural killer cells, NOD 1. Mice, NOD. 2.of Diabetes Insulin-Depenmice[DNLM: are subject to Inbred development a wideMellitus, spectrum of neoplasms. dent. 3. Disease Models, Animal.quite QY 60.R6 1997]strains, are relatively Among these, osteosarcomas, rare N761 in inbred RC660.N63 common in1997 NOD/Lt mice. Thymic lymphomas affect 100% of NOD619'.93--dc21 97-31480 scid/ scid mice, but this development is circumvented in NODRag/ Rag CIPdescribed mice. The defective innate immune functions in NOD mice,

in chapters 2 through 4 of this volume combined with single gene mutations eliminating adaptive immune function (e.g., scid and Rag) is proving to be one of the most important biomedical applications of the N O D EDICAL gen om e. As d escr ibed in ch ap ter 7, th ese dou bly immunocompromised stocks of mice are being further genetically modiNTELLIGENCE fied to abet the growth of normal and neoplastic human cells and tisNIT mice are dubbed “Hu-SCID” for humanized severe comsues. These bined immunodeficiency mice. Recent investigations have demonstrated the value of these mice for the study of human infectious diseases, including AIDS, filiariasis and malaria. The genomes of these NOD HuSCID mice are continually being modified by transgenic insertion of human genes to promote the growth of human hematopoietic cells, coupled with simultaneous elimination of selected murine genes that impair human cell development and survival. Continued modification of these mice may facilitate identification of children with pre-autoimmune diabetes by tracking adoptively transferred peripheral blood T cells to the host islets. The writers of the component chapters recognize that, because of the intense research activity involving NOD mice, the literature described will soon be outdated. What the authors have attempted, however, is to present, in an overview format, the literature current at the time of writing. Hence, information in this volume should permit the reader to incorporate the most recent published findings into a conceptual frameTheframework Jackson Laboratory work. When a conceptual is presented, some bias is inherent. The views expressed are based upon each author’s personal experiBar Harbor, Maine, U.S.A. ence working with the NOD model. As scientific knowledge increases, concepts will evolve. We acknowledge and gratefully thank our families who have supported us even when theUniversity NOD mouse distracted us from spending of has Florida more time with them. The staff at Landes are also thanked Gainesville, Florida, Bioscience U.S.A. for their patience and forbearance when deadlines were not met. Finally, the people who comprise The Juvenile Diabetes Foundation International “family” deserves a special “thank you” since many, if not most, of the pilot studies performed on the NOD mouse outside of Japan were supported by the generosity of this very special Foundation.

M I U

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NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer and Other Diseases Edward Leiter

Mark Atkinson

Edward Leiter, Ph.D. Mark Atkinson, Ph.D. R.G. LANDES COMPANY

AUSTIN , TEXAS U.S.A.

ABBREVIATIONS NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer and Other Diseases

AIDS Acquired immunodeficiency syndrome AMLR Autologous mixed lymphocyte reaction APC Antigen presenting cell BCG Bacille Calmette-Guerin BSA Bovine serum albumin CFA Complete Freund’s adjuvant CTS Cataract Shionogi DTH Delayed type hypersensitivity EBV Epstein Barr virus GABA Gamma aminobutyric acid GAD Glutamic acid decarboxylase Hsp Heat shock protein HIV Human immunodeficiency virus IFN Interferon Ig Immunoglobulin IL Interleukin Idd Insulin dependent diabetes susceptibility loci, mouse ISBN: 1-57059-466-X IDDM Insulin dependent diabetes mellitus LPS Lipopolysaccharide NK Natural killer MHC Major histocompatibility complex NOD Nonobese diabetic NON Nonobese nondiabetic NOR Nonobese resistant RBC Red blood cell RT PCR Reverse transcriptase polymerase chain reaction PBMC Peripheral blood mononuclear cell PGE2 Prostaglandin E-2 PKC Protein kinase C scidmice and related Severe combined immunodeficiency NOD strains: research applications in diabetes, SMLR mixed leukocyte reaction AIDS, cancer, and Syngeneic other diseases / edited by Edward Leiter, Mark Atkinson. STZ Streptozocin p. cm. -- (Medical intelligence unit) SPF1-57059-466-X Specific pathogen free ISBN (alk. paper) TCR T cell receptor 1. Diabetes--Animal models. 2. Mice as laboratory animals. Th Edward. II. T helper I. Leiter, Atkinson, Mark, 1961- . III. Series. TNF Tumor necrosis [DNLM: 1. Mice, Inbred NOD.factor 2. Diabetes Mellitus, Insulin-DepenVDJC Variable, Diverse, Joining, Constant dent. 3. Disease Models, Animal. QY 60.R6 N761 1997] regions RC660.N63 1997 619'.93--dc21

97-31480 CIP

CHAPTER 1

NOD Mice and Related Strains: Origins, Husbandry and Biology Introduction Edward H. Leiter

onobese diabetic (NOD) is a recently generated inbred strain with a unique susceptibility to spontaneous development of autoimmune, insulin dependent diabetes mellitus (IDDM). Since their initial description in 1980 by Dr. Susumu Makino, NOD mice have been widely distributed and studied. These mice have provided important new immunogenetic and pathophysiologic insights into autoimmune disease and its prevention. The NOD genome is currently one of the most extensively characterized genomes of extant inbred strains. The strain is widely utilized in the analysis of how polygenically controlled immune defects confer susceptibility to IDDM. Multiple genes within its unique H2g7 major histocompatibility (MHC) haplotype confer the major component of IDDM susceptibility, consistent with the major contributions of MHC to autoimmune diseases, including IDDM, in humans. NOD mice have proven valuable in immunogenetic analysis to elucidate the pathogenic interactions between MHC and non-MHC diabetogenic loci, termed Idd loci (for Insulin Dependent Diabetes). However, interest in this strain is not limited to diabetes researchers. NOD mice develop a wide variety of organ-specific leukocytic infiltrates in addition to insulitis, and are susceptible to a broad spectrum of neoplasms. As detailed in the chapter by Greiner and Shultz in this volume, stocks of NOD mice congenic for the severe combined immunodeficiency ( scid) mutation are proving exceptionally useful for

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NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer and Other Diseases, edited by Edward Leiter and Mark Atkinson. © 1998 R.G. Landes Company.

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NOD Mice and Related Strains: Research Applications in Diseases

growing both normal and neoplastic human tissues. The origin of the NOD mouse strain is fortuitous, the lucky combination of a prepared mind and serendipity. The fact that NOD mice were initially not released to the international research community willingly, and that certain NOD-related strains of obvious scientific value are still embargoed, highlights how corporate involvement in the development of an animal model of biomedical significance can adversely affect scientific progress. The history of this strain’s development in Japan, as well as the development of related strains, will be recounted briefly below. DEVELOPMENT OF THE NOD AND RELATED STRAINS Although the NOD strain was developed in Japan, its heritage is international. NOD and related strains are derived from “Swiss” mice. “Swiss” mice were outbred, originating in Paris, but called “Swiss” because of the importation to the Rockefeller Institute for Medical Research in New York by Dr. Clara Lynch in 1926 of 2 albino males and 7 females from a colony maintained in Lausanne, Switzerland. The Rockefeller colony served as a source of “Swiss” mice to both research institutions and commercial breeders in the United States. SWR/ J and SJL/J represent two standardized strains derived from inbreeding progeny of Lynch’s “Swiss” mice. In 1947, Dr. Theodore S. Hauschka at the Institute for Cancer Research in Philadelphia received randombred “Swiss” mice from a commercial dealer and established the Ha/ ICR outbred stock. The stock was purposely kept outbred for cancer research purposes to reflect the genetically heterogeneous human population. In 1957, a commericial supplier, Charles River Laboratories, took Ha/ICR breeding stock and today markets their progeny as CD-1®. Both ICR and CD-1®mice have been widely used in research in Japan since the Second World War and are distributed there as Jcl:ICR by CLEA, Japan, and CD-1®:Crj (Charles River, Japan), respectively. In some publications, the ICR strain descriptor is occasionally (and erroneously) identified as “Imperial Cancer Research” instead of Institute for Cancer Research.2 The process of inbreeding ICR mice to produce new inbred strains in Japan continues to the present. Two of the more recently produced strains are ALS and ALR, selected respectively for susceptibility and resistance to the diabetogenic agent, alloxan.3

NOD Mice and Related Strains: Origins, Husbandry and Biology

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The NOD and certain of the NOD-related inbred strains were developed from Jcl:ICR progenitors at the Shionogi Research Laboratories in Aburahi, Japan by Dr. Makino.1,4,5 Selection for cataract development was the initial goal of the breeding program. Inbreeding was begun in 1966 by Ohotori6 with progeny from an outbred Jcl:ICR female mouse exhibiting cataracts and microphthalmia. This selective breeding program led to the development of a strain in which all m ice develop cataracts (now designated CTS, for Cataract Shionogi), as well as a cataract-free control strain separated at the 4th generation of inbreeding (designated NCT, both now beyond 100 generations of brother x sister matings). At the 6th generation of inbreeding (F6), two additional sublines, both free of cataracts, were initiated with the intent of developing a model for spontaneous diabetes development. At F6, Makino noticed some individuals exhibited high fasting blood glucose levels. By selective breeding of F6 sibs and their progeny that exhibited this phenotype, Makino hoped to develop a new inbred strain exhibiting spontaneous diabetes. At the same time, he recognized the need for a euglycemic control strain, and simultaneously inbred F6 sibs and their progeny exhibiting normal fasting blood glucose. In 1974, at the 20th generation of inbreeding (F20), a female spontaneously developed overt IDDM associated with heavy leukocytic infiltrations within the pancreatic islets (termed insulitis). Paradoxically, this female was not found in the line being selected for fasting hyperglycemia. Instead, the female with spontaneous IDDM was found in the line being inbred as a diabetes-free euglycemic control line. The progeny of this diabetic female were the founders of the Nonobese diabetic, or NOD strain. Since the line originally selected for high fasting glucose never progressed to overt diabetes at the time that IDDM appeared in the euglycemic control (now the NOD) line, the former strain was designated NON, for Nonobese normal,1 and more recently, redesignated as Nonobese nondiabetic.7 Obviously, it is quite serendipitous that Makino co-selected a euglycemic “control” line, and that his screening of this line was sufficiently rigorous so that the “exceptional” female was discovered and her progeny then selected to produce the NOD strain. Further adding to the luster of Makino’s achievement was that he maintained a husbandry environment sufficiently pathogen-free such that the diabetic phenotype could develop (see the section below on the critical effects of environment on the penetrance of the diabetic phenotype in NOD

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NOD Mice and Related Strains: Research Applications in Diseases

mice). It should be noted that while subsequent inbreeding of ICR mice in Japan has led to other inbred strains, including the ILI8 (ICRL line-Ishibe), NSY (Nagoya-Shibata-Yasuda),9 ALS (alloxan-induced diabetes susceptible),3 and ALR (alloxan-induced diabetes resistant),3 none of the latter have developed spontaneous, autoimmune IDDM. Thus, Makino’s achievement is truly remarkable. NOD DISTRIBUTION AND THE POLITICS OF INTERNATIONAL COMPETITION Makino’s first two publications describing the NOD strain 1,10 appeared in Experimental Animals, a Japanese journal not widely seen by the diabetes and immunology research communities outside of Japan. A letter was sent by this author to Dr. Makino in December of 1980 asking whether it might be possible to import CTS and its related diabetic and non-diabetic substrains to The Jackson Laboratory for genetic analysis. A reply from Dr. Makino indicated that his employer, the Shionogi Company, a large Japanese pharmaceutical concern, had formed a “Committee on NOD Mice” and that the committee had decided to restrict distribution of these new strains within Japan. Investigations on diabetogenesis in NOD mice were carried out almost exclusively in Japan between 1980 and 1984 among scientists that constituted an “NOD Mouse Study Group” which met annually and published the proceedings of their annual meetings. During this period, the T cell basis for the autoimmune insulitis in NOD mice was firmly established. The culmination of this “All Nippon” approach to scientific progress was a volume on the NOD mouse, Insulitis and Type 1 Diabetes: Lessons from the NOD Mouse,11 published in 1986. This volume essentially summarized most of the work done in Japan on NOD mice during the four year “embargo” period. Two events opened the door to NOD’s worldwide immigration. The first was a set of letters in 1983 to the Shionogi Company from editors of the major Western diabetes-focused journals, Diabetes and Diabetologia, indicating that no further manuscripts on the NOD model would be accepted until the distribution barrier was dropped. The second event was the “unscheduled” migration of NOD mice to institutions in Australia (the NOD/Wehi strain progenitors, 1984) and the United States. A colony established at the University of California, Los Angeles by Dr. Yoko Mullen was the source of the current NOD/ Ym, NOD/Mrk, and NOD/MrkTac (Taconic Farms, Inc.) substrains. NOD and NON brought by Dr. M. Hattori from a colony in Kyoto to

NOD Mice and Related Strains: Origins, Husbandry and Biology

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the Joslin Diabetes Center in Boston in 1984 (NOD/Jos and NON/ Jos) also provided two breeding pairs of NOD and three breeding pairs of NON mice to the author at The Jackson Laboratory (the nucleus of NOD/Lt and NON/Lt). Because of the exceptional breeding performance of NOD mice, it was soon possible to supply pedigreed breeding pairs of NOD/Lt mice to numerous investigators in the United States, Canada, Europe and Australia. By 1986, the Central Laboratory for Experimental Animals (CLEA, Japan) was receiving NOD/Shi breeding stock from the Shionogi Company’s source colony for international distribution. Research grants funded by the Juvenile Diabetes Foundation International provided many investigators with the financial support necessary to begin their investigations on the NOD mouse, such that by the time of the publication of the information obtained by the “NOD Study Group” in Japan,11 the international literature was burgeoning with publications on the im munopathogenesis of diabetes in the NOD mouse. In the five year period between 1986 and 1990, there were 192 entries on the NOD mouse in the bibliographic database of the National Library of Medicine (MEDLINE); in the ensuing five years between 1990-1995, there have been in excess of 669 entries in this database. A scientific meeting sponsored by the Juvenile Diabetes Foundation International in 1989 brought Japanese and non-Japanese NOD researchers together for the first time to share the benefits of international scientific cooperation. Currently, in addition to NOD/Shi distributed by CLEA, Japan, inbred NOD substrains are distributed in the United States by The Jackson Laboratory (NOD/LtJ and NOD/LtSz- scid) and Taconic Farms, Inc. (NOD/MrkTac). In Europe, NOD mice are distributed by Bomholtgard, Denmark. NON/LtJ and NOR/LtJ, an NOD-related strain (see below) are distributed by The Jackson Laboratory. At the time of this writing, open distribution of CTS/Shi mice is still forbidden by the Shionogi Company despite multiple publications by Shionogi investigators in the international literature. NOD STRAIN CHARACTERISTICS Many of the observations provided in this section were obtained by observation of the author’s own substrain, NOD/Lt, at The Jackson Laboratory. Many details of the immunopathogenesis of IDDM will be omitted from this section, since they comprise the focus of later chapters in this volume.

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NOD Mice and Related Strains: Research Applications in Diseases

SPONTANEOUS DEVELOPMENT OF INSULIN DEPENDENT DIABETES MELLITUS As would be inferred from their name, the most notable strain characteristic of NOD mice is their unique susceptibility to spontaneous development of IDDM in the absence of any cytopathic pancreatotropic viruses or other viruses. The frequency of diabetes achieved in any colony is critically dependent upon the specific pathogen-free (SPF) status of the colony.12-14 Whereas the Kilham rat virus is known to be a diabetogenic trigger in certain strains of inbred rats,15 exposure of NOD mice to a variety of murine pathogenic viruses prevents rather than triggers IDDM.12-14,16 During the initial period of NOD investigations in Japan, destruction of pancreatic b cells was firmly established as a T cell-mediated process (reviewed in ref. 5; also see other chapters in this volume). Insulitis initiates as leukocytic aggregates at the perimeters of islets (“peri-insulitis”). CD4+ T cells are the predominant leukocyte, followed by smaller numbers of CD8+ T cells, B cells and macrophages.17,18 As reviewed in chapter 3 both CD4+ and CD8+ T cell subsets are essential mediators of the spontaneously-developing disease. B cells and macrophages also are necessary contributors to pathogenesis. Autoantibodies, presumably maternal in origin, have been detected on b cells from 3-week-old NOD/Uf mice.19 As insulitic destruction of islets progresses, autoantibodies to a variety of b cell antigens, including insulin and glutamic acid decarboxylase (GAD) are generated. Although these autoantibodies are not central to the process of b cell destruction, the absence of IDDM in a congenic stock of B cell-deficient NOD/Lt mice establish that B cells are essential antigen presenting cells for both initiation and amplification of T cell responses to b cell autoantigens.20 The extent and severity of insulitis, as it progresses from a non-destructive peri-insulitis to a destructive intra-islet infiltration, is used as a semi-quantitative method to assess the efficacy of various antidiabetic therapies. A guide for scoring the various stages of insulitis is available.21 Although the incidence of IDDM in NOD/Shi is much higher in females (>80% ) than in males ( 250 mg/dl) and glycosuria (>1/4%) without any administration of insulin. Plasma insulin levels of young normoglycemic NOD mice of both sexes usually range between 1-1.5 ng/ml (25-38 µU/ml). When chronic hyperglycemia is established, plasma insulin levels fall below the range of assay detection (200 days) mice of both the NOD/Wehi and NOD/Lt substrains.98 In the low diabetes incidence NOD/Wehi substrain, 14/17 non-diabetic mice were affected by 550 days. TUMORS Aging NOD mice have been described as a veritable “Pandora’s Box” of aging-associated pathologies, including tumors.99 The description below will be limited to the authors’ NOD/Lt colony. It is rare for an NOD/Lt mouse that has not developed diabetes to survive beyond 1.5 years. Consistent with the presence of multiple immunodeficiencies in NOD/Lt mice, including the lack of functional NK cells, a broad variety of neoplasias were observed.13,99 In a relatively small sampling population of 54 diabetes-free mice, of which only 39 were older than eight months of age, the following neoplasms (number of cases in parenthesis) were detected: lymphomas and lymphosarcomas (5), osteosarcom as an d osteochon drosarcom as (3), myoepitheliocarcinoma (1), rhabdomyosarcoma (1), mammary carcinoma (1) and hepatoma (1). Only one type of tumor was detected in a single individual. As mentioned above and as discussed in chapter 7 by D.G reiner and L.D. Shultz, thymic lymphomas are the major cause of mortality in NOD/LtSz- scid/ scid mice maintained under SPF conditions.

REPRODUCTIVE AND DEVELOPMENTAL BIOLOGY NOD mice are excellent breeders. They mate at a young age and, for an inbred strain, produce exceptionally large litters (i.e., 9-14 pups). Little pre-weaning mortality in litters is experienced as long as the dam remains IDDM-free. In high incidence colonies, females seldom

NOD Mice and Related Strains: Origins, Husbandry and Biology

19

deliver more than two litters before females develop IDDM. Offspring of diabetic dams are larger at birth, making NOD mice a model for diabetic pregnancy.100 Testicular alterations in diabetic males have also been reported, including germ cell degeneration, tubular fibrosis and calcification, and disruption of spermatogenesis.101 High incidences of developmental anomalies have also been reported in embryos of diabetic dams.102,103 Injection of a single dose of 50 µl of Complete Freund’s Adjuvant into one hind footpad of each breeder female and male prevents IDDM onset in NOD/Lt females until after at least two and usually more litters have been born and weaned.13 Although NOD females are good natural ovulators, they do not superovulate well in response to the standard doses of gonadotropins used to superovulate other inbred strains, and the embryos obtained following treatment with gonadotropins are fragile and do not survive microinjection/reimplantation. Thus, while transgenes can be inserted directly into the NOD genome instead of that of hybrid strain combinations, yields of founder transgenic mice are low. Further, NOD preimplantation embryos do not progress beyond the two-cell stage when cultured in vitro in conventional culture media, but will develop to blastocyst in more advanced media.21 The important features of these media are reduced sodium, increased potassium, decreased glucose and phosphate, and addition of EDTA (Dr. John Eppig, The Jackson Laboratory, personal communication). CHARACTERISTICS OF NOD-RELATED STRAINS Because certain of the NOD-related strains described above are often used for genetic, immunologic or physiologic comparison to NOD, a brief description of their strain characteristics will be included here, while salient genetic features of each strain will be covered in the next chapter. NON (N ONOBESE N ONDIABETIC) The inbred strain developed from the ICR-derived line with the high fasting blood glucose levels has been designated NON (Nonobese nondiabetic).5 Because of its relatedness to the NOD strain, NON mice are extremely useful for genetic analyses since they apparently share with NOD, some, but not all of the diabetogenic loci predisposing to diabetes. 104,105 Aging NON/Lt m ice develop sm all foci of perivascular/periductular leukocytic infiltrates into the pancreas that do n ot progress to destr u ctive in su litis. Focal in filtr ates in th e

20

NOD Mice and Related Strains: Research Applications in Diseases

submanidbular salivary glands are also increasingly common in older NON/Lt mice. Although NON mice do not develop autoimmune diabetes, and never become hyperglycemic under non-fasting conditions, NON/Lt males at The Jackson Laboratory develop a marked obesity by 20 weeks of age.106 This may be caused by low levels of the hormone leptin in adipocytes and in serum.107 Moreover, NON mice of both sexes are intolerant to glucose loading, and develop severe glomerulosclerotic kidney lesions.108 As NON/Lt mice age, immunoregulatory anomalies become apparent. Although T cell numbers and function are normal in young NON/Lt mice, by 20 weeks of age, T-lymphocytopenia, particularly in the CD8+ subset, is demonstrable in the spleen.109 This loss of T cells is reflected by a decline in T cell mitogen responses, and probably is associated with an NON strainspecific defect in the Tap1 (transporter associated with antigen processing) gene.110 A 113 bp insertion approximately 1 kb upstream of the Insulin 2 ( Ins2) locus on Chromosome 7 in NON/Shi has been reported.111 However, it was subsequently found that this represented a wild-type sequence.112 NON/Lt mice are not hearing impaired like NOD/Lt, but are blind due to the presence of the retinal degeneration ( rd) mutation on proximal Chromosome 5 associated with integration of a xenotropic proviral genome ( Xmv25). Thus, NON mice are not necessarily the best controls for establishing certain “normative” behavioral, physiologic or immunologic baselines. However, because the genome of the NON strain has been as extensively characterized for simple sequence repeat polymorphisms as has NOD, this strain is extremely useful for genetic outcross experiments with NOD.105,113 CTS (CATARACT SHIONOGI) AND NCT (N ON CATARACT) This strain, initially selected for cataracts with microphthalmia, has fixed an autosomal recessive mutation producing T-lymphopenia and T-lymphocytopenia that expresses at the level of the thymus.4648 The mutation may well be the mouse homolog of the lymphopenia ( Lyp) gene in diabetes-prone BB rats.114 If so, it should map to mouse Chromosome 6 in linkage with the neuropeptide Y locus based upon homology with the map position of rat linkage markers.115 Although thymus and thymocyte development appear normal, a defect in ability of mature thymocytes to emigrate to the periphery produces a profound peripheral T-lymphopenia and lymphocytopenia evident

NOD Mice and Related Strains: Origins, Husbandry and Biology

21

at weaning.46-48 CTS mice fail to reject NOD skin grafts. Peripheral B cell numbers are normal. Mature thymocytes express the Mel-14 antigen and exhibit normal homing to the lymph nodes when explanted thymocytes were injected intravenously.48 This peripheral T-lymphocytopenia, then, represents the mirror image of the T-lymphoaccumulation characteristic of NOD mice. CTS thymocytes exhibit strong mitogenic responses upon Concanavalin A or anti-CD3 activation 48 whereas NOD thymocytes are anergic. The NON strain is intermediate between NOD and CTS in that peripheral T cell numbers are normal at weaning, but decline rapidly with aging, with the loss of the CD8+ subset being especially marked. The CTS strain expresses a rare H2 haplotype, H2ct, which shares with NOD the same unique MHC class II and one of the unique class III ( Hsp70) alleles, but differs at class I genes, some of which are unique to CTS.116 NCT is a cataract-free control strain separated from CTS at the 4th generation of inbreeding. There is no mention in the Japanese publications describing the CTS strain as to whether NCT mice carrying the T-lymphocytopenia gene, nor have details of its MHC been published. Both NCT and CTS apparently develop perivascular/periductular infiltrates at low frequency in females as they age.8 ILI (ICR-L LINE-ISHIBE) This strain was independently derived from outbred Jcl:ICR mice. Serologic analysis shows that ILI mice share common MHC class I and class II alleles with NOD.8 ILI mice, like NON mice, become obese with aging (M. Hattori, Joslin Diabetes Center, personal communication). They are apparently not T-lymphocytopenic since unlike CTS mice, they rapidly reject NOD skin grafts.8 ILI mice develop perivascular/periductular infiltrates, but not insulitis nor IDDM. ILI and NOD lymphocytes failed to respond reciprocally in a mixed lymphocyte reaction.8 Splenocytes from diabetic NOD donors transfer insulitis, but not IDDM, into ILI- nu / nu recipients. ILI could potentially serve as an excellent non-diabetic control strain for many types of immunologic studies conducted in NOD mice. NOR/Lt NOR/Lt is a recombinant congenic stock produced following an outcross between NOD/Lt and C57BLKS/J, and inbreeding initiated at the second backcross to NOD.117,118 These mice are albino and

22

NOD Mice and Related Strains: Research Applications in Diseases

carry NOD alleles at approximately 88% of all polymorphic markers typed, including the H2g7 haplotype as well as NOD markers for Idd susceptibility genes on Chromosomes 2, 3, and elsewhere. The NOR/Lt stock exhibits the same degree of hearing loss as NOD/LtJ mice. However, NOR mice only very rarely develop IDDM spontaneously, although they exhibit T-lymphoaccumulation, sialoadenitis and perivascular/periductular leukocytic infiltrates in the pancreas that progress to insulitis in a few islets as the mice age. Analysis of congenic stocks has demonstrated that C57BL-derived genome on Chromosome 2 provides a major component of the diabetes resistance.118 Young NOR mice of both sexes are resistant to cyclophosphamide induced IDDM. However, as they age, 20-30% can be rendered diabetic following administration of two injections of 200 mg/kg cyclophosphamide injected two weeks apart (this laboratory, unpublished). NOR/Lt mice exhibit a more robust syngeneic mixed lymphocyte reaction than do NOD/Lt mice, and certain aberrant antigen presenting cell responses to interferon gamma observed in NOD/Lt mice are not exhibited by NOR/Lt.50 ALS, ALR (ALLOXAN SUSCEPTIBLE, ALLOXAN RESISTANT) These two inbred strains have been recently derived by inbreeding of outbred CD-1 mice with selection for susceptibility (ALS) or resistance (ALR) to diabetes induced within a week after administration of 45-47 mg/kg alloxan.3 Other strain characteristics have not been described, although a preliminary genetic comparison has been reported.119 Six-month old ALR, but not ALS mice, are completely deaf (Dr. Ken Johnson, The Jackson Laboratory, personal communication). IQI/JIC This strain was produced by inbreeding ICR mice in Japan. Treatment of these mice with mercuric chloride induced antinucleolar autoantibodies.120 Similar to a report for the NOD strain,121 the presence of large numbers of thymic B cells has recently been reported in IQI/Jic mice.122 At the time of this writing, there has been no genetic or immunogenetic characterization of these mice other than the anecdotal report of a slowly progressive sialitis in females.122

Closely related to NOD, same MHC class II genes, different class I genes

closely related to NOD, MHC-identical

progenitor stock for NOD and related strains

Swiss-derived like ICR and NOD, genetically very different from but inbred and without NOD, including MHC immunodeficiencies; available

NOD-derived recombinant exhibits some but not all of NOD’s congenic stock; same MHC, immune dysfunctions. differs at relatively few non-MHC loci.

CTS/Shi

ILI/Jic

ICR (usually available as CD-1)

SWR/J

NOR/Lt

randomly bred

not commonly available

early developing T-lymphocytopenia; unavailable

Closely related to NOD; diabetes Develops obesity, impaired glucose resistant MHC tolerance, immunodeficiencies, difficult to breed.

NON/Lt

Disadvantage

Advantage

Strain

Table 1.1. Diabetes-free or resistant control strains for NOD mice References

53

117-118

Analysis to establish which Idd genes control aberrant immunopheno-types essential to pathogenesis.

116

8

46-48

control for immune functions that are aberrant in NOD

analysis of population frequency of rare genetic polymorphisms present NOD

Genetic analysis of non-MHC -linked Idd genes

Genetic analysis of the contributory role of MHC class II as well as other H2g7 alleles in the Idd1 complex

Resistant MHC 7, 13 Genetic analysis of Idd genes, potential model for type II diabetes

Best Use

NOD Mice and Related Strains: Origins, Husbandry, and Biology 23

no endogenous T- or B-lymphocyte functions

NOD. CB17-scid and NOD.CB.17 -scid Emv30null

NOD.B10H2b

diabetes-resistant MHC from SWR/J, available diabetes-resistant MHC from C57BL/10J, available

NOD-SWR-H2q

NON.NOD-H2g7 diabetogenic MHC from NOD/Lt; available

develops high incidence of thymoma with age; slower onset in Emv30null stock

delineation of the role of T cell 7,125-126 subsets; source of NOD islets free of insulitis; growth of human tissues

63

References

exhibits some but not all of NOD’s all MHC congenic stocks are 50-51 immune dysfunctions extremely useful in dissecting the role of MHC exhibits some but not all of NOD’s versus non-MHC genes 50-51 immune dysfunctions in pathogenesis and in identifying aberrant immunoexhibits some but not all of NOD’s phenotypes under MHC control 123 immune dysfunctions exhibits some but not all of NOD’s 81, 124 immune dysfunctions

analysis of T-helper cell repertoire development; presentation of β cell antigens

Best Use

NOD.NON-H2nb1 diabetes-resistant MHC from NON/Lt; available

Disadvantage NOD mice not tolerant to I-E+ antigen presenting cells from these mice

Advantage

one gene difference confers NOD-Tg(Ead)Lt Line 5 transgenics diabetes-resistance; several independent lines available in Europe, U.S., and Japan

Strain

Table1.2. Diabetes-free or resistant controls for NOD: available transgenic and congenic stocks

24 NOD Mice and Related Strains: Research Applications in Diseases

NOD.NON-Thy1a useful T cell allotypic marker for adoptive transfer studies into NOD-scid recipients (which are Thy1b)

127

77

130

123,128-129

delineation of essential pathogenic role of B lymphocytes

delineation of role of MHC class I and CD8+ T cells in pathogenesis

same as scid mice

retarded onset of diabetes, requiring various types of adoptive that more mice be aged to provide transfer studies diabetic donors

not tolerant to Ig+ cells

NOD.Igµnull

B-lymphocyte deficient, available

B2mnull stock not tolerant to class I+ cells; scidB2mnulls stock is tolerant.

no endogenous T- or B-lymphocyte no thymic lymphomas functions; not yet widely available reported

NOD.B2mnull no MHC class I on cell surfaces in NOD.scidB2mnull the absence of b2-microglobulin, CD8+/-deficient, available

NOD.Rag2

NOD Mice and Related Strains: Origins, Husbandry, and Biology 25

26

NOD Mice and Related Strains: Research Applications in Diseases

APPROPRIATE CONTROLS FOR NOD MICE The question of the appropriate control to use for experimentation with NOD mice often arises. With the exception of the NON/Lt, SWR/J, and NOR/Lt stocks that are available from The Jackson Laboratory, most of the other NOD-related strains have not been distributed outside of Japan. Table 1.1 lists some of the potential strains and congenic stocks that have been used to establish experim ental “baseline” parameters in the absence of the insulitis and diabetes characteristic of NOD mice. The most appropriate choice for a control strain depends upon the nature of the investigation being undertaken (endocrinologic, immunologic, physiologic, etc.). Table 1.1 and Table 1.2 summarize the more well-defined control strains or stocks. ACKNOWLEDGMENTS This writing has been supported by the National Institutes of Health grants DK 36175 and DK27722, and a grant from The Juvenile Diabetes Foundation International. The author thanks Drs. David Serreze, Len Shultz and Linda Wicker for critical reviews. REFERENCES 1. Makino S, Kunim oto K, Muraoka Y, Mizushim a Y, Katagiri K, Tochino Y. Breeding of a non-obese, diabetic strain of mice. Exp Anim 1980; 29:1-8. 2. Ikegami H, Eisenbarth GS, Hattori M. Major histocompatibility complex-linked diabetogenic gene of the nonobese diabetic mouse. Analysis of genomic DNA amplified by the polymerase chain reaction. J Clin Invest 1990; 85:18-24. 3. Ino T, Kawamoto Y, Sato K, et al. Selection of mouse strains showing high and low incidences of alloxan-induced diabetes. Exp Anim 1991; 40:61-67. 4. Makino S, Hayashi Y, Muraoka Y, Tochino Y. Establishment of the nonobese-diabetic (NOD) mouse. In: Sakamoto N, Min HK, Baba S, ed. Current Topics in Clinical and Experimental Aspects of Diabetes Mellitus. Amsterdam: Elsevier 1985:25-32. 5. Kikutani H, Makino S. The murine autoimmune diabetes model: NOD and related strains. In: Dixon FJ, ed. Advances in Immunology NY: Academic Press, 1992; 50:285-322. 6. Ohotori H, Yoshida T, Inuta T. “Small eye” and “cataract”, a new dominant mutation in the mouse. Exp Anim 1968; 17:91-96. 7. Makino S, Yamashita H, Kunimoto K, et al. Breeding of the NON mouse and its genetic characteristics. In: Sakamoto N, Hotta N, Uchida K, ed. Current Concepts of a New Animal Model: The NON mouse. Tokyo: Elsevier Science Publishers BV, 1992:4-10.

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8. Hattori M, Fukuda M, Ichikawa T, et al. A single recessive non-MHC diabetogenic gene determines the development of insulitis in the presence of an MHC-linked diabetogenic gene in NOD mice. J Autoimmun 1990; 3:1-10. 9. Ueda H, Ikegami H, Yamato E, et al. The NSY mouse: A new animal model of spontaneous NIDDM with moderate obesity. Diabetologia 1995; 39:503-508. 10. Makino S, Kunimoto K, Muraoka Y, Katagiri K. Effect of castration on the appearance of diabetes in NOD mouse. Exp Anim 1981; 30:137-140. 11. Tarui S, Tochino Y, Nonaka K. Insulitis and Type 1 Diabetes. Lessons from the NOD Mouse.Tokyo: Academic Press, 1986:294. 12. Leiter EH. The role of environmental factors in modulating insulin dependent diabetes. In: deVries R, Cohen I, van Rood JJ, eds. Current Topics in Immunology and Microbiology. The Role of Microorganisms in Non-infectious Disease. Berlin: Springer Verlag, 1990: 39-55. 13. Leiter EH. The nonobese diabetic mouse: a model for analyzing the interplay between heredity and environment in development of autoimmune disease. ILAR News 1993; 35:4-14. 14. Ohsugi T, Kurosawa T. Increased incidence of diabetes mellitus in specific pathogen-eliminated offspring produced by embryo transfer in NOD mice with low incidence of the disease. Lab Anim Sci 1994; 44(4):386-388. 15. Ellerman KE, Richards CA, Guberski DL, et al. Kilham rat virus triggers t-cell-dependent autoimmune diabetes in multiple strains of rat. Diabetes 1996; 45(5):557-562. 16. Bowman MA, Leiter EH, Atkinson MA. Autoimmune diabetes in NOD mice: a genetic programme interruptible by environmental manipulation. Immunol Today 1994; 15:115-120. 17. Miyazaki A, Hanafusa T, Yamada K, et al. Predominance of T lymphocytes in pancreatic islets and spleen of pre-diabetic non-obese diabetic (NOD) mice: a longitudinal study. Clin Exp Immunol 1985; 60:622-630. 18. Jarpe AJ, Hickman MR, Anderson JT, et al. Flow cytometric enumeration of mononuclear cell populations infiltrating the islets of Langerhans in prediabetic NOD mice: development of a model of autoimmune insulitis for type 1 diabetes. Regional Immunol 1991; 3:305-317. 19. Shieh D-C, Cornelius J, Winter W, Peck A. Insulin-dependent diabetes in the NOD mouse model. I. Detection and characterization of autoantibody bound to the surface of pancreatic beta cells prior to development of the insulitis lesion in prediabetic NOD mice. Autoimmunity 1993;15:123-135. 20. Serreze D, Chapman H, Varnum D, et al. B lymphocytes are essential for the initiation of T cell-mediated autoimmune diabetes: analysis of a new speed congenic stock of NOD.Igm null mice. J Exp Med 1996; 184(5):2049-2053.

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21. Leiter E. The NOD mouse: a model for insulin dependent diabetes mellitus. In: Shevach EM, Coico R, ed. In: Current Protocols in Immunology. New York: John Wiley & Sons, Inc., 1997: vol 3, in press. 22. Makino S, Muraoka Y, Kishimoto Y, Hayashi Y. Genetic analysis for insulitis in NOD mice. Exp Anim 1985; 34:425-432. 23. Leiter EH. Lessons from the animal models: the NOD mouse. In: Palmer JP, ed. Diabetes Prediction, Prevention, and Genetic Counselling. London: John Wiley & Sons, 1996:201-226. 24. Baxter AG, Adams MA, Mandel TE. Comparison of high- and lowdiabetes incidence NOD mouse strains. Diabetes 1989; 38:1296-1300. 25. Fox HS. Androgen treatment prevents diabetes in nonobese diabetic mice. J Exp Med 1992;175:1409-1412. 26. Serreze DV, Leiter EH. Genetic and pathogenic basis for autoimmune diabetes in NOD mice. Current Opin Immunol 1994; 6:900-906. 27. Coleman DL, Kuzava JE, Leiter EH. Effect of diet on the incidence of diabetes in n on -obese diabetic (NOD) m ice. Diabetes 1990; 39:432-436. 28. Logothetopoulos J, Valiquette N, Madura E, Cvet D. The onset and progression of pancreatic insulitis in the overt, spontaneously diabetic, young adult BB rat studied by pancreatic biopsy. Diabetes 1984; 33:33-36. 29. Gaskins HR, Prochazka M, Hamaguchi K, Serreze DV, Leiter EH. Beta cell expression of endogenous xenotropic retrovirus distinguishes diabetes susceptible NOD/Lt from resistant NON/Lt mice. J Clin Invest 1992; 90:2220-2227. 30. Coleman DL. Acetone metabolism in mice: increased activity in mice heterozygous for obesity gen es. Proc Natl Acad Sci USA 1980; 77:290-293. 31. Ohneda A, Kobayashi T, Nihei J, et al. Insulin and glucagon in spontaneously diabetic non-obese mice. Diabetologia 1984; 27:460-463. 32. Rosmalen J, Jansen A, Homo-Delarche F, et al. Effect of prophylacticinsulin treatment on the number of antigen presenting cells and macrophages inthe pancreas of NOD mice. Is the prevention of diabetes based on b -cell rest? J Autoimmunity 1995; 21(1):28-29. 33. Akhtar I, Gold JP, Pan LY, et al. CD4(+) beta islet cell-reactive T cell clones that suppress autoimmune diabetes in nonobese diabetic mice. J Exp Med 1995; 182(1):87-97. 34. Serreze D. Autoimmune diabetes results from genetic defects manifest by antigen presenting cells. FASEB J 1993; 7:1092-1096. 35. Liblau RS, Singer SM, McDevitt HO. Th1 and Th2 CD4(+) T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol Today 1995;16(1):34-38. 36. Gombert J, Tancredebohin E, Hameg A, et al. IL-7 reverses NK1(+) T cell-defective IL-4 production in the non-obese diabetic mouse. Int Immunol 1996;8(11):1751-1758.

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37. Fujino-Kurihara H, Fujita H, Hakura A, et al. Morphological aspects on pancreatic islets of non-obese diabetic (NOD) mice. Virchows Arch, Cell Pathol 1985; 49:107-120. 38. Jaramillo A, Gill BM, Delovitch TL. Minireview: insulin dependent diabetes mellitus in the non-obese diabetic mouse: a disease mediated by T cell anergy? Life Sci 1994; 55(15):1163-1177. 39. Garchon H-J, Luan J-J, Eloy L, Bédossa P, Bach J-F. Genetic analysis of immune dysfunction in non-obese diabetic (NOD) mice: mapping of a susceptibility locus close to the Bcl-2 gene correlates with increased resistance of NOD T cells to apoptosis induction. Eur J Immunol 1994; 24:380-384. 40. Colucci F, Cilio CM, Lejon K, et al. Programmed cell death in the pathogenesis of murine IDDM: resistance to apoptosis induced in lymphocytes by cyclophosphamide. J Autoimmun 1996; 9(2):271-276. 41. Penha-Goncalves C, Leijon K, Persson L, Holmberg D. Type 1 diabetes and the control of dexamethazone-induced apoptosis in mice m aps to the sam e region on chrom osom e 6. Gen om ics 1995; 28(3):398-404. 42. Serreze D, Leiter E. Insulin Dependent Diabetes Mellitus (IDDM) in NOD Mice and BB Rats: Origins in Hematopoietic Stem Cell Defects and Implications for Therapy. In: Shafrir E, ed. Lessons from Animal Diabetes. V. London: Smith-Gordon, 1995: 59-73. 43. Many M-C, Drexhage HA, Denef J-F. High frequency of thymic ectopy in thyroids from autoimmune prone nonobese diabetic female mice. Lab Invest 1993; 69(3):364-367. 44. Prochazka M, Gaskins HR, Shultz LD, Leiter EH. The NOD- scid mouse: a model for spontaneous thymomagenesis associated with immunodeficiency. Proc Natl Acad Sci, USA 1992; 89:3290-3294. 45. Kataoka S, Satoh J, Fujiya H, et al. Immunologic aspects of the nonobese diabetic (NOD) mouse. Abnormalities of cellular immunity. Diabetes 1983; 32:247-253. 46. Yagi H, Suzuki S, Matsumoto M, Makino S, Harada M. Immune deficiency of the CTS mouse. I. Deficiency of in vitro T cell-mediated immune response. Immunol Invest 1990; 19:279-295. 47. Yagi H, Nagata M, Takeuchi M, et al. Immune deficiency of the CTS m ouse. II. Im paired in vivo T cell-m ediated im m une response. Immunol Invest 1990;19:493-505. 48. Yagi H, Matsumoto M, Nakamura M, et al. Defect of thymocyte emigration in a T cell deficiency strain (CTS) of the mouse. J Immunol 1996; 157:3412-3419. 49. Shimada A, Charlton B, Rohane P, et al. Immune regulation in type 1 diabetes. J Autoimmun 1996; 9(2):263-269. 50. Serreze DV, Gaskins HR, Leiter EH. Defects in the differentiation and function of antigen presenting cells in NOD/Lt mice. J Immunol 1993;150:2534-2543.

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51. Serreze DV, Gaedeke JW, Leiter EH. Hematopoietic stem cell defects underlying abnormal macrophage development and maturation in NOD/Lt mice: defective regulation of cytokine receptors and protein kinase C. Proc Natl Acad Sci USA 1993; 90:9625-9629. 52. Langmuir P, Bridgett M, Bothwell A, Crispe I. Bone marrow abnormalities in the non-obese diabetic mouse. Internat Immunol 1993; 5:169-177. 53. Serreze DV, Leiter EH. Defective activation of T suppressor cell function in Nonobese Diabetic mice. Potential relation to cytokine deficiencies. J Immunol 1988; 140:3801-3807. 54. Serreze DV, Hamaguchi K, Leiter EH. Immunostimulation circumvents diabetes in NOD/Lt mice. J Autoimmunity 1990; 2:759-776. 55. Jacob CO, Aiso S, Michie SA, et al. Prevention of diabetes in nonobese diabetic mice by tumor necrosis factor (TNF); similarities between TNF-a and interleukin 1. Proc Natl Acad Sci USA 1990; 87:968-972. 56. Xie T, Hofig A, Yui M, et al. Spontaneous prostaglandin synthase-2 (Pgs2) gene expression in macrophages of NOD and congenic mice. Autoimmunity 1995; 21(1):17A. 57. Xie T, Reddy S, Hofig A, et al. Regulation of prostaglandin synthase2 (Pgs-2) in NOD macrophages. Autoimmunity 1996; 24(1):23A. 58. Wicker LS, Todd JA, Prins J-B, et al. Resistance alleles in two nonMHC-linked insulin dependent diabetes loci on chromosome 3, Idd3 and Idd10, protect NOD m ice from diabetes. J Exp Med 1994; 180:1705-1713. 59. Prins J-B, Todd J, Rodriques N, et al. Linkage on chromosome 3 of autoimmune diabetes and defective Fc receptor for IgG in NOD mice. Science 1993; 260:695-698. 60. Luan J, Monteiro R, Sautes C, et al. Defective Fc gamma RII gene expression in m acrophages of NOD m ice—genetic linkage with upregulation of IgG1 and IgG2b in serum. J Immunol 1996; 157(10): 4707-4716. 61. Marsh CB, Pope H A, Wewers MD. FCg recptor cross-lin kin g downregulates IL-1 receptor antagonist and induces IL-1b in mononuclear phagocytes stim ulated with endotoxin or Staphylococcus aureus. J Immunol 1994; 152:4604-4611. 62. Serreze DV, Leiter EH, Kuff EL, et al. Molecular mimicry between insulin and retroviral antigen p73. Development of cross-reactive autoantibodies in sera of NOD and C57BL/KsJ- db/db mice. Diabetes 1988; 37:351-358. 63. Hanson MS, Cetkovic-Cvrlje M, Ramiya V, et al. Quantitative thresholds of MHC Class II I-E expression on hematopoietically derived APC in transgenic NOD/Lt Mice determine level of diabetes resistan ce an d in dicate m echan ism of protection . J Im m un ol 1996; 157:1279-1287. 64. Lenschow D, Herold K, Rhee L, et al. CD28/B7 regulation of Th1 and Th2 subsets in the development of autoimmune diabetes. Immunity 1996; 5(3):285-293.

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65. Tsumura H, Komada H, Ito Y, Shimura K. In vitro and in vivo interferon production in NOD mice. Lab Anim Sci 1989; 39:575-578. 66. Rapoport M, Zipris D, Lazarus A, et al. IL-4 reverses thymic T cell anergy and prevents the onset of diabetes in NOD mice. J Exp Med 1993; 178:87-99. 67. Rabinovitch A. Immunoregulatory and cytokine imbalances in the pathogenesis of IDDM. Diabetes 1994; 43(5):613-621. 68. Lord CJ, Bohlander SK, Hopes EA, et al. Mapping the diabetes polygene Idd3 on mouse chromosome 3 by use of novel congenic strains. Mamm Genome 1995; 6(9):563-570. 69. Chesnut K, Shie J-X, Cheng I, Muralidharan K, Wakeland EK. Characterization of candidate genes for IDD susceptibility from the diabetes-prone NOD mouse strain. Mamm Genome 1993; 4:549-554. 70. Suzuki T, Yamada T, Takao T, et al. Diabetogenic effects of lymphocyte transfusion on the NOD or NOD nude mouse. In: J. Rygaard NB, N. Graem, M. Sprang-Thomsen, ed. Immune-Deficient Animals in Biomedical Research. Basel: Karger, 1987:112-116. 71. Bendelac A, Boitard C, Bedossa P, et al. Adoptive T cell transfer of autoimmune nonobese diabetic mouse diabetes does not require recruitment of host B lymphocytes. J Immunol 1988; 141:2625-2628. 72. Carrasco-Marins E, Shimizu J, Kanagawa O, Unanue E. The class II MHC I-Ag7 molecules from Nonobese Diabetic mice are poor peptide binders. J Immunol 1996; 156:450-458. 73. Leijon K, Hammarström B, Holmberg D. Non-obese diabetic (NOD) mice display enhanced immune responses and prolonged survival of lymphoid cells. Int Immunol 1994; 6:339-345. 74. Hu Y, Nakagawa Y, Purushotham KR, Humphreys-Beher MG. Functional changes in salivary glands of autoimmune disease-prone NOD mice. Am J Physiol 1992; 263:E607-E614. 75. Humphreys-Beher MG, Brinkley L, Purushotham KR, et al. Characterization of antinuclear autoantibodies present in the serum from nonobese diabetic (NOD) mice. Clin Immun Immunopath 1993; 68(3):350-356. 76. Skarstein K, Wahren M, Zaura E, Hattori M, Jonsson R. Characterization of T-cell receptor repertoire and anti-ro/ssa autoantibodies in relation to sialadenitis of NOD mice. Autoimmun 1996; 22(2):9-16. 77. Christianson SW, Shultz LD, Leiter EH. Adoptive transfer of diabetes into immunodeficient NOD- scid/scid mice: relative contributions of CD4+ and CD8+ T lymphocytes from diabetic versus prediabetic NOD.NON-Thy-1a donors. Diabetes 1993; 42:44-55. 78. Kerr M, Lee A, Wang P, et al. Detection of insulin and insulin-like growth factors I and II in saliva and potential synthesis in the salivary glan ds of m ice. Biochem ical Pharm acology 1995; 49(10): 1521-1531. 79. Ito S, Suzuki T, Isemura S, et al. ‘Salivary peptide P-C’ of human pancreatic B-cells shares only partly immunoreactivity with salivary

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peptide P-C indicating a new B-cell protein which is different from insulin. Acta Endocrinol Copenh 1989; 120(1):62-8. Prochazka M, Serreze DV, Worthen SM, Leiter EH. Genetic control of diabetogenesis in NOD/Lt mice: development and analysis of congenic stocks. Diabetes 1989; 38:1446-1455. Wicker LS, DeLarto NH, Pressey A, Peterson LB. Genetic control of diabetes and insulitis in the nonobese diabetic mouse: analysis of the NOD.H-2b and B10.H-2nod strains. In: Alt F, Vogel H, ed. Molecular Mechanisms of Immunological Self-Recognition. New York: Academic Press, 1993:173-181. Li X, Golden J, Faustman DL. Faulty major histocompatability complex class II I-E expression is associated with autoimmunity in diverse strains of mice. Diabetes 1993; 42:1166-1172. Robinson CP, Yamamoto H, Peck AB, Humphreysbeher MG. Genetically programmed development of salivary gland abnormalities in the NOD (nonobese diabetic)-scid mouse in the absence of detectable lymphocytic infiltration: a potential trigger for sialoadenitis of NOD mice. Clin Immunol Immunopathol 1996; 79(1):50-59. Robinson CP, et al. Expression of parotid secretory protein (PSP) in murine lacrimal glands and its possible function as a bacterial binding protein in exocrine secretions. Am J Physiol 1997; 275(Gastrointestinal Liver Physiol 35):G863-G871. Bernard NF, Ertug F, Margolese H. High incidence of throiditis and anti-throid autoantibodies in NOD mice. Diabetes 1991; 41:40-46. Krug J, Williams AJK, Beales PE, Doniach I, Gale EAM, Pozzilli P. Parathyroiditis in the non-obese diabetic mouse-a new finding. J Endocrinol 1991; 131:193-196. Many M, Maniratunga S, Varis I, et al. Two-step development of Hashimoto-like thyroiditis in genetically autoimmune prone nonobese diabetic mice: effects of iodine-induced cell necrosis. Journal of Endocrinology 1995; 147:311-320. Wicker LS, Todd JA, Peterson LB. Genetic control of autoimmune diabetes in the NOD mouse. Ann Rev Immunol 1995; 13:179-200. Doi T, Hattori M, Agodoa LYC, et al. Glomerular lesions in nonobese diabetic mouse: before and after the onset of hyperglycemia. Lab Invest 1990; 63:204-212. Yang CW, Hattori M, Vlassara H, et al. Overexpression of transforming growth-factor-beta-1 messenger-RNA is associated with upregulation of glom eru lar ten ascin an d lam in in gen e-expression in nonobese diabetic mice. J Am S Neph 1995; 5:1610-1617. Leiter EH. Multifactorial control of autoimmune insulin dependent diabetes in NOD mice: Lessons for inflammatory bowel disease. In: Sutherland LR, Collins SM, Martin P, ed. Inflammatory Bowel Diseases: Basic research, clinical implications, and trends in therapy. London: Kluwer Academic Publishers, 1994:24-34. Shimada A, Rohane P, Fathman CG, Charlton B. Pathogenic and protective roles of CD45RB(low) CD4(+) cells correlate with cytokine

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profiles in the spontaneously autoimmune diabetic mouse. Diabetes 1996; 45(1):71-78. Gerling IC, Freidman H, Greiner DL, Shultz LD, Leiter EH. Multiple low dose streptozotocin-induced diabetes in NOD- scid /scid mice in the absence of functional lymphocytes. Diabetes 1994; 43:433-440. Baxter AG, Horsfall AC, Healey D, et al. Mycobacteria precipitate an SLE-like syndrome in diabetes-prone NOD mice. Immunology 1994; 83(2):227-231. Mahler M, Leiter E, Birkenmeier E, Bristol I, Elson C, Sundberg J. Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis. Am J Physiol 1997; submitted. Amor S, Baker D, Groome N, Turk JL. Identification of major encephalitogenic epitope of proteolipid protein (residues 56-70) for the induction of experimental allergic encephalomyelitis in Biozzi AB/H and nonobese diabetic mice. J Immunol 1993; 150:5666-5672. Reifsnyder P, Liu O, Anderson N, et al. Genomic characterization of new recombinant congenic stocks between CBA/LsLt and NOD/Lt. Mamm Genome; manuscript in preparation. Baxter AG, Mandel TE. Hemolytic anemia in non-obese diabetic mice. Eur J Immunol 1991; 21:2051-2055. Leiter EH . The NOD m ouse m eets the “Nerup H ypothesis”. Is diabetogenesis the result of a collection of common alleles present in unfavorable combinations? In: Vardi P, Shafrir E, ed. Frontiers in Diabetes Research: Lessons from Animal Diabetes III. London: SmithGordon, 1990:54-58. Formby B, Schmid-Formby F, Jovanovic L, Peterson CM. The offspring of the female diabetic “Nonobese Diabetic” (NOD) mouse are large for gestational age and have elevated pancreatic insulin content: a new animal model for human diabetic pregnancy. Proc Soc Exp Biol Med 1987; 184:291-294. Tarleton G, Gondos B, Form by B. Testicular alterations in the nonobese diabetic mouse. Endocr Pathol 1990; 1:85-93. Tatewaki R, Otan i H , An do S, et al. Chrom osom e an alysis of postimplantation stage embryos for studying possible causes of developmental abnormalities in nonobese diabetic mice. Biol Neonate 1991; 60:395-402. Otani H, Tanaka O, Tatewaki R, et al. Diabetic environment and genetic predisposition as causes of congenital malformations in NOD mouse embryos. Diabetes 1991; 40:1245-1250. Leiter EH. The genetics of diabetes susceptibility in mice. FASEB J 1989; 3:2231-2241. McAleer MA, Reifsnyder P, Palmer SM, et al. Crosses of NOD mice with the related NON strain: a polygenic model for type I diabetes. Diabetes 1995; 44:1186-1195. Committee on Immunologically Compromised Rodents. In: Immunodeficient Rodents. A Guide to their Immunobiology, Husbandry,

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and Use. Washington: National Research Council, National Academy Press 1989:103-104. Igel M, Becker W, Herberg L, Joost H-G. Evidence that reduced leptin levels, but n ot an aberran t sequen ce of leptin or its receptor, contributre to the obesity syndrome in NON mice. Horm Metab Res 1996; 28:669-673. Tochino Y, Kanaya T, Makino S. Microangiopathy in the spontaneously diabetic nonobese mouse (NOD mouse) with insulitis. In: Abe H, Mitsuru H, ed. Diabetic Microangiopathy. Tokyo: Univ of Tokyo Press, 1983:423-432. Leiter E, Prochazka M, Coleman DL, et al. Genetic factors predisposing to diabetes susceptibility in mice. In: Jaworsk M, Molnar G, Rajotte R, Singh B, ed. The Im m unology of Diabetes Mellitus. Amsterdam: Elsevier, 1986:29-36. Pearce RB, Trigler L, Svaasand EK, Peterson CM. Polymorphism in the mouse Tap-1 gene. J Immunol 1993; 151:5338-5347. Sawa T, Ohgaku S, Morioka H, Yano S. Molecular cloning and DNA sequence analysis of preproinsulin genes in the NON mouse, an animal model of human non-obese, non-insulin dependent diabetes mellitus. J Mol Endocrinol 1990; 5:61-67. Pearce R. Clarification of the Ins2 gene sequence: relevance to glucose in toleran ce in NON/Lt m ice. Mam m alian Gen om e 1996; 7:143-144. Prochazka M, Leiter EH, Serreze DV, Coleman DL. Three recessive loci required for insulin-dependent diabetes in NOD mice. Science 1987; 237:286-289. Awata T, Kanazawa Y. Genetic-markers for insulin-dependent diabetes-mellitus in Japanese. Diabet Res Clin Prac 1994; 24(S):S83-S87. Jacob HJ, Pettersson A, Wilson D, et al. Genetic dissection of autoimmune type 1 diabetes in the BB rat. Nature Genet 1992; 2:56-60. Ikegami H, Makino S, Yamato Y, et al. Identification of a new susceptibility locus for insulin dependent diabetes mellitus by ancestral haplotype congenic mapping. J Clin Invest 1995; 96:1936-1942. Prochazka M, Serreze DV, Frankel WN, Leiter EH. NOR/Lt; MHCmatched diabetes-resistant control strain for NOD mice. Diabetes 1992; 41:98-106. Serreze DV, Prochazka M, Reifsnyder PC, et al. Use of recombinant congenic and congenic strains of NOD mice to identify a new insulin depen den t diabetes resistan ce gen e. J Exp Med 1994; 180: 1553-1558. Sekiguchi F, Ishibashi K, Katoh H, et al. Genetic profile of alloxaninduced diabetes-susceptible mice (ALS) and resistant mice (ALR). Exp Anim 1990; 39:269-272. Saegusa J, Kiuchi Y, Itoh T. Antinucleolar autoantibody induced in mice by mercuric chloride. Strain differences in susceptibility. Exp Anim 1990; 39:597-599.

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121. Savino W, Boitard C, Bach J-F, Dardenne M. Studies on the thymus in Nonobese Diabetic Mouse I. Changes in the microenvironmental compartments. Lab Invest 1991; 64:405-417. 122. Saegusa J, Yasuda A, Kubota H. IQI/Jic mice have thymic B cells. Exp Anim 1996; 45:353-360. 123. Serreze D, Gallischan W, Snider D, et al. MHC Class I-mediated antigen presentation and induction of CD8+ cytotoxic T-cell responses in autoimmune diabetes-prone NOD mice. Diabetes 1996; 45:902-908. 124. Wicker LS, Todd JA, Peterson L. Genetic control of autoimmune diabetes in the NOD mouse. Ann Rev Immunol 1995; 13:179-200. 125. Shultz LD, Schweitzer PA, Christianson SW, et al. Multiple defects in innate and adaptive immunological function in NOD/LtSZ- scid mice. J Immunol 1995; 154:180-191. 126. Serreze DV, Leiter EH, Hanson MS, et al. Emv30null NOD-scid mice: an improved host for adoptive transfer of autoimmune diabetes and gr owth of h u m an lym p h oh em atop oietic cells. Diabetes 1995; 44:1392-1398. 127. Soderstrom I, Bergm an ML, Colu cci F, Lejon K, Bergqvist I, Holmberg D. Establishment and characterization of RAG-2 deficient non-obese diabetic mice. Scand J Immunol 1996; 43:525-30. 128. Serreze DV, Chapman HD, Gerling IC, et al. Initiation of autoimmune diabetes in NOD/Lt mice is MHC class I-dependent. J Immunol 1997; 158:3978-3986. 129. Wicker LS, Leiter EH, Todd JA, et al. b2 microglobulin-deficient NOD mice do not develop insulitis or diabetes. Diabetes 1994; 43:500-504. 130. Serreze DV, Chapman HD, Varnum DS, et al. B lymphocytes are essential for the initiation of T cell-mediated autoimmune diabetes: analysis of a new ‘‘speed congenic’’ stock of NOD.Igm null mice. J Exp Med 1996; 184(5):2049-2053.

CHAPTER 2

Genetics and Immunogenetics of NOD Mice and Related Strains Edward H. Leiter

INTRODUCTON

ince the NOD mouse is distinguished from mice of other inbred strains in terms of its unique predisposition to develop autoimmune, insulin dependent diabetes mellitus (IDDM), genetic analysis has tended to focus upon genes controlling development of aberrant autoimmune responses. Indeed, most of the chapters in this volume attest to the extensive knowledge gained about the immune system of NOD mice. Yet the NOD mouse is of considerable value to mouse and human geneticists interested in mapping complex traits that may be totally unrelated to autoimmune disease in general and IDDM in particular. In most cases, a newly-developed inbred strain is of less utility to a geneticist than long-standing inbred strains because of the lack of genetic characterization of the former. However, because of its importance to autoimmunity research, the NOD strain, or more precisely DNA from NOD mice, represents one of the “select few” 11 strains included in the Massachusetts Institute of Technology (MIT) Genome Center’s survey of over 6,000 PCR-typed and geneticallymapped simple sequence repeat (SSR) polymorphisms. Hence, NOD and its related NON strain, despite their recent origins, represent two of the most genetically well-characterized inbred strains. The SSR

S

NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer and Other Diseases, edited by Edward Leiter and Mark Atkinson. © 1998 R.G. Landes Company.

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markers, distributed throughout the genome, cover most of the 20 mouse chromosomes with the exception of the Y chromosome at a density of ~3cM. Commerically-available PCR primer sets are available for each of these marker loci. Strain-specific polymorphic allele sizes are available on-line (www.mit.genome.edu). SSR chromosomal map positions are integrated into the genetic map of the mouse. Information for a given SSR-based polymorphic locus is available online (www.informatics.jax.org or www.resgen.org), and its map position is constantly being updated as new linkage data become available for identified structural genes mapped to the same chromosomal region. Commercially-available yeast artificial chromosome (YAC) contig libraries based upon these SSR markers permit physical ordering of DNA sequences found within polymorphic regions. NOD mice differ at approximately 50% of the >6,000 polymorphic SSR-containing loci when compared to other commonly-used, non-Swiss derived inbred mouse strains such as C57BL/6J (B6), C3H/HeJ (C3H) and CBA/J. The simple-to-perform PCR technology allows the investigator to select a defined NOD set of polymorphisms distributed over the 20 chromosomes, and follow their segregation through multiple outcross/intercross or outcross/backcross cycles (by PCR-typing of DNA that can be easily prepared from peripheral blood). This technique is known as a genome wide scan. DNAs from progeny are scanned for distortions in the frequency of polymorphic parental alleles between affected and unaffected progeny in the case of a discontinuous trait such as IDDM, or for linkage to a high or a low response in a continously varying quantitative trait. As discussed in the previous chapter, NOD mice breed relatively early; dams produce uncommonly large litters for an inbred strain, and are excellent mothers, nursing nearly all of their pups to weaning. The developmental biologist should note that the excellent maternal instincts of NOD females makes the pseudopregnant NOD female an excellent host for blastocyst transfer when chimeric mice are being produced (Dr. Robyn Slattery, John Curtin School of Medicine, Canberra, Australia, personal communication). This excellent breeding performance, combined with the increasing numbers of congenic and transgenic stocks, as well as of defined gene “knock-outs” being developed on the NOD inbred strain background, is proving especially useful for establishing genetic linkages for multigenically controlled traits. If the mouse geneticist is trying to map the gene con-

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trolling a monogenic trait, he immediately thinks of initiating an interspecific outcross to maximize genetic polymorphisms between the strain susceptible to the disorder and a resistant strain. However, as will be described below, for complex multifactorial diseases like IDDM or inflammatory bowel disease, this strategem is often counterproductive because the trait is not detectable or detectable only at very low frequency in either an F2 or first or second backcross (BC1, BC2) generation. If intraspecific crosses are performed instead, the frequency of trait expression can be expected to be higher. To illustrate, in the previous chapter, it was mentioned that NOD mice develop a progressive hearing loss that is multigenically controlled whereas CBA/J mice do not. Following an interspecific outcross between NOD and a wild-derived inbred strain, such as SPRET/Ei or CAST/Ei (derived from Mus spretus and Mus castaneus, respectively), there is a high probability that the frequency of the deafness trait may be so low that very large numbers of F2 mice would have to be produced, or multiple backcrosses would have to be performed before the penetrance of the deafness phenotype made the genetic analysis feasible. In contrast, if NOD were outcrossed to CBA, a higher penetrance of the disease might be anticipated. Further, recombinant congenic stocks in which defined NOD chromosomal regions are fixed on a predominantly CBA genetic background exist. Although a geneticist might eschew the use of the NOD genome for mapping purposes based upon fear that mice might be lost from a study through development of IDDM, this is an easily obviated problem. As detailed in the preceding chapter, MHC congenic stocks of NOD mice can be used that do not develop IDDM. Thus, the remarkable breeding performance of this inbred strain, coupled with its well-characterized genome, should be kept in mind for use in the genetic dissection of complex traits such as obesity, cardiovascular disease, and inflammatory bowel disease. NOD AND THE GENETICS OF AUTOIMMUNITY THE GENETIC BASIS OF IDDM The fact that NOD mice and BB/Wor-DP rats develop spontaneous IDDM as a result of selective breeding confirms the primary role of genetics in establishing disease susceptibility in these rodent models. However, not all NOD mice or BB-DP rats will develop IDDM if the intrinsic and extrinsic environment is not optimum. Because of

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this complex, multifactorial control, it is most accurate to refer to NOD mice simply by the abbreviation “NOD”, and not as “nonobese diabetic”, since, in most vivaria, not all NOD mice develop clinical features of diabetes. In humans, comparison of disease concordance frequency between monozygotic versus dizygotic twins is a classic way to establish the relative contributions of heredity versus environment. In the case of IDDM in humans, the higher concordance rates for monozygotic twins (~30-50%) versus dizygotic twins (~5%) clearly establishes a primary etiopathologic contribution for disease-predisposing genetic factors. However, since concordance rates for IDDM in monozygotic twins rarely exceeds 50%, either random somatic genetic changes are important in development of a fully susceptible individual (e.g., in the constitution of T cell receptor and immunoglobulin repertoires), or the penetrance of the diabetogenic genes is strongly influenced by environment, or both. Further complicating elucidation of the genetic contributions to development of a complex, multifactorial disease represented by IDDM in humans is the fact that the disease is genetically quite heterogeneous. Only about 20% of new cases of IDDM occur in families in which another family member is affected; the remainder are “sporadic”, suggesting that new combinations of potentially diabetogenic collections of genes are constantly being generated in a randomly breeding human population. Disease heterogeneity is reflected by the finding that not all cases of IDDM have an autoimmune etiology. Even within the demonstrated cases of autoimmune IDDM, where genes within the major histocompatibility (MHC) human leukocyte antigen (HLA) complex on Chromosome 6p21 are clearly major determinants of susceptibility, heterogeneity in terms of the predisposing HLA alleles, as well as in the non-HLA genetic linkages is encountered within and between racial and ethnic groups.1 Given this background of genetic complexity in humans, the NOD mouse must be viewed as a single case study because an inbred mouse essentially represents a single individual reproduced in multiple copies. Same sex individuals can exchange organ and tissue grafts without rejection. However, within the strain, males and females will be distinguished by gender-dimorphic patterns of gene expression, including maternal versus paternal imprinting of genes, as well as by sex chromosome-controlled differences. One of the latter is the socalled H-Y antigen, which precludes the transfer of NOD male tissues

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into female recipients.2 Gender-independent variations between individuals within the strain will also arise through stochastic somatic processes associated with immune repertoire development, including rearrangement of immunoglobulin and T cell receptor genes, as well as differential amplification of the adaptive immune system in response to variable exposure to environmental antigens. Finally, as discussed in the previous chapter, random mutation in an individual may become fixed in a breeding colony if it improves fitness, reproductive performance, etc., and such genetic shifts will eventually lead to substrain divergence.3 W HAT MAKES THE NOD GENOME U NIQUELY PERMISSIVE FOR SPONTANEOUS IDDM D EVELOPMENT? The reader of this book should have no difficulty finding current reviews describing the latest advances in knowledge regarding the immunogenetic basis of IDDM susceptibility in NOD mice. The following reviews are recommended at the time of this writing.4-7 All workers in the field agree that the unique H2g7 MHC haplotype of NOD mice is the predominant contributor of IDDM susceptibility in NOD mice. Depending upon the strain paired with NOD in genetic segregation analysis, this complex locus is estimated to contribute upwards of 40% of the relative risk for diabetes development.8 This is fully consistent with what is known about the genetics of autoimmune IDDM in humans, wherein diabetes-predisposing HLA alleles also are the strongest known determinants of genetic susceptibility to IDDM.9 Before the specific details of the diabetogenic MHC and nonMHC loci are summarized, we will consider the more general question of why the NOD strain, among all the extant mouse strains, is the only one to develop IDDM spontaneously. As described in the preceding chapter, the ICR-derived ILI strain shares the same diabetogenic H2g7 haplotype as NOD, and the CTS strain shares class II MHC alleles, yet neither ILI nor CTS mice nor other strains of mice congenic for H2g7 develop IDDM.10 Two possibilities have been debated. The first is that the NOD strain has accumulated a set of rare mutations in both MHC and nonMHC genes that, collectively, set this strain apart from virtually all other extant strains. A listing of some of the non-MHC genetic loci reported to be rare or defective in NOD is provided in Table 2.1. The second possibility, termed the “Nerup hypothesis”11 after Dr. Jørn

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Table 2.1. Important Genetic Polymorphisms Distinguishing NOD Mice Gene (chromosome)

Phenotype

Fcγr2 (low affinity Fc receptor gamma II, Chr 1)

multiple mutations in promoter regions led to low expression in macrophage, impaired binding of IgG1 and IgG2b.

Ref. 98

null mutation (Hc0 allele) in the gene so that NOD 99 lacks the C5a component such that NOD serum does not support complement dependent lysis. Fcγr1 (high affinity multiple mutations in both cytoplasmic and 80,10 Fc receptor gamma 1, extracellular domains affect both surface expression Chr 3) and ligand binding, as well as impaired clearance of IgG2a. Il2 (interleukin-2, NOD expresses the Il2b allele, the product of which 55, Chr 3) may be less potent (due to glycosylation) than the 101 Il2a allele product. Slc1a1 (formerly Nhe1 ubiquitously expressed membrane ion exchanger 56 solute carrier family 9, maintaining pH homeostasis; NOD has low activity sodium/hydrogen allelle. exchanger, isoform 1, Chr 4) Nk1 (natural killer cell NOD mice fail to develop functional NK1+ T cells 102, antigen-1, Chr 6) and both NOD and NOD-scid mice lack functional 103 NK killing activity. NOD mice exhibit rare poly– morphisms in the NK1-linked mouse homolog to the human gene, NKR-P1, encoding an NK signal transduction molecule (Leiter lab., unpublished) Art2a, 2b (ADP-riboNOD has rare restriction fragment length 104, syltransferase, polymorphism, somewhat reduced expression 105 formerly Rt6, Chr 7) levels in spleens of young but not old mice. Mrv6 (Mouse MAIDS defective endogenous proviral genome; no virus-related proviral evidence for expression in NOD thymus or islets. 76 gene), Chr 14 Ly6c (lymphocyte impaired expression on bone marrow, splenocytes, 106 antigen 6C, Chr 15) and lymph node cells correlates with mutations in the 5” flanking region. H2g7 complex, Chr 17 H2-K common allele, but acquires diabetogenic function 31,33 with rest of haplotype Lmp2 same allele as H2r,q, 109 Tap1 unusual RFLV in intron; tryptophan replaces 23,110 cysteine at residue 174 of cDNA Lmp7 same allele as H2r,cas4, 109 g7 H2-A rare amino acid substitutions in Ab chain 16 H2-E nonsense mutation in Ea chain, no surface expression 14

Hc (hemolytic complement, Chr 2)

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Gene (chromosome)

Phenotype

Hsp70 Tnfa H2-D

unusual RFLV suggest novel allele 22 reduced transcription, secretion in some studies 107 common allele, but acquires diabetogenic function 31-33 with rest of haplotype

Qa region Xmv66 Ptgs2 (prostaglandin synthase 2, Chr 1) Reg (rat regenerating, islet derived, mouse homolog 1, 2, Chr 3 & 12)

Ref.

deletions of some Qa family genes indicated novel 108 xenotropic proviral genome distal to H2-K 8 cytokine-inducible cyclooxygenase with anomalous 65,66 high constitutive expression in NOD macrophage. high levels of expression in prediabetic and diabetic 112 NOD pancreas.

Nerup of Denmark, who first raised the issue for debate,12 is that the NOD genome does not contain diabetes-specific genes, but rather, by chance, has accumulated unfavorable combinations of relatively common alleles. Accumulated evidence at this point suggests that the NOD strain is both a collection of some relatively rare diabetogenic alleles working disharmoniously with a larger collection of common alleles. THE H2g7 HAPLOTYPE: A SCAFFOLDING FOR INITIATION OF INSULITIS AND DIABETES MHC class I and class II molecules expressed on antigen presenting cells (APC) select the repertoire of T cells intrathymically, and regulate their functions in the periphery such that, in normal mice, Tc ell autoreactivity against cells in glandular tissues is prevented.13 Genetic segregation analysis initially identified the H2 region as containing a gene or genes controlling susceptibility to IDDM in NOD mice.14,15 A provisional nomenclature was proposed wherein chromosomal regions containing such genes were sequentially identified as Idd loci.15 The region linked to H2 on Chromosome 17 was provisionally designated Idd1. A similar provisional nomenclature has since been adopted for human IDDM susceptibility conferring loci, with the homologous HLA region designated as IDDM1.1 MHC haplotype designations in mice are based in part upon allele typing at the H2K and H2D markers at the proximal and distal ends of the MHC complex. H2g denotes a recombinant haplotype containing a H2Kd and a H2Db allele. The H2g7 haplotype assigned to NOD mice encompasses additional unique and/or rare alleles within the class II and class III region of the complex. The notation I-Ag7 denotes a class II

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molecule encoded by a common Aad allele and a very rare Abg7 allele in which a nested set of five nucleotide substitutions between position 248-252 converts a conserved proline residue at amino acid position 56 to histidine and a common aspartic acid residue at position 57 to serine.16 I-Ag7 is present in the related CTS and ILI strains, and has been found in randomly screened outbred ICR mice17 as well as in the Biozzi high antibody responder strain.18 The Abg7 of NOD is therefore homologous to “diabetogenic” HLA- DQβ non-aspartic acid 57 containing alleles in humans.19 MHC class II molecules (generally only expressed on APC) are essential for the initiation and maintenance of both cell-mediated and humoral immune responses. The Abg7 amino acid substitutions are located on parts of the molecule that affect the binding of peptides to the antigen-binding cleft, with the consequence that the I-Ag7 peptide binding cleft is able to selectively bind and present peptides with acidic residues at their C-terminus.20 A second “diabetogenic” mutation is represented by the Eab allele in the H2g7 complex (homologous to the human HLA - DR region). This allele is relatively common in inbred strains such as B6 that fail to express cell surface IE molecules due to a deletion within the first exon of the Ea gene that prevents transcription.14 The Eb allele in H2g7 is Ebd 21 and is expressed normally, but in the absence of Eα chains, no I-E heterodimers form at the cell surfaces of APC. The class III region distal to the Ebd locus contains a unique heat shock protein allele ( Hsp70).22 Proximal to the Ab locus, the H2g7 haplotype also contains rare alleles at Tap1 and Tap2 (for transporters associated with antigen processing, and previously designated Ham1 and Ham2).23 Confirmation of the diabetogenic contributions of the unique I-Ag7 molecule in the absence of cell surface I-E molecules was provided by transgenic experiments (reviewed by Slattery and Miller 24 and by Cooke et al25). In brief, these studies confirmed that both the proline to histidine substitution at residue 56, and the aspartic acid to serine substitution at position 57 of the Aβ chain contribute indepen den tly to IDDM susceptibility. Sim ilarly, in ser tion of Ead transgenes leading to I-E cell surface expression also protected against insulitis and IDDM development. Protection was apparently not mediated by negative selection within the thymus of diabetogenic effectors.26,27 Rather, alterations in peptide presentation by APC are indicated by the finding that protection is associated with a shift in the cytokine profile toward reduced T cell production of interferon gamma

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45

and increased production of cytokines associated with T-helper 2 functions.26,28 This deviation in T cell functional activities apparently leads to peripheral suppression of autoim m une effectors. The I-Ag7 heterodimer is reportedly unstable on cell surfaces of NOD APC; transgenic expression of I-E molecules does not correct this anomaly.29 High levels of I-E expression on NOD APC result in lower levels of T cell proliferative responses in a recall assay in vitro following priming in vivo with putative β cell autoantigens such as glutamic acid decarboxylase.26 These I-E effects do not seem to reflect competition with I-Ag7 for binding of diabetogenic peptides, but rather, may reflect further quantitative reductions in the absolute numbers of I-Ag7 molecules expressed on APC.26 The respective diabetogenic contributions of the H2g7 class II region genes partially answer the question posed at the beginning of this section. The two diabetogenic mutations in Abg7 are uncommon whereas the “diabetogenic” mutation in the Eab gene is relatively common. Hence, genetic predisposition to IDDM in NOD mice indeed is comprised in one instance by the presence of a common null mutation ( Eab) that is not inherently diabetogenic. However, when an expressed allele is inserted by transgenesis, the I-E region is typed as an Idd locus because the I-E molecules epistatically suppress the diabetogenic contributions of the I-Ag7 locus. Epistasis is a form of gene interaction wherein one gene interferes with or otherwise modifies the phenotypic influence of another nonalleleic gene in the same individual. THE U SEFULNESS OF CONGENIC STOCKS The classic technique in mouse genetics for demonstrating the role of a specific genetic locus in the control of a given phenotype was pioneered by Nobel laureate Dr. George Snell at The Jackson Laboratory in the 1950s. Snell investigated the role of H2 (then thought to be a single gene called Histocompatibility-2!) from a donor strain onto a recipient strain. After eight backcrosses, approximately 99.2% of the total genome (comprising ~100,000 expressed genes) is recipient-derived, with the major concentration of donor genes being linked together on the specifically introgressed chromosomal segment. This classic technique provided further confirmation of the primary role of MHC in controlling beta cell autoimmunity in NOD mice. Replacement of the diabetogenic H2g7 haplotype with a protective MHC haplotype ( H2b, H2q or H2nb1) completely eliminated islet-invasive

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insulitis and spontaneous IDDM development in NOD mice.4,5 This fact, coupled with evidence (described below) that no single non-MHC locus has a comparable effect, firmly establishes this complex locus as the major component of susceptibility. Although immunogenetic analysis has concentrated on the diabetogenic contributions of the MHC class II region of the H2g7 haplotype, current evidence suggests that the haplotype as a whole must be considered as contributing to susceptibility. The most compelling evidence comes from the congenic transfer of the unique MHC haplotype of the related CTS/Shi strain onto the NOD/Shi genetic background. The MHC of CTS ( H2ct) mice apparently contains the same class II alleles as NOD, but distinct class I loci, indicating that loci between these markers may differ as well. When this CTS haplotype was transferred onto the NOD inbred background and compared in homozygous state to segregants homozygous for the H2g7 haplotype, a lower incidence of diabetes and insulitis was observed in the H2ct homozygous mice than in segregants homozygous for H2g7.30 The reduced diabetogenic potency of the H2ct thus provides strong support for the concept that, while the class II region is clearly important to disease development, other loci within the extended H2g7 haplotype also contribute. One of these has been dubbed Idd16, although the position of the locus in the complex was not established.31 Even though the MHC class I alleles of NOD mice ( H2Kd, H2Db) are common in non-autoimmune prone strains, they acquire in NOD mice a diabetogenic function—the selection and targeting of CD8+ T cells essential for initiation of the diabetogenic process.32 Thus, the class I alleles also contribute to the overall susceptibility represented by the extended H2g7 haplotype. The NOD allele at the β2-microglobulin locus ( B2m a) is an excellent illustration of the complexity of diabetes genetics in this mouse. It is not uncommon, nor is it defective in NOD mice. However, current unpublished evidence indicates that it may be one of several genes within the Idd13 locus on Chromosome 2 that promotes diabetogenesis by affecting the conformation of MHC class I molecules.33 If the role of B2m a as a diabetogenic contributor is confirmed by transgenic insertion of B2m a and B2m b alleles separately into a congenic stock of NOD/Lt- B2m null mice, then this will be a confirmation of the “Nerup hypothesis” that common alleles can acquire diabetogenic functions in certain unfortunate combination with other genes.

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RARE ALLELES DO NOT NECESSARILY EQUATE WITH DIABETOGENIC ALLELES: THE TAP GENE IMBROGLIO If common alleles such as H2Kd, H2Db, Eab and B2m a can subserve a diabetogenic function when combined with rare diabetogenic genes such as Abg7, the inverse also holds true—rare alleles do not necessarily equate with diabetogenic genes. The intra-MHC Tap genes in H2g7 provide an object lesson in this regard. It was noted previously that both the Tap1 and Tap2 alleles in H2g7 were uncommon based upon restriction fragment length polymorphisms.23 The products of these loci are members of a superfamily of ATP-dependent transport proteins. They form heterodimers that transport processed antigenic peptide fragments generated in endosomal compartments into the lumen of the endoplasmic reticulum. These processed peptides are then complexed with MHC class I molecules and are subsequently translocated to the cell surface for presentation to CD8+ T cells.34 Many mutant mouse and human cell lines lacking the ability to form stable MHC class I-peptide complexes carry mutations which map to regions encoding Tap1 or homologous genes.34 Accordingly, if comparable mutations existed in the NOD’s Tap loci that impaired antigen presentation, they too could represent Idd loci. Both the author’s laboratory23 and another laboratory35 observed and reported the same unique polymorphism in a non-coding region of the NOD Tap1 gene. However, subsequent analysis of NOD’s Tap1 and Tap2 coding regions showed completely normal sequences.36 Several groups have shown normal levels of Tap mRNA expression in NOD splenic leukocytes.23,36,37 The Tap gene products of the H2g7 haplotype did not differ from those encoded within four other MHC haplotypes in affinity for ATP, kinetics of peptide uptake, and substrate specificity.38 These facts notwithstanding, Faustman et al35,39 have asserted that presumed Tap1 gene defects likely explain the diabetogenic contribution of MHC in mice and humans. These workers had difficulty in observing Tap1 transcripts from NOD spleen by Northern analysis,35,39 and further reported that splenocytes from the NOD mice in their colony were expressing diminished constitutive levels of cell surface MHC class I molecules. These diminished class I levels were inferred to be the consequence of a null mutation in the Tap1 gene. It was further proposed that the low class I expression was the basis for failure of NOD mice to develop immune tolerance to endogenous antigens, and thus represented the molecular basis for the MHC-associated susceptibility.

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This latter premise was buttressed by the observation of glucose intolerance in association with low grade islet-associated leukocytic infiltrates in older 129↔C57BL/6 chimeric mice rendered MHC class I deficient by a “knockout” of the β2-microglobulin ( B2m ) gene.35 In a subsequent study, the Tap gene function and MHC class I mediated antigen presentation was reported by this group to be impaired only in diabetes-prone NOD females, but not in more resistant males.40 This claim of low class I expression has been widely refuted by numerous laboratories.20,36,41,42 Since the T-lymphoaccumulation described in the preceding chapter produces a reduction in the ratio of splenic B cells to T cells, the perceived underexpression of class I molecules in fact represented this strain-characteristic reduction in the numbers of splenic B cells (with larger cell volumes). Reduction in Bc ell numbers is not inherently diabetogenic; on the contrary, NOD mice with a targeted mutation in the immunoglobulin heavy chain ( Igh6tm1Cgm ) gene do not develop B cells, and are insulitis and diabetes-resistant as a consequence of the loss of this population of APC.43 Further, four different laboratories44-47 have analyzed independentlyproduced stocks of NOD mice demonstrating MHC class I- and CD8+ T cell-deficiency following congenic introduction of a disrupted B2m gene. These congenic NOD.B2m null stocks were all insulitis- and IDDM-resistant, firmly refuting the contention by Faustman et al35 that T cells become inherently diabetogenic when maturing in a MHC class I-deficient environment. A defect in IFN γ-upregulation of MHC class I has been reported in NOD/Lt peritoneal macrophages.42 However, this is a Tap gene-independent trans-effect entailing defective signaling via transcriptional activators, as demonstrated by the finding that the H2g7-identical NOR/Lt strain, with the same Tap1 sequence as NOD, shows normal IFN γ regulation of MHC class I.42 Finally, another assertion made by the Faustman group,48 that constitutive levels of MHC class I expression are diminished on peripheral blood leukocytes (PBL) from human IDDM patients, has also failed to be confirmed.49 This discussion should convince the reader that caution should be exercised to avoid over-interpreting the clinical significance of rare genomic polymorphisms. Tap gene polymorphisms are certainly useful to human geneticists. Although primary associations between human TAP2 (and not TAP1) allelic variants and IDDM have been suggested,50 these appear to result from linkage disequilibrium with diabetogenic class II alleles.51,52 Availability of Tap polymorphisms, however, helps to better identify genetic subtypes of IDDM in humans.53

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49

GENETIC ANALYSIS OF INSULITIS: IDDM SUSCEPTIBILITY INHERITED AS A THRESHOLD LIABILITY As described above, analysis of transgenic and congenic stocks of NOD mice in which the H2g7 haplotype has been altered or replaced has shown that multiple Ioci within or tightly-linked to this MHC are essential for insulitis development. NOR/Lt, a closely-related recombinant congenic stock sharing approximately 88% of its genome with NOD (including H2g7) perfectly illustrates the requirement for an interaction between MHC and non-MHC ldd loci. NOR/Lt mice are resistant to development of destructive insulitis, even though T-lymphoaccumulation in the pancreas still occurs.54 Genetic modeling based upon the large numbers of Idd loci identified by segregation analysis8,54-56 indicates that a polygenic threshold liability (or multiplicative) model best fits the experimental results.8,57 This is also the case for polygenic, spontaneously developing lupus-like syndromes in mice.58 The alternative model would be a genetic heterogeneity model in which widely variable combinations of the Idd loci would be capable of triggering insulitis sufficiently aggressive to mediate clinical symptoms. Penetrance of the phenotype of clinical IDDM requires destruction of approximately 90% of the β cell mass in the pancreas. Most NOD mice of both sexes will develop an islet-invasive insulitis as they age, yet not all develop overt clinical symptoms of diabetes. The NOD/Wehi substrain is an excellent example of this phenomenon.59 A genetic program for development of autoimmune IDDM is contained in hematopoietic stem cells of NOD mice.60,61 As discussed in chapter 6 by M.A. Atkinson, environmental factors strongly influence the extent to which this program is expressed. Early genetic analysis in Japan and the United States (reviewed in Kikutani and Makino10) showed a much higher frequency of insulitis than of clinical IDDM in backcross progeny following outcross of NOD to other inbred strains, suggesting that only a few of the diabetogenic NOD genes controlled development of islet-destructive insulitis. Insulitis is properly assessed not as a discontinuous trait (+ or -), but rather as a continuously varying trait with phenotypes scored between a range of “benign” peri-insulitis to fully-destructive intra-islet insulitis. Perception of the number of “insulitis” genes segregating in a given cross is a function of the partner strain used in an intraspecific outcross.61 When interspecific outcross with Mus spretus is employed, the genetic control of insulitis is seen to be considerably more complex than when

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intraspecific crosses are utilized.62 This serves to emphasize that an inbred partner strain, especially if it is Swiss-derived, may share with NOD a number of susceptibility genes not shared by the wild-derived strain. As will be discussed further below, the partner strain may also contribute diabetes acceleration genes in addition to resistance genes. The susceptibility genes, regardless of whether they originate from NOD or from the partner strain used in outcross, are assumed to express at the level of the immune system rather than in pancreatic β cells. For example, NOD alleles of genes controlling the rate and level of T cell apoptosis have been implicated,63 as have genes controlling the biochemical functions of peri-insular leukocytes.64-66 If certain of these were estrous cycle dependent, as is apparently the case for one candidate gene, prostaglandin E2 synthase-2 ( Ptgs2) on Chromosome 1 (see chapter 4 by M. Clare-Salzler), a mechanism for the gender-dimorphic destructiveness of the insulitic process in females versus males would be provided. GENETIC SEGREGATION ANALYSIS AS A FIRST STEP IN IDENTIFICATION OF NON-MHC IDD LOCI Outcross of NOD to mice of various other inbred strains have identified over 16 loci on 13 different chromosomes that influence the diabetogenic process in hybrids.67 By definition, any locus which affects susceptibility/resistance to IDDM development in NOD mice is an Idd locus. By this broad definition, any mutant locus (spontaneous or targeted disruption by homologous recombination) introduced into the NOD genome that prevents or accelerates IDDM development is an Idd locus. By this definition, there are well over 100 loci that require the presence of the undisturbed NOD allele to promote IDDM development. These would include spontaneous or targeted mutations at the scid, Rag, nu, Igh, and B2m loci since mutations affecting function of all of these loci are capable of preventing IDDM in NOD mice. H2g7 exerts a codominant permissive effect for insulitis development against which the non-MHC loci are identified.60,68 Widespread, destructive patterns of insulitis do not develop in F2 segregants which have inherited zero copies of H2g7, while it is prevalent in both H2g7 heterozygous and homozygous segregants. Similarly, with few exceptions (reviewed by Wicker et al,4 e.g., NOD.H2h2, and NOD.H2h4 as well as NOD.H2ct 30) replacement of H2g7 by other MHC haplotypes

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51

suppresses development of destructive insulitis. Given the predominant role of H2g7 in setting the basal “threshold” liability, the most informative segregation analyses of non-MHC Idd loci have employed outcross of NOD with H2g7-congenic stocks of B10, B6 or NON.8,55,69 Segregation analysis has primarily concentrated on genome wide scan of diabetic versus non-diabetic progeny in either a first backcross (BC1) or F2 generation. A contingency Chi-square analysis is performed to identify significant deviations between expected and observed frequencies of diabetic versus non-diabetic segregants at all typed genetic markers. A first backcross generation offers the advantage that, if many genes are segregating, a sufficient number of NOD susceptibility genes will be present in homozygous state to permit a sufficiently high spontaneous IDDM incidence necessary for statistical comparison to non-diabetic segregants. However, NOD loci whose contributions to susceptibility are strongly dominant cannot be assessed in a BC1 generation since no significant difference in frequency would be expected between homozygous versus heterozygous BC1 segregants. To assess dominant contributions of a diabetogenic Idd locus from either parental strain, an F2 analysis is required since comparisons between heterozygotes versus both parental homozygotes can be made. As will be noted by examining the listing of currently-identified chromosomal regions carrying Idd loci, both BC1 and F2 analysis has uncovered the fact that the diabetes-resistant parental genome also contributes susceptibility loci to hybrid progeny (Table 2.2). Genes effecting selection of the T cell repertoire would be excellent candidates for Idd genes. Notable in this regard are the products produced by endogenous mammary tumor virus ( Mtv) proviral loci. These retroviral loci encode proteins that mimic superantigens by interacting with the Vβ portion of the T cell receptor, effecting deletions of specific Vβ clonotypes.70 The NOD/Lt genome carries Mtv3 on Chromosome 11,71 Mtv17 on Chromosome 471 and Mtv31 on the Y chromosome.72 The NOD/Crc substrain is segregating for a new Mtv, Mtv45, also on proximal Chromosome 11.73 However, genetic segregation analysis using either NON/Lt or B6 or B10 mice as outcross partner strains has not yet implicated any of these Mtv loci. The effect of a superantigen-like factor controlling transient shifts in the Vβ8.3 CD8 T cell subset has been proposed as the basis for the differential female:male susceptibility.74 However, as described in chapter 3 by D.V. Serreze, this subset is not required for diabetogenesis. The

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NOD genome contains a wide variety of other classes of proviral elements (summarized by Gaskins et al75). A MAIDS (mouse AIDS)-like proviral genome ( Mrv6) has been mapped to Chromosome 14, but not in the region associated with the Idd8 locus.76 NOD mice express the Fv1n (N-tropic) allele on Chromosome 4 controlling susceptibility to infection by naturally occuring ecotropic leukemia viruses. SPEED CONGENICS Another form of segregation analysis is accomplished through construction of a “speed congenic” stock. This entails outcrossing NOD with the strain thought to carry a gene controlling IDDM development (potentially a congenic chromosomal segment from a diabetes-resistant strain or stock, or a genetically disrupted gene such as B2m or the immunoglobulin heavy chain gene ( Igµ). The F1 hybrid is backcrossed to NOD, but before the second backcross is initiated, BC1 individuals are typed for the known NOD-derived chromosomal segments presumed to carry Idd loci (listed in Table 2.2). Individuals are then selected for the next backcross that are homozygous for the greatest number of NOD-derived Idd loci, while heterozygous for allogeneic markers demarcating the introgressing congenic segment or for a marker homologously recombined into a gene as part of a gene targeting strategy (e.g., a neomycin resistance gene). In this situation, the assumption is that the introgressed resistance allele carried through mutliple backcrosses in heterozygous state will be protective when intercrossed to generate segregants homozygous for the locus. Generally, at least nine cycles of backcrossing following outcross (N10) are required to eliminate residual heterozygosity at markers on nonselected chromosomes, and 20 generations of backcrossing are desirable. However, by employing the speed congenic method, a high inciden ce of IDDM can be dem on strated after on ly six cycles of backcrossing (in N7F1 segregants). The intercross generates segregants homozygous or heterozygous for the resistance (or disrupted) allele, as well as homozygous for the NOD susceptibility (or non-disrupted) allele. As an example, N7 segregants homozygous or heterozygous for the NOD allele at the Igµ locus exhibit high incidences of IDDM, confirming that sufficient numbers of NOD diabetogenic genes have been accumulated in the stock to reconstitute the susceptible genotype. This is the necessary finding if the effect of the “knock-out” is to be assessed. In the example cited, the complete absence of B cells ef-

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fected by the Igµ “knock-out” completely suppressed diabetogenesis.43 Particular caution needs to be taken when introgressing a disrupted alllele (usually derived from the 129/Sv or 129/Ola genome providing the embryonal stem (ES) cells used in the targeting procedure). This introgression process not only brings in the targeted gene mutation, but also a considerable number of strain 129 alleles flanking the “knock-out” locus. These linked genes could modify IDDM susceptibility independently of any effect of the disrupted gene to which they are linked. A striking example of the latter was the erroneous conclusion that a disrupted T cell receptor (TCR)alpha gene introgressed into NOD mice blocked development of autoimmunity, when in fact, protection was derived from another strain 129-derived resistance allele on Chr 14, possibly at the Idd 8 or Idd12 locus.77 The only way to control for this is to introgress in parallel with the “knock-out” gene a congenic segment from the 129 genome carrying the wildtype allele. CONGENIC AND SUBCONGENIC STOCKS INDICATE THE PRESENCE OF MULTIPLE NON-MHC SUSCEPTIBILITY LOCI ON NUMEROUS CHROMOSOMES The congenic or speed congenic method represents the most utilized method to date not only for confirming the existence and strength of an Idd locus or a candidate gene, but for defining candidate gene or locus function in the control of immunophenotypes aberrantly regulated in standard NOD mice. Introgression into the NOD genome of a congenic segment conferring resistance is the more common approach, but the reciprocal may also be done (introgressing the NOD allele onto a resistant background). When the latter approach is taken, a bicongenic stock containing either H2g7 plus a single nonMHC Idd region, or else mutliple Idd genes on a single non-MHC region usually needs to be produced to see a shift in insulitis level.78 Congenic mapping allows more precise positioning of an Idd locus, since with each backcross cycle, individuals can be selected in which the length of the original congenic segment has been reduced by recombination. This results in a series of interval specific congenic stocks; if diabetes resistance or some other donor-strain specific subphenotype is retained within a specific interval, then the Idd locus (or loci) are assumed to be within the interval. The Idd3 locus on proximal Chromosome 3 has been fine-mapped to within less than a

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one centimorgan segment containing the Il2 candidate gene by this technique.79 With the exception of the MHC class II genes, the identities of the non-MHC loci are unknown, although, as indicated above, all non-MHC Idd regions contain interesting candidate genes to tantalize the interested investigator. Idd loci on 5 NOD chromosomes have been reported to control the insulitic process directly (see Wicker et al for a review4). Although only single Idd loci were originally detected by exclusion mapping on these NOD chromosomes, further analysis of interval specific subcongenic stocks has led to the realization that most chromosomes carrying what was originally mapped as a single Idd locus, in fact, carries more. Because of the strong contribution to insulitis development by H2g7, inability to demonstrate that a specific chromosomal region does not affect the extent of insulitis in a NOD congenic stock is not always reason to exclude the region as containing insulitis determinants. H2g7 expressing bi- or polycongenic stocks would be more informative in this regard, particularly when the role of an NOD-derived “insulitis” locus is being examined on a diabetes resistant background such as B6.78 Analysis of B6 congenic stocks carrying NOD-derived susceptibility loci have led to the suggestion that Chromosomes 1 and 17 regulate mononuclear cell trafficking into the islets, while other loci are involved in regulation of tolerance to islet cell autoantigens.78 To illustrate the complex interactions between Idd genes, only one locus on Chromosome 3 ( Idd3) was initially detected.69 However, the complexity of the Chromosome 3 contribution to diabetogenesis was appreciated when Chromosome 3 subcongenic stocks were produced. At least two widely separated loci ( Idd3 and Idd10 ) on Chromosome 3 are now known to interact epistatically in bicongenic stocks to modulate the frequency and destructiveness of insulitis.4 As discussed above, the Idd3 region has been narrowed to a 0.3 cM region containing the Il2 locus.79 Although the defective NOD Fc gamma receptor-1 ( Fcgr1) allele was originally proposed as an Idd10 candidate gene,80 it has recently been excluded by fine mapping (Dr. Linda Wicker, personal communication). Even more Idd genes are likely to be identified on this chromosome (Dr. Linda Wicker, personal communication). Chromosome 1 is one of the longer mouse chromosomes. Although separate loci controlling insulitis/diabetes [ Idd5, near the IL-1 receptor ( Il1r) and the cytotoxic T lymphocyte associated protein-4 ( CTLA4) gene] and peri-insulitis (“Nod1”, near Bcl2, a protooncogene

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Table 2.2. Currently-identified Idd loci controlling IDDM in NOD mice Locus(Chromosome, Contribution of Comments linkage marker) NOD allele to IDDM

Idd1 = H2g7 (17)

susceptibility

Idd2 (9, Thy1)

susceptibility

Idd3 (3, Il2))

susceptibility

Idd4 (11, Acrb)

susceptibility

Idd5 (1, Il1r, Ctla4) susceptibility, (1, Bcl2) insulitis susceptibility, peri-insulitis Idd6 (6, D6Nds1)

susceptibility

Idd7 (7, Ckmm) Idd8 (14, Plau)

resistance resistance resistance

Idd9 (4, Nppa)

susceptibility

Idd10 (3, Tshb)

susceptibility

Idd11 (4, Nhe1)

susceptibility

Idd12 (14, Plau)

susceptibility

Idd13 (2, Il1)

susceptibility

Idd14 (13, D13Mit61) Idd15 (5, Xmv65)

susceptibility susceptibility

Idd16 (17, MHC)

susceptibility

MHC class I and II loci contribute; unique Tap and Hsp70 alleles affects timing of IDDM onset; effect more pronounced in outcross with NON than with B10 or B6 controls frequency and severity of insulitis; may be the Il2 gene itself affects timing of IDDM onset in B10, but not NON outcross analysis Bcl2 locus may control reduced susceptibility of NOD T cells to apoptotic cell death. Segregates in outcross with C57 strains, but not NON (which also develops peri-insulitis) NON contributes a gene in this region that is more diabetogenic than the NOD allele at Idd6. B10 allele is protective Both NON and C57BL strains contribute a diabetogenic allele Diabetogenic allele contributed by B10, but not NON diabetogenic effect clearly demonstrable in F2 Protective effect in B10, but negligible in NON outcross Observed in outcross/backcross with B6 and SJL Observed in outcross/backcross with B6 and SJL; possibly the same locus as Idd8, but B6 and SJL alleles are not diabetogenic Protective allele observed in outcross with related NOR/Lt stock discovered in F2 cross with NON discovered in F2 cross with NON; Xmv65 a xenotropic proviral locus possibly encoding a defective retrovirus expressed in NOD beta cells based upon observation that NOD-H2ct congenic stock exhibits lower IDDM incidence than H2g7; position proximal or distal to class II loci unclear

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associated with resistance to programmed cell death) have been identified, this chromosome is laden with many more loci implicated in autoimmune disease. Currently, CTLA4 is receiving attention because it lies in a region homologous to a human IDDM locus. Table 2.3 indicates the known human IDDM linkages and potential homologs in the mouse. It is certain that many more insulitis/diabetes controlling genes will eventually be identified in humans and in mice. The ability to identify an Idd locus in mice is wholly contigent upon the genome of the partner strain used in the initial outcross. Other than H2g7, the only non-MHC locus that consistently appears in multiple NOD outcrosses is the Idd3 locus. CONNECTING Idd LOCI WITH IMMUNOPHENOTYPES Insulitis is too broad a phenotype to provide critical insight as to how a given Idd allele may be functioning. Hence, more specific immune anomalies must be identified in the NOD genome that are corrected or ameliorated when a given NOD allele is replaced by a resistance allele in an interval-specific congenic stock. Certain of the defects in functional maturation of marrow derived NOD macrophages (discussed in detail in chapter 1) appear to be corrected in NOD mice congenic for B6 resistance alleles at both Idd3 and Idd10. Marrow-derived macrophages from this diabetes-resistant congenic stock exhibit normalized growth and functional responses to myeloid growth factors previously reported to be an NOD strain-specific defect.42,81 Further, marrow from these Idd3/ Idd10 congenic mice does not transfer IDDM into lethally irradiated NOD/Lt mice, unlike standard NOD/Lt marrow. Associated with the developmental defects of these marrow-derived macrophages is an aberrant release of bioactive IL-1 following lipopolysaccharide (LPS) stimulation. In comparison to B6 marrow-derived macrophages, this is manifest as a lower release of IL-1β coupled with very high levels of IL-1 receptor gene transcription coupled with release of an IL-1 antagonist (D.V. Serreze and E.H. Leiter, unpublished observations). This NOD strain-specific phenotype is ameliorated in Chromosome 3 congenic mice in which the defective NOD Fcrg1 allele, previously suggested as a possible Idd10 candidate gene,80 was replaced by a wild-type B6 allele. However, this congenic stock, maintained by Drs. Linda Wicker and Larry Peterson (Merck Research Laboratories, Rahway, NJ) carries diabetogenic NOD alleles proximal and distal to the small region of B6 genome around Fcgr1, and is not protected from IDDM. Thus, correction of this de-

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Table 2.3. Comparative mapping of human and mouse Idd genes Human Locus (Chr) Marker/ Candidate

Potential NOD Idd homolog (Chr)

Marker

IDDM1 (6p21) IDDM2 (11p15.5) IDDM3 (15q26) IDDM4 (11q13) IDDM5 (6q25) IDDM6 (18q) IDDM7 (2q31-33) IDDM8 (6q25-27)

HLA-DQB1 INS/VNTR D15S107 FGF3 ESR D18S64 D2S326 D6S264

H2g7

IDDM9 (3q21-q25) IDDM10 (10p11.2-q11.2) IDDM11 (14q24.3-q31) IDDM12 (2q31-33) IDDM13 (2q34) GCK (7p) IDDM15 (6q21)

D3S1303 GAD2

Idd1 (17) none yet identified (distal 7) ?Idd2 (middle 9) none yet identified (distal 7) none yet identified (proximal 10) ? “Idd5 “(1) ? “Idd5” (1) none yet identified (proximal 10 or 17) none yet identified (middle 6) none yet identified (proximal 2)

D14S67

none yet identified (middle 12)

IGFBP-2,5 GCK D6S283

?”Idd5 “ (1) ?”Idd5 “ (1) none yet identified (proximal 11) ?Idd14 (13)

?Cyp19

Bcl2 Ctla4, Il1r

Bcl2 Igfbp-5 D13Mit61

Idd5 placed in quotation marks because there appear to be multiple Idd loci on mouse Chr 1

viant immunophenotype is apparently dissociated from other B6 alleles on distal Chromosome 3 conferring IDDM protection. Genetic segregation analysis has also been used to identify immunophenotypes controlled by Idd loci. Currently lumped together as “Idd5”, two loci on Chromosome 1 (one or more proximal, near the IL-1 receptor ( Il1r) and CTLA loci controlling insulitis and diabetes, and one or more distal, near the Bcl2 proto-oconcogene controlling insulitis and peri-insulitis respectively have been identified 55,82 Idd genes on Chr 9 ( Idd2) and Chr 11 ( Idd4) appear to act as “timing” genes,55 determining the rate of activation of cytopathic effectors in the insulitic infiltrates. A gene in the Idd4 region has been linked to thymocyte proliferative unresponsiveness in NOD mice.64 A gene on Chromosome 6 presumably near and possibly the same as Idd6, controls dexamethasone-induced apoptosis in NOD thymocytes.83 A locus controlling the sex-dependent elevation of CD4+ T cells ( Tlf, for T lymphocyte fraction) circulating in peripheral blood segregated with IDDM in an NOD x NON outcross.84 This locus is near, and possibly the same as Idd2 on Chromosome 9.84 The Tlf locus is androgen-sen-

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sitive, correlating in backcross analysis with the stronger protective action of the NON allele at Idd2 in males than in females.84 Mice congenic for the NON-derived Idd2 containing segment of Chromosome 9 are more resistant to cyclophosphamide-induced IDDM compared to standard NOD/Lt sex-matched controls (this laboratory, unpublished). The interferon gamma inducing factor (i.e., IL-18) gene has been mapped to the Idd2 region and is abnormally upregulated in NOD but not BALB/c macrophages following cyclophosphamide administration.85 DO NOD ISLETS EXPRESS STRAIN-SPECIFIC Idd GENES? It remains an open question as to whether any of the Idd genes described above exhibit a β cell restricted pattern of expression. Intracisternal type A retroviral genomes are expressed in NOD β cells, but it is expression of xenotropic forms that distinguish NOD from related strains.86 A gene whose product is expressed in NOD islets and typed by proliferation of a cytopathic islet-reactive CD4+ T cell line from NOD spleen has been mapped to Chromosome 6 near the Idd6 region.87 β cell expression of a variety of proviral genomes present in NOD mice could represent targets of β-cytotoxic T cells. Humoral responses to endogenous retroviral gene products are typically found in autoimmune-prone strains of mice, including NOD.75,88 Initiation of insulitis in NOD islets may represent a response to β cell expression of xenotropic retroviral antigens encoded by two endogenous proviral loci, Xmv65 and Xmv66.75 The Xmv65 locus on proximal Chr 58 segregates with IDDM in an (NOD x NON.H2g7)F2 cross ( Idd15 marker). Xmv66 has been mapped just distal to H2g7 (this laboratory, unpublished) and thus would be a potential candidate for Idd16.31 Finally, certain genes not normally expressed by NOD β cells can be induced either by conditions of neoplastic transformation or by cytokines, especially interferon gamma. One of these is Emv30, an endogenous ecotropic proviral genome whose expression is induced in transformed NOD β cells.89 A peculiar antigenic specificity induced in NOD macrophages and islet cells by gamma interferon is typed by anomalous binding of 28-13-3, an “irrelevant” H2Kb monoclonal antibody (NOD mice express H2Kb). The antigen, termed the “occult” antigen, is MHC-linked, with complex trans-regulation implicated.90 Although it is unknown whether the new serologic specificity represents induction of a “spare” class I gene, or simply reflects altered conformation of the standard NOD class I genes due to binding of new

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peptides, the phenomenon itself further illustrates the possibility that exposure of NOD β cells to high concentrations of cytokines may elicit altered forms of “self”. NOD β cells have also been reported to express low voltage activated calcium channels.91 This expression is reportedly abnormal, leading to higher levels of intracellular calcium (and possibly enhanced excitability at higher glucose concentrations) than found in β cells of a control strain.91 LESSONS FROM GENETICS OF IDDM IN NOD MICE FOR THE GENETIC PREDICTION OF IDDM IN HUMANS Currently, pre-type I autoimmune diabetes in humans is detected by testing for autoantibody development to a variety of islet cell autoantigens, coupled with the demonstration of impaired insulin secretory capacity. In combination with evidence for impaired β cell function, detection of diagnostic high levels of 2-3 of these antibodies (e.g., with specifities to glutamic acid decarboxylase, insulin, and other candidate autoantigens such as IA-2) 92 essentially means that the autoimmune destruction of β cells has already been initiated. Identification of genotypes conferring high risk for IDDM in infants at birth would greatly enhance the likelihood that intervention therapy would be successful if initiated prior to the onset of β cell damage. Since “high risk” HLA class II genes are so prevalent in Caucasian populations, neonatal screening for these genes at a population level are not predictive per se of future IDDM development. However, if there were a relatively limited set of high risk non-MHC susceptibility loci that were commonly associated with high risk HLA haplotypes, genetic risk assessment of prediabetes would be greatly enhanced. The original hope was that elucidation of the locations of the major nonMHC Idd genes in NOD mice would directly lead to identification of homologous counterparts in the human genome (designated IDDM loci). This direct extrapolation has not been realized.67 Recent analysis in human multiplex families reported evidence for IDDM linkage at 18 chromosomal regions.93 The most frequently detected IDDM linkage outside of HLA ( IDDM1) in humans is IDDM2, associated with different alleles of a variable number tandem repeat (VNTR) located upstream of the insulin ( INS) locus.94 Evidence exists that shorter alleles express at lower levels in fetal thymus, such that less tolerance to (pro)insulin may be induced while the immune repertoire is being generated.95,96 An Idd homolog near the Ins2 gene on mouse Chromosome 7 has not yet been identified in any outcross

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between NOD and diabetes-resistant strains, perhaps because mice have two insulin genes so that (pro)insulin levels in fetal thymus are not contingent on one locus only. Combining linkage analysis in NOD mice with identification of the immunophenotypes controlled by the Idd loci may allow identifcation of similar immune defects essential to IDDM development in humans, even though the genetic events underlying to the defect may be different. An important insight gained from the genetic analysis of IDDM susceptibility in NOD mice (and illustrated in Fig. 2.1) is that the NOD genome contains but one subset of a much larger set of potential Idd genes predisposing to autoimmune disease. This is illustrated in Fig. 2.1, showing that the risk of IDDM development increases as H2g7 is combined with increasing numbers of non-MHC Idd loci. When the NOD-specific diabetogenic interactions are disrupted by outcross, and then reassorted into diabetogenic combinations through intercross or backcross, the same fixed set of IDDM susceptibility modifiers defining the NOD genome need not be fully reconstituted to elicit IDDM. One of the most interesting discoveries (see refs. 8, 55), made by outcross of NOD with IDDM-resistant strains such as B10 and NON, is that the “normal” parental strain contributes susceptibility as well as resistance alleles (e.g., B10 derived susceptibility alleles at Idd7 and Idd8, and NON derived susceptibility alleles at Idd6 and Idd7, but not at Idd8). The NON strain was, in fact, originally selected for impaired glucose homeostasis. Indeed, NON/Lt mice become obese with age and exhibit impaired glucose tolerance. Accordingly, NON genes contributing to glucose intolerance apparently synergize deleteriously with the collection of NOD-derived Idd susceptibility genes to increase the penetrance of the disease phenotype. Similarly, following outcross of NOD to an inbred Mus spretus mouse, undefined M. spretus alleles precipitated a non-insulin dependent form of diabetes in males,97 emphasizing not only the heterogeneity in the genetics of diabetes, but also in its pathophysiology. In addition, the activities of certain immune mediators such as Natural Killer (NK) cells and lytic complement which could possibly participate in autoimmune destruction of pancreatic β cells, are deficient in NOD, but not B10 or NON mice. Idd susceptibility loci from these “resistant” strains might include genes promoting more normal activation of immune responses defective in NOD, expanding the repertoire by which β cells may be destroyed in hybrid mice. A possible illustration

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Fig. 2.1. Stepwise logistic regression analysis shows that the probability of developing diabetes increases as a function of the number of deleterious nonMHC genotypes contributed by each parental genome. Data are from genotyping 245 diabetic and nondiabetic first backcross progeny (to NOD) following intercross between NOD/Lt x NON/Lt.H2g7. The threshold susceptibility conferred by H2g7 homozygosity is present in individuals typed as “zero” for the non-MHC loci entered into this analysis. A deleterious gene was considered present when a backcross individual was homozygous for any of the NOD-derived susceptibility markers or hetero-zygous for the NON-derived Idd7 susceptibility locus marker, Ckmm. Reprinted in modified form by permission from McAleer et al (1995). Crosses of NOD mice with the related NON strain: a polygenic model for type I diabetes. Diabetes 44:1186-1195.

of this is that congenic replacement of a defective NOD Fcgr2 allele on distal Chromosome 1 accelerates rather than retards diabetogen– esis.98 The finding that the genome of the NOD mouse represents but one subset of a larger spectrum of potentially pathogenic genes combinations certainly indicates that the genetics of IDDM in randomlyreproducing human populations will be no less complicated.

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ACKNOWLEDGMENTS This writing has been supported by NIH grants DK 36175 and DK27722, and a grant from The Juvenile Diabetes Foundation International. The author thanks Drs. David Serreze, Len Shultz, and Linda Wicker for critical reviews. REFERENCES 1. Merriman T, Todd J. Genetics of insulin-dependent diabetes; nonmajor histocompatibility genes. Horm Metab Res 1996; 28(6):289-293. 2. Chandler P, Fairchild S, Simpson E. H-Y responses of non-obese diabetic (NOD) mice. J Immunogenet 1988; 15:321-330. 3. Bailey DW. How pure are inbred strains of mice? Immunol Today 1982; 3:210-214. 4. Wicker LS, Todd JA, Peterson LB. Genetic control of autoimmune diabetes in the NOD mouse. Ann Rev Immunol 1995; 13:179-200. 5. Serreze DV, Leiter EH. Insulin Dependent Diabetes Mellitus (IDDM) in NOD Mice and BB Rats: Origins in Hematopoietic Stem Cell Defects and Implications for Therapy. In: Shafrir E, ed. Lessons from Animal Diabetes. V London: Smith-Gordon, 1995:59-73. 6. Slattery R. Transgenic approaches to understanding the role of MHC genes in insulin dependent diabetes mellitus. II. The non-obese diabetic (NOD) mouse. Baillieres-Clin-Endocrinol-Metab 1991; 5(3): 449-54. 7. Vyse TJ, Todd JA. Genetic analysis of autoimmune disease. Cell 1996; 85(3):311-318. 8. McAleer MA, Reifsnyder P, Palmer SM et al. Crosses of NOD mice with the related NON strain: a polygenic model for type I diabetes. Diabetes 1995; 44:1186-1195. 9. Nepom GT. Class II antigens and disease susceptibility. Annu Rev Med 1995; 46:17-25. 10. Kikutani H, Makino S. The murine autoimmune diabetes model: NOD and related strains. In: Dixon FJ, ed. Adv Immunol NY: Academic Press, 1992; 51:285-322. 11. Leiter EH . The NOD m ouse m eets the “Nerup H ypothesis”. Is diabetogenesis the result of a collection of common alleles present in unfavorable combinations? In: Vardi P, Shafrir E, ed. Frontiers in Diabetes Research: Lessons from Animal Diabetes III. London: SmithGordon, 1990:54-58. 12. Nerup J, Mandruppoulsen T, Helqvist S et al. On the pathogenesis of IDDM. Diabetologia 1994; 37(Suppl 2):S82-S89. 13. Serreze D. Autoimmune diabetes results from genetic defects manifest by antigen presenting cells. FASEB J 1993; 7:1092-1096. 14. Hattori M, Buse JB, Jackson RA et al. The NOD mouse: recessive diabetogenic gene in the major histocompatability complex. Science 1986; 231:733-735.

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45. Serreze DV, Leiter EH, Christianson GJ, et al. MHC class I deficient NOD- B2m null mice are diabetes and insulitis resistant. Diabetes 1994; 43:505-509. 46. Sumida T, Furukawa M, Sakamoto A et al. Prevention of insulitis and diabetes in beta(2)-microglobulin-deficient non-obese diabetic mice. Int Immunol 1994; 6(9):1445-1449. 47. Katz J, Benoist C, Mathis D. Major histocompatibility complex class I molecules are required for the development of insulitis in non-obese diabetic mice. Eur J Immunol 1993; 23:3358-3360. 48. Fu Y, Nathan DM, Li F, Li X, Faustman DL. Defective major histocompatibility complex Class I expression on lymphoid cells in autoimmunity. J Clin Invest 1993; 91:2301-2307. 49. Hao W, Gladstone P, Engardt S et al. Major histocompatibility complex class I molecule expression is normal on peripheral blood lymphocytes from patients with insulin-dependent diabetes mellitus. J Clin Invest 1996; 98(7):1613-1618. 50. Caillat-Zucman S, Bertin E, Timsit J et al. TAP1 and TAP2 transporter genes and predisposition to insulin dependent diabetes mellitus. CR Acad Sci, Paris 1992; 315:535-539. 51. Ronningen KS, Undlien DE, Ploski R et al. Linkage disequilibrium between TAP2 variants and HLA class II alleles; no primary association between TAP2 variants and insulin dependent diabetes mellitus. Eur J Immunol 1993; 23:1050-1056. 52. Kawaguchi Y, Ikegami H, Fukuda M et al. Absence of association of TAP and LMP genes with type 1 (insulin dependent) diabetes mellitus. Life Sci, 1994; 54:2049-2053. 53. Maugendre D, Alizadeh M, Gauthier A et al. Genetic-heterogeneity between type 1A and type 1B insulin-dependent diabetes-mellitus— H LA class- II an d TAP gen e an alysis. Tissu e An tigen s 1996; 48(5):540-548. 54. Serreze DV, Prochazka M, Reifsnyder PC et al. Use of recombinant congenic and congenic strains of NOD mice to identify a new insulin depen den t diabetes resistan ce gen e. J Exp Med 1994; 180: 1553-1558. 55. Ghosh S, Palmer SM, Rodrigues NR et al. Polygenic control of autoimmune diabetes in nonobese diabetic mice. Nature Genet 1993; 4:404-409. 56. Morahan G, McClive P, Huang D, Little P, Baxter A. Genetic and physiological association of diabetes susceptibility with raised Na+/H+ exchange activity. Proc Natl Acad Sci, USA 1994; 91:5898-5902. 57. Risch N, Ghosh S, Todd JA. Statistical evaluation of multiple-locus linkage data in experimental species and its relevance to human studies: application to Nonobese Diabetic (NOD) mouse and human insulin-dependent diabetes mellitus (IDDM). Am J Human Genet 1993; 53:702-714.

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58. Morel L, Rudofsky U, Longmate J et al. Polygenic control of susceptibility to murine systemic lupus erythematosus. Immunity 1994; 1:219-229. 59. Baxter AG, Koulamanda M, Mandel TE. High and low diabetes incidence nonobese diabetic (NOD) mice. Origins and characterization. Autoimmunity 1991; 9:61-67. 60. Leiter EH, Serreze DV. Antigen presenting cells and the immunogenetics of autoimmune diabetes in NOD mice. Regional Immunol 1992; 4:263-273. 61. Leiter EH. The genetics of diabetes susceptibility in mice. FASEB J 1989; 3:2231-2241. 62. De Gouyon B, Melanitou E, Richard MF et al. Genetic analysis of diabetes and insulitis in an interspecific cross of the nonobese diabetic m ouse with M us spretus. Proc Natl Acad Sci, USA 1993; 90:1877-1881. 63. Garchon HJ, Bedossa P, Eloy L, Bach JF. Identification and mapping to chromosome-1 of a susceptibility locus for peri-insulitis in nonobese diabetic mice. Nature 1991; 353:260-262. 64. Gill BM, Jaramillo A, Ma LL et al. Genetic linkage of thymic T-cell proliferative unresponsiveness to mouse chromosome 11 in NOD mice: a possible role for chemokine genes. Diabetes 1995; 44(6): 614-619. 65. Xie T, Reddy S, Hofig A et al. Regulation of prostaglandin synthase2 (Pgs-2) in NOD macrophages. Autoimmunity 1996; 24 (suppl 1): 23A. 66. Xie T, Hofig A, Yui M et al. Spontaneous prostaglandin synthase-2 (Pgs2) gene expression in macrophages of NOD and congenic mice. Autoimmunity 1995; 21(1):17A. 67. Leiter EH. Lessons from the animal models: the NOD mouse. In: Palmer JP, ed. Diabetes Prediction, Prevention, and Genetic Counselling. London: John Wiley & Sons, 1996: 201-226. 68. Wicker LS, Miller BJ, Fischer PA, Pressey A, Peterson LB. Genetic control of diabetes and insulitis in the nonobese diabetic mouse. Pedigree analysis of a diabetic H-2nod/b heterozygote. J Immunol 1989; 142:781-784. 69. Todd JA, Aitman TJ, Cornall RJ et al. Genetic analysis of autoimmune type 1 diabetes mellitus in mice. Nature 1991; 351:542-547. 70. Frankel WN, Rudy C, Coffin JM, Huber BT. Linkage of Mls genes to endogenous mammary tumor viruses of inbred mice. Nature 1991; 349:526-528. 71. Knight AM, Dyson PJ. Detection of DNA polymorphisms between two inbred mouse strains-limitations of restriction fragment length polymorphisms (RFLPs). Mol Cell Probes 1990; 4:497-504. 72. Prochazka M, Leiter EH. Identification of a novel mouse mammary tumor proviral locus ( Mtv-31) on chromosome Y. Mouse Genome 1990; 87:111.

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73. Fairchild S, Rosenwasser O, Dyson P, Tomonari K. Tcrb-V3+ T cell deletion and a new mouse mammary tumor provirus, Mtv-44. Immunogenetics 1992; 36:189-194. 74. McDuffie M, Ostrowska A. Superantigen-like effects and incidence of diabetes in NOD mice. Diab 1993; 42:1094-1098. 75. Gaskins HR, Prochazka M, Hamaguchi K, Serreze DV, Leiter EH. Beta cell expression of endogenous xenotropic retrovirus distinguishes diabetes susceptible NOD/Lt from resistant NON/Lt mice. J Clin Invest 1992; 90:2220-2227. 76. Tsumura H, Reifsnyder P, Leiter E. Mapping of a murine AIDS virus-related proviral gene (Mrv6) in NOD/Lt mice to chromosome 14. Mamm Genome 1996. 77. Elliott JI, Altmann DM. Non-obese diabetic mice hemizygous at the T cell receptor alpha locus are susceptible to diabetes and sialitis. Eur J Immunol 1996; 26(4):953-956. 78. Yui M, Muralidharan K, Moreno-Altamirano B, Perrin G, Chestnut K, Wakeland E. Production of congenic mouse strains carrying NODderived diabetogenic genetic intervals: an approach for the genetic dissection of complex traits. Mamm Genome 1996; 7:331-334. 79. Lord CJ, Bohlander SK, Hopes EA et al. Mapping the diabetes polygene Idd3 on mouse chromosome 3 by use of novel congenic strains. Mamm Genome 1995; 6(9):563-570. 80. Prins J-B, Todd J, Rodriques N et al. Linkage on chromosome 3 of autoimmune diabetes and defective Fc receptor for IgG in NOD mice. Science 1993; 260:695-698. 81. Serreze DV, Gaedeke JW, Leiter EH. Hematopoietic stem cell defects underlying abnormal macrophage development and maturation in NOD/Lt mice: defective regulation of cytokine receptors and protein kinase C. Proc Natl Acad Sci USA 1993; 90:9625-9629. 82. Garchon H-J, Luan J-J, Eloy L, Bédossa P, Bach J-F. Genetic analysis of immune dysfunction in non-obese diabetic (NOD) mice: mapping of a susceptibility locus close to the Bcl-2 gene correlates with increased resistance of NOD T cells to apoptosis induction. Eur J Immunol 1994; 24:380-384. 83. Penha-Goncalves C, Leijon K, Persson L, Holmberg D. Type 1 diabetes and the control of dexamethazone-induced apoptosis in mice m aps to the sam e region on chrom osom e 6. Gen om ics 1995; 28(3):398-404. 84. Pearce RB, Formby B, Healy K, Peterson CM. Association of an androgen-responsive T cell phenotype with murine diabetes and Idd2. Autoimmunity 1995; 20(4):247-258. 85. Rothe H, Jenkins N, Copeland N, Kolb H. Active stage of autoimmune diabetes is associated with the expression of a novel cytokine, IGIF, which is located near Idd2. J Clin Invest 1997; 99:469-474. 86. Leiter EH, Hamaguchi K. Viruses and diabetes: diabetogenic role for endogenous retroviruses in NOD mice? J Autoimmunity 1990; (3 suppl):31-40.

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87. Dallas-Pedretti A, McDuffie M, Haskins K. A diabetes-associated T-cell autoantigen maps to a telomeric locus on mouse chromosome 6. Proc Natl Acad Sci, USA 1995; 92:1386-1390. 88. Serreze DV, Leiter EH, Kuff EL, et al. Molecular mimicry between insulin and retroviral antigen p73. Development of cross-reactive autoantibodies in sera of NOD and C57BL/KsJ- db/db mice. Diabetes 1988; 37:351-358. 89. Hamaguchi K, Gaskins HR, Leiter EH. NIT-1, a pancreatic β cell line established from a tran sgen ic NOD/ Lt m ou se. Diabetes 1991; 40:842-849. 90. Leiter E, Christianson G, Serreze D, Ting A, Worthen S. MHC antigen induction by interferon γ on cultured mouse pancreatic β cells and macrophages. Genetic analysis of strain differences and discovery of an “occult” class I-like antigen in NOD/Lt mice. J Exp Med 1989; 170:1243-1262. 91. Wang L, Bhattacharjee A, Fu J, Li M. Abnormally expressed lowvoltage-activated calcium channels in beta-cells from NOD mice and a related clonal cell line. Diabetes 1996; 45(12):1678-1683. 92. Gottlieb PA, Eisenbarth GS. Mouse and man: multiple genes and multiple autoantigens in the aetiology of type i DM and related autoimmune disorders. J Autoimmun 1996; 9(2):277-281. 93. Davies JL, Kawaguchi Y, Bennett ST, et al. A genome-wide search for h u m an type 1 diabetes su sceptibility gen es. Natu re 1994; 371:130-136. 94. Owerbach D, Gabbay K. The search for IDDM susceptibility genes: The next generation. Diabetes 1996; 45(5):544-551. 95. Pugliese A, Zeller M, Fernandez Jr A, et al. The insulin gene is transcribed in the human thymus and transcription levels correlate with allelic variation at the INS VNTR- IDDM2 susceptibility locus for type 1 diabetes. Nature Genetics 1997; 15:293-297. 96. Vafiadis P, Bennett S, Todd J, et al. Insulin expression in human thymus is modulated by INS VNTR alleles at the IDDM2 locus. Nature Genetics 1997; 15:289-292. 97. Hattori M, Yamato E, Matsumoto E, et al. Occurrence of pretype I diabetes (pre-IDDM) and type II diabetes (NIDDM) in BC1 [(NOD x Mus spretus)F1 x NOD] mice. In: Shafrir E, ed. Lessons from Animal Diabetes VI. Boston: Birkhaüser, 1996; VI:83-95. 98. Luan J, Monteiro R, Sautes C, et al. Defective Fc gamma RII gene expression in m acrophages of NOD m ice—genetic linkage with upregulation of IgG1 and IgG2b in serum. J Immunol 1996; 157(10): 4707-4716. 99. Baxter AG, Cooke A. Complement lytic activity has no role in the pathogenesis of autoimmune diabetes in NOD mice. Diabetes 1993; 42:1574-1578.

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100. Gavin A, Hamilton J, Hogarth P. Extracellular mutations of Nonobese Diabetic mouse FcγRI modifiy surface expression and ligand binding. J Biol Chem 1996; 271:17091-17098. 101. Chesnut K, Shie J-X, Cheng I, et al. Characterization of candidate genes for IDD susceptibility from the diabetes-prone NOD mouse strain. Mamm Genome 1993; 4:549-554. 102. Shultz LD, Schweitzer PA, Christianson SW, et al. Multiple defects in innate and adaptive immunological function in NOD/LtSZ- scid mice. J Immunol 1995; 154:180-191. 103. Giorda R, Rudert WA, Vavassori C, et al. NKR-P1, a Signal Transdu ction Molecu le on Natu ral Killer Cells. Scien ce 1990; 249: 1298-1300. 104. Cetkovic-Cvrlje M, Leiter E. Mono-ADP ribosyltransferase genes and diabetes in NOD mice: is there a relationship? In: Haag F, KochNolte F, ed. ADP-Ribosylation IN Animal Tissues: Structure, Function and Biology of Mono(ADP-Ribosyl)Transferase and Related Enzymes. New York: Plenum Press, 1997:217-227. 105. Prochazka M, Gaskins HR, Leiter EH, et al. Chromosomal localization, DNA polymorphism, and expression of Rt-6, the mouse homologue of rat T cell lymphocyte differentiation marker RT6. Immunogenetics 1991; 33:152-156. 106. Philbrick WM, Maher SE, Bridgett MM, Bothwell ALM. A recombination event in the 5’ flanking region of the Ly-6C gene correlates with impaired expression in the NOD, NZB and ST strains of mice. EMBO 1990; 9:2485-2492. 107. Bazzoni F, Beutler B. Comparative expression of TNF- α alleles from normal and autoimmune-prone MHC haplotypes. J Inflamm 1995; 45:106-114. 108. Lund T, Shaikh S, Kendall E, et al. RFLP analysis of the MHC class III region defines unique haplotypes for the non-obese diabetic, cataract Shion ogi an d the n on -obese n on -diabetic m ou se strain s. Diabetologia 1993; 36:727-733. 110. Nandi D, Iyer MN, Monaco JJ. Molecular and serological analysis of polymorphisms in the murine major histocompatibility complex-encoded proteasome subunits, LMP-2 and LMP-7. Exp Clin Immunogenet 1996; 13(1):20-9. 111. Marusina K, Iyer M, Monaco J. Allelic variation in the mouse Tap-1 and Tap-2 transporter genes. J Immunol 1997; 158:5251-5256. 112. Baeza NJ, Moriscot CI, Renaud WP, Okamoto H, Figarella CG, Vialettes BH. Pancreatic regenerating gene overexpression in the nonobese diabetic mouse during active diabetogenesis. Diabetes 1996; 45(1):67-70.

CHAPTER 3

The Identity and Ontogenic Origins of Autoreactive TLymphocytes in NOD Mice David V. Serreze

INTRODUCTION

nsulin dependent diabetes mellitus (IDDM) in both humans and the NOD mouse model is caused by autoimmune destruction of pancreatic β cells by T lymphocytes.1-4 The identity of T cells mediating autoimmune β cell destruction in IDDM, and the mechanisms that allow these effectors to be generated, has been a matter of intense investigation and controversy. This chapter will focus on studies in the NOD mouse which have provided insight into these questions.

I

OVERVIEW OF T CELL ONTOGENY AND SELECTION T lymphocytes are derived from presursor cells of bone marrow origin that subsequently differentiate within the thymus. Mature Tl ymphocytes that emigrate from the thymus and seed the blood and peripheral lymphoid organs can be divided into two major subsets based on cell surface expression of the CD4 or CD8 markers.5,6 The ability of an individual T cell within either subset to recognize a specific antigen is imparted by clonally distributed T cell receptor (TCR) molecules that are expressed as α/ β chain heterodimers on the NOD Mice and Related Strains: Research Applications in Diabetes, AIDS, Cancer and Other Diseases, edited by Edward Leiter and Mark Atkinson. © 1998 R.G. Landes Company.

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cell surface.7,8 While the total mouse genome contains on the order of 105 genes, at least 109 different TCR molecules can be generated.7 This broad diversity is generated as T cells differentiate in the thymus by splicing and rejoining of germline DNA sequences that encode various components of the TCR α and β chains followed by pairing of the resulting gene products.9 The germline sequence for the murine TCR β chain resides on chromosome 6 and consists of approximately 30 variable (V) region gene segments divided in 20 subfamilies of 1-3 members each, and two consecutive downstream clusters each consisting of 1 diversity (D), 6 joining (J) and 1 constant (C) region gene segment.10 The germline sequence for the TCR α chain on mouse chromosome 14 lacks D region segments, but contains approximately 100 V region gene segments, 50 J region segments, and 1 C region segment.10 The particular germline segments which are incorporated into any given VDJ ( β chain) or VJ ( α chain) gene rearrangement generates structural variability at the N-terminus of both resulting molecules, which upon pairing contributes to the antigenic specificity of the TCR.10 An additional factor contributing to the heterogeneity of rearranged TCR α and β chains is that any given V and J coding region can be linked by differing splice sequences.7 These V to J linkage sequences (which incorporate a D segment in the TCR β chain) are termed CDR3 regions and are also major contributors to the antigenic specificity of the rearranged TCR molecule.11 The constant region domain at the C-terminus of rearranged TCR α/ β molecules noncovalently associates with the CD3 complex at the cell surface. Antigen binding to the rearranged TCR α/ β molecules induces the CD3 complex to transmit signals that activate intracellular protein kinase C (PKC) mediated second messanger pathways that trigger appropriate T cell effector functions.12 Once a TCR gene rearrangem en t has occu r red in any differen tiatin g T cell, su bsequ en t rearrangment of TCR α and β sequences remaining in the germline configuration is suppressed through a mechanism termed allelic exclusion, although this process is less complete for α than β chain sequences.13,14 Mice also generate a small number of T cells (< 5% of the total popu lation ) that express rear ran ged TCR γ / δ chain heterodimers encoded by germline DNA sequences on chromosomes 10 and 14, respectively.9 These γ/ δ T cells will not be discussed further in this review since they have not yet been implicated in the pathogenesis of autoimmune IDDM in NOD mice.

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The TCR α/ β chain heterodimers generated by the recombinatorial processes described above, recognize antigens which consist of peptide fragments bound to molecules encoded within the major histocompatability complex (MHC).15 There are two primary types of MHC gene products. MHC class I gene products ( H2K and H2D in mice) are expressed on virtually all cell types and present peptides of intracellular origin, such as those derived from replicating viruses, to T cells that express TCR α/ β chain heterodimers complexed with CD8 molecules. Such MHC class I restricted CD8+ T cells usually exert a cytotoxic function. In contrast, MHC class II gene products (I-A and I-E in mice) which exist as α/ β chain heterodimers, are generally only expressed on thymic epithelial cells and bone marrow derived antigen presenting cells (APC) such as B-lymphocytes, macrophages, and dendritic cells. APC take up extracellular proteins and degrade them into peptide fragments which are then bound to MHC class II molecules for presentation to T cells that express TCR α/ β chain heterodimers complexed with CD4 molecules. Following antigenic activation, MHC class II restricted CD4+ T cells usually provide helper functions that amplify other components of the immune response including cytotoxic CD8+ T cells and antibody production by B-lymphocytes. Neither MHC class I or class II molecules have an inherent capacity to discriminate between peptides derived from foreign pathogens or normal endogenous proteins.8 Thus, to prevent the development of deleterious autoimmune responses, it is necessary to destroy or inactivate any T cells that express a rearranged TCR that recognizes endogenous peptides bound to self MHC molecules. This normally occurs through several different mechanisms which select from the theoretical total pool of ~109 TCR clonotypes, a subset of effectors that can respond to foreign, but not endogenous peptides bound to self MHC molecules. One such tolerogenic mechanism occurs in the thymus.7,8 The TCR molecules of T cell precursors differentiating within the thymus interact with peptides presented by MHC gene products expressed on both thymic epithelial cells and hematopoietically derived APC. These interactions result in the positive selection of T cells capable of recognizing foreign antigens presented by self-MHC gene products. Immature T cells whose TCR engages endogenous peptides bound to self-MHC molecules are normally negatively selected in the thymus through an activation driven cell death

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process known as apoptosis. Several recent studies have demonstrated that the threshold of T cell activation required to induce negative selection in the thymus is quantitatively greater than that required for positive selection.16,17 However, not all autoreactive T cells are negatively selected in the thymus. Such autoreactive T cells can undergo apoptotic cell death in the periphery when stimulated to a sufficiently high activation threshold by APC presenting large quantaties of the appropriate antigen.18-22 The activities of other autoreactive T cells escaping intrathymic deletion are suppressed in the periphery by immunoregulatory T cells which also must be stimulated by a highly activated APC in order to become functional.23 Another mechanism by which APC can downregulate autoimmune responses is by inducing a change in the cytokine profile produced by CD4+ T cells reacting against self peptides.24-26 Autoimmune tissue destruction appears to be promoted when self peptide reactive CD4+ T cells produce a Th1 pattern of cytokines including interleukin-2 (IL-2) and gammainterferon ( γIFN). These Th1 cytokines amplify cytotoxic CD8+ T cell functions, and also support macrophage activation and delayed type hypersensitivity (DTH) responses. In contrast, autoimmune tissue destruction appears to be blocked when self peptide reactive CD4+ T cells produce a Th2 pattern of cytokines including IL-4, IL-5, IL-6, IL-10 and IL-13, which provide help for the activation of B lymphocyte mediated humoral immunity. Of these cytokines, IL-4 appears to be most important in switching CD4+ T cells from a Th1 to Th2 response profile. Generally CD4+ T cells switch from a Th1 to a Th2 profile as a function of increasing antigen dose presented by APC. The cytokine profile produced by CD4+ T cells can also vary depending upon the type of APC providing antigenic stimulation, with Blymphocytes tending to promote the activation of Th2 responses. The particular MHC class II gene product that an APC uses to present a given antigen can also determine whether a Th1 or Th2 CD4+ T cell response is elicited.27 These T cell response patterns can also be effected by APC produced cytokines, with macrophage production of IL-12 or IL-1 promoting Th1 and Th2 activation respectively.28-31 A constant for all of these immunoregulatory mechanisms, is that high levels of T cell stimulation tend to promote tolerance, while lower levels tend to promote immunological effector responses. Thus, any genetic defects that compromise the stimulatory capacity of APC and/ or impairs T cell responsiveness could preferentially diminish any or all of these tolerogenic mechanisms without fully abrogating immu-

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nological effector responses. As discussed later in this chapter, such defects are present in both APC and T cells from NOD mice and may be central to the development of autoimmune IDDM. THE DIABETOGENIC ROLE OF CD4+ VERSUS CD8+ T CELLS IN NOD MICE In NOD mice, T cell infiltration of the pancreatic islets (insulitis) initiates at approximately 5 weeks of age, with the first appearance of overt diabetes in females generally occuring at 12-14 weeks of age.2,32 Among the major controversies in the etiopathogenesis of IDDM in NOD mice is the phenotypical and functional relationship of the autoreactive T cells that initiate β cell destruction, to those present at the onset of overt disease which can mediate rejection of subsequently implanted islet grafts. The class II region genes of the NOD H2g7 MHC haplotype encode the rare I-Ag7 gene product, but no expressible I-E molecules.33-36 Since the genes that encode this unusual MHC class II region provide a primary component of IDDM susceptibility in NOD mice (see chapter 2 by E. Leiter), it is not surprising that CD4+ T cells are major contributors to autoimmune β cell destruction. However, there has been much debate as to whether I-Ag7 positive APC activate a CD4+ T cell response that mediates autoimmune IDDM in an independent manner, or whether this process also requires contributions from CD8+ T cells that recognize antigens presented on the surface of pancreatic β cells by the relatively common class I gene products (e.g., Kd and D b) of the H2g7 haplotype. Supporting this latter concept was a report that both CD4+ and CD8+ T cells must be transferred from adult NOD donors to accelerate IDDM onset in neonatal or young sub-lethally irradiated syngeneic recipients.37,38 Similarly, using a series of β cell autoreactive CD4+ and CD8+ T cell clones isolated from the insulitic lesion of adult NOD mice, Reich et al39 found that both T cell subsets must be co-transferred to accelerate IDDM onset in young recipients. In contrast, other investigators have proposed that APC from NOD mice process soluble antigens common to all β cells, and bind these to their unusual I-Ag7 MHC class II molecules for presentation to CD4+ T cells which then mediate autoimmune β cell destruction in a manner analogous to a DTH response. This hypothesis was based on the finding that diabetic NOD mice rapidly reject leukocyte depleted islet, but not pituitary grafts, from allogeneic BALB/c donors.40 The same laboratory subsequently reported that diabetic NOD mice retain leukocyte depleted BALB/c islet grafts if the

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recipients are first depleted of CD4+ T cells by monoclonal antibody treatment.41,42 This was taken as evidence that diabetogenic T cells in NOD mice are not restricted by MHC class I molecules expressed on the target β cell. Indeed, cloned lines of CD4+ T cells isolated from spleens of overtly diabetic NOD mice can also passively transfer IDDM and mediate islet graft rejection.43-46 However, it is important to note that the CD4+ T cells which independently transfer IDDM or mediate islet graft destruction were originally derived from overtly diabetic donors. This is significant since the repertoire of diabetogenic T cells present at the onset IDDM in NOD mice may not be reflective of the autoreactive effectors that actually initiate autoimmune β cell destruction. Major insights into the effector mechanisms responsible for the initiation of autoimmune β cell destruction have been provided by studies in which various T cell populations have been passively transferred into a stock of NOD mice made T and B-lymphocyte deficient by congenic transfer of the severe combined immunodeficiency ( scid) mutation.47-49 Since these NOD- scid mice lack functional T-lymphocytes, they remain diabetes free. An obvious advantage of using T cell deficient NOD- scid mice as recipients for passive transfer studies, is that the transferred T cell population cannot activate effectors endogenous to the host. Furthermore, processed β cell autoantigens are present on the I-Ag7 MHC class II molecules of intra-islet APC from NOD- scid mice.50 This indicates that NOD islets contain APC with I-Ag7 MH C class II m olecu les that are preloaded with β cell autoantigens prior to the infiltration of diabetogenic CD4+ T cells. When CD4+ T cells isolated from the spleens of overtly diabetic NOD donors are transferred into NOD- scid recipients, both insulitis and overt IDDM develop within 3-4 weeks.48 Similarly, some cloned lines of islet-reactive CD4+ T cells isolated from the spleens of overtly diabetic NOD mice can also transfer IDDM to NOD- scid recipients.51 IDDM also develops at a high frequency in a stock of NOD mice in which allelic exclusion has resulted in > 95% of the T cells expressing rearranged TCR α and β chain transgenes derived from one of these CD4+ β cell autoreactive T cell clones.52 Collectively, these results would seem to support the hypothesis that β cell autoantigens bound to IAg7 MHC class II molecules on intra-islet APC of NOD mice elicit a DTH, like response by CD4+ T cells that is sufficient to induce IDDM. However, passive transfer studies using NOD- scid recipients have provided solid evidence that while populations of CD4+ T cells present

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Fig. 3.1. Initiation and amplification of T cell mediated autoimmune β cell destruction in NOD mice.

in diabetic NOD mice are sufficient to transfer IDDM, as well as to mediate islet graft rejection,43,45,46 these effectors are only generated after β cell necrosis has been initiated by a process dependent upon MHC class I restricted CD8+ T cells. This was demonstrated by the finding that while CD4+ T cells isolated from young prediabetic NOD donors can “home” to NOD- scid islets, they cannot initiate IDDM in the absence of CD8+ T cells.48 Furthermore, it has been recently demonstrated that following in vitro activation on islet cells that express transgene encoded B7-1 co-stimulatory molecules, β cell autoreactive CD8+ Tc ell clones isolated from standard NOD mice can rapidly transfer IDDM to NOD- scid recipients in the absence of CD4+ T cells.53 Several laboratories have unequivocally demonstrated the essential role of MHC class I restricted CD8+ T cells for initiating autoimmune β cell destruction, by developing stocks of NOD mice which

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fail to develop this T cell subset. This was done by congenically transferring a β2-microglobulin ( B2m ) gene that was functionally inactivated by homologous recombination onto the NOD inbred background. Intact B2m is required to transport MHC class I molecules to cell surfaces.54,55 Thus, NOD.B2m null mice fail to express cell surface MHC class I molecules, and hence do not positively select CD8+ T cells.56–58 These NOD.B2m null mice fail to develop either insulitis or IDDM. This conclusively demonstrates that the relatively common MHC class I gene products encoded by the H2g7 haplotype of NOD mice exert an autoimmune function that is essential for initiating autoimmune β cell destruction. This function most likely entails the selection of autoreactive CD8+ T cells and their targeting to pancreatic β cells. A recent study has demonstrated such MHC class I restricted autoreactive CD8+ T cells are likely to initiate pancreatic β cell destruction through a Fas/Fas-ligand, rather than a perforin mediated mechanism.59 The initiation of β cell necrosis in NOD mice by a process requiring MHC class I restricted CD8+ T cells, apparently results in the release of previously sequestered antigens which subsequently activate and amplify many additional effector T cell populations (Figure 3.1). Thus, by the time overt diabetes has developed, NOD mice have accumulated a greatly expanded repertoire of β cell autoreactive T cells. Some of the CD4+ T cells that accumulate in overtly diabetic NOD mice, or are present in stocks that express rearranged TCR α and β chain transgenes from these effectors, can clearly mediate autoimmune β cell destruction, but their mechanisms of action do not accurately reflect those of the T cells which actually initiate pathogenesis in young prediabetic NOD mice. A recent study has found that the cascade of autoreactive T cell responses required for the progression to overt IDDM in NOD mice is dependent upon the presence of B lymphocytes which may serve as a critical APC population at some point(s) in this process.60 TCR GENE REARRANGEMENTS ASSOCIATED WITH β CELL AUTOREACTIVITY IN NOD MICE As described above, the repertoire of T cells that initiate autoimmune β cell destruction in NOD mice is less diverse than that present at the onset of overt hyperglycemia. Many investigators have analyzed whether the T cells proposed to initiate autoimmune β cell destruction in NOD mice express a fixed and finite set of rearranged TCR α and β chain genes. Many of these efforts have been pursued with the

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hope that autoimmune IDDM is initiated by a T cell that expresses a single rearranged TCR clonotype that recognizes a single pancreatic β cell peptide. This has been reported to occur in other autoimmune syndromes such as experimentally induced allergic encephalitis.61 If autoimmune IDDM is also initiated by a single T cell clonotype, it has been proposed that it may be possible to inhibit the disease process in high risk individuals by pharmacological treatment with a peptide that specifically blocks the TCR of this autoreactive effector. As an alternative approach, it has also been proposed that it may be possible to prevent IDDM development through various protocols that induce tolerance to the autoantigenic peptide recognized by the initiating T cell clonotype. However, the development of such therapies will become more complicated with each increase in the number of Tc ell clonotypes found to initiate autoimmune β cell destruction in IDDM. Indeed, the studies described below indicate that while it is much less diverse than that present at the onset of overt hyperglycemia, the spectrum of T clonotypes contributing to the initiation of auto- immune β cell destruction in NOD mice is not monoclonal. The germline TCR α and β chain sequences of NOD mice are identical to those most commonly found in other inbred laboratory mouse strains.62,63 Thus, the development of T cells that initiate autoimmune β cell destruction in NOD mice cannot be ascribed to mutations in the TCR α or β chain germline sequences. This conclusion is further supported by the finding that IDDM still develops in a stock of NOD mice congenic for the germline TCR β chain sequences characterizing the SWR or C57L/J inbred strains.64,65 Significantly, in the SWR and C57L/J strains approximately one-half of the normal TCR Vβ germline elements are deleted including Vβ8, which had previously been implicated in an antibody depletion study to be a component of T cell clonotypes initiating autoimmune β cell destruction in NOD mice.66 This indicates either that the TCR β chain elements absent in the SWR and C57L/J strains, but present in NOD, are not required to generate β cell autoreactive T cells, or that there is great plasticity in the repertoire of T cell clonotypes capable of initiating autoimmune IDDM. Numerous lines of evidence support the latter possibility. The first line of evidence indicating that autoimmune β cell destruction is not initiated by a fixed set of T cell clonotypes was the finding that IDDM develops at a high frequency in a stock of NOD mice expressing rearranged TCR α and β chain transgenes specific for

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a non-relevant antigen.67 Due to allelic exclusion >98% of the peripheral T cells in this stock express the rearranged TCR β transgene. However, as described earlier, allelic exclusion is less complete for TCR α than β chain germline sequences. Because of this, ~17% of the rearranged TCR α chain mRNA transcripts in this stock are not encoded by the transgene, but rather by endogenous α chain germline sequences. Even though they are greatly limited in scope compared to standard NOD mice, some portion of the rearranged TCR α chains derived from endogenous germline sequences are apparently capable of pairing with the single transgene derived TCR β chain to generate diabetogenic T cell clonotypes. Recent evidence indicates that the limited TCR recombinatorial processes available to this transgenic stock of NOD mice still enables them to generate a relatively broad spect r u m of d iabetogen ic T cells th at recogn ize m u ltip le β cell autoantigens.68 The mosaic of TCR clonotypes that initiate and then amplify autoimmune β cell destruction in NOD mice has also been assessed using the technique of reverse-transcription polymerase chain reaction (RT-PCR) analysis. Such analyses have indicated that even at 3 weeks of age, islet infiltrating T cells of NOD mice utilize TCR genes in a polyclonal fashion.69,70 As an alternative approach, other investigators have isolated β cell autoreactive T cell clones from NOD mice and then correlated their patterns of TCR gene utilization with their ability to passively transfer IDDM. As shown in Table 3.1, both CD4+ and CD8+ T cell clones with the capacity to passively transfer IDDM utilize a wide spectrum of TCR genes. Interestingly, this approach has also identified T cell clones which block IDDM development. However, as observed with autoreactive effector populations, these protective clones do not appear to be characterized by a fixed pattern of TCR gene utilization. The nature of these IDDM protective T cell clones will be discussed later in this chapter. The studies described above clearly indicate that the T cells which initiate autoimmune IDDM in NOD mice do not incorporate a fixed set of V, D and J elements into their rearranged TCR molecules. On the other hand, there have been reports that a relatively limited set of CDR3 sequences and V, D and J elements are incorporated into the diabetogenic TCR molecules of NOD mice.76,79 However, the TCR sequences identified in these studies were not totally monoclonal in nature and differed from each other (Table 3.2). One factor that may influence the array of TCR gene rearrangements that are detected among diabetogenic T cells in NOD mice is whether the sequenced

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Table 3.1. β cell autoreactive T cell clones isolated from NOD mice Ref.

Source of T cell clones

CD4+ clone name

(43, 71) diabetic NOD BDC 2.4 spleen BDC 4.6 BDC 4.12 BDC 5.2 BDC 5.10 BDC 6.3 BDC 6.9 (39) diabetic NOD unnamed islets (72) diabetic NOD PR-3 islets (73) 8 wk old NOD IS-37SD islets (74) diabetic NOD 9.2.2 spleen 9.2.6 9.2.8 (75, 76) diabetic and None prediabetic NOD islets (77) prediabetic 1.19 NOD islets 2.20 2.35 2.40 4.1 4.4 (78) 4 wk old NOD-5 NOD islets NOD-14 NOD-21

Vβ/Vα TCR gene usage

Effect on CD8+ IDDM in clone passive name transfer

Vβ/Vα TCR gene usage

Effect IDDM in passive transfer

ND BDC 2.5 ND 19/? 6/12 ND ND 4/13.1 ND*

ND None 4/? promotes ND ND promotes ND ND promotes promotes unnamed

—

—

ND*

promote

2/ND

blocks

—

—

ND

promotes IS-2.15

11/ND

blocks

11/ND 11/ND 11/ND —

promotes 9.1.1.17 promotes 9.1.1.33 promotes — NY 2.3 NY 8.3

6/ND 6/ND

neutral neutral

6/ND ? 8.3/ND 8.3/ND 2/ND ? 14/10 8.1/4 8.1/1

promotes None promotes promotes promotes promotes promotes blocks None blocks blocks

None

11/4 promote 8.1/n1.1 promote —

—

—

—

*Originally reported as Vβ5 but subsequently withdrawn, ND-not determined, ?-Unable to equate with any previously identified TCR segment.

TCR genes are isolated from single or pooled islets. This was demonstrated by the finding that while T cells infiltrating an individual NOD islet only utilize one to six independent CDR3 sequences, a much broader diversity of such sequences are detected when comparing multiple T cell infiltrated islets from the same mouse.80 Thus, in NOD mice the diabetogenic T cell infiltrates within each individual islet

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Table 3.2. TCR gene sequencing results from reports of limited heterogeneity among β cell autoreactive T cells in NOD mice Ref. Source of TCR genes analyzed

T cell subset

Vα/Jα # Different Vβ/Jβ TCR # Different TCR gene CDR3 splice gene usage CDR3 splice usage sequences sequences linking Vα to Jα linking Vβ to Jβ

(76) T cell clones CD8+ from prediabetic and diabetic NOD islets

(76) Islets from 30- to 40-day old NOD mice

(82) Islets from 14day-old NOD mice

1/20n n1.1/34 n1.2/30n 2/33 2/26n 2/25n 2/23n n2/25n 3/47n 3/20n 4/15 4/10n 4/33 4/47n 4/26n 5/34 5/7 8/8 8/32 8/34 10/20n 10/42n CD4+ ND plus CD8+

1 1 1 2 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 ND

ND

ND

ND

5.2/1.5 8.1/2.7 8.3/1.1 8.3/2.1 9/2.4 9/2.3 10/2.7 10/1.1 14/1.4 14/1.3 16/2.7 x/1.3

1 1 1 1 1 1 1 1 1 1 1 1

1/ND 2/ND 3/2.1 3/2.2 3/2.4 3/2.5 5/ND 6/ND 7/2.1 7/2.4 7/2.5 8.1/2.1 8.2/1.1 8.2/2.5 8.2/2.6 13/2.1 13/ND 13/2.5

— — 7 3 4 8 — — 9 4 6 1 1 1 1 3 1 3

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appear to develop autonomously from a limited but differing set of initiating clonotypes. The great plasticity in the repertoire of T cells capable of initiating autoimmune β cell destruction in NOD mice also suggests that this strain is characterized by broad based rather than antigen-specific immunotolerogenic defects. This conclusion is further supported by the fact that NOD mice also develop autoimmune pathologies directed against a number of tissues other than pancreatic β cells.81 PANCREATIC β CELL AUTOANTIGENS TARGETED BY DIABETOGENIC T LYMPHOCYTES IN NOD MICE A single peptide-MHC antigenic complex can be recognized by T cells expressing multiple TCR gene rearrangements.83-86 Thus, while the T cells which initiate autoimmune IDDM in NOD appear to utilize a relatively broad spectrum of TCR gene rearrangements, this does not automatically mean that these effectors recognize an equally diverse set of β cell autoantigens. The breadth of β cell antigens targeted by autoreactive T cells in NOD mice has been the subject of many investigations (Table 3.3). Proposed to be among the initial antigens targeted are the 65 kD and 67 kD isoforms of glutamic acid decarboxylase (GAD). Their potential role as primary β cell autoantigens is supported by two reports that CD4+ T cells in spleens of very young (3-4-week-old) prediabetic NOD mice demonstrate a spontaneous Th1 like response directed against two epitopes of GAD65 spanning amino acid positions 509-528 (p34) and 524-543 (p35).87,88 In older NOD mice with more pronounced insulitic lesions, CD4+ T cell reactivity was found to have spread to other epitopes within the GAD molecules, as well as to additional β cell autoantigens including insulin, heat shock proteins, peripherin and carboxypeptidase-H. In addition to insulin and carboxypeptidase-H, other unidentified β cell secretory granule proteins are also targeted by CD4+ T cell clonotypes activated in the latter stages of the NOD autoimmune cascade.89,90 Interestingly, one of these unknown β cell granule antigens is encoded by a gene on the region of NOD chromosome 6 that contains the IDDM susceptibility locus Idd6 (see chapter 2 by E. Leiter). The failure of NOD mice to establish T cell tolerance to the early β cell autoantigen GAD, can be overridden by injecting large quantities of this protein intrathymically88 or intravenously87 into 3-4-week-old recipients. These treatments not only blocked the development of primary NOD T cell responses to GAD, but also the spread of T cell

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reactivity to secondary β cell autoantigens, and most importantly, inhibited the development of IDDM. Thus, the appearance of CD4+ T cell reactivity to GAD appears marks a key turning point in the development of autoimmune IDDM in NOD mice. While the appearance of CD4+ T cell reactivity to GAD may represent a key milestone in the pathogenesis of IDDM, it is unlikely to represent the earliest β cell autoreactive T cell response in NOD mice. This is supported by the finding that both GAD isoforms are intracellular proteins,94 and thus should be immunologically presented by

Table 3.3. Survey of β cell autoantigens recognized by NOD T cells Ref.

β cell protein or fraction recognized by NOD autoreactive T cells

(87)

GAD65

(88)

(91)

(89) (90)

(77, 92)

(93)

Epitope within T cell subtype β cell protein responding to recognized by indicated β cell autoreactive autoantigen T cells

p34 (AA 509-528) p35 (AA 524-543) ND ND ND ND ND ND ND ND ND

CD4+ CD4+ ND ND ND CD4+ CD4+ CD4+ CD4+ CD4+ ND

Hsp65 CPH Insulin GAD65 GAD67 Peripherin CPH Hsp60 30-60kD β cell cytosol fraction β cell insulin ND CD4+ BDC 2.5 granule (see Table 3.1) NOD Chr. 6 ND CD4+ BDC 6.9 encoded β cell (see Table 3.1) insulin granule Insulin B-chain recognize CD4+ by 22/40 islet (AA 9-23) reactive clones with diverse TCR gene utilization Proinsulin B-chain (AA 9-23) CD4+

Age of NOD mice exhibiting earliest T cell response to indicated β cell autoantigen

Anatomical source of T cells responding to indicated β cell autoantigen

3-4 weeks 3-4 weeks 6 weeks 8 weeks 12-15 weeks 4 weeks 4 weeks 6 weeks 6 weeks 6 weeks 8 days

Spleen Spleen Spleen Spleen Spleen Spleen Spleen Spleen Spleen Spleen Spleen

Overt diabetic

Spleen

Overt diabetic

Spleen

4 weeks

Pancreatic islets

7-10 weeks

Spleen

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MHC class I molecules that trigger CD8+ rather than CD4+ T cell responses. Therefore, it is likely that a CD4+ T cell response to GAD is only elicited after β cell lysis is initiated by T cells responding to other antigens. Indeed, splenic T cells from 8-day-old NOD mice reportedly respond to antigens contained within a 30-60 kD β cell cytosolic fraction but not to recombinant GAD.91 While it was not stated, the fact that the β cell extracts used in this study were soluble antigens indicates that they most likely stimulated CD4+ rather than CD8+ T cells from the 8-day-old NOD donors. As described earlier, the initiation of autoimmune β cell destruction in NOD mice clearly requires contributions from MHC class I restricted CD8+ T cells in addition to MHC class II restricted CD4+ T cells. To date there have been no reports identifying MHC class I restricted antigens recognized by CD8+ T cells contributing to any phase of autoimmune β cell destruction in NOD mice. The identification of such MHC class I restricted antigens would be of great significance, since they are likely to be recognized by T cell clonotypes that contribute to the earliest initiation phases of autoimmune β cell destruction in IDDM. An important, but often overlooked factor that may effect whether a given β cell antigen is defined as a primary or secondary target of the autoimmune attack in NOD mice, is the anatomical site from which the responding T cells are isolated. This is illustrated by an analysis of NOD T cell responses to insulin. As described above, splenic T cells demonstrating spontaneous reactivity to insulin were not detected in NOD mice younger than 12 weeks of age, a time point well after the establishment of autoimmune β cell destruction.87 Similarly, peripheral lymph node T cells from NOD mice only respond to exogenous insulin priming after significant levels of β cell necrosis has already occurred.95 However, other investigators have found insulin autoreactive CD4+ T cell clones can be isolated from pancreatic islets of NOD mice as young as four weeks of age.77,92 Greater than 50% (22/40) of the β cell autoreactive CD4+ T cell clones isolated by these investigators recognize an epitope within the insulin B-chain spanning amino acids 9-23. Despite recognizing this single antigenic epitope, these NOD insulin autoreactive T cell clones utilize a diverse array of TCR genes. This supports the concept that while NOD mice generate a very broad repertoire of β cell autoreactive T cell clonotypes, the array of antigens that they recognize may be relatively limited in scope. However, since NOD mice generate such a broad spectrum of

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β cell autoreactive T cell clonotypes, and the lesion within each islet

develops autonomously, there is likely to be variability in the sequence that antigens are targeted. MECHANISTIC BASIS FOR THE DEVELOPMENT OF AUTOREACTIVE T CELLS IN NOD MICE Since knowledge gained from the NOD mouse indicates that the spectrum of T cell clonotypes which mediate autoimmune β cell destruction is broad and they recognize multiple antigens, the best hope for an IDDM prophylactic therapy is one that corrects the immunoregulatory dysfunctions that underlie the generation and activation of these effectors. As described earlier, autoreactive T cells are normally physically deleted either in the thymus or periphery, functionally suppressed by immunoregulatory T cells, or rendered nonpathogenic by a shift in their cytokine production pattern from a Th1 to Th2 profile. For each of these immunoregulatory mechanisms, the threshold of T cell activation required to induce tolerance is higher than that required to trigger an effector response. Thus, any defects that compromise the stimulatory capacity of APC and/or impair Tcell responsiveness could preferentially diminish tolerogenic mechanisms without fully abrogating immunological effector functions. Such dysfuctions are present in both APC and T cells from NOD mice, and may be major contributors to the development of autoimmune IDDM. The unusual H2g7 MHC haplotype of NOD mice contributes to several APC dysfunctions that may lead to the development of β cell autoreactive T cells. IDDM rarely develops (70% by 200 days of age Normal

Growth of Human CEM Tumor Cells Engraftment of Human Peripheral Lymphocytes Engraftment of Human Hemopoietic Stem Cells

Delayed

8.5 months Impaired Defective Severely Reduced Absent 400 mg/dl) within 7-10 days. These mice are subsequently able to accept syngeneic, allogeneic, and/or xenogeneic islet grafts (unpublished observations). Adoptive transfer of allogeneic or xenogeneic immunocompetent lymphocytes into the islet-bearing mice will allow the pathogenesis of islet graft rejection to be investigated in the absence of host immune system contribution. NOD/LtSz-scid MICE AS ISLET GRAFT RECIPIENTS As noted above, NOD/LtSz- scid mice can be readily rendered hyperglycemic following the injection of multiple low doses or a single high dose of streptozocin. This now enables NOD/LtSz- scid mice to function as a universal recipient of islet grafts and immune cells to study the pathogenesis of diabetes and graft rejection. In previous studies, C.B-17- scid mice have been used to examine the rejection of islet allografts and xenografts.66-68 In these reports, chemically diabetic C.B-17- scid mice were transplanted with islets from C57BL/6 mice or allogeneic CBA islets. The C.B-17- scid mice were then injected with spleen cells from untreated Igh-1b congenic BALB/c mice, or from BALB/c mice tolerized to allogeneic C57BL/6 islet grafts by transplantation of C57BL/6 islet grafts pre-cultured to remove APC activity. C.B-17- scid mice receiving BALB/c spleen cells from mice tolerant to C57BL/6 islets promptly rejected the CBA, but not the C57BL/6 islet grafts. C.B-17- scid mice also accept xenogeneic rat islet grafts, and these mice bearing xenogeneic islet grafts have been used as a model to examine the cellular requirements for xenograft islet rejection.66-68 The results using C.B-17-scid mice suggest that this model paradigm may also be useful for investigation of the immune rejection of functioning human islets in vivo. When combined with the enhanced human cell engraftment observed in NOD/LtSz- scid mice (described below), this model system may be useful for investigating the interaction of a mature or developing human immune system with human islets in vivo in a small animal model.

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HUMAN CELL ENGRAFTMENT IN NOD/LtSz- scid MICE ENGRAFTMENT OF MATURE H UMAN LYMPHOCYTES First described in 1988, intraperitoneal injection of mature human peripheral blood leukocytes into C.B-17- scid mice (termed HuPBL-SCID) results in engraftment of the human cells.29 Human cell engraftment typically represents 1-5% of the cells present in the recipient spleen and blood 4-8 weeks after transfer of the cells. Engraftment levels then decrease with time, with few to no human cells detectable by 12-16 weeks after injection. Human cell engraftment can be increased by irradiation and/or treatment of the recipient with anti-asialo GM1, suggesting a role for NK cells in the resistance of the C.B-17- scid mouse to the engrafted human cells.69-71 This model system has been used extensively to examine secondary human immune responses in vivo, to analyze immune activity of lymphocytes obtained from autoimmune patients, and to investigate human-specific infectious diseases such as human immunodeficiency virus-1 (HIV-1). In the specific example of diabetes, Dyrberg et al demonstrated that adoptive transfer of lymphocytes from diabetic patients resulted in the production of islet related autoantibodies following challenge of the C.B-17- scid recipient with rat islets.72 The engraftment of human peripheral blood lymphocytes in C.B-17- scid mice, however, appears to be predominated almost exclusively by the expansion of antigen specific cells following challenge and by a strong human anti-mouse MHC class II reaction.73,74 Although a few reports have suggested that a human primary immune response can be generated in Hu-PBL-SCID mice,69,75-77 this remains extremely difficult to reliably reproduce. Based on the observation that NOD/LtSz- scid mice display numerous defects in innate immunity,18 including deficiencies in NK cell activity and macrophage function, it has been reasoned that NOD/LtSz- scid mice would be an improved host for human lymphocyte engraftment. Engraftment of human spleen cells in NOD/LtSzscid mice were initially compared to that observed in C.B-17- scid mice. In contrast to the low levels of human cell engraftment observed in C.B-17- scid mice in the spleen and peripheral blood 4-8 weeks after injection of human spleen cells, high levels of engraftment were observed in NOD/LtSz- scid mice.78 The increased levels of engraftment, up to 90% of the spleen cells being of human origin, were associated with elevated levels of human Ig in the serum, migration of high numbers of human cells to peripheral lymphoid and non-lymphoid tis-

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sues, and evidence of stable engraftment for up to 24 weeks. In addition, human anti-mouse red blood cell antibodies were observed, and antibodies against filarial antigens could be induced following challenge with Brugia malayi L3 larvae. These results suggested that NOD/LtSz- scid mice were much better recipients of human lymphocytes than were C.B-17- scid mice, and represented an improved model for study of human lymphocytes in scid mice. To generalize this observation, human peripheral blood mononuclear cell (PBMC) engraftment was compared between NOD/LtSzscid mice and a number of other genetic stocks of scid mice, including C.B- 17- scid , C3H / H eJ- scid , C57BL/ 6J- scid , N K1.1- d ep leted C57BL/6- scid, and DBA/2- scid mice.79 In all cases, human PBMC engraftment in NOD/LtSz- scid mice was 5-10-fold higher than in any of the other genetic stocks of scid mice examined. The increased engraftment levels in NOD/LtSz- scid mice were associated with histological evidence of infiltration of human lymphocytes into the lung and liver, and elevated levels of human Ig in the circulation.79 These results demonstrated that ablation of NK cell activity (NK-depleted C57BL/6J- scid mice), loss of hemolytic complement (DBA/2J- scid m ice) or defects in lipopolysacchar ide (LPS) respon siven ess (C3H/HeJ- scid mice) failed to increase engraftment levels of human cells in mice expressing the scid mutation on other genetic backgrounds to the levels observed in NOD/LtSz- scid mice. Combinations of these defects, or abnormalities of NOD/LtSz- scid mice not yet described, are most likely important in the improved engraftment of mature human cells in this mouse strain. ENGRAFTMENT OF HEMOPOIETIC STEM CELLS Based on the improved engraftment of mature human lymphoid cells in NOD/LtSz- scid mice, investigators have tested the ability of human hemopoietic stem cells to engraft in this model system. The ability of human bone marrow to engraft in irradiated C.B-17- scid mice was first described in 1988.30 The engraftment levels obtained were low, and represented only a few percent of host bone marrow cells. Differentiation of the stem cells into mature progeny could be driven by the administration of exogenous human cytokines, including c-kit ligand and PIXY321 (a fusion protein of IL-3 and granulocyte-macrophage colony-stimulating factor) and erythropoietin.30 The authors observed that the growth factor-treated mice contained both m ultipoten tial an d com m itted hum an myeloid an d er ythroid

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progenitors. This model has been extended to examine engraftment of primitive hematopoietic cells from β-thalassemia and sickle cell anemia patients.80 In this report, NOD/LtSz- scid mice were included as recipients, and the engraftment levels in the bone marrow of these mice increased to at least 40% in the animals tested. NOD/LtSz- scid mouse recipients of human bone marrow consistently contained higher levels of human cell engraftment than were observed in C.B-17- scid mice.80 In a second model of human hemopoietic stem cell engraftment, investigators have injected human cord blood into irradiated scid mice.81 Cord blood is a source of human CD34+ CD38– cells, a population of primitive hemopoietic stem cells.82,83 In addition, cord blood injected mice do not require administration of exogenous human growth factors for engraftment of the human stem cells. This is thought to be due to the presence of immature T cells in the cord blood inoculum that secrete the necessary human cytokines for proliferation and differentiation of the human stem cells. Again, in cord blood injected C.B-17- scid mice, engraftment levels of human cells in the bone marrow of the host were low.81 Even in irradiated BALB/c- scid mice carrying transgenes for human IL-3, granulocyte-macrophage-colony stimulating factor (GM-CSF), and c-kit ligand, engraftment levels of human cord blood cells were low, and could only be detected in 50% of the engrafted mice.84 However, injection of cord blood into irradiated NOD/LtSz- scid mice resulted in engraftment levels of 5-95% in all 11 animals examined in this report. In our laboratories, we have demonstrated that NOD/LtSz-scid mice are improved recipients of human cord blood cells as compared directly with C.B-17- scid mice, and that human cell engraftment in the bone marrow reaches levels of 80-90% in many NOD/LtSz- scid recipients within six weeks after injection.85 Engraftment levels in C.B-17- scid mice were 5- to 10-fold lower following injection of the same cord blood cells. The engraftment of human cord blood cells in the bone marrow of NOD/LtSz- scid mice was associated with the presence of human CD34+ cells, and engraftment was observed to persist for at least 15 weeks. In addition, it was shown that human cord blood cells would engraft in unconditioned NOD/LtSz- scid recipients following multiple day injections of unfractionated cord blood cells.85 This latter observation suggests that irradiation preconditioning for engraftment of human hemopoietic stem cells in scid mice may not

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be necessary, an especially important observation since scid mice have defects in DNA repair and increased susceptibility to ionizing radiation.20-24 There is increasing use of NOD/LtSz- scid mice as recipients of human stem cells. Confirmation that engraftment of human stem cells is more robust in NOD/LtSz- scid mice has appeared,86-89 and the injection of purified human CD34+ cord blood progenitor cells has been initiated using NOD/LtSz- scid mice as the preferred recipients.87,88,90 This model has also been extended to examine the proliferative potential of cells capable of initiating human acute leukemia. Hum an acu te myeloid leukemia cells transplanted into irradiated NOD/LtSz- scid mice in the presence of stem cell factor, IL-3 and granulocyte-macrophage colony stimulating factor readily expanded in the adoptive recipients.91 This may represent an excellent model system for the study of leukemia initiating cells in an in vivo environment, and provide a pre-clinical model for the analysis of efficacy of treatment modalities. EPSTEIN BARR VIRUS (EBV)-RELATED TUMORS Im m unocom prom ised patients such as those undergoing chronic immunosuppression or patients with acquired immunodeficiency (AIDS) often develop EBV-induced B cell lymphomas.92 The Hu-PBL-SCID mouse readily develops EBV-induced B cell lymphomas when a higher number of human cells ( ≥50 x 106) are injected.93,94 The majority of mice eventually succumb to these tumors. Since human cells engraft at higher levels in NOD/LtSz- scid mice than in C.B-17- scid mice,78,79,85 it was of interest to determine the incidence of EBV-related tumors in this murine model. Upon injection of 50x10 6 human PBMC into NOD/LtSz- scid mice and into C.B-17- scid mice, it was observed that the incidence of EBV-related tumors in the NOD/LtSz- scid mice was significantly lower.95 This was interpreted to suggest that greater engraftment of CD8+ T cells resulted in enhanced virus-specific cytotoxic T cell activity, impeding the development of the EBV-induced tumors. This also suggests that the enhanced engraftment of human cells observed in NOD/LtSz- scid mice might be increased even more by injection of higher numbers of human cells without the associated increase in EBV-induced tumors that are observed in C.B-17- scid mice when higher numbers of human cells are injected.

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HUMAN IMMUNODEFICIENCY VIRUS-1 (HIV-1) INFECTION OF HU-PBL-NOD-scid MICE The development and optimization of a small animal model for the investigation of HIV-1 remains an important goal of researchers studying the infectivity and pathogenesis of this virus. The utility of the Hu-PBL-SCID model for HIV-1 infection studies was demonstrated soon after the first description of the model system.96 The caveats associated with this model include the low levels of human cell engraftment that are observed, and the low incidence of infectivity that is present following injection of HIV-1 into the human cell engrafted C.B-17- scid mice.97 In addition, the C.B-17- scid mouse has normal macrophage function, elevated NK cell activity, and circulating hemolytic complement, all of which may be important in viral toxicity in vivo. To investigate the effect of host strain background on human PBMC engraftment, Hesselton et al studied the infectivity rate of human PBMC engrafted NOD/LtSz- scid mice.79 Corresponding with the 5- to 10-fold higher levels of human cell engraftment observed in the NOD/LtSz- scid mice as compared to C.B-17- scid mice, the authors also observed an increase in the frequency of infection following inoculation of HIV-1. Four weeks after infection, almost 80% of the human cell-engrafted NOD/LtSz- scid mice harbored replicating virus, while only 40% of the C.B-17- scid mice that received the same cells and virus stock had detectable levels of replicating virus in their spleens.79 These studies were performed with primary isolates of HIV-1 obtained from pediatric patients rather than from laboratory tissue culture-adapted strains of virus which demonstrate enhanced growth rates in Hu-PBL-SCID mice. In additional studies, it has been observed that injection of cytopathic strains of HIV-1 into human cell-engrafted NOD/LtSz- scid mice results in a significant decrease of human cell engraftment at four weeks post infection, with a loss of both CD4+ and CD8+ T cells (Hesselton et al, personal communication). These results suggest that investigation of human HIV-1 pathogenesis, antiviral drug efficacy, and HIV-1-related gene therapy protocols might be facilitated by the utilization of the NOD/LtSz- scid mouse model. The NOD/LtSz- scid mouse has also been used as a source of fetal thymus for human stem cell/murine fetal thymic organ cultures. When human stem cells are engrafted in scid mice, few to no developing T cells of human origin have been observed.98 To attempt to study the infectivity and pathogenesis of HIV-1 on developing human T

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cells in vitro, human cord blood cells were seeded onto NOD/LtSzscid fetal thymi, and infected with a non-cytopathic strain of HIV-1.99 The developing human T cells in the xenogeneic chimeric thymic organ were readily infected with the virus, and infected cultures were dramatically diminished in the number of human T cells that developed. This in vitro model system promises to be useful for analyses of the effects of HIV-1 on developing human T cells in a defined organ culture system. NOD/LtSz-scid MICE AS MODELS FOR HUMAN TUMOR GRAFTS Athymic nu/nu mice and C.B-17- scid mice have been used as recipients of human tumors, organs and tissues due to their inability to mount adaptive immune responses to the human xenografts.100-105 To investigate whether the NOD/LtSz- scid mouse might have an en h an ced abilit y to su p p or t growth of xen ogen eic tu m or s, NOD/LtSz- scid mice and C.B-17- scid mice were injected with the human T lymphoblastoid cell tumor line, CEM.18 As was observed when the engraftment of human lymphocytes was compared between NOD/LtSz- scid mice and C.B-17- scid mice, the CEM T cell tumor also showed enhanced growth in NOD/LtSz- scid mice. By 4 weeks after the injection of 1 x 106 tumor cells, the spleen and peripheral blood of the NOD/LtSz- scid mouse had 4- to 5-fold higher numbers of human cells than did C.B-17- scid mice.18 This observation suggests that engraftment of many of the human tumors, organs, and tissues that have been difficult to study in C.B-17- scid mice may be more easily engrafted and investigated in NOD/LtSz- scid mice. Coupled with the enhanced ability of NOD/LtSz- scid mice to support human lymphoid cell engraftment, this mouse model may now allow the in vivo investigation of the interaction of the human immune system with the target tissue or tumor of choice. USE OF NOD/LtSz-scid MICE IN PARASITIC RESEARCH The study of human specific parasites in murine models has been hampered by the presence of a host immune system, and the specificity of the parasite for tissues or cells in the host species. The development of the C.B-17- scid mouse circumvented in part these obstacles, by eliminating the host adaptive immune system, and allowing the engraftment of the human target tissues. The former characteristic is clearly illustrated by studies that have utilized C.B-17- scid mice in the

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investigation of the human lymphatic filarial parasite, Brugia malayi. Following injection of infective B. malayi L3 larvae, this nematode has been found to grow into adults, mate, and produce microfilaria in C.B-17- scid mice.106 Since B. malayi fails to infect immunocompetent mice, the apparent human specificity of the parasite is in part due to the ability of the murine adaptive immune system to eliminate the L3 larvae. It has also allowed the components of the murine adaptive immune system that are important in the resistance of normal mice to the human filarial parasite to be investigated.107,108 Based on the numerous defects in innate immunity in NOD/LtSz- scid mice as compared to C.B-17- scid mice, it was postulated that the human parasite would show enhanced growth in NOD/LtSz- scid mice. However, when B. malayi was injected into both strains of scid mice, the NOD/LtSzscid mouse showed increased resistance to the growth of the filarial nematode, and few to no adult worms were observed surviving in these mice (Rajan et al, personal communication). These observations suggest that either NOD/LtSz- scid mice have an enhanced innate immune parameter function different than that present in C.B-17- scid mice to which B. malayi is particularly susceptible, or that NOD/LtSz- scid mice might lack a growth factor important for the survival of B. malayi in vivo. The determination of the difference in susceptibility of NOD/LtSz- scid mice and C.B-17- scid mice to infection with B. malayi may provide important insights into the growth requirements of the parasite, innate immune function of NOD/LtSzscid m ice, and additional differences between C.B-17- scid and NOD/LtSz- scid mice that may be important in their relative ability to support human cell engraftment and their susceptibility to other infectious agents. A second parasite of particular interest is that of Plasmodium falciparum . This is the causative agent of malaria in man, and is the most pathogenic of the four human Plasmodial species that have been identified.109 The target cell for this organism is the human red blood cell (RBC), and the specificity of this parasite-target interaction prevents the establishment of a small animal model where murine cells can be infected with this organism. Attempts to establish significant levels of circulating human red blood cells in C.B-17- scid mice beyond a few hours have failed.110 Injection of human RBC into NOD/LtSz- scid mice results in sustained RBC levels in the circulation that can be maintained by daily intra-peritoneal human RBC supple-

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mental injections. When the initial injection of human RBCs are infected with the human malarial parasite P. falciparum , parasites could be detected for up to seven days in the circulation.111 When splenectomized NOD/LtSz- scid mice were used to prevent the splenic trapping and removal of human RBCs from the circulation, the injection of infected RBCs resulted in the circulation of mature sexual stage parasites (gametocytes). The infected NOD/LtSz- scid mice were able to transmit the mature gametocytes to Anopheline mosquitoes feeding on the mice, resulting in the development of oocysts in the mosquito midguts.111 These results demonstrate that the NOD/LtSz- scid mouse may be an enhanced model for study of human blood-specific parasites, and may also provide an excellent model for the study of human red blood cell disorders of lymphohemopoietic origin. As noted above, β-thalassemia and sickle cell anemia have recently been studied using the C.B-17- scid mouse.80 While low levels of human cell engraftment in the bone marrow was observed in C.B-17- scid mice, higher levels were observed in NOD/LtSz- scid mice. NEW MODELS OF NOD/LtSz-IMMUNODEFICIENT MICE NOD/LtSz-scid-B2m null mice: NOD/LtSz- scid mice, although deficient in NK cell activity, nonetheless display detectable NK cell function that can be elevated by administration of poly I:C.18 In addition, in the human PBL engraftment studies, a major component of the engrafted cells are postulated to be xenoreactive to MHC antigens.73,74 To address these limitations, genetic crosses were performed to produce NOD/LtSz mice doubly homozygous for the scid mutation and the β2microglobulin null ( B2m null) allele. Since β2m is required for cell surface expression of MHC class I, B2m null mice are MHC class I negative.112,113 As before, the innate immune defects apparent in the NOD/Lt parental strain were observed in the NOD/LtSz- scid-B2m null mutant strain of mouse.114 In these mice, no NK cell activity could be detected, even after stimulation with poly I:C. Upon engraftment of human PBL, these mice were found to support elevated levels of human T cell engraftment as compared with NOD/LtSz- scid mice. The increased engraftment was associated with a major increase in the percentage and numbers of human CD4+ T cells.114 This increase led to a normalization of the CD4:CD8 ratio of human T cells in NOD/LtSz- scid-B2m null mice. Thus, the lack of MHC class I expression led to an increase in the expansion of human CD4+ T cells. The

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higher levels of human CD4+ T cells and the normalization of the CD4:CD8 ratio suggests that this system may be an excellent model for studies of immune responses and HIV pathogenesis. In addition to the higher levels of human T cell engraftment, NOD/LtSz- scid-B2m null mice also display two additional characteristics not observed in NOD/LtSz- scid mice. First, due to the lack of β2m , NOD/LtSz- scid-B2m null mice also rapidly clear IgG.114 B2m appears to be required for normal IgG circulation, and the absence of β2m leads to a defective Fc receptor for IgG that is hypothesized to normally protect IgG from catabolism via a salvage pathway.115,116 Second, as in all β2m deficient mice,117,118 NOD/LtSz- scid-B2m null mice displayed a parenchymal iron overload.114 The high levels of iron in the liver of NOD/LtSz-scid-B2m null mice resemble the human syndrome of familial hemochromatosis.119 The use of the NOD/LtSz-scid-B2m null mouse as a model for human hemochromatosis may allow insights into the pathogenesis and etiology of this human disorder in which little of the pathogenetic mechanisms are currently known. NOD-RAG-2null mice: Another approach to generate an immunodeficient NOD mouse has been to backcross the RAG-2null locus onto the NOD strain.120 These mice, termed NOD/Bom- rag2null, do not develop functionally mature T or B lymphocytes, and fail to develop either insulitis or diabetes. Adoptive transfer of spleen cells from diabetic NOD donors rapidly induced diabetes in the NOD/LtSz-scidB2m null mice recipients.120 It will be important to determine the innate immune functions, the development of thymic lymphomas, and the ability of this new genetic stock of mice to support human cell engraftment. CONCLUSIONS The above observations demonstrate that the NOD/LtSz- scid mouse may be an improved model for many of the experimental systems currently using the C.B-17- scid mouse. The NOD/LtSz- scid mouse will be of significant importance in the study of the autoimmune syndrome of NOD/Lt mice, and in the model systems that utilize engraftment of human lymphohemopoietic cells into scid mice. The defects in innate immunity, including lack of hemolytic complement, abnormalities in APC development and function, and severe deficiencies in NK cell activity, render the NOD/LtSz- scid mouse a superior recipient for both murine autoimmune effector cells and

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human lymphohemopoietic cells. In addition, the defects in innate immunity increase the utility of this murine stock of mice in studies of the growth of human tumors and human-specific parasites. Constraints associated with the utility of the NOD/LtSz- scid mouse continue to be the high incidence of thymic lymphoma development, and the resultant short lifespan of the animal as result of these tumors. However, continuing efforts to identify the cause of the thymic lymphomagenesis, and to address the issues of irradiation sensitivity, anti-murine xenoreactivity by engrafted human cells, and the lack of easily demonstrable human primary immune responses in human cell engrafted NOD/LtSz- scid mice are resolving these concerns. REFERENCES 1. Bosma GC, Custer RP, Bosma MJ. A severe combined immunodeficiency mutation in the mouse. Nature 1983; 301:527-530. 2. An on ym ou s. Mou se gen om e database (MGD), m ou se gen om e informatics project, The Jackson Laboratory, Bar Harbor, Maine. World Wide Web (http://www.informatics.jax.org). 1996. 3. Bosma GC, Davisson MT, Ruetsch NR et al. The mouse mutation severe combined immune deficiency ( scid) is on Chromosome 16 [published erratum appears in Immunogenetics 1989;29(3):224]. Immunogenetics 1989; 29:54-57. 4. Kirchgessner CU, Patil CK, Evans JW et al. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science 1995; 267:1178-1183. 5. Blunt T, Gell D, Fox M, Taccioli GE et al. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci USA 1996; 93:10285-10290. 6. Fried LM, Koumenis C, Peterson SR et al. The DNA damage response in DNA-dependent protein kinase-deficient SCID mouse cells: replication protein A hyperphosphorylation and p53 induction. Proc Natl Acad Sci USA 1996; 93:13825-13830. 7. Jeggo PA, Jackson SP, Taccioli GE. Identification of the catalytic subunit of DNA dependent protein kinase as the product of the mouse scid gene. Curr Top Microbiol Immunol 1996; 217:79-89. 8. Miller RD, Hogg J, Ozaki JH et al. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus. Proc Natl Acad Sci USA 1995; 92:10792-10795. 9. Bosma MJ, Carroll AM. The SCID mouse mutant: definition, characterization, and potential uses. Annu Rev Immunol 1991; 9:323-350. 10. Lieber MR, Hesse JE, Lewis S. et al. The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination. Cell 1988; 55:7-16.

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11. Malynn BA, Blackwell TK, Fulop GM et al. The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism. Cell 1988; 54:453-460. 12. Taylor PC. The use of SCID mice in the investigation of human autoimmune diseases. 1994:1-127. 13. Habu S, Kimura M, Katsuki M et al. Correlation of T cell receptor gene rearrangements to T cell surface antigen expression and to serum im m unoglobulin level in scid m ice. Eur J Im m unol 1987; 17:1467-1471. 14. Shores EW, Sharrow SO, Uppenkamp I et al. T cell receptor-negative thym ocytes from SCID m ice can be in duced to en ter the CD4/CD8 differentiation pathway. Eur J Immunol 1990; 20:69-77. 15. Hardy RR, Kemp JD, Hayakawa K. Analysis of lymphoid population in scid mice; detection of a potential B lymphocyte progenitor population present at normal levels in scid mice by three color flow cytometry with B220 and S7. Curr Top Microbiol Immunol 1989; 152:19-25. 16. Bosma GC, Fried M, Custer RP et al. Evidence of functional lymphocytes in some (leaky) scid mice. J Exp Med 1988; 167:1016-1033. 17. Nonoyama S, Smith FO, Bernstein ID et al. Strain-dependent leakiness of mice with severe combined immune deficiency. J Immunol 1993; 150:3817-3824. 18. Shultz LD, Schweitzer PA, Christianson SW et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz- scid mice. J Immunol 1995; 154:180-191. 19. Mosier DE, Stell KL, Gulizia RJ et al. Homozygous scid/scid;beige/ beige mice have low levels of spontaneous or neonatal T cell-induced B cell generation. J Exp Med 1993; 177:191-194. 20. Hendrickson EA, Qin XQ, Bump EA et al. A link between doublestrand break-related repair and V(D)J recombination: the scid mutation. Proc Natl Acad Sci (USA) 1991; 88:4061-4065. 21. Biedermann KA, Sun JR, Giaccia AJ et al. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA dou ble-str an d br eak r epair . Pr oc Natl Acad Sci ( USA) 1991; 88:1394-1397. 22. Tanaka T, Yamagami T, Oka Y et al. The scid mutation in mice causes defects in the repair system for both double-strand DNA breaks and DNA cross-links. Mutation Research 1993; 288:277-280. 23. Fulop GM, Phillips RA. The scid mutation in mice causes a general defect in DNA repair. Nature 1990; 347:479-482. 24. Disney JE, Barth AL, Shultz LD. Defective repair of radiation-induced chromosomal damage in scid/scid mice. Cytogenetics Cell Genetics 1992; 59:39-44. 25. Bancroft GJ, Schreiber RD, Unanue ER. T cell-independent macrophage activation in scid mice. Curr Top Microbiol Immunol 1989; 152:235-242.

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205

Index

INDEX A Acquired immunodeficiency syndrome (AIDS), 189 Activation induced cell death (AICD), 103, 112-113 Activation threshold, 74 Adoptive transfer, 177, 181-183, 185-186, 194 Alloxan, 2, 4, 22, 130 alloxan resistant (ALR) mice, 2, 4, 22 alloxan susceptible (ALS), 2, 4, 22 -induced diabetes, 4 Antigen administration intra-peritoneal, 9 intravenous, 21, 84, 154-155 oral, 154, 158, 160-161 prophylactic, 89-90, 157-158, 160-161, 183 thymic, 154-155 Antigen presenting cells, 6, 10, 12, 15, 24, 43, 73, 101 Apoptosis, 11, 14, 50, 57, 74, 89, 103, 105, 109 Autoantibodies, 6, 13-16, 18, 22, 59, 121-123, 147, 160, 186 Autoantigen, 6, 15, 45, 54, 59, 76, 79-80, 83-84, 87, 89-90, 105, 121-123, 130, 153-156, 161-162 Autologous mixed lymphocyte reaction, 156 Azathioprine, 146, 150, 152

B β cell, 6, 9-11, 14-15, 21-22, 24, 45, 48-50, 52, 58-60, 71, 75-90, 106-107, 109, 112, 121, 123-125, 128-130, 132-133, 146, 148-149, 153-162, 174-175, 178, 181-182, 189 B lymphocytes, 25, 78, 107, 109, 181, 194 B7, 77, 124, 129, 131, 150, 152 Bacille Calmette Gue’rin, 150 Backcross, 18, 21, 38-39, 45, 49, 51-53, 55, 58, 60-61, 105, 176-179, 194 BB rat, 8-9, 20, 157-158 Bcl/XL, 105 Bcl2, 14, 54, 55, 57, 124

Bone marrow, 12-13, 42, 71, 73, 87-88, 108, 132-133, 150, 157, 178-179, 187-188, 193 Bovine serum albumin, 155 Breeding, 2-5, 7, 18, 21-22, 38-41, 149, 173, 176 Brugia malayi, 187, 192

C C.B-17-scid, 173, 175-180, 185-194 Caesarean transfer, 149 Carboxypeptidase, 83, 123, 155 Castration, 8, 132, 151, 159 Cataract Shionogi, 3, 20 CD-1 mice, 22 CD25, 103, 112, 125 CD28, 104-106, 112, 131, 152 CD3, 11, 21, 72, 131, 151-152, 154 CD4, 6, 10, 13, 17, 57-58, 71, 73-78, 80-85, 87-88, 123-125, 127-135, 148, 151-152, 156, 174, 181-184, 190, 193 CD8, 6, 10, 20-21, 25, 46-48, 51, 71, 73, 75, 77, 78, 80-82, 85, 124-125, 127-132, 148, 151, 174, 181-184, 189-190, 193 CD80, 101-102, 104-106, 108, 112 CD86, 101, 104-106, 112 Chemical diabetes, 184 Clones, 10, 75-77, 80-82, 84-85, 123-124, 126, 129-130, 132, 155-156, 178, 183 Co-stimulatory molecules, 101-102, 107-108, 112 Colitis, 17 Complete Freund’s adjuvant, 19, 126, 151, 156 Concanavalin A, 14, 21 Congenic mouse, 107 Congenic stocks, 18, 22, 26, 39, 45, 49, 51, 53 Constitutive, 17, 43, 47-48, 110, 112 CTLA-4, 54, 151 Cyclophosphamide induced diabetes, 130-131 Cyclosporin, 146, 151-152 Cytokines, 10, 44, 58-59, 74, 101, 104, 126-127, 130, 152, 154, 156-157, 159, 187-188

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Deafness, 17, 39 Delayed type hypersensitivity, 74 Dendritic cells, 73, 88-89, 104, 106, 108-110, 112-113, 151, 156 Developmental biology, 18 Diabetes prevention trial, 160 Diet, 8, 16, 109, 148, 151, 158-159, 162 DNA repair, 174-175, 189

Housing requirements, 175 Human cell, 47, 180, 185-195 Human immunodeficiency virus (HIV), 186, 190 Human islet, 185 Human leukocyte antigen, 40 Human peripheral blood mononuclear cell, 187 Hyperglycemia, 3, 8-9, 128, 148-149, 184-185

E

I

Effector cell, 10, 44-45, 57, 71-80, 83, 8690, 101, 121, 124, 133, 135, 152, 155, 182-184, 194 Engraftment, 177, 181, 185, 186-191 Environment, 3, 7-8, 39-41, 48-49, 149 Epstein Barr virus (EBV), 189 European Nicotinamide Diabetes Intervention Trial, 160

I-A, 14, 43-45, 73, 75-76, 86-87, 152, 183 I-E, 15-16, 24, 44-45, 73, 75, 87 Idd1, 13, 23, 43, 55, 57 Idd2, 55, 57-58 Idd3, 13, 53-56 Idd4, 55, 57 Idd5, 54-55, 57 Idd6, 55, 57-58, 60 Idd7, 55, 60-61 Idd8, 52, 55, 60 Idd9, 55 Idd10, 13, 54-56 Idd11, 55 Idd12, 53, 55 Idd13, 46, 55 Idd14, 55, 57 Idd15, 55, 58 Idd16, 46, 55, 58 IDDM1, 43, 57, 59 IDDM2, 57, 59 IDDM3, 57 IDDM4, 57 IDDM5, 57 IDDM6, 57 IDDM7, 57 IDDM8, 57 IDDM9, 57 IL-1, 12-13, 56-57, 74, 88, 108, 157, 159, 179 IL-2, 13-14, 74, 87, 103, 105, 112, 127, 129, 131, 154, 157 IL-2 receptor, 103 IL-3, 13, 187-189 IL-4, 10, 13-14, 103, 105, 126-127, 131-132, 134, 159 IL-7, 10, 134-135 IL-10, 13, 101, 103, 109, 126-127, 132, 154, 157 IL-12, 101, 131

D

F Fas, 11, 14, 78, 103, 112 Fas ligand, 11, 103 Fc, 42, 54, 194

G Gender dimorphism, 7 Gene rearrangements, 78, 81, 83 Genetic polymorphisms, 39, 42 Genetic susceptibility, 41 Glutamic acid decarboxylase, 6, 45, 59, 83, 104, 121, 151, 155 Glycosuria, 8-9 Gonadectomy, 7-8 Granulocyte-macrophage colonystimulating factor, 187

H H2ct, 21, 46, 50, 55 H2g7, 41-51, 53-58, 60-61, 102, 104 Harderian gland, 11 Heat shock protein, 44, 83, 123, 151, 155 Hemochromatosis, 194 Hemolytic anemia, 18 Hemolytic complement, 177, 179-180, 187, 190, 194 Hormones, 110, 159

207

Index

Immunodeficiency, 1, 10, 173, 186, 189-190 Immunoglobulin, 41, 48, 52, 173 Immunostimulation, 150, 152-153, 156 Immunosuppression, 147, 150, 152, 189 Indomethacin, 110, 113 Innate immunity, 175, 177-178, 186, 192, 194 Insulin, 1, 6, 9-10, 13, 15, 37, 59-60, 71, 83-85, 121-123, 127-130, 145-146, 151, 154-155, 157-161, 177, 183 Insulin dependent diabetes mellitus, 1, 6, 37, 121, 145-146, 155, 177 Insulitis, 1, 3-4, 6-7, 10-11, 15-16, 19, 21-22, 24, 26, 44-46, 48-51, 53-58, 106-109, 111-112, 122-127, 148, 150, 154, 157, 161, 178, 181, 184, 194 Interferon gamma, 12-13, 22, 44, 58, 140 Interleukin, 42, 103, 124-125, 150, 154, 179 Intra-islet, 6, 10, 49, 183 IQI, 22 Islet, 3, 6, 8-10, 22, 24, 42-43, 45, 48-49, 54, 58-59, 75-77, 80-86, 89, 102, 104, 108-109, 112-113, 121-133, 135, 147148, 150, 154, 156, 158, 160-161, 182183, 185-186

K Ketoacidosis, 9, 147 Kilham rat virus, 6

L L-selectin, 151-152 Lacrimal gland, 11, 15-16 Leakiness, 174, 178, 182 Lipopolysaccharide, 56 Litters, 18-19, 38, 176 Lymphoaccumulation, 6, 10-11, 14, 17, 21-22, 48-49 Lymphohemopoietic, 179 Lymphomas, 11, 18, 25, 146

M Macrophage, 6, 12-14, 42-43, 48, 56, 58, 106, 108,-113, 152, 157, 159 Mammary tumor virus (Mtv), 51 Mel-14, 21 Memory, 107

Metabolic activity, 152, 154, 157-158 MHC, 1, 13, 15-16, 18, 21, 23-25, 39-41, 43-47, 49-51, 53-54, 56, 58-61, 102-108, 112, 149 MHC class I, 21, 25, 43, 46-48, 55 MHC class II, 14-15, 21, 23, 44, 46, 54, 104, 107, 112, 150-152 Mouse strains, 38, 41, 103, 111 Myelin basic protein, 103, 126

N Natural killer cell, 14, 42, 60, 175, 179 Negative selection, 44, 74 Neoplasia, 18 Nerup hypothesis, 41, 46 Nicotinamide, 151, 160-161 Nitric oxide, 101, 109, 131, 160-161 Non cataract, 20 Nonobese diabetic, 1, 3, 40, 102, 145 Nonobese nondiabetic, 3, 19

O Original description, 15 Oxygen radicals, 109

P p277, 151, 155 Pancreas, 10-11, 15, 19, 22, 43, 49, 113, 122, 125-128, 133, 152 Parasite, 191-194 Peri-insulitis, 6, 49, 54-55, 57 Phenotype, 3, 24, 39, 45, 49, 56, 60, 103, 174 Plasmodium falciparum, 192 Pneumocystis carinii, 175-176 Polymerase chain reaction, 80, 126 Polymorphisms, 37, 38, 39, 42, 47-48 Positive selection, 73-74 Pregestimil, 8, 151, 158-159 Prostaglandin E 2, 109 Protein kinase C, 72 Protein kinase, DNA activated catalytic polypeptide, 173

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R Receptors, 13, 107 Red blood cell, 175, 179, 187, 192-193

S Salivary gland, 11, 15, 20, 125 Severe combined immunodeficiency, 1, 76, 173 Shionogi Research Laboratories, 3 Sialoadenitis, 15-16, 22 Simple sequence repeat polymorphisms, 20 Sjögren’s syndrome, 15 Soy, 159 Specific pathogen free, 148, 151, 175 Speed congenic, 52-53 Streptozotocin, 17, 184-185 Submandibular salivary glands, 15 Sulfamethoxazole, 175 Suppression, 45, 110, 132, 147, 150, 157 Susumu Makino, 1 Syngeneic mixed leukocyte reaction, 87

T-lymphoaccumulation, 10-11, 22, 48-49 Threshold liability, 49 Thymectomy, 11, 131-132, 134, 151 Thymus, 10-12, 14, 20, 42, 44, 59-60, 71-74, 86, 130-135, 153, 177, 190 Thyroid, 11, 16 Thyroiditis, 16-17 Time course, 15 Tolerance, 9, 23, 47, 54, 59-60, 74, 79, 83, 86, 104, 106-107, 111, 113, 122, 152-157, 161, 182 Transplantation, 133, 150, 161, 182, 185 Transporter associated with antigen processing, 20 Tumor necrosis factor (TNF), 126, 151, 153 Tumors, 18, 177, 180, 189, 191, 194-195

V Variable, 41, 49, 149, 154, 159 Variable number tandem repeat (VNTR), 59 Virus, 6, 42, 51-52, 73, 149-151, 156, 186, 189-191

T T cell receptor, 40-41, 51, 53, 71, 101, 103, 124, 126, 151, 174, 184 T cells, 71-90, 101-105 T helper, 154 T lymphocyte, 54, 57

W Wheat, 159