T-Cell Autoimmunity and Multiple Sclerosis (Neuroscience Intelligence Unit 5) 1570595674, 9781570595677, 9780585408637

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

5

Marco Londei

T-Cell Autoimmunity and Multiple Sclerosis

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

NEUROSCIENCE INTELLIGENCE UNIT 5

T-Cell Autoimmunity and Multiple Sclerosis Marco Londei Kennedy Institute of Rheumatology London, England, UK

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

NEUROSCIENCE INTELLIGENCE UNIT T-Cell Autoimmunity and Multiple Sclerosis R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1999 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:1-57059-567-4 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

T-cell autoimmunity and multiple sclerosis/[edited by] Marco Londei p. cm. -- (Neuroscience intelligence unit) Includes biographical references and index ISBN 1-57059-567-4(alk. paper) 1. Multiple sclerosis -- Immunological aspects. 2. T cells. 3. Autoimmunity. I. Londei, Marco. II. Series. [DNLM: 1. Multiple sclerosis -- immunology. 2. Autoimmunity -- physiology 3. T-Lymphocytes -- physiology. WL 360 T111 1999] RC377.T23 1999 616.8'34079--dc21 DNLM/DLC 99-27041 for Library of Congress CIP

CONTENTS 1. The Role of γδ T-Cells in Multiple Sclerosis ............................................ 1 Gennaro De Libero The TCR γδ .......................................................................................... 1 Structure of the Human TCR γδ .......................................................... 2 Antigen Specificity of Human γδ T-Cells ............................................... 2 Recognition of Phosphorylated Nonpeptidic Metabolites .................... 3 Recognition of Heat Shock Proteins ....................................................... 5 Recognition of Cell Surface Molecules ................................................... 6 Effector Functions of γδ T-Cells ............................................................. 8 Molecules Regulating Activation of γδT-Cells ....................................... 8 Possible Role of γδ T-Cells in Autoimmune Diseases ........................... 9 Tissue Distribution of γδ T-Cells in MS Patients .................................. 9 TCR gd Repertoire in Peripheral Blood, CSF and Brain Tissue of MS Patients .................................................................................... 10 Antigen Specificities of γδ T-Cells Isolated from MS Patients ............ 11 The Role of γδ T-Cells in Experimental Allergic Encephalomyelitis (EAE) .................................................................. 12 Conclusion ............................................................................................. 13 2. T-Cell Autoimmunity and EAE in Nonhuman Primates ..................... 19 Bert A. ‘t Hart, Ronald E. Bontrop, and Antonio Uccelli Similarity of MHC and TCR Genes in Macaques, Marmosets and Humans ...................................................................................... 20 The MHC and TCR Systems of Macaques and Common Marmosets ......................................................................................... 21 EAE in Macaques ................................................................................... 22 EAE in Marmosets ................................................................................. 23 Conclusion ............................................................................................. 25 3. MBP-Reactive T Cells in Multiple Sclerosis .......................................... 29 Katarzyna D. Bieganowska, Lara J. Ausubel, and David A. Hafler MBP-Reactive T Cells From MS Patients are Activated In Vivo ........ 29 MBP-Reactive T Cells in Some MS Patients are Clonally Expanded ...................................................................... 30 High Frequency of MBP-Reactive T Cells in MS Patients .................. 30 Plasticity of Antigen Recognition by MBP-Reactive T Cells ............... 31 Cytokine Production Pattern by MBP-Reactive T Cells ..................... 32

4. Genetic Engineering of Brain-Specific T Cells for Treatment of Diseases in the Central Nervous System ............................................ 37 A. Flügel and H. Wekerle General Aspects of Gene Therapeutic Approaches in CNS Diseases ................................................................................. 37 Brain-Specific T Cells as a Possible Therapeutic Tool ......................... 39 Gene Delivery Techniques for T Lymphocytes .................................... 43 Therapeutical Achievements with Genetically Engineered T Cells ................................................................................................ 46 Conclusion ............................................................................................. 48 5. T Cells and Cytokines ............................................................................. 59 Enrico Maggi Type 1 and Type 2 T Helper Cells: Two Polarized Forms of the Specific Immune Response .................................................... 59 Surface Molecules Preferentially Associated with Human TH1 or TH2 Cells .............................................................................. 60 Tc1 and Tc2 Cells and Their Functions ............................................... 62 TH1/ TH2-Polarizing Signals ............................................................... 62 Intracellular Signaling Involved in the TH1/TH2 Cell Development .............................................................................. 67 Role of the TH1/TH2 Cells in Autoimmune Disorders ...................... 69 Inbalance of TH1/TH2 Cells in Multiple Sclerosis .............................. 74 Conclusion ............................................................................................. 77 6. Cytokines in Multiple Sclerosis and Its Experimental Models ............. 91 Tomas Olsson General Features of Cytokines, with Implications for Their Study and Interpretation ................................................... 91 Polarization of the Cytokine Profile ..................................................... 94 Role of Cytokines in EAE ...................................................................... 94 Cytokine Expression and Target Immune Reactivity ........................ 100 Cytokines in MS .................................................................................. 102 7. Antigen-Specific T-Cell Responses in Autoimmune Demyelinating Disease .......................................................................... 113 Johannes M. van Noort and Sandra Amor Myelin Antigens ................................................................................... 114 Factors that Govern the Encephalogenicity of Myelin Antigens ........................................................................................... 116 Experimental Autoimmune Encephalomyelitis in Rodents .............. 117 Antigen-Specific T-Cell Responses in MS .......................................... 124 8. Immunopathogenesis of MS ................................................................. 133 M. Vergelli, L. Massacesi, and H.F. McFarland The MS Lesion ..................................................................................... 133 MS Pathogenesis .................................................................................. 135 Conclusion ........................................................................................... 142

EDITORS Marco Londei Kennedy Institute of Rheumatology London, England, UK

CONTRIBUTORS Sandra Amor Department of Immunology The Rayne Institute St. Thomas’ Hospital London, England, UK Chapter 8 Lara J. Ausubel Laboratory of Molecular Immunology Center for Neurologic DiseasesBrigham and Women’s Hospital Harvard Medical School Boston, Massachusetts, USA Chapter 3 Katarazyna D. Bieganowska Laboratory of Molecular Immunology Center for Neurologic Diseases Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts, USA Chapter 3 Ronald E. Bontrop Department of Immunobiology Biomedical Primate Research Center Rijswijk, The Netherlands Chapter 2 Gennaro De Libero Experimental Immunology Department of Research University Hospital Basel, Switzerland Chapter 1

Alexander Flügel Department of Neuroimmunology Max-Planck-Institut for Neurobiology Martinsried, Germany Chapter 4 David A. Hafler Laboratory of Molecular Immunology Center for Neurologic Diseases Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts, USA Chapter 3 Enrico Maggi Immunoallergology and Respiratory Disease Unit Department of Internal Medicine University of Florence Florence, Italy Chapter 5 L. Massacesi Department of Neurological and Psychiatric Sciences University of Florence Florence, Italy Chapter 9 H.F. McFarland Neuroimmunology Branch National Institute of Neurological Disorders and Strokes National Institutes of Health Bethesda, Maryland, USA Chapter 9

Tomas Olsson Neuroimmunology Unit Department of Medicine Center for Molecular Medicine Karolinska Hospital Stockholm, Sweden Chapter 6 Bert A.’t Hart Department of Immunobiology Biomedical Primate Research Center Rijswijk, The Netherlands Chapter 2 Antonio Uccelli Department of Neurological Sciences University of Genova Genova, Italy Chapter 2

Johannes M. van Noort Division of Immunological and Infectious Diseases TNO Prevention and Health Leiden, The Netherlands Chapter 8 M. Vergelli Department of Neurological and Psychiatric Sciences University of Florence Florence, Italy Chapter 9 H. Wekerle Department of Neuroimmunology Max-Planck-Institute for Neurobiology Martinsried,Germany Chapter 4

PREFACE Research on T cells in multiple sclerosis and related animal models has always been regarded as at the cutting edge of autoimmunity. It is difficult to explain why MS and EAE have been so important in leading this field. The present venture aims to produce a book for scientists experienced or fresh to this field. It is our expectation that we have achieved this objective and thus provided the reader with a text both easy to read and to consult. We hope we have done so in a balanced way, although inevitably not all the aspects relating to T-cell biology in MS can be covered. I have to thank all the colleagues involved in this project for their contributions, and I hope that our effort will provide a useful tool for scientists with an interest in MS and in autoimmunity in general. Marco Londei London, England, UK

CHAPTER 1

The Role of γδ T-Cells in Multiple Sclerosis Gennaro De Libero

M

ultiple sclerosis is an inflammatory disease of the brain tissue with the hallmarks of extensive, discrete and progressive myelin loss and neuronal damage which are responsible for the irreversible neurological disability experienced by the vast majority of the patients. The immune-mediated pathogenesis of the initial and late lesions is a commonly accepted belief (reviewed in refs. 1-3) The current view is that the putative autoimmune response is characterized by a coordinated attack to different structures of the CNS, which leads in a later phase to permanent demyelination and axonal loss. This view is complicated by recent findings showing that several predisposing genes are associated with an increased risk of developing the disease.4 The exact functions of the predisposing genes are not known, and they might vary among different groups of patients. Inflammation in the brain is considered the main pathogenetic event resulting from the autoimmune response.5 Different cell populations have been implicated and several effector mechanisms are likely to be involved in the different phases of the disease. The main task of current research is to understand which are the relevant cell populations responsible for the inflammatory reaction and what are the roles of the different effector mechanisms. This chapter focuses on the population of T lymphocytes bearing the T-cell receptor (TCR) γδ. First, the molecular characteristics of human TCR γ and δ genes are described, with a particular emphasis on the great potential diversity which can be generated in the TCR γδ repertoire. Second, the functional properties of these cells are summarized on the basis of the antigen specificities documented so far. Third, the studies concerning the characterization of γδ T cells in MS patients are reviewed and their possible involvement in the disease is discussed.

The TCR γδ T lymphocytes recognize antigens with two types of surface receptor: the TCR αβ, expressed on ~90 % of circulating T cells, is constituted of α and β chains; and the TCR γδ which is expressed on ~10 % of circulating T cells and is constituted of γ and δ chains. The TCR γ gene was isolated first,6 while the γδ T-cells were isolated two years later.7 Numerous studies have addressed the structure and the genomic organization of the TCR γ and δ genes, the functional capabilities of these cells and their requirements for development in the thymus and other lymphoid organs (reviewed in refs. 8, 9). However, the physiological role of γδ T cells is still poorly understood. This is mainly caused by fragmented information on the nature of the antigens which stimulate these cells. The investigation of γδ T-cell function is also hampered by the fact that mouse models of γδ immune responses cannot be T Cell Autoimmunity and Multiple Sclerosis, edited by Marco Londei. ©1999 R.G. Landes Company.

2

T-Cell Autoimmunity and Multiple Sclerosis

transposed to the human immune system, because of important functional differences among γδ T-cells and structural differences of TCR γ and δ chains in the two species.

Structure of the Human TCR γδ Both TCR γ and δ genes undergo rearrangement during ontogeny as do TCR α, β and Ig heavy and light chain genes. Rearrangement at these loci is a tightly regulated process which depends on timing regulated programs and on the physical distribution of the TCR genes on the chromosome. During rearrangement a variable (V) gene segment is joined with diversity (D) and joining (J) segments which fall before the constant (C) regions of the TCR genes. The human γ locus is located on chromosome 7 and comprises two constant gene segments (Cγ1 and Cγ2) and five J elements. Six functional human Vγ genes have been identified,10 distributed into four subgroups designated VγI-VγIV, showing only 23-44% amino acid identity. No D elements have been found in human γ genes. However, variability exists at many Vγ -Jγ junctions due to imprecision of the joining process and to the addition during recombination of nucleotides not encoded in the germ line (N regions). Considering the limited repertoire of Vγ and Jγ genes, plus the lack of D elements, junctional diversity may account for most of the γ chain diversity. The human δ locus is located on chromosome 14, between Jα and Vα gene segments. Four Jδ and three Dδ are located 5' to a single Cδ in germline DNA.8 The three Dδ regions can be read in all reading frames and are flanked on both sides by recombination signal sequences. This particular feature allows the possible use of all the three D regions together in the same δ chain. Such a possibility is not present in other rearranging gene families and confers an extremely high variability in the junctional region of the δ chain. The human Vδ repertoire differs from the Vα gene repertoire, despite the fact that same V genes are used for Vα or Vδ. The number of human Vδ chains found expressed on the cell surface is limited to 6,11 while the number of Vα genes approximates 100. Comparison of the various Vδ sequences shows a high degree of amino acid homology.11 Using Vδ8 as a prototype of Vδ chains, six Vδ genes expressed in γδ T-cell clones show more than 40% similarity. The bulk of Vα subfamilies that have never been found expressed on TCR δ chains show striking sequence differences. The most divergent Vδ gene is Vδ2, which is the least homologous to Vδ8 (22%). The Vδ2 gene does not have a homologous gene in the mouse and is used by the majority of circulating human γδ T-cells. Interestingly, γδ T-cells using the Vδ2 chain acculumulate at the edges of the plaques in relapsing MS lesions (see below). With relatively few V genes, no Dγ, and a few J segments, γ and δ genes are the least diverse of the rearranging receptor gene families. Despite this small number of individual gene segments, the theoretical combinations of different γδ receptors are >1018, two orders of magnitude higher than the theoretical number of TCR αβ receptors. This extremely high variability is concentrated in the junctional regions of the γ chain and mainly of the δ chain.12,13 This property is unique among the rearranging genes and might be instrumental for antigen recognition by γδ T-cells. However, this hypothesis is not supported by experimental findings so far and knowledge about the molecular rules of TCR γδ-ligand interaction is very preliminary.

Antigen Specificity of Human γδ T-Cells The antigens which stimulate human γδ T-cells continue to be a matter of intensive investigation. Several ligands, soluble proteins and surface molecules, have been shown to stimulate γδ T-cells. These are very rare antigen specificities and we suspect that these are of no physiological relevance. Only one class of ligands is known to stimulate a high number of human γδ T-cells in a consistent manner in all donors. These are phosphorylated

The Role of γδ T-Cells in Multiple Sclerosis

3

nonpeptidic metabolites, and this antigen specificity will be reviewed first. Next, studies showing other types of antigen reactivities which are less frequent and perhaps less important in MS are discussed.

Recognition of Phosphorylated Nonpeptidic Metabolites At first, it appeared that the γδ T lymphocyte population was a mere curiosity that might not warrant much consideration. These cells constitute a small subset (1-10%) in the circulating lymphocyte pool and in most human tissues.8 Later studies showed that in most of the cases γδ T-cells do not express the CD4 and CD8 molecules. In the majority of normal donors, 50-80% of circulating γδ T-cells express disulfide linked Vγ9/Vδ2 chains14 with highly diverse junctional regions.15 Gamma-delta T-cells do not recognize antigens in the same manner as αβ T-cells. Vγ9/Vδ2 T-cells react to a series of bacterial and parasite cell extracts,15-19 without classical MHC restriction.15,20 The seminal work of Pfeffer et al21 showed that the bacterial products which stimulate Vγ9/Vδ2 T-cells are resistant to protease digestion, thus suggesting for the first time that human γδ T-cells might recognize nonpeptidic ligands. Partial structural characterization of these bacterial compounds showed that they contain phosphate residues important for their stimulatory capacities. 22,23 Some of them also contain 5'-deoxythymidine triphosphate22 or 5'-uridine triphosphate nucleotide moieties24 associated to unidentified structures responsible for the biological activity. Vγ9/Vδ2 cells are also stimulated by phosphorylated nonpeptidic metabolites, which are the first stimulatory ligands whose structure was fully identified.25-28 Among these molecules, the most active compounds have an isoprene backbone and a pyrophosphate group. Some of them are, instead, phosphorylated monosaccharides. In Table 1.1 the stimulatory compounds are listed. These metabolites are produced by both eukaryotic and prokaryotic cells and participate in the oligonucleotide, carbohydrate and lipid metabolism. Recognition by Vγ9/Vδ2 cells is increased in the presence of human feeder cells,15 and does not require antigen processing.20 Normal cells produce these compounds but do not stimulate Vγ9/Vδ2 lymphocytes, most likely because cells with a normal metabolism generate limited amounts of stimulatory metabolites or because these highly charged molecules do not diffuse into intact membranes. It is possible that transport of these molecules is altered by viral and bacterial infection, or by tumor transformation, allowing their surface appearance.28 Indeed, Vγ9/Vδ2 T-cells recognize and kill target cells after bacterial29 or viral infection,30,31 and are cytotoxic for different types of tumor cells.15,16 Recognition of nonpeptidic ligands has two remarkable characteristics. First, these ligands have pleiotropic antigenicity, since they stimulate all Vγ9/Vδ2 T-cells from all individuals tested so far. Second, each Vγ9/Vδ2 T-cell clone broadly crossreacts with all the stimulatory ligands. Thus, several antigens stimulate many γδ T-cells. This property may enable prompt recruitment of all Vγ9/Vδ2 T-cells as soon as one of the ligands is released in the microenvironment. Comparison of the frequency of circulating Vγ9/Vδ2 T-cells specific for nonpeptidic phosphorylated ligands (~10-1) to that of αβ T-cells specific for a defined peptide-MHC complex (~10-3-10-4), suggests that γδ T-cells are not a minor subpopulation of T lymphocytes and are likely to fulfill an important function. Vγ9/Vδ2 T-cells have the proper numbers and specificities to behave as sentinel cells which, being readily activated, may locally promote early phases of inflammation.28

T-Cell Autoimmunity and Multiple Sclerosis

4

Table 1.1. Ligands stimulating Vγ9/Vδ2 T cells Natural Source

Structure Identification

P Groups

m.w.

Ref.

TubAg1

M. tuberculosis

no

1

-

22

TubAg2

M. tuberculosis

no

1

-

22

TubAg3

M. tuberculosis

no

3

-

22

TubAg4

M. tuberculosis

partial

3

-

22

MEP

no

yes

1

126

25

Methyl-P

no

yes

1

112

25

n-Propyl-P

no

yes

1

140

25

Isopropyl-P

no

yes

1

140

25

sec-Butyl-P

no

yes

1

154

25

β-Hydroxyethyl-P

no

yes

1

142

25

Phosphoglycolic acid

yes

yes

1

156

25

P-non peptidic

M. smegmatis

no

?

-

25

P-non peptidic

M. fortuitum

no

?

-

25

P-non peptidic

M. tuberculosis

no

?

-

23

IPP

M. smegmatis

yes

2

246

26,27

IPP-CH2OH

M. fortuitum

partial

2

276

26

DMAPP

yes

yes

2

246

26,27

Farnesyl-PP

yes

yes

2

382

26

Geranyl-PP

yes

yes

2

314

26

Geranyl-geranyl-PP

yes

yes

2

450

26

Monoethyl-2'-dTTP

?

yes

3

510

26

Compound

5

The Role of γδ T-Cells in Multiple Sclerosis

Table 1.1. Ligands stimulating Vγ9/Vδ2 T cells (cont.) Natural Source

Structure identification

P Groups

m.w.

Ref

Monoethyl-2',3'-dTPP

?

yes

3

494

26

Monoethyl-2'-dUTP

?

yes

3

512

26

M. tuberculosis

partial

2

262

28

DPG

yes

yes

2

266

27

G-3-P

yes

yes

1

172

27

Xylose-1-P

yes

yes

1

200

27

Ribose-1-P

yes

yes

1

200

27

Compound

PP-non peptidic

P=phosphate; MEP=monoethylphosphate; P-non peptidic=phosphorylated non peptide; IPP=isopentenylpyrophosphate; DMAPP=dimethylallylpyrophosphate; PP=pyrophosphate; 2'-dTTP=2'deoxythymidine triphosphate; 2',3'-dTTP=2',3'-dideoxythymidine triphosphate; 2'-dUTP=d'deoxyuridine triphosphate; DPG=diphosphoglyceric acid; G-3-P=glycerol-3phosphate.

Recognition of Heat Shock Proteins Recognition of heat shock proteins (HSP) is a property of a subset of mouse γδ T-cells.32 This antigen reactivity is directed against a mycobacterial HSP65 peptide,33 is not restricted by MHC molecules and involves both TCR γ and δ chains.34 When injected in vivo, the peptide, and not the intact HSP, induces the activation of murine γδ T-cells, without any stimulation of αβ T-cells.35 Despite these convincing studies, it is still not clearly established whether HSP-specific mouse γδ T-cells are activated during immune responses in experimental bacterial and viral infections or in autoimmune disease models. Whether human γδ T-cells recognize the same class of molecules remains an open question. Indeed, in spite of many attempts, formal proof of this type of antigen recognition by human γδ T cells is missing. This issue is of great importance in the study of the possible role of γδ T-cells in MS, since many reports have proposed the hypothesis that recognition of HSP could take place in brain lesions. For this reason, the published data concerning recognition of HSP by human γδ T-cells are here extensively reviewed and discussed. A single γδ line reacting with recombinant M. bovis 65 kD HSP and restricted by unidentified self MHC molecules has been described.36 This type of restriction is quite unusual for γδ T-cells and remains unique, since it has not been detected in further investigations. In another study, Daudi Burkitt’s lymphoma cells were shown to induce expansion of Vγ9/Vγδ2 T-cells from freshly isolated bulk peripheral blood mononuclear cells (PBMC). Proliferation of these γδ T-cells was partially inhibited by a rabbit antiserum or a mAb specific for a human mitochondrial HSP. 16,37 As these antibodies also immunoprecipitated a protein from the surface of Daudi cells, the authors concluded that a surface HSP was recognized by the TCR γδ.

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T-Cell Autoimmunity and Multiple Sclerosis

Other studies have also provided pieces of evidence suggesting recognition of HSP by human γδ T-cells. Vγ9/Vδ2 clones proliferating to recombinant mycobacterial HSP70 in the presence of autologous PBMC as feeder cells have been described.38 However, when recombinant HSP were used to establish HSP-specific lines, only in some cases were γδ T-cells expanded, and γδ T-cell clones with these specificities were never isolated, leading the authors to conclude that it is very difficult to establish HSP-reactive γδ T-cell clones.39 In another study, when lymphocytes from endomyocardial biopsies of heart transplanted patients were stimulated in vitro with HSP65 and autologous feeder cells, Vδ1 cells preferentially expanded.40 In all these investigations with human T cells two different approaches have been exploited: 1. Recombinant HSP have been used to stimulate proliferation of γδ T cells; and 2. Anti-HSP antibodies have been used to inhibit proliferation of γδ T cells. However, both these types of experiments do not prove a direct interaction of the TCR γδ with HSP. Indeed, in all the experiments where γδ T-cells were stimulated with recombinant HSP, bulk PBMC were used as APC. PBMC contain HSP-reactive cells such as monocytes and αβ T cells, which release cytokines upon stimulation with HSP. These cytokines may facilitate proliferation of γδ T cells, independently from the antigen specificity of the TCR γδ.41 This is not a remote possibility, because expansion of human γδ T cells after stimulation with crude mycobacterial extracts requires the concomitant activation of CD4 αβ T cells.42,43 Also the experiments where anti-HSP antibodies partially inhibited proliferation of γδ T-cells may have different explanations. For example, the activation of class II-restricted and peptide-specific TCR αβ clones is inhibited by a rabbit antiserum raised against a mitochondrial HSP.44 The inhibition was attributed to the role of this HSP in antigen presentation to αβ T cells, and not to the blocking of a putative TCR-HSP interaction. A second possible flaw of studies using anti-HSP antibodies is that bulk populations of APC were used, thus making the identification of the cells interacting with the inhibitory anti-HSP antisera difficult. Anti-HSP antibodies could inhibit the activation of PBMC used as feeder cells, thus limiting their release of factors necessary for γδ T-cell expansion. Conclusive experiments could have been performed by using APC not releasing growth factors and not reacting with anti-HSP antibodies to stimulate γδ T-cell clones rather than bulk T-cell lines. Recognition and killing by γδ clones of target cells pulsed with HSP could also provide more informative results. In a few cases these experiments were performed and failure to show direct HSP recognition by γδ T cells was reported (refs. 38, 45 and our own data). All together these experimental results show that it is extremely difficult to isolate human γδ T-cells recognizing HSP. Therefore, in the absence of more convincing data supporting a cognate interaction of the human TCR γδ with HSP, this reactivity remains only hypothetical.

Recognition of Cell Surface Molecules Some human γδ T cells react against various surface molecules. These reactivities are rare and we will arbitrarily describe only those that could have importance in MS. Alloreactive γδ clones against classical MHC class I46-48 and class II molecules49,50 have been isolated. These cells are very rare and can be detected only with particular stimulator-responder combinations.51 The fact that the frequency of alloreactive γδ T-cells in peripheral blood is very low, as compared to αβ T-cells, indicates that the TCR γδ repertoire is not selected for reactivity with classical MHC molecules. It has been suggested that γδ T-cells might recognize non-polymorphic structures presenting small peptides or carbohydrates as antigens.8,9,52 In particular, MHC class I-like molecules have been suspected as possible restriction elements, and human γδ clones reacting

The Role of γδ T-Cells in Multiple Sclerosis

7

against molecules encoded by unknown genes in the HLA locus have been isolated.53,54 Another MHC-like molecule recognized by human γδ T cells is CD1c.55,56 Recently, human γδ T-cells recognizing bacterial glycolipids and restricted by CD1 molecules have been isolated.57 Since in chronic active MS lesions the CD1b molecule is upregulated on perivascular inflammatory cells,58 it is tempting to speculate that in MS patients recognition of lipid molecules in the context of CD1-presenting molecules may participate in lesion development. These reactivities would broaden the types of self antigens responsible for local T-cell activation in autoimmune diseases. Human γδ T-cells may also react with EBV-transformed B cells and with normal activated B cells.59,60 All the responder γδ T cells bear different Vδ1 chains paired to heterogeneous Vγ chains.61 B7 and CD39 molecules expressed by activated B cells appear to be crucial for this stimulation. These findings have raised the hypothesis that a superantigen-like recognition is involved in this type of antigen reactivity. Interestingly, polyclonal γδ T cells in inflamed tissues of arthritis patients, in the intestinal mucosa of celiac patients and in the CSF of MS patients predominantly express Vδ1, and in the same tissues many activated B cells are also present. Vδ1 cells might have either a pathogenetic role contributing to the accumulation of inflammatory cytokines or, alternatively, they might exert a regulatory role by local inhibition of recently activated autoimmune B cells. Human γδ T-cells may also react against virus-infected targets. High numbers of γδ T-cells have been found in CSF of patients with Mumps meningitis,62 but not of patients with noninflammatory neurological diseases, suggesting selective expansion of reactive γδ T-cells. Vγ9/Vδ2 clones recognize Herpes simplex-infected targets without being restricted by classical HLA molecules.30,31,63 Since the same γδ clones also kill target cells infected with Vaccinia virus, these γδ T-cells might recognize a cellular ligand induced or modified by acute viral infection rather than a viral product. This type of antigen specificity is important in MS, since it has been repeatedly suggested that viral infections could play a role in its pathogenesis.64 The low expression of HLA molecules in the brain would not be a limiting factor for activation of γδ T-cells. The last series of γδ T-cell reactivities here reviewed are those against tumor cells. In general, in vitro expanded human γδ T-cells have potent cytolytic activity against tumor targets, which is comparable with the activity of lymphokine activated killer (LAK) cells.65,66 Most of the cytotoxic γδ clones bear Vδ1 chains. Another tumor-specific γδ reactivity, different from LAK activity, resembles recognition of superantigens and is directed against Daudi Burkitt’s lymphoma cells.16 Despite intensive efforts, the stimulatory tumor ligands remain unknown. In conclusion, the antigen recognition by γδ T-cells is different from that of αβ T-cells. When TCR γδ specificity is directed against small nonpeptidic metabolites, neither processing nor MHC restriction is required. Unexpectedly, despite the TCR γδ being characterized by the highest number of potentially diverse junctional regions, the broad specificity for nonpeptidic antigens correlates with the used Vγ and Vδ chains, rather than with clonally distributed TCR junctional regions. This finding raises the question of how the TCR interacts with several nonpeptidic ligands which share negative charges but not a common structure. Perhaps the stimulatory phosphorylated metabolites induce a conformational change in the TCR chains, which thus acquire the capacity to contact nonpolymorphic molecules on the surface of adjacent cells. The cognate interaction with a nonpolymorphic molecule might necessitate the conserved regions of Vγ9 and Vδ2 chains, without the participation of the diverse junctional regions.

8

T-Cell Autoimmunity and Multiple Sclerosis

Effector Functions of γδ T-Cells Like αβ T-cells, γδ T-cells can be grouped in different functional subsets. γδ clones are potent cytotoxic cells7 and secrete different lymphokines, thus suggesting that they may fulfill both effector and regulatory functions.67 For example, γδ T-cells isolated from leprosy patients release soluble factors that can positively influence the production of granulomas.68 Another important function is protection during infections. By releasing proinflammatory lymphokines, γδ T-cells may facilitate activation of macrophages in the early phases of infection. This is the case in mice infected with Candida albicans, where γδ T-cells increase the nitric oxide production by macrophages and enhance resistance to mucosal candidiasis.69 In addition, by killing infected target cells they might contribute to the burden of the microorganisms, as reported with cytotoxic CD8 cells in mycobacterial and listerial infections in mice.70-72 γδ T-cells may also regulate the effector phases of the immune response. In mice, after inhalation of soluble antigens γδ T-cells potently inhibit the induction of IgE secretion, possibly by blocking the maturation of Th2 cells.73 Thus, γδ T-cells might have a prominent role in protection against primary allergic sensitization to environmental antigens. Gamma-delta T-cells from mice infected with Listeria monocytogenes produce Th1 type cytokines, while γδ T-cells from Nyppostrongylus brasiliensis-infected animals produce Th2 type cytokines.74 In both infections γδ T cells are among the first T cells to be activated. As the effector phase of the immune response is influenced by the cytokine milieu in which the initial antigen priming occurs, γδ T-cells may have the important regulatory role of determining the immune response in later phases. The additional important function of regulating isotype switching and IgG production has been attributed to human γδ-T cells.17,75,76

Molecules Regulating Activation of γδ T-Cells Adhesion and costimulatory molecules play important roles in γδ T-cell activation, as shown by inhibition using mAbs specific for CD2, CD11a, CD28 and CD80.77 It also appears that additional γδ specific costimulatory molecules exist, contributing to the activation of γδ− and not of αβ T-cells. This function may be exerted by three new proteins detected on the surface of a Burkitt’s lymphoma.78 In sheep and cattle, surface molecules preferentially expressed by γδ T-cells have been identified and their genes cloned.79 These proteins belong to a family of scavenger receptors with unknown function. In some cases, γδ T-cells express unique adhesion molecules. For instance, bovine γδ T-cells bind E-selectin via a novel glycoprotein receptor which is upregulated by TNFα.80 Human γδ T cells also express MHC class I-binding molecules shared with NK cells. These molecules have Ig- or lectin-like structures and, depending on the type of intracytoplasmic tails, they have activatory or inhibitory activities. The majority of γδ T-cells in the peripheral blood express the inhibitory forms of these receptors, which have the function of setting a higher activation threshold.81 These receptors finely regulate γδ T-cell responses by inhibiting cell activation in the presence of low amounts of ligands. In preliminary studies, γδ T-cells from CSF also display this type of molecule (our unpublished data). However, it will important to identify which form (inhibitory or activatory) is expressed and to study their presence on lymphocytes in MS plaques to assess whether MHC class I molecules, which are upregulated during inflammation, tune γδ T-cell activation in the lesions.

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Possible Role of γδ T-Cells in Autoimmune Diseases

A pathogenetic role of γδ T-cells in acute autoimmune reactions has been suggested,8,9 but direct evidence for this function is scarce. Instead, it has been repeatedly shown that γδ T-cells increase in number and are activated during diseases characterized by a chronic inflammatory state. Increased numbers of γδ T-cells have been reported in patients with rheumatoid arthritis,82 autoimmune thrombocytopenia,83 autoimmune neutropenia84 and MS. To understand the role of γδ T-cells in MS it is important to consider the peculiar localization of the lesions, the nature of infiltrating cells, and the heterogeneity of the lesions. These points raise three important questions: 1. How do γδ T cells and other lymphocytes reach the brain tissue? Cells of the lymphoid tissue are sparse in the normal CNS, which is considered an immunological privileged organ. It is not clear how this tissue remains excluded from immune cells, what is the role of the blood-brain barrier and what are the initial events which incite the inflammatory process. 2. Which are the antigens stimulating γδ T cells in the brain? Are these molecules predominantly expressed in MS patients or are they ubiquitous, but immunogenic only in some patients? 3. Is there a different function for γδ T cells in different forms of MS? Careful histological examination has shown heterogeneity of lesion patterns.85 These differences might result from multiple pathogenetic events causing activation of different types of autoimmune cells. Answers to these questions may facilitate definition of the exact role of γδ T-cells in MS, keeping open the possibility that they might be important in the pathogenesis of the disease as well as in its control. In the next paragraphs, evidence for the possible involvement of γδ T-cells in the pathogenesis of MS and in the regulation of the immune response in MS patients is presented.

Tissue Distribution of γδ T-Cells in MS Patients In MS patients the number and distribution of γδ T-cells have been the first parameters to be investigated. In peripheral blood, the number of circulating γδ T-cells has been found to be increased in some patients with early disease onset, but decreased in those in chronic phases.86 These findings have suggested the possible participation of γδ T-cells in the pathogenesis of the early phase of the disease, at least in some patients. No correlation has been found between the number of γδ T-cells and disease type, clinical course, disease duration and disability score, likely because an altered cell number is a parameter which is difficult to maintain in an organ-specific autoimmune disease like MS. Gamma-delta T-cells from peripheral blood of MS patients preferentially expand after IL-2 stimulation in vitro as compared with those of patients with other neurological diseases. These findings indicate that a high number of circulating γδ T-cells express the IL-2 receptor, a marker of T-cell activation.38 Phenotype analyses have shown that Vγ9/Vδ2 T-cells constitute the major circulating γδ population in MS patients,87 as in normal donors.14 Gamma-delta T-cells present in CSF have also been extensively investigated. A concordant correlation between increased numbers of CD5+ B cells and CD4-8- γδ T cells in CSF has been described in MS and aseptic meningitis.88 The highest numbers have been observed in patients with relapsing-remitting MS. However, a careful second study performed on a high number of patients has shown that in MS CSF the number of γδ T cells is lower as compared to CSF taken from patients with non-inflammatory neurological diseases. Intriguingly, the percentage of CD45RO+ γδ T-cells, which belong to the memory

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population, was decreased. These findings have been explained with a possible sequestration of activated γδ T-cells within active MS lesions.89 Local activation in the brain or selective CSF recruitment of activated T cells has been also suggested by the increased expression of the T-cell activation marker CD69.90 These results are in agreement with the increased frequency of γδ T-cells responsive to IL-2 stimulation in MS CSF as compared to normal CSF or CSF from patients with other neurological diseases.38 Another study has similarly shown expansion of γδ T-cells after IL-2 stimulation in subjects with recent disease onset, but not in patients with chronic MS or other neurological diseases.91 Although none of these findings provide direct evidence that γδ T-cells participate in MS pathogenesis, they confirm that γδ T-cells in CSF express cell activation markers predominantly during recent disease onset or acute relapsing phases. A suggestive evidence of the possible role of γδ T-cells in MS has been provided by histological studies of brain lesions. An intimate contact between oligodendrocytes and γδ T-cells has been described by several investigators.92-94 Accumulation of γδ T-cells is predominant at the margin of acute, demyelinating plaques.93 Most of these cells use the Vγ9/Vδ2 TCR, while Vδ1 T-cells are less frequent and scattered within the white matter. An enrichment of Vδ1 T cells has been observed mainly in chronic plaques.92 These observations have suggested the hypothesis of an active recognition by the TCR γδ of new ligands expressed on the surface of reactive oligodendrocytes.92

TCR γδ Repertoire in Peripheral Blood, CSF and Brain Tissue of MS Patients The analysis of the TCR γδ repertoire has been used as an alternative approach to study whether these cells participate to the pathogenesis of MS. These investigations, although indirect and cumbersome, turned out to be very informative as long as the γδ-stimulatory antigens remain unknown. In peripheral blood, a monoclonal population using the same Vδ5-Jδ1 rearrangement has been found in all analyzed Vδ5+ clones.95 The clonal nature of this rearrangement has been proven by gel electrophoresis and sequencing analysis. Since this population has been detected in seven out of nine MS patients studied, it is possible that these cells might have an important role. Oligoclonal Vδ1+ cells have been found in CSF cell lines expanded after a short culture with IL-2.91 This finding has not been confirmed in other studies.38,87,96 In particular, Nick et al analyzed the TCR γδ repertoire in cell lines independently established from the same CSF to avoid skewing of the repertoire as a consequence of in vitro expansion.87 Phenotypic analyses of both γδ cell lines and clones performed with Vγ- and Vδ-specific mAbs have confirmed that Vδ1 is the most frequently used Vδ chain in CSF of MS patients and has shown a preferential association with Vγ chains belonging to the VγI family. In contrast with the findings of Shimonkevitz et al,91 sequence analysis revealed heterogeneity of both TCR δ and γ junctional regions. In conclusion, these reports agree on the finding that in CSF, differently from peripheral blood, there is a predominance of γδ T cells expressing the Vδ1 chain, independently of the type of neurological inflammatory disease. However, the conclusions on the clonality of these cells are opposite. It is likely that this discrepancy is due to the different approaches used to isolate γδ clones from CSF. Sequence analyses of TCR γ and δ genes have also been performed on brain tissue after PCR amplification of the low amounts of TCR-specific RNA in biopsies. Polyclonal Vγ or Vδ sequences have been detected in chronic MS lesions.97 On the contrary, in acute lesions oligoclonal sequences of Vδ1, Vδ2 and Vγ9 have been observed, suggesting the possibility of local proliferation of γδ T-cells.93 Oligoclonal Vδ2 sequences have been also reported in chronic-active plaques from nine MS patients and not in CNS tissue from patients with

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other inflammatory neurological diseases.98 In this study a predominant monoclonal Vδ2-Jδ3 gene rearrangement has been described. Intriguingly, the same authors also found the same sequences in peripheral blood cell lines from MS and tuberculosis patients after stimulation with a recombinant mycobacterial HSP70.99 Furthermore, cells with Vδ2-Jδ3 rearrangements are less frequent in the circulating pool of MS patients and of autoimmune control patients than normal donors.100 Since the differences increase in patients with exacerbated disease, preferential trapping of these γδ T-cells to the injured tissue has been suggested. Thus, Vδ2-Jδ3 bearing cells might represent an important population which deserves further studies. The isolation of γδ clones expressing a Vδ2-Jδ3 chain and the identification of their antigen specificities are essential investigations toward disclosing the exact role of these cells in MS. The finding of oligoclonal or even monoclonal populations from small tissue biopsies has to be interpreted with caution when PCR technique is used to amplify very small amounts of TCR sequences. Indeed, it is possible that, due to the limited amounts of starting material, one or a few sequences become predominant, thus creating a false representation of the TCR repertoire. Unexpectedly, the studies describing monoclonal populations of γδ T cells have always shown identical Vδ sequences. This is surprising since the Vδ junctional sequence is the most variable among the rearranging genes of the immunoglobulin superfamily and the likelihood of independently generating the same TCR δ rearrangement is statistically extremely low. Therefore, excluding a bias due to technical reasons, these findings strongly support clonal expansion of these γδ T-cells.

Antigen Specificities of γδ T Cells Isolated from MS Patients Tissue damage in early MS lesions is selective, since it consists of a loss of oligodendrocytes and myelin from circumscribed regions of central nervous tissue. This finding led to the hypothesis that lesion formation is the result of an immune response against an unidentified myelin or oligodendrocyte antigen. Studies with electron microscopy support this possibility, since they have shown lymphocyte-oligodendrocyte interaction, nuclear coartaction and membrane blebbing of oligodendrocytes. However, there is also evidence that myelin loss is not always preceded by oligodendrocyte destruction.85 In some cases, myelin loss is found even in the presence of very minor alterations of oligodendrocytes, thus implying that at least in those cases, the primary pathogenetic events are not directed against this type of cell. Since the interaction between lymphocytes and oligodendrocytes has also been described in early plaques, a lot of interest has generated the finding that human γδ T-cells kill recently established oligodendrocyte cell lines.45 Expression of a γδ ligand by human glial cells is further supported by the observation that oligodendrocytes, which are MHC class II negative and weakly class I positive, induce the expansion of Vγ9/Vδ2 T cells from normal donors.101 HSP do not seem involved in cognate interaction with the TCR γδ, since cell lines which are HSP negative are recognized as well.102 The nature of the oligodendrocyte antigen responsible for γδ T-cell activation remains unknown. It shares some properties with the antigens present on the surface of Daudi and RPMI 8226 cells, which stimulate the same γδ population in the absence of MHC restriction, and it is tempting to speculate that these antigens belong to the same group of phosphorylated nonpeptidic metabolites which stimulate Vγ9/Vδ2 T cells.28 These latter compounds could accumulate at the edges of degenerating MS lesions and thus specifically activate γδ T-cells. Indeed, Vγ9/Vδ2 clones isolated from the CSF of MS patients display high reactivity against isopentenylpyro-phosphate (our own unpublished results). In addition, in MS patients an altered isoprenoid biosynthesis has been reported, leading to reduced ubiquinone levels in the serum.103 Ubiquinone is a downstream product of the biosynthesis of isopentenylpyrophosphate, which in turn is generated from isopentenylpyrophosphate.104

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Thus, it is possible that alterations in ubiquinone biosynthesis might lead to the accumulation of precursor phosphorylated metabolites capable of stimulating γδ T-cells. A second possibility is that, following the first tissue injury, cells in the process of apoptosis and those already irreversibly damaged release phosphorylated metabolites which usually have only an intracellular localization in intact cells. When released, these ligands may locally activate γδ T-cells which might participate in the progression of the lesion and not in its primary establishment. A second group of molecules suspected to be involved in activation of γδ T-cells in MS lesions are HSP. The possibility that γδ T cells could recognize HSP present in MS lesions was suggested on the basis of a series of histological studies describing the colocalization of γδ lymphocytes with cells positive with anti-HSP65,92 anti-HSP60, anti-HSP90,93 and anti-HSP70 antibodies.94 HSP are induced and overexpressed as a consequence of many different signals received by glial cells and their localization in cells adjacent to γδ T cells cannot be taken as proof of a direct interaction with the TCR γδ. So far it has not been possible to isolate T cells from the lesions and test their antigen specificity. Therefore, this type of antigen recognition by γδ T-cells present in MS lesions remains a fascinating hypothesis not yet supported by experimental findings. Reactivity of γδ T-cells against proteins like MBP, MOG, MAG, or αB-crystallin has never been reported. These proteins are suspected to be important self antigens stimulating αβ T-cells in MS patients.1 On the contrary, bacterial superantigens which activate γδ T-cells may activate also γδ T-cells isolated from MS CSF.105 Several γδ clones using Vδ1 or Vδ2 chains are stimulated by the staphylococcal enterotoxins A and B and by the toxic shock syndrome toxin 1. These reactivities could have relevance during bacterial infections, because they could lead to activation of autoimmune γδ T-cells. Analysis of antigen specificities of γδ clones isolated from CSF of MS patients has shown that some Vδ1 and Vδ2 clones recognize and kill glioblastoma, astrocytoma or monocytic cell lines.87 This recognition is not dependent on the particular Vγ/Vδ heterodimer used, because other clones isolated from the same CSF with the same TCR variable chains, but different junctional sequences, do not display this reactivity. Remarkably, the tumor-reactive γδ T-cells have not been detected in the peripheral blood using PCR oligotyping, but they have been found in other CSF lines independently established from the same MS patient. Thus, γδ T-cells reacting against brain-derived antigens might have been locally expanded. Also in this case, recognition of the tumor-associated antigens is not MHC restricted. In conclusion, our knowledge of the antigens recognized by γδ T-cells in MS is not very advanced. Little is known about the specificities of Vδ2+ cells and almost none about those of Vδ1+ cells, which are the most abundant in CSF. This ignorance impedes our understanding of the exact role of γδ T-cells in MS.

The Role of γδ T-Cells in Experimental Allergic Encephalomyelitis (EAE) Studies on the role of γδ T-cells in the development and regulation of EAE in mice have provided a series of opposite results and conflicting conclusions. In some studies, γδ T-cells have been ascribed an important role in the pathogenesis of the disease. An intralesional accumulation of Vγ6 positive cells in early onset EAE has been observed.106 The pathogenetic activity of these γδ T-cells is supported by the observation that immunization with a Vγ6 peptide induces an immune response against this Vγ chain and a delay of the disease onset.106 The possible pathogenetic role of γδ T-cells has been also shown in studies describing disease-related changes in the γδ T-cell numbers in CNS.107 The number of γδ T-cells remains low in the spleen at all stages, while in the CNS it increases at the height of the acute attack, falls during the recovery phase, but rises again during the

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chronic phase. Depletion of γδ T-cells immediately before the onset of acute disease, or during the chronic stage, causes significant reduction in the severity of the clinical signs associated with a decrease in the percentage of γδ T-cells in the CNS. Histological studies in depleted animals have confirmed a significant reduction in inflammation and demyelination during the acute stage. These findings support the conclusion that γδ T-cells play an important role in the pathogenesis of EAE in mice during both acute and progressive phases of the disease. Opposite conclusions have been drawn from other studies where γδ T-cell depletion with TCR γδ-specific mAbs led to disease aggravation and recurrence.108 These findings suggest a regulatory role, rather than a pathogenetic one, in EAE. Along this line, it has been reported that in mice lacking the αβ T-cell population after gene recombination, EAE is not induced, thus showing that γδ T-cells alone are not able to elicit this disease.109 As different experimental protocols were used in these studies, it is difficult to compare such conflicting results. Taken together, these findings suggest that γδ T-cells participate in the autoimmune response and that their activation in different stages of the inflammatory process may result in different final effects.

Conclusion In conclusion, γδ T-cells appear activated more often in the early phases of MS, their number is also increased in patients with early disease and their distribution in plaques changes during different phases of MS. Furthermore, in some patients oligoclonal γδ populations appear in the circulating pool, in CSF and in the brain lesions. All together, these findings are suggestive of γδ T-cell activation during this autoimmune disease. However, despite the enormous set of data, the important question of what the exact role of γδ T-cells is in MS has not yet been answered. Several causes may explain this lack of information. One is that the ligands recognized by the various γδ T-cell subpopulations in different organs remain mysterious. A second important issue is that γδ T-cells might play different roles in different stages of MS. It is possible that after a primary inflammatory event, cells present in MS lesions express new ligands capable of stimulating a particular form of the TCR γδ. In this case, γδ-cells might be less important in the establishment of the initial lesions but more active in the persistence of the tissue damage, thus being responsible for the recurrence of the inflammatory process. Alternatively, if the γδ stimulatory ligands are expressed by the cells responsible for the autoimmune attack, γδ T-cells might directly affect these autoimmune cells and protect the brain tissue from further damage. It is also not known whether γδ T-cells have a role in all forms of MS. If the various histological patterns of MS lesions have different pathogeneses, it is possible to envisage that only some pathogenetic events lead to the expression of the γδ ligands and to the activation of γδ T-cells in the brain. Understanding the exact function of γδ T-cells in MS and, more generally, in autoimmune diseases has implications with respect to either important basic aspects of the immune response or the design of novel therapeutic approaches. It is presumed that future drug treatments will rely more often on inhibition of defined cellular populations and selective blocking of biochemical cell activation pathways. Thus, a therapy which is useful in some forms of disease might be useless or even harmful in others. Disclosing the function of γδ T-cells in each of these cases, with the possibility to monitor and target their function in vivo, is a challenge for the future.

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Acknowledgments I thank Drs. D. Leppert, M. Londei and L. Mori for reviewing the manuscript. This work was supported by Swiss National Fond Grant N. 31-45518.95 and EEC Grant N. BMH4-CT96-0893

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46. Ciccone E, Viale O, Pende D et al. Specificity of human T lymphocytes expressing a γδ T- cell antigen receptor. Recognition of a polymorphic determinant of HLA class I molecules by a γδ clone. Eur J Immunol 1989; 19(7):1267-1271. 47. Spits H, Paliard X, Engelhard VH et al. Cytotoxic activity and lymphokine production of T-cell receptor (TCR)-αβ+ and TCR-γδ+ cytotoxic T-lymphocyte (CTL) clones recognizing HLA-A2 and HLA-A2 mutants. Recognition of TCR-γδ+ CTL clones is affected by mutations at positions 152 and 156. J Immunol 1990; 144(11):4156-4162. 48. Del Porto P, D’Amato M, Fiorillo MT et al. Identification of a novel HLA-B27 subtype by restriction analysis of a cytotoxic γδ T-cell clone. J Immunol 1994; 153:3093-3100. 49. Bosnes V, Qvigstad E, Lundin KE et al. Recognition of a particular HLA-DQ heterodimer by a human γδ T-cell clone. Eur J Immunol 1990; 20(7):1429-1433. 50. Flament C, Benmerah A, Bonneville M et al. Human TCR-γδ alloreactive response to HLA-DR molecules. Comparison with response of TCR-αβ. J Immunol 1994; 153:2890-2904. 51. Kabelitz D, Bender A, Schondelmaier S et al. Human cytotoxic lymphocytes. V. Frequency and specificity of γδ+ cytotoxic lymphocyte precursors activated by allogeneic or autologous stimulator cells. J Immunol 1990; 145(9):2827-2832. 52. Strominger JL. The γδ T-cell receptor and class Ib MHC-related proteins: Enigmatic molecules of immune recognition. Cell 1989; 57(6):895-898. 53. Lam V, DeMars R, Chen BP et al. Human T-cell receptor-γδ-expressing T-cell lines recognize MHC-controlled elements on autologous EBV-LCL that are not HLA-A, -B, -C, -DR, -DQ, or -DP. J Immunol 1990; 145(1):36-45. 54. Spits H, Paliard X, De Vries J. Antigen-specific, but not natural killer, activity of T-cell receptor-γδ cytotoxic T-lymphocyte clones involves secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase and influx of Ca2+ ions. J Immunol 1989; 143(5):1506-1511. 55. Porcelli S, Brenner MB, Greenstein JL et al. Recognition of cluster of differentiation 1 antigens by human CD4-CD8- cytolytic T lymphocytes. Nature 1989; 341(6241):447-450. 56. Faure F, Jitsukawa S, Miossec C et al. CD1c as a target recognition structure for human T lymphocytes: Analysis with peripheral blood γδ cells. Eur J Immunol 1990; 20(3):703-706. 57. Beckman EM, Melian A, Behar SM et al. CD1c restricts responses of mycobacteria-specific T cells. Evidence for antigen presentation by a second member of the human CD1 family. J Immunol 1996; 157(7):2795-803. 58. Battistini L, Fischer FR, Raine CS et al. CD1b is expressed in multiple sclerosis lesions. J Neuroimmunol 1996; 67(2):145-151. 59. Häcker G, Kromer S, Falk M et al. Vδ1+ subset of human γδ T cells responds to ligands expressed by EBV-infected Burkitt lymphoma cells and transformed B lymphocytes. J Immunol 1992; 149(12):3984-3989. 60. Orsini DLM, Res PCM, Van Laar JM et al. A subset of Vδ1+ T cells proliferates in response to Epstein-Barr virus-transformed B cell lines in vitro. Scand J Immunol 1993; 38:335-340. 61. Orsini DL, van Gils M, Kooy YM et al. Functional and molecular characterization of B cell-responsive Vδ1+ γδ T cells. Eur J Immunol 1994; 24(12):3199-3204. 62. Bertotto A, Spinozzi F, Gerli R et al. γδ T lymphocytes in mumps meningitis patients. Acta Paediatr 1995; 84(11):1268-1270. 63. Maccario R, Comoli P, Percivalle E et al. Herpes simplex virus-specific human cytotoxic T-cell colonies expressing either γδ or αβ T-cell receptor: role of accessory molecules on HLA-unrestricted killing of virus-infected targets. Immunology 1995; 85(1):9-56. 64. Cook SD, Rohowsky-Kochan C, Bansil S et al. Evidence for a viral etiology of multiple sclerosis. In: Cook SD, ed. Handbook of Multiple Sclerosis. 2nd ed. New York: Marcel Dekker, Inc. 1996:97-118 65. Borst J, van de Griend RJ, van Ostveen JW et al. A T-cell receptor γδ/CD3 complex found on cloned functional T lymphocytes. Nature 1987; 325(6106):683-688. 66. Brenner MB, McLean J, Scheft H et al. Two forms of the T-cell receptor γ protein found on peripheral blood cytotoxic T lymphocytes. Nature 1987; 325(6106):689-694. 67. Morita CT, Verma S, Aparicio P et al. Functionally distinct subsets of human γδ T cells. Eur J Immunol 1991; 21(12):2999-3007.

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68. Modlin RL, Pirmez C, Hofman FM et al. Lymphocytes bearing antigen-specific γδ T-cell receptors accumulate in human infectious disease lesions. Nature 1989; 339(6225):544-548. 69. Jones-Carson J, Vazquez-Torres A, van der Heyde HC et al. γδ T cell-induced nitric oxide production enhances resistance to mucosal candidiasis. Nature Medicine 1995; 1:552-557. 70. De Libero G, Flesch I, Kaufmann SH. Mycobacteria-reactive Lyt-2+ T-cell lines. Eur J Immunol 1988; 18(1):59-66. 71. Flynn JL, Goldstein MM, Triebold KJ et al. Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc Natl Acad Sci USA 1992; 89(24):12013-12017. 72. Kägi D, Lederman B, Bürki K et al. CD8+ T cell-mediated protection against an intracellular bacterium by perforin-dependent cytotoxicity. Eur J Immunol 1994; 24:3068-3072. 73. McMenamin C, Pimm C, McKersey M et al. Regulation of IgE responses to inhaled antigen in mice by antigen-specific γδ T cells. Science 1994; 265:1869-1871. 74. Ferrick DA, Schrenzel MD, Mulvania T et al. Differential production of interferon-γ and interleukin-4 in response to Th1- and Th2-stimulating pathogens by γδ T cells in vivo. Nature 1995; 373(6511):255-257. 75. Hacker G, Adam S, Wagner H. Interaction between γδ T cells and B cells regulating IgG production. Immunology 1995; 84(1):105-110. 76. Horner AA, Jabara H, Ramesh N et al. Gamma-delta T lymphocytes express CD40 ligand and induce isotype switching in B lymphocytes. J Exp Med 1995; 181(3):1239-1244. 77. Takamizawa M, Fagnoni F, Mehta DA et al. Cellular and molecular basis of human γδ T-cell activation. Role of accessory molecules in alloactivation. J Clin Invest 1995; 95(1):296-303. 78. Nelson EL, Kim HT, Mar ND et al. Novel tumor-associated accessory molecules involved in the γδ cytotoxic T-lymphocyte-Burkitt’s lymphoma interaction. Cancer 1995; 75(3):886-893. 79. O’Keeffe MA, Metcalfe SA, Glew MD et al. Lymph node homing cells biologically enriched for γδ T cells express multiple genes from the T19 repertoire. Int Immunol 1994; 6(11):1687-1697. 80. Walcheck B, Watts G, Jutila MA. Bovine γδ T cells bind E-selectin via a novel glycoprotein receptor: First characterization of a lymphocyte/E-selectin interaction in an animal model. J Exp Med 1993; 178:853-863. 81. Carena I, Shamshiev A, Donda A et al. MHC class I molecules modulate activation treshold and early signalling of TCR γδ stimulated by nonpeptidic ligands. J Exp Med 1997; 186(10):in press. 82. Brennan FM, Londei M, Jackson AM et al. T cells expressing γδ chain receptors in rheumatoid arthritis. J Autoimmun 1988; 1(4):319-326. 83. Ashihara E, Shimazaki C, Hirata T et al. Autoimmune thrombocytopenia following peripheral blood stem cell autografting. Bone Marrow Transplant 1993; 12(3):297-299. 84. Scott CS, Richards SJ, Sivakumaran M et al. Persistent clonal expansions of CD3+ TCR γδ+ and CD3+ TCR αβ+ CD4-CD8- lymphocytes associated with neutropenia. Leuk Lymphoma 1994; 14(5-6):429-440. 85. Lucchinetti CF, Brück W, Rodriguez M et al. Distinct patterns of multiple sclerosis pathology indicates heterogeneity in pathogenesis. Brain Pathology 1996; 6:259-274. 86. Ejima M, Ota K, Tanaka H et al. Peripheral blood γδ T cells in multiple sclerosis. Rinsho Shinkeigaku 1993; 33(11):1131-1134. 87. Nick S, Pileri P, Tongiani S et al. T-cell receptor γδ repertoire is skewed in cerebrospinal fluid of multiple sclerosis patients: Molecular and functional analyses of antigen-reactive γδ clones. Eur J Immunol 1995; 25(2):355-363. 88. Correale J, Mix E, Olsson T et al. CD5+ B cells and CD4-8- T cells in neuroimmunological diseases. J Neuroimmunol 1991; 32(2):123-132. 89. Droogan AG, Crockard AD, Hawkins SA et al. Gamma-delta T-cell distribution in cerebrospinal fluid and peripheral blood of patients with multiple sclerosis. J Neurol Sci 1994; 126(2):172-177. 90. Perrella O, Carrieri PB, De MR et al. Markers of activated T lymphocytes and T-cell receptor γδ+ in patients with multiple sclerosis. Eur Neurol 1993; 33(2):152-155.

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91. Shimonkevitz R, Colburn C, Burnham JA et al. Clonal expansions of activated γδ T cells in recent-onset multiple sclerosis. Proc Natl Acad Sci USA 1993; 90(3):923-927. 92. Selmaj K, Brosnan CF, Raine CS. Colocalization of lymphocytes bearing γδ T-cell receptor and heat-shock protein hsp65+ oligodendrocytes in multiple sclerosis. Proc Natl Acad Sci USA 1991; 88(15):6452-6456. 93. Wucherpfennig KW, Newcombe J, Li H et al. Gamma-delta T-cell receptor repertoire in acute multiple sclerosis lesions. Proc Natl Acad Sci USA 1992; 89(10):4588-4592. 94. Muller N, Frenzel KH, Schwarz M et al. Expression of human heat-shock protein 70 antigens and γδ T-cell receptor antigens in human central nervous tissue. Ann N Y Acad Sci 1994; 741(305):305-315. 95. Nowak JS, Michalowska WG, Januszkiewicz D et al. Limited junctional diversity of Vδ5-Jδ1 rearrangement in multiple sclerosis patients. Mol Chem Neuropathol 1997; 30(1-2):95-100. 96. Mix E, Fiszer U, Olsson T et al. Vδ1 gene usage, interleukin-2 receptors and adhesion molecules on γδ+ T cells in inflammatory diseases of the nervous system. J Neuroimmunol 1994; 49(1-2):59-66. 97. Hvas J, Oksenberg JR, Fernando R et al. Gamma-delta T-cell receptor repertoire in brain lesions of patients with multiple sclerosis. J Neuroimmunol 1993; 46(1-2):225-234. 98. Battistini L, Selmaj K, Kowal C et al. Multiple sclerosis: Limited diversity of the Vδ2-Jδ3 T-cell receptor in chronic active lesions. Ann Neurol 1995; 37(2):198-203. 99. Battistini L, Salvetti M, Ristori G et al. Gamma-delta T-cell receptor analysis supports a role for HSP 70 selection of lymphocytes in multiple sclerosis lesions. Mol Med 1995; 1(5):554-562. 100. Liedtke W, Meyer G, Faustmann PM et al. Clonal expansion and decreased occurrence of peripheral blood γδ T cells of the Vδ2Jδ3 lineage in multiple sclerosis patients. Int Immunol 1997; 9(7):1031-1041. 101. Freedman MS, D’Souza S, Antel JP. Gamma-delta T cell-human glial cell interactions. I. In vitro induction of γδ T-cell expansion by human glial cells. J Neuroimmunol 1997; 74(1-2):135-142. 102. Freedman MS, Bitar R, Antel JP. Gamma-delta T-cell human glial cell interactions. II. Relationship between heat shock protein expression and susceptibility to cytolysis. J Neuroimmunol 1997; 74(1-2):143-148. 103. Steen G, Axelsson H, Bowallius M et al. Isoprenoid biosynthesis in multiple sclerosis. Acta Neurol Scand 1985; 72:328-335. 104. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Science 1990; 343:425-430. 105. Stinissen P, Vandevyver C, Raus J et al. Superantigen reactivity of γδ T-cell clones isolated from patients with multiple sclerosis and controls. Cell Immunol 1995; 166(2):227-235. 106. Olive C. Modulation of experimental allergic encephalomyelitis in mice by immunization with a peptide specific for the γδ T-cell receptor. Immunol Cell Biol 1997; 75(1):102-106. 107. Rajan AJ, Gao YL, Raine CS et al. A pathogenic role for γδ T cells in relapsing-remitting experimental allergic encephalomyelitis in the SJL mouse. J Immunol 1996; 157(2):941-949. 108. Kobayashi Y, Kawai K, Ito K et al. Aggravation of murine experimental allergic encephalomyelitis by administration of T-cell receptor γδ-specific antibody. J Neuroimmunol 1997; 73(1-2):169-174. 109. Elliott JI, Douek DC, Altmann DM. Mice lacking αβ+ T cells are resistant to the induction of experimental autoimmune encephalomyelitis. J Neuroimmunol 1996; 70(2):139-144.

CHAPTER 2

T-Cell Autoimmunity and EAE in Nonhuman Primates Bert A. ‘t Hart, Ronald E. Bontrop, and Antonio Uccelli

M

ultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). The reactivity of the immune system is directed towards the myelin layer that surrounds the axons in the CNS white matter. An intact myelin sheath is essential for the proper functioning of the axon. Demyelination impairs the pulse conduction across the axon and results in defective sensory and motor functioning. Myelin antigen-reactive T cells infiltrating the CNS have been identified as initiating inflammation, whereas antibodies have a critical role in demyelination.1,2 The ethiopathogenic event that triggers the autoimmune reaction is not known, but it may be connected with the genetic background of the individual and the immune response to certain infectious pathogens.3,4 Immune mediation of MS pathogenesis appears from the histology of the lesion within the CNS white matter and the detection of a specific immune response in MS patients, such as the presence of oligoclonal immunoglobulins in the cerebrospinal fluid and clonally expanded MBP-specific T-cell populations in the peripheral blood. The similarity of the human disease to the animal model experimental autoimmune encephalomyelitis (EAE) further emphasizes the likelihood that MS is an autoimmune disease.5 EAE can be induced in different genetically susceptible strains of laboratory animals, such as mice, rats, guinea pigs and nonhuman primates.5-7 The experimental disease can be induced by immunization with whole myelin or purified myelin antigens, such as myelin basic protein (MBP), myelin/oligodendrocyte glycoprotein (MOG) or proteolipid protein (PLP). The antigens are injected as emulsions in complete adjuvant. In some models permeabilization of the blood-brain barrier by additional stimulation with Bordetella pertussis particles or pertussis toxin is considered necessary. There is now increasing evidence that the immune response to different myelin antigens results in different forms of neuropathology. For example, immunization of susceptible rodent strains with MBP or PLP induces a monophasic predominantly inflammatory EAE, whereas immunization with MOG results in a relasping/remitting EAE with abundant demyelination.8,9 Many investigators use inbred rat and mouse strains for their EAE research. The obvious advantage of a mouse EAE model is that the basic immunology and genetics of most laboratory mouse strains have been very well characterized and all essential reagents are available. Another advantage of a rat EAE model is the size of the animal, which is better suited to collecting larger amounts of blood, e.g., for longitudinal monitoring of immunopathogenic processes and for CNS imaging studies. Due to the high expense and the need for specialized knowledge and facilities, only a few investigators have the opportunity to study EAE in nonhuman primates. As an illustration, of the 788 publications on EAE that T-Cell Autoimmunity and Multiple Sclerosis, edited by Marco Londei. ©1999 R.G. Landes Company.

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were listed in Medline over the period 1990-1997, 44% deal with EAE in mice, 54% with EAE in rats and only 1.0 % with EAE in monkeys. Recent advances in the capacity to manipulate, control and understand the pathogenesis of EAE has led to effective and sophisticated approaches for the treatment of this experimental disease. In spite of some encouraging preliminary results obtained from inbred rodent species, there is no definitive evidence to justify the therapeutic value of clinical trials currently in progress for MS. It is obvious that the closer the model is to the human disorder, the more relevant is the information to be obtained. The high degree of similarity observed between major histocompatibility complex (MHC) and T-cell receptor (TCR) genes in distinct primate species are consistent with their close phylogenetic relationship.10-15 Hence, the evolutionarily determined similarity of genes of the immune system, as well as the inbred condition of rodents, makes nonhuman primates valuable species to study the role of disease-inducing T cells in autoimmune diseases. The following section discusses the immunological relationship of man with some monkey species in which EAE has been established and studied, namely two macaque species (rhesus = Macaca mulatta and cynomolgus = M. fascicularis) and the common marmoset (Callithrix jacchus). Available data on the EAE models in macaques and marmosets is then discussed.

Similarity of MHC and TCR Genes in Macaques, Marmosets and Humans Macaques are Old World monkeys (Catarrhini) with their geographical habitat in North Africa and the southern part of Asia, ranging from Afghanistan and India to Thailand and south China. Marmoset species belong to the family Callitrichidae and are New World monkeys (Platharrhini), originating from the neotropical forests of South America. In Figure 2.1 a simplified schematic representation of the phylogenetic relationships between different primate species is depicted. The hominoids and the Old and New World monkey species are thought to have shared a common ancestor that lived approximately 35 and 55 million years ago, respectively. When plotted on the same scale, the radiation to primates and rodents (mice and rats) took place approximately 70 million years ago. It is generally accepted that, because of this fact, the genetic degree of similarity between the primate species, including man, is much higher than between humans and rodents. As discussed above and in other chapters of this book, myelin-reactive T cells have a key role in the pathogenesis of MS and EAE, both as effector cells and in the orchestration of almost all immunological processes in the disease. T-cell activation is initiated by the formation of a trimolecular complex comprised of the T-cell antigen receptor (TCR) and processed antigen particles (peptides) presented in the context of a MHC molecule.16,17 For this purpose MHC class I and II molecules are equipped with a peptide binding site. The hallmark of the MHC system in vertebrates is its extensive degree of polymorphism. It is thought that this polymorphism minimizes the chance that a population may be exterminated by a single pathogen. Considerable debate relates to whether MHC polymorphisms predate speciation or have occurred after speciation and are thus relatively recent. In the case of primates, most MHC class II loci and lineages are thought to have evolved along a trans-species mode of evolution.10 The degree of similarity between some MHC class II and TCR molecules in humans and nonhuman primates can be so high that a functional trimolecular complex between APC and T cells from different primate species can be formed, resulting in T-cell activation.18-22 In contrast, most MHC class I lineages seem to accumulate variation faster than class II by means of frequent exchange of polymorphic segments. As a consequence, most MHC class I lineages originate after speciation. Detailed analysis of TCR

T-CellAutoimmunityandEAEinNonhumanPrimates

21

Fig. 2.1. Diagram showing the evolutionary relationship between primate species. and MHC involvement in nonhuman primate EAE models are of crucial importance for a better understanding of the immunopathogenesis of MS.

The MHC and TCR Systems of Macaques and Common Marmosets The MHC system of the rhesus macaque (MHC Mamu) was initially investigated by detailed serological analysis. Utilizing allospecific antisera, the evolutionary equivalents of 14 HLA-A and at least 15 B locus alleles have been identified. Similarly, at least 10 HLA-DR like specificities have been determined. In the past decade the characterization of the Mamu class II region has made significant progress due to the introduction of molecular biology techniques. Nucleotide sequence studies were not only instrumental in characterizing the various Mamu DR, DQ and DP genes, but were also important in describing a large number of allelic varieties.10 These studies have demonstrated that humans and rhesus macaques are closely related given that both species share many class II loci and lineages. For example, some rhesus macaques encode class II molecules that are highly similar to the HLA-DR3 equivalents in humans.18 A detailed description of the class I region is currently in progress. At present we know that rhesus macaques have genes that are orthologous to HLA-A, -B, -E, -F and -G, while the equivalents of HLA-C seem to be absent. The T-cell receptor repertoire of rhesus macaque appears to be very similar to that of humans.11,12,19a Detailed analysis of the TCR-VA and -VB repertoire demonstrated that both species seem to share the same set of gene segments. The copy number of the gene segments

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may differ slightly between both species. Based on these studies it is likely that the TCR system has been relatively stable over the last 35 million years of primate evolution. The common marmoset has a bona fide MHC class I and II region. The first analyses demonstrate that the MHC Caja class I region encodes molecules which cluster with the molecules of the HLA-G lineage (Antunes, unpublished data). Similar observations have been made for the cotton-top tamarin (Sanguinus oedipus), another New World primate species.23a Detailed nucleotide sequencing analysis demonstrated that the Caja class II region genes encode at least the evolutionary equivalents of the HLA-DR and -DQ molecules. As compared to other thoroughly investigated primate species, the Caja class II genes seem to express low or moderate degrees of polymorphism at the population level.23b

Marmoset TCR TCR genes in Callithrix jacchus have been recently characterized. Using an inverse-PCR approach a large number of rearranged TCRB chain genes (2 TCRBC, 13 BJ, 2 BD and 15 BV) have been identified.13,14 A high degree of similarity was found between human and marmoset V-D-J-CB chain gene sequences, ranging from 82.6 to 93.4%, suggesting a close phylogenetic relationship between the two species. It should be noted that in C. jacchus gene segments from the TCRBC1-BJ1 cluster are used for functional rearrangement more frequently than BC2-BJ2 genes (77%). This is in contrast to the preferential usage of BC2-BJ2 genes known to occur in humans and mice and may suggest that different mechanisms are involved during TCR rearrangement in marmosets. The length of the CDR3 region, which is similar to that of humans, together with the presence of purifying selection acting on framework residues, is consistent with the fact that TCR genes display a high degree of stability among primates. Similar conclusions have been drawn from comparison of TCR genes of other primate species and their human counterparts.12,15 It is therefore likely that the selective pressures described are consistent across primate species barriers, resulting in an extensively conserved TCR repertoire.

EAE in Macaques Disease Course and Pathology EAE has been induced in two closely related macaque species, M. mulatta and M. fascicularis, by a single immunization with myelin or MBP emulsified in complete adjuvant. The EAE course in most macaques is mostly hyperacute or acute and often has a lethal outcome. A chronic EAE course is rarely observed, but can be established by experimental manipulation of the disease with immunosuppressive drugs such as steroids. Some investigators claim that EAE induced with autologous MBP is less severe with a more chronic course.24 In a subgroup of M. mulatta we have been unable to induce EAE with bovine MBP. These EAE-resistant monkeys all lack one particular MHC-DPB1 allele, suggesting that the Mamu-DP locus is involved in the regulation of EAE susceptibility.25 The histopathology of EAE in macaques is atypical for MS, but rather resembles an acute disseminated encephalomyelitis. This form of EAE is marked by severe inflammation, forming lesions which are often hemorrhagic with necrosis and which often contain many neutrophils. Lesions with various degrees of demyelination, axonal destruction and necrosis can be found in the CNS. In different studies the lesion development and characteristics have been monitored using new imaging techniques which are also utilized in the diagnosis of MS, in particular magnetic resonance imaging (MRI).26-28

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23

Immunology

The association of EAE susceptibility with an MHC class II allele (Mamu-DPB1*01),25 the appearance of myelin-reactive T cells during active EAE.29,30 the beneficial effect of anti-CD4 antibodies on EAE outcome31 and the induction of (mild) EAE by adoptive transfer of an autologous MBP-specific T-cell line of the Th1 phenotype32 provide strong evidence that T cells, and especially those of the helper phenotype, play a central role in the disease. Although in our panel study no association between EAE susceptibility and one particular Mamu DR allele has been found in M. mulatta, a possible role of Mamu-DR molecules in EAE pathogenesis cannot be excluded. Firstly, MBP recognition by the above-mentioned encephalitogenic T helper 1 cell line was Mamu-DR restricted.32 Secondly, it was found that presentation of MBP to a HLA-DR3 restricted and MBP-specific T helper 1 cell line isolated from a MS patient could be performed by Mamu-DR3-positive antigen presenting cells. The activated T-cell line produced a similar cytokine profile as upon activation with HLA-DR-matched human APC. These observations suggest that the activation of the EAE-mediating T cells may be controlled by at least two MHC class II loci, namely Mamu-DR and -DP. Few data are available on the epitope specificity and V-gene usage in TCR molecules of T-cells in macaque EAE. Slierendregt et al25 have isolated a Mamu-DPB1*01-restricted T-cell clone from an EAE-affected rhesus macaque. Analysis of TCR of this line showed functional transcripts of VA1.2 rearranged to JA10, VA1.3 to JA6, VB3 to JB2.4 and VB6.7 to JB2.3. This line was recognizing a peptide encompassing the sequence 61-82 of human MBP. Meinl et al32 have isolated three rhesus MBP-specific CD4-positive T-cell lines from three unrelated healthy rhesus macaques. The fine specificity of the three lines was mapped using overlapping 15-mer peptides from human MBP. The one EAE-inducing T-cell line recognized none of the peptides. One non-EAE-inducing line recognized residues 61-82, being the main Mamu-DPB1*01-binding residue. The third line recognized residues 80-99 and 86-105.

EAE in Marmosets Disease Course and Pathology The successful induction of EAE in marmosets has been documented only recently for the first time by Hauser and coworkers.33,34 The published data and those obtained in our laboratory indicate a striking similarity between this EAE model and human MS with respect to clinical course, MRI and histopathology. EAE in marmosets is induced by immunization with human myelin in complete adjuvant and is characterized by the formation of white matter lesions with inflammation and demyelination. The incidence of succesful EAE induction is surprisingly high, which may be explained by the limited number of allelic variations within the Caja-DR locus.23b The original protocol by Massacesi et al33 also includes two intravenous injections of inactivated Bordetella pertussis particles to enhance the blood-brain barrier permeability. However, in our experience, administration of Bordetella is not necessary for induction of severe demyelinating EAE and may even adversely affect lesion pathology.35 Myelin-induced EAE in marmoset monkeys is typically characterized by a relapsingremitting or chronic progressive course. Using MR-imaging techniques, abnormalities were detectable in early EAE, mainly located around the ventricles but later also scattered in the parenchyma of the white matter.35 Histopathology analysis shows that most lesions in the brain are selectively demyelinated with remarkable sparing of axons. Large demyelinated areas within the white matter are formed by confluency of smaller perivascular lesions. However, in older lesions also axonal destruction can be found. The white matter lesions that are formed in EAE induced with Bordetella pertussis may have a more destructive

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appearance and axonal structures can be severely damaged. In addition to the CNS, inflammatory infiltrates are also found in the spinal roots, although peripheral nerves, such as the sciatic nerve, are usually not affected. The white matter infiltrates consist mainly of mononuclear cells, although in EAE induced with Bordetella pertussis granulocytes may also be found. For a detailed characterization of the infiltrates, cryosections of brain and spinal cord were made during periods of clinically active EAE and stained with a large panel of monoclonal antibodies. In the perivascular infiltrates high expression of CD40 and CD40 ligand were found, characterizing respectively activated macrophages and T lymphocytes.36 Moreover, we found strong reactivity with antibodies raised to human cytokines, such as IL-1α and β, interferon α and γ, IL-10, IL-12 and TNF-α. These data are highly suggestive of active immunological processes during lesion formation.

Immunology The New World monkey common marmoset Callithrix jacchus (C. jacchus) is an unique outbred primate species that develops in utero as genetically distinct twins or triplets sharing bone marrow-derived elements through a common placental circulation. As a consequence, permanent and stable chimerism develops, allowing the adoptive transfer of suspected encephalitogenic T-lymphocyte lines between fraternal siblings without generating an alloresponse. Exploiting this unique situation, adoptive transfer experiments have been performed between siblings (Fig. 2.2).33,34 Thus, it has been established that MBP-specific T cells have a pivotal role in the pathogenesis of EAE in nonhuman primates. By analysis of MBP specific T-cell clones, it was shown that genetically different marmosets recognize different epitopes of the molecule by means of a diverse TCR repertoire.14 The transferred EAE was characterized by inflammation in the CNS, mainly as perivascular cuff of infiltrated mononuclear cells, but without demyelination. Similar to the situation in rodents, in marmoset monkeys autoimmunity to major myelin proteins like MBP and PLP also mediates inflammation, whereas demyelination seems primarily associated with autoimmunity to one of the minor myelin antigens, in particular MOG.8,37 This was clearly demonstrated by the cotransfer of MBP-specific T cells with anti-MOG antibodies. While transfer of MBP-specific T cells alone induces mainly inflammation, cotransfer of the antibodies induced substantial demyelination. This finding emphasizes the important role of humoral autoimmunity in EAE pathogenesis38 which, compared to cellular autoimmunity, is receiving too little attention in the literature. The protection of monkeys against EAE by administration of the cAMP-specific type IV phosphodiesterase inhibitor Rolipram indicates that Th1 cytokines such IFN-γ and TNF-α play an important role in EAE induction.39 In accordance with this, deviation of the immune response in MOG-immunized monkeys to a Th2 state by intravenous administration of recombinant MOG protected the monkeys to EAE. In lymph nodes and PBMC of MOG-treated monkeys, enhanced mRNA levels for anti-inflammatory Th2 cytokines IL-10 and IL-6 and reduced mRNA levels for proinflammatory cytokines like TNF-α and IFN-γ were found. During the period of MOG administration, MOG-reactive T cells could not be detected, but MOG-specific antibodies were formed. Strikingly, after discontinuation of MOG treatment, proliferative T-cell responses suddenly appeared and serum levels of anti-MOG antibodies increased dramatically, concurrent with development of lethal EAE.40 Neuropathology analysis of monkeys with exacerbated EAE showed widespread white matter lesions with more severe inflammation and more substantial demyelination that placebo-treated monkeys. Based on these data, it can be concluded that Th2 cytokines may play an important role in advanced stages of EAE by induction of autoantibodies mediating demyelination.

T-CellAutoimmunityandEAEinNonhumanPrimates

lymph node cells

25

Myelin antigen

splenocytes

Monkey 1

Monkey 2

Active EAE induction

Passive transfer of EAE

Fig 2.2. Adoptive transfer EAE in marmosets. This figure is based on a diagram depicted in Massacesi L, Genain CP, Lee-Parritz D et al. Actively and passively induced experimental autoimmune encephalomyelitis in common marmosets: a new model for multiple sclerosis. Ann Neurol 1995; 37:519-530.

Conclusion Obviously, the special nature of nonhuman primates puts limitations on their use as laboratory animals. Especially when a severely disabling disease is induced, the ethical justification must be carefully considered. In our view, the justification of a nonhuman primate EAE model for human MS is twofold. First, safety and efficacy testing of biological molecules (monoclonal antibodies, cytokines, MHC-binding peptides etc.) as therapy for MS asks for suitable animal models. A plethora of biological molecules with therapeutic potential is designed to react with high specificity to their target molecules in humans, such as (monoclonal) antibodies, cytokines or cytokine antagonists and MHC-binding peptides. The molecular distance between the human and rodent immune systems makes rodent EAE models invalid for the safety and efficacy testing of these molecules. In such cases, nonhuman primate EAE models should be chosen. The relevance of a valid animal model for safety evaluation of new therapies has recently been strongly underlined by Genain and coworkers.40 Second, the molecular and functional organization of the human immune system is more similar to nonhuman primates than to rodent species. A valid nonhuman primate EAE model for MS can therefore be of great help to unravel the immunological basis of MS pathogenesis and lesion formation.

Acknowledgments The authors wish to thank Mrs. Michelle Uccelli for critically reading the manuscript, Mr. Henk van Westbroek for the artwork and Mrs. Mea van der Sman for secretarial assistance.

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References 1. Olsson T. Immunology of multiple sclerosis. Curr Opin Neurol Neurosurg 1992; 5:195-202. 2. Lassmann H, Brunner C, Bradl M et al. Experimental allergic encephalomyelitis: The balance between encephalitogenic T lymphocytes and demyelinating antibodies determines size and structure of demyelinated lesions. Acta Neuropathol 1988; 75:566-576. 3. Ebers GC, Sadovnick AD. The role of genetic factors in multiple sclerosis susceptibility. J Neuroimunol 1994; 54:1-17. 4. Wucherpfennig K. Autoimmunity in the central nervous system: Mechanisms of antigen presentation and recognition. Cell Immunol Immunopathol 1994; 72:293-306. 5. Steinman L. Multiple sclerosis and its animal models: The role of major histocompatibility complex and the T-cell receptor repertoire. Springer Semin Immunopathol 1992; 14:79-93. 6. Rose LM, Richards T, Alvord EC Jr. Experimental allergic encephalomyelitis in nonhuman primates: A model of multiple sclerosis. Lab Anim Sci 1994; 44:508-512. 7. Rose LM, Richards TL, Petersen R et al. Remitting-relapsing EAE in nonhuman primates: A valid model of multiple sclerosis. Clin Immunol Immunopathol 1991; 59:1-15. 8. Linington C, Engelhardt B, Kapocs G et al. Induction of persistently demyelinated lesions in the rat folowing the repeated adoptive transfer of encephalitogenic T cells and demyelinating antibody. J Neuroimmunol 1992; 40:219-224. 9. Genain CP, Nguyen M-H, Letvin NL et al. Antibody facilitation of multiple sclerosis-like lesions in a nonhuman primate. J Clin Invest 1995; 96:2966-2974. 10. Bontrop RE, Otting N, Slierendregt BL et al. Evolution of major histocompatibility complex polymorphisms and T-cell receptor diversity in primates. Immunol Rev 1995; 143:43-62. 11. Levinson G, Hughes AL, Letvin NL. Sequence and diversity of rhesus monkey T-cell receptor beta chain genes. Immunogen 1992; 35:75-88. 12. Jaeger EM, Bontrop RE, Lanchbury JS. Structure, diversity and evolution of T-cell receptor Vβ gene repertoire in primate. Immunogenetics 1994; 40:184-191. 13. Uccelli A, Oksenberg JR, Jeong M et al. Characterization of the TCRB chain repertoire in the New World monkey Callithrix jacchus. J Immunol 1997; 158:1201-1207. 14. Uccelli A, Rombos A, Nguyen M et al. Diversity of MBP-reactive T cells in primate EAE. Neurology 1995; 45:A210. 15. Allen TM, Lanchbury JS, Hughes AL et al. The T-cell receptor β chain-encoding gene repertoire of a new world primate species, the cotton-top tamarin. Immunogen 1996; 45:151-160. 16. Brown JH, Jardetzky TS, Gorga JC et al. The three dimensional structure of the human MHC class II histocompatibility antigen HLA-DR1. Nature 1993; 364:33-39. 17. Germain RN. MHC-dependent antigen processing and antigen presentation: Providing ligands for T-lymphocyte activation. Cell 1994; 76:287-299. 18. Geluk A, Elferink DG, Slierendregt BL et al. Evolutionary conservation of major histocompatibility complex-DR/peptide/T cell interactions in primates. J Exp Med 1993; 177:979-987. 19. a) Jaeger EEM, Bontrop RE, Lanchbury JS. Nucleotide sequences, polymorphism and gene deletion of T-cell receptor β-chain constant regions of Pan troglodytes and Macaca mulatta. J Immunol 1993; 151:5301-5309. 19. b) Bontrop RE, Elferink DG, Otting N et al. Major histocompatibility complex class II-restricted antigen presentation across a species barrier: Conservation of restriction determinants in evolution. J Exp Med 1990; 172:53-59. 20. Cooper S, Kowalski H, Erickson AL et al. The presentation of a hepatitis C viral peptide by distinct major histocompatibility complex class I allotypes from two chimpanzees. J Exp Med 1996; 183:663-668. 21. Meinl E,’t Hart BA, Bontrop RE et al. Transspecies activation of a myelin basic protein specific human T-cell clone using antigen presenting cells from MHC-compatible rhesus monkeys. Int Immunol 1994; 7:1489-1495. 22. ‘t Hart BA, Elferink DG, Drijfhout JW et al. Liposome-mediated peptide loading of MHC-DR molecules in vivo. FEBS Letts 1997; 150:91-95.

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23. a) Watkins DI, Chen ZW, Hughes AL et al. Evolution of the MHC class I genes of a New World primate from ancestral homologues of human nonclassical genes. Nature 1990; 346:60-63. 23. b)Antunes SG, de Groot NG et al. The common marmoset: A New World primate species with limited Mhc class II variability. Proc Natl Acad Sci USA 1998; 95:11745-11750. 24. Shaw C-M, Alvord EC, Hruby S. Chronic remitting-relapsing experimental allergic encephalomyelitis induced in monkeys with homologous myelin basic protein. Ann Neurol 1988:738-748. 25. Slierendregt BL, Hall M, ‘t Hart BA et al. Identification of an Mhc-DPB1 allele involved in susceptibility to experimental autoimmune encephalomyelitis in rhesus macaques. Int Immunol 1995; 7:1671-1679. 26. Richards TL, Alvord EC Jr, Peterson J et al. Experimental allergic encephalomyeltis in nonhuman primates: MRI and MRS may predict the type of brain damage. NMR Biomed 1995; 8:49-58. 27. Heide AC, Richards TL, Alvord EC Jr et al. Diffusion imaging of experimental allergic encephalomyeltis. Magn Reson Med 1993; 29:478-484. 28. Stewart WA, Alvord EC Jr, Hruby S et al. Magnetic resonance imaging of experimental allergic encephalomyelitis in primates. Brain 1991; 114:1069-1096. 29. Van Lambalgen R, Jonker M. Experimental allergic encephalomyelitis in the rhesus monkey. I. Immunological parameters in EAE resistant and susceptible monkeys. Clin Exp Immunol 1987; 67:100-107. 30. Massacesi L, Joshi N, Lee-Parritz D et al. Experimental allergic encephalomyelitis in cynomolgus monkeys. Quantitation of T-cell responses in peripheral blood. J Clin Invest 1992; 90:399-404. 31. Van Lambalgen R, Jonker M. Experimental allergic encephalomyelitis in the rhesus monkey. II. Treatment of EAE with anti-T lymphocyte subset monoclonal antibodies. Clin Exp Immunol 1987b; 67:305-312. 32. Meinl E, Hoch RM, Dornmair K et al. Differential encephalitogenic potential of myelin basic protein-specific T cell isolated from normal rhesus macaques, Am J Pathol 1997; 150:445-453. 33. Massacesi L, Genain CP, Lee-Parritz D et al. Chronic relapsing experimental autoimmune encephalomyelitis in new world primates. Ann Neurol 1995; 37:519-530. 34. Genain CP, Lee-Parritz D, Nguyen MH et al. In healthy primate, circulating autoreactive T cells mediate autoimmune disease. J Clin Invest 1994; 94:1339-1345. 35. ‘t Hart BA, Bauer J, Muller H-J et al. Histopathological characterization of magnetic resonance imaging-detectable brain white matter lesions in a primate model of multiple sclerosis. A correlative study in the experimental autoimmune encephalomyelitis model in common marmosets (Callithrix jacchus ). Am J pathol 1998; 153:649-663. 36. Laman JD, van Meurs M, Schellekens MM et al. Expression of accessory molecules and cytokines in acute EAE in marmoset monkeys (Callithrix jacchus). J Neuroimmunol 1998; 86:30-45. 37. Genain CP, Nguyen MH, Letvin NL et al. Antibody facilitation of multiple sclerosis-like lesions in a nonhuman primate. J Clin Invest 1995; 96:2966-2974. 38. Willenborg DO, Prowse SJ. Immunoglobulin-deficient rats fail to develop experimental allergic encephalomyelitis. J Neuroimmunol 1983; 5:99-109. 39. Genain CP, Robers T, Davis R et al. Prevention of autoimmune demyelination in nonhuman primates by a cAMP-specific phophodiesterase inhibitor. Proc Natl Acad Sci USA 1995; 92:3601-3605. 40. Genain CP, Abel K, Belmar N et al. Late complications of immune deviation therapy in a nonhuman primate. Science 1996; 274:2054-2057.

CHAPTER 3

MBP-Reactive T Cells in Multiple Sclerosis Katarzyna D. Bieganowska, Lara J. Ausubel, and David A. Hafler

M

ultiple sclerosis (MS) is a chronic inflammatory disease characterized by lymphocytic infiltration of T cells of the CNS white matter.1-5 T cells with high affinity receptors recognizing myelin basic protein (MBP) and proteolipid protein (PLP) are part of the normal T-cell repertoire. Although T cells that recognize myelin basic protein are present in healthy subjects as well as in MS patients, there are quantitative and qualitative differences among them.

MBP-Reactive T Cells from MS Patients are Activated In Vivo It has been demonstrated that MBP-reactive T cells are present in normal individuals. However, in experimental autoimmune encephalomyelitis (EAE), it has been found that only activated myelin-reactive T cells can induce the disease. Thus, it has been of great importance to characterize the in vivo activation state of MBP-reactive T cells in MS patients and controls. Allegretta et al6 have used an HPRT – mutant assay for identification of activation state of MBP-reactive T cells. This assay allows a selection of cells with a mutated HPRT gene. Mutation of this gene occurs naturally in activated cells during multiple divisions, and its presence is a mark of active proliferation. This approach showed that MBP-reactive T cells from MS patients accumulated HPRT mutations while MBP-reactive T cells from control subjects did not, thus indicating that in control subjects MBP-reactive T cells were resting, in contrast to actively proliferating MBP-reactive T cells from MS patients. Another approach to studying the activation state of MBP-reactive T cells was taken by Zhang et al.7 Based on the fact that antigen stimulated T cells express high affinity receptors for interleukin 2 (IL-2) and can proliferate in response to IL-2, it was postulated that if MBP-reactive T cells are present in an IL-2R-expressing population, these cells would expand in in vitro culture in the presence of IL-2 alone. This study showed that in MS patients the frequency of T-cell clones recognizing MBP after primary culture with IL-2 was significantly higher than in control subjects. Moreover, it was demonstrated that 7% of all IL-2-responsive cells derived from cerebrospinal fluid (CSF) of MS patients showed MBP reactivity, which represented 10 times higher frequency for these cells when compared to paired blood samples. Interestingly, MBP-reactive T cells were not detectable in CSF of patients with other neurological disorders (OND). Thus, results presented above showed a clear difference in the activation state of MBP-reactive T cells in MS patients and suggested a pathogenic role of autoreactive T cells in humans affected with MS.

T Cell Autoimmunity and Multiple Sclerosis, edited by Marco Londei. ©1999 R.G. Landes Company.

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MBP-Reactive T Cells in Some MS Patients are Clonally Expanded A series of experiments was designed to characterize the pool of T cells that recognize MBP. MBP recognition could be mounted by a polyclonal population of T cells, or the response could be narrowed to expansion of only a few T-cell clones. The clonal origin of a T cell can be identified by sequencing T-cell receptor (TCR) α and β chains that are used for antigen recognition. Three hypervariable regions known as complementarity determining regions (CDRs) are present in the α and β chains of a T-cell receptor. CDR1 and CDR2 are encoded within V region genes. The CDR3 is at the junction between the V-J domain for the α chain and the V-D-J junction domain for the β chain. The CDR3 sequence is a unique marker of a given T cell because of a large number of combinatorial possibilities for V-(D)-J segment rearrangement and additional diversification introduced by random additions and deletions of nucleotides at the junctions. Thus, the identical TCR rearrangement sequences obtained from different MBP-reactive T-cell clones from the same individual will indicate a clonal expansion of these cells in vivo. MBP-reactive T-cell clones were isolated from peripheral blood of MS patients and control subjects at different timepoints. Subsequent analysis of TCR rearrangements revealed that in two out of seven patients tested, the response to MBP was mounted by a clonally expanded population of T cells.8 Repeated analysis of MBP-reactive T cells in two patients showed that expanded clones persisted in vivo during the subsequent 4 year period tested.

High Frequency of MBP-Reactive T Cells in MS Patients That the response to MBP in MS patients was persistently mounted by a clonally expanded population of T cells led us to develop methods to directly estimate the frequency of MBP-reactive T cells by measuring mRNA transcripts encoding the α and β TCR chains ex vivo in peripheral blood without in vitro manipulation.9 Previously applied methods to estimate the frequency of antigen-specific T cells were based on the ability of T cells to proliferate in response to a given antigen, thus requiring the culture of cells in vitro. These measurements of T-cell frequencies by short-term T-cell cloning and thymidine incorporation, as is used in limiting dilution analysis (LDA), do not allow for correct estimates of activated antigenreactive T cells. A direct approach was used in which transcripts encoding for TCR α and β chains were amplified by polymerase chain reaction (PCR) from whole mononuclear cells and then subsequently tested for a specific CDR3 sequence expressed by previously characterized MBP-reactive T-cell clones.9 In two MS patients tested, in contrast to frequencies of one in 105 to 106 as measured by LDA, estimates of the T-cell frequencies expressing TCR chain transcripts associated with MBP recognition were as high as one in 300 and one in 1000. Previously characterized MBP-reactive T cells from control subjects could not be identified, implying that these T cells did not persist in vivo. The activation state of clonally expanded MBP-reactive T cells in MS patients was further examined by measuring the frequency of amplified TCR transcripts in a population of T cells positive or negative for IL-2 receptor (IL-2R) expression. These analyses performed on two timepoints (3 mo apart) have shown that two different MBP-reactive T-cell clones could be diferentially activated in vivo. While the frequency of a TCR sequence present in one clone was equally distributed among IL-2R-positive and -negative populations on the two timepoints tested, the frequency of a sequence associated with another MBP-reactive T-cell clone fluctuated among IL-2R-positive and -negative populations. Since it has been demonstrated that antigen stimulation of activated T cells expressing IL-2R induces apoptosis mediated by expression of Fas (CD95) and Fas ligand on the T-cell surface,10-17 we further showed that antigen stimulation of peripheral blood T cells in the presence of blocking anti-CD95 antibodies selectively inhibited the loss of MBP-reactive T cells present in IL-2R positive

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populations. Thus, culture of peripheral blood T cells with MBP p85-99 appeared to induce Fas-mediated apoptosis of activated T cells. The high frequency of MBP-reactive T cells may reflect chronic stimulation of MBPreactive T cells in the CNS. It is also possible that repeated challenges by cross-reactive microbes may induce selective T-cell activation over time. The MBP-reactive T-cell clones expressing different TCR had similar dose response curves to MBP p85-99, yet exhibited markedly different fine specificities for peptides with different TCR contact residues. Moreover, these MBP p85-99-reactive T-cell clones have been shown to recognize different crossreactive viruses.18 As only one of the MBP p85-99-reactive T-cell populations was activated at a second time point tested, as measured by IL-2Rα chain expression, these data suggest that at this timepoint the MBP p85-99-reactive T cells expressing the CDR3 sequence were activated by a crossreactive antigen and not the native MBP p85-99 sequence. Fluctuation of the MBP p85-99 specific clone with another CDR3 sequence among IL-2Rα-positive and -negative populations over a time of three months could also support such a possibility. Use of this approach to examine other subjects over longer periods of time may allow the determination of events that lead to the activation of autoreactive T cells in humans. Nevertheless, these analyses of MBP-reactive T cells provide the first direct evidence for clonal expansion of MBP-reactive T cells in patients with MS and demonstrate that direct amplification of TCR chains can be used to quantitate circulating autoreactive T cells. Moreover, these data demonstrate that at least a subpopulation of patients with MS can have a very high frequency of activated autoreactive T cells which undergo Fas-mediated apoptosis upon antigen stimulation.

Plasticity of Antigen Recognition by MBP-Reactive T Cells T cells recognize a complex formed by peptide antigens and major histocompatibility complex (MHC) molecules.19-21 In recent years, a wealth of information has been produced examining this complex. Based on X-ray crystallography, the structure of several MHC/peptide complexes has been solved.22-27 Functionally, the determination of MHC allelic motifs, the sequencing of naturally processed MHC ligands, and the evaluation of MHC site-directed mutants have all helped in the elucidation of the three dimensional orientation of this complex (reviewed in ref. 28). By contrast, little is known about the T-cell receptor (TCR) structure engaging the MHC/peptide complex. In particular, it is unknown what TCR/MHC structure would allow recognition of two distinct peptides that share little sequence homology, yet would at the same time allow the TCR the specificity to differentiate individual atoms on a molecule.29 The autoantigen myelin basic protein (MBP) is an extensively studied autoantigen in experimental models of autoimmunity as well as in the autoimmune disease multiple sclerosis (MS).30,31 It is one of the few antigens whose immunodominant epitopes have been mapped in humans.32-34 Moreover, as crossreactivity of T cells to MBP and viral/bacterial antigens has been implicated in the pathogenesis of MS,18,35-37 it was of interest to study the degree of flexibility in the T-cell response to this antigen. T-cell clones were generated against either the immunodominant region 85-99 of human MBP or an altered peptide of MBP in which one of the predominant TCR contact residues, that at position 93, was changed from lysine to arginine. T-cell proliferation and cytokine secretion of the T-cell clones in response to a series of peptides substituted at either one or both of the predominant TCR contact residues (positions 91 and 93) were examined. Any single amino acid substitution at these predominant TCR contact positions abrogated reactivity as measured by proliferation or cytokine secretion. However, a conservative substitution at position 93 of the peptide induced a change in the receptor such that a substitution of the amino acid side chain at position 91 could then induce T-cell

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activation equivalent to that elicited by the original stimulating peptide. These data are best explained by a model in which the TCR possesses at least two functional pockets; the exact nature of the side chain engaging one TCR functional pocket may change the apparent selectivity of the other TCR functional pocket. This suggests a remarkable degree of plasticity of theTCR/MHC structure and can potentially explain a number of immunologic phenomena. First, the ability of peptide antigens to induce structural changes in the TCR or MHC provides an explanation for the ability of a T-cell clone to recognize two distinct peptides that share little sequence homology, yet simultaneously to differentiate individual atoms on the same antigenic peptide. This concept further suggests that an individual TCR can recognize more antigenic complexes than previously thought and indicates that potentially vast numbers of exogenous antigens may be capable of crossreacting with self antigens. This is of obvious importance in understanding the development of autoimmunity. Moreover, it is also possible that there are structural changes in the TCR which allow a single T cell to recognize both allogeneic MHC/peptide and self MHC/peptide complexes. Lastly, it would appear likely that this capacity of recognizing multiple ligands may better explain how a limited number of peptides in the thymus can select for T cells that react to the wide variety of peptide antigens possible. To investigate further the plasticity of a peptide recognition by MBP-reactive T cells, a new method was introduced by Hemmer et al.38 Their approach introduced combinatorial peptide libraries to identify crossreactive ligands for MBP-reactive T-cell clones. The libraries with defined peptide length were generated by random synthesis for each position. Thus, the library represents a pool of peptides with all possible amino acid combinations at each position. Therefore, it can be defined how many ligands can be recognized by a single T cell. By screening the response of a MBP-reactive T-cell clone to 220 undecapeptide sublibraries, a high number of ligands were identified. Some of them were homologous to human and bacterial proteins and were able to induce even higher response than the MBP peptide. In agreement with previous observations, it has been shown that many amino acid exchanges in the native MBP peptide are tolerated and can even induce a stronger response. Moreover, alterations of primary and secondary TCR contact points abolished T-cell response, but the response could be restored by modifications at other positions. Thus, there is a high degeneracy in antigen recognition and a large number of ligands can trigger the response of a T cell. This may have important implications in induction of autoimmune disease. Epitopes of an infectious agent such as bacteria or viruses which induce immune response in the host may share homology with self proteins of the host. The immune response directed against foreign microbes may activate autoreactive T cells and thus lead to autoimmunity. These data may also explain the high frequency of MBP-reactive T cells in MS patients.

Cytokine Production Pattern by MBP-Reactive T Cells It is known that naive T helper cells, upon activation by antigen, will differentiate into distinct functional groups characterized by the types of cytokines they secrete.39,40 Th1 cells secrete activating cytokines such as IL-2, IFN-γ, TNF-α, and IL-12, and these cytokines activate macrophages and induce delayed type hypersensitivity (DTH). In contrast, Th2 cells secrete regulatory cytokines including IL-4, IL-5, and IL-10, which can suppress cell-mediated immunity and induce class switching of antibody, leading to IgE production (reviewed in ref. 41). Some cells will release cytokines from both of these categories and are known as Th0 type cells. The cytokines in each group crossregulate each other. For example, a Th1 cytokine such as IL-12 induces IFN-γ production from Th precursor cells and promotes their differentiation into Th1 type cells.42,43 The production of IFN-γ has been shown to downregulate the production of Th2 type cytokines such as IL-4.44 Likewise, IL-4 is a major

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growth factor for Th2 cells and inhibits IFN-γ production.45-48 Interestingly, IL-4 inhibits the expression of the IL-12 receptor β2 subunit on early Th2 cells, thus promoting commitment of these cells to the Th2 pathway. On the other hand, IFN-γ treatment of early Th cells maintains IL-12R β2 expression.49 Much of the work on the role of cytokines in autoimmune disease stems from work in animal models of autoimmunity. In the rodent model of multiple sclerosis (MS) known as experimental autoimmune encephalomyelitis (EAE), it has been found by many that autoreactive T cells which can induce disease are Th1 type cells which secrete cytokines highly stimulatory to the cellular immune response, such as IFN-γ and TNF-α.50,51 In the nonobese diabetic (NOD) mouse model of diabetes, Th1 type autoreactive T cells can induce disease. Yet, when these same T cells are induced to secrete Th2 type cytokines such as IL-4 and IL-10 which can downregulate the cellular immune response after in vitro culture, they can no longer induce disease.52 Similarly, recovery from EAE has been associated with IL-4 and TGF-β1 secretion.53 Other animal studies have also demonstrated that adoptive transfer of autoantigen specific Th2 type cells (or Th3 type cells that secrete transforming growth factor TGF-β) protects mice from the development of EAE.54,55 However, it is clear that this simple notion of Th1 and Th2 cytokines either regulating or suppressing animal models of autoimmune disease has higher levels of complexity. For example, both IFN-γ and TNF-α deficient mice can still develop EAE56 as well as diabetes,57 although in the NOD model mice had a delayed onset of disease. This is not surprising considering the great redundancy in cytokine function. It is quite likely that other cytokines such as TNF-α and IL-2 are able to activate the immune system in such a way as to induce disease without the need for IFN-γ. Studies of the cytokine secretion of human autoreactive T-cell clones specific for the immunodominant epitope of MBP 83-99 demonstrated heterogeneity in cytokine secretion without a strict separation into either a Th1 or Th2 pattern.58 However, in the same study, it was found that MBP-reactive T-cell clones derived from control subjects had lower IFN-γ/IL-4 ratios, thus suggesting a bias towards the Th1 type response in MS patients. A similar observation was made while studying cytokine response of proteolipid protein-specific T-cell clones, where disease activity was correlated with higher frequency of Th1 type cells.59 Since Th1 but not Th2 cells induce disease in animal models of MS and Th2 type cytokines downregulate secretion of Th1 cytokines, it has been thought that induction of Th2 type cells could provide some sort of protection or treatment for individuals with autoimmune disease with a prominent Th1 response. The question thus arises: How could we manipulate the cytokine secretion pattern of autoreactive T cells? It has been shown by Windhagen et al that a peptide altered at a secondary TCR contact residue could induce MBP-reactive T cells which secreted both Th1 and Th2 cytokines (Th0) to secrete TGF-β.60 Recent data from our laboratory indicate that APLs may work to induce Th2 type cytokines in autoreactive T-cell clones from humans.61 T cells were isolated from two patients with multiple sclerosis directly from peripheral blood by stimulation either with native MBP p85-99 or with APLs in which substitutions of the lysine were made at position 93, a TCR contact residue. We found that the APL 93A, in which the lysine normally found at position 93 was changed to an alanine, could alter the cytokine profile of autoreactive T cells, switching them from a Th0 phenotype secreting high amounts of IL-4 and IFN-γ into Th2 cells which no longer secreted significant amounts of IFN-γ.61 In fact, recent trials using the APL 93A to alter the cytokine pattern of pathogenic, autoreactive MBP T-cells have begun in humans (Steinman L, personal communication). It is difficult to predict the outcome of administration of APLs in humans with autoimmune disease. It is likely that the ability of APLs to effectively treat patients with autoimmune disease will depend on the specific T-cell repertoire of the individual patients.

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It may also be important to examine whether a particular APL is crossreactive with the native self antigen, and thus a weak agonist for autoreactive T cells, before using such a peptide in treating patients with autoimmune disease. Moreover, it has been recently shown in adoptive transfer systems that Th2 type cells are able to induce EAE62 or acute pancreatis and diabetes63 in immunocompromised mice. These results suggest that Th2 type cells have a potential for inducing autoimmune disease; thus Th2-based therapy in autoimmune disease in humans should be cautiously evaluated.

References 1. Adams RD, Sidman RL. Introduction to Neuropathology. 1st ed. New York: McGraw-Hill, 1968:149-170. 2. Prineas J W, Raine CS. Electron microscopy and immunoperoxidase studies of early multiple sclerosis lesions. Neurology 1976; 26:29-32. 3. Prineas JW, Wright RG. Macrophages, lymphocytes, and plasma cells in the perivascular compartment in chronic multiple sclerosis. Lab Invest 1978; 38:409-421. 4. Booss J, Esiri MM, Tourtellotte WW. Immunohistochemical analysis of T-lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis. J Neurol Sci 1983; 62:19-32. 5. Hafler DA, Weiner HL. Immunologic mechanisms and therapy in multiple sclerosis. Immunol Rev 1995; 144:75-107. 6. Allegretta M, Nicklas JA, Sriram S et al. T cells responsive to myelin basic protein in patients with multiple sclerosis. Science 1990; 247:718-721. 7. Zhang J, Markovic S, Raus J et al. Increased frequency of IL-2 responsive T cells specific for myelin basic protein (MBP) and proteolipid protein (PLP) in peripheral blood and cerebrospinal fluid (CSF) of patients with multople sclerosis. J Exp Med 1994; 973-84:973-984. 8. Wucherpfennig KW, Zhang J, Witek C et al. Clonal expansion and persistence of human T cells specific for an immunodominant myelin basic protein peptide. J Immunol 1994; 52:5581-5592. 9. Bieganowska KD, Ausubel LJ, Modabber Y et al. Direct ex vivo analysis of activated, fas sensitive autoreactive T cells in human autoimmune disease. J Exp Med 1997; 185:1585-1594. 10. Alderson MR, Tough TW, Davis-Smith T et al. Fas ligand mediates activation-induced cell death in human T lymphocytes. J Exp Med 1995; 181:71-77. 11. Critchfield JM, Racke MK, Zuniga-Pflucker JC et al. T-cell deletion in high antigen dose therapy of autoimmune encephalomyelitis. Science 1994; 263:1139-1143. 12. Griffith TS, Brunner T, Fletcher SM et al. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 1995; 270:1189-1192. 13. Zheng L, Fisher G, Miller RE et al. Induction of apoptosis in mature T cells by tumor necrosis factor. Nature 1995; 377:348-351. 14. Ju ST, Panka DJ, Cui H et al. Fas (CD95)/FasL interactions required for programmed cell death after T-cell activation. Nature 1995; 373:444-448. 15. Brunner T, Mogil RJ, LaFace D et al. 1995. Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas. Nature 1995; 373:441-444. 16. Dhein J, Walczak H, Baumler C et al. Autocrine T-cell suicide mediated by Apo-1(Fas/D95). Nature 1995; 373:438-441. 17. Lenardo MJ. Fas and the art of lymphocyte maintenence. J Exp Med 1996; 183:721-724. 18. Wucherpfennig KW, Strominger JL. Molecular mimicry in T cell-mediated autoimmunity: Viral peptides activate human T-cell clones specific for myelin basic protein. Cell 1995; 80:695-705. 19. Zinkernagel R, Doherty P. Immunological surveillance against altered self components by sensitized T lymphocytes in lymphocytic choriomeningitis. Nature 1974; 251:547-548. 20. Katz DH, Hamaoka T, Benacerraf B. Cell interactions between histoincompatible T and B lymphocytes. II. Failure of physiologic cooperative interactions between T and B lymphocytes from allogeneic donor strains in humoral response to hapten-protein conjugates. J Exp Med 1973; 137:1405-1418.

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21. Rosenthal AS, Shevach EM. Function of macrophages in antigen recognition by guinea pig T lymphocytes. I. Requirement for histocompatible macrophages and lymphocytes. J Exp Med 1973; 138:1194-1212. 22. Bjorkman PJ, Saper MA, Samraoui B et al. Structure of the human class I histocompatibility antigen, HLA-A2. Nature 1987; 329:506-512. 23. Madden DR, Gorga JC, Strominger JL et al. The three-dimensional structure of HLA-B27 at 2.1. Å resolution suggests a general mechanism for tight peptide binding to MHC. Cell 1992; 70:1035-1048. 24. Fremont DH, Matsumura M, Stura EA et al. Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb. Science 1992; 257:919-927. 25. Matsumura M, Fremont DH, Peterson PA et al. Emerging principles for the recognition of peptide antigens by MHC class I molecules. Science 1992; 257:927-934. 26. Zhang WY, Young ACM, Imarai M et al. Crystal structure of the major histocompatibility complex class I H-2Kb molecule containing a single viral peptide: Implications for peptide binding and T-cell receptor recognition. Proc Natl Acad Sci USA 1992; 89:8403-8407. 27. Brown JH, Jaretzsky TS, Gorga JC et al. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 1993; 364:33-39. 28. Chien Y, Davis MM. How αβ T-cell receptors ‘see’ peptide/MHC complexes. Immunol Today 1993; 14:597-602. 29. Evavold BD, Sloan-Lancaster J, Wilson KJ et al. 1995. Specific T-cell recognition of minimally homologous peptides: Evidence for multiple endogenous ligands. Immunity 2:655-663. 30. Zamvil S, Nelson P, Trotter J et al. 1985. T-cell clones specific for myelin basic protein induce chronic relapsing paralysis and demyelination. Nature 1985; 317:355-358. 31. Mokhtarian F, McFarlin DE, Raine CS. Adoptive transfer of myelin basic protein-sensitized T cells produces chronic relapsing demyelinating disease in mice. Nature 1984; 309:356-358. 32. Pette M, Fujita K, Wilkinson D et al. Myelin autoreactivity in multiple sclerosis: Recognition of myelin basic protein in the context of HLA-DR2 products by T lymphocytes of multiple sclerosis patients and healthy donors. Proc Natl Acad Sci USA 1990; 87:7968-7972. 33. Martin R, Howell MD, Jaraquemada D et al. A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis. J Exp Med 1991; 173:19-24. 34. Ota K, Matsui M, Milford EL et al. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 1990; 346:183-187. 35. Damian RT, Molecular mimicry: Antigen sharing by parasite and host and its consequences. Am Nat 1964; 98:129-149. 36. Oldstone MBA. Molecular mimicry and autoimmune disease. Cell 1987; 50:819-820. 37. Fujinami RS, Oldstone MBA. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: Mechanism for autoimmunity. Science 1985; 230:1043-1046. 38. Hemmer B, Fleckenstein BT, Vergelli M et al. Identifcation of high potency mcrobial and self ligands for a human autoreactive class II-restricted T-cell clone. J Exp Med 1997; 185:1651-1659. 39. Mosmann TR, Cherwinski H, Bond MW et al. Two types of murine helper T-cell clones. I. Definition according to profiles of lymhokine activities and secreted proteins. J Immunol 1986; 136:2348-2357. 40. Janeway CA, Carding S, Jones B et al. CD4+ T cells: Specificity and function. Immnol Rev 1988; 101:39-80. 41. Nicholson LB, Kuchroo VK. Manipulation of the Th1/Th2 balance in autoimmune disease. Curr Opin Immunol 1996; 8:837-842. 42. Manetti R, Parronchi P, Grazia M et al. Natural killer cell stimulatory factor (IL-12) induces T helper type 1 (Th1)-specific immune responses and inhibits the develpment of IL-4 producing Th cells. J Exp Med 1993; 177:1199-1204. 43. Sypek JP, Chung CL, Mayor SE et al. Resolution of cutaneous leishmaniasis: Interleukin 12 initiates a protective T helper type 1 immune response. J Exp Med 1993; 177:1797-1802.

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44. Gajewski TF, Fitch FW. Anti-proliferative effect of IFN-γ in immunoregulation. I. IFN-γ inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. J Immunol 1988; 140:4245-4252. 45. Chatelain R, Varkila K, Coffman RL. IL-4 induces a Th2 response in Leishmania major-infected mice. J Immunol 1992; 148:1182-1187. 46. LeGros G, Ben-Sasson SZ, Seder R et al. Generation of IL-4-producing cells in vivo and in vitro. IL-2 and IL-4 are required for in vitro generation of IL-4-producing cells. J Exp Med 1990; 172:921-929. 47. Betz M, Fox BS. Regulation and development of cytochrome c-specific IL-4 producing T cells. J Immunol 1990; 145:1046-1052. 48. Seder RA, Paul WE, Davis MM et al. The presence of interleukin-4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T-cell receptor transgenic mice. J Exp Med 1992; 176:1091-1098. 49. Szabo SJ, Dighe AS, Gubler U et al. Regulation of the interleukin (IL)-12R β2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J Exp Med 1997; 185:817-824. 50. Zamvil SS, Steinman L. The T lymphocyte in experimental allergic encephalomyelitis. Ann Rev Immunol 1990; 8:579-621. 51. Kuchroo VK, Martin CA, Greer JM et al. Cytokines and adhesion molecules contribute to the ability of myelin proteolipid protein-specific T-cell clones to mediated experimental allergic encephalomyelitis. J Immunol 1993; 151:4371-4382. 52. Katz JD, Benoist C, Mathis D. T helper cell subsets in insulin-dependent diabetes. Science 1995; 268:1185-1188. 53. Khoury SJ, Hancock WW, Weiner HL. Oral tolerance to myelin basic protein and natural recovery from experimental allergic autoimmune encephalomyelitis are associated with downregulation of inflammatory cytokines and differential upregulation of transforming growth factor-β, interleukin-4 and prostaglandin E expression in the brain. J Exp Med 1992; 176:1355-1364. 54. Kuchroo VK, Prabhu Das M, Brown JA et al. B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: Application to autoimmune disease therapy. Cell 1995; 80:707-718. 55. Chen Y, Kuchroo VK, Inobe J-i et al. Regulatory T-cell clones induced by oral tolerance: Suppression of autoimmune encephalomyelitis. Science 1994; 265:1237-1240. 56. Ferber IA, Brocke S, Taylor-Edwards C et al. Mice with a disrupted IFN-γ gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J Immunol 1996; 156:5-7. 57. Hultgren B, Huang X, Dybdal N et al. Genetic absence of γ-interferon delays but does not prevent diabetes in NOD mice. Diabetes 1996; 45:812-817. 58. Hemmer B, Vergelli M, Calabresi P et al. Cytokine phenotype of autoreactive T-cell clones specific for the immunodominant myelin basic protein peptide (83-99). J Neurosci Res 1997; 45:852-862. 59. Correale J, Gilmore W, McMillan M et al. Patterns of cytokine secretion by autoreactive proteolipid protein-specific T-cell clones during the course of multiple sclerosis. J Immunol 1995; 154:2959-2968. 60. Windhagen A, Scholz C, Hollsberg P et al. Modulation of cytokine patterns of human autoreactive T-cell clones by a single amino acid substitution of their peptide ligand. Immunity 1995l; 2:373-380. 61. Ausubel LJ, Krieger JI, Hafler DA. Changes in cytokine secretion induced by altered peptide ligands of myelin basic protein peptide 85-99. J Immunol, in press. 62. Lafaille JJ, Van de Kerre F, Hsu AL et al. Myelin basic protein specific T helper 2 (Th2) cells cause experimental autoimmune encephalomyelitis in immunodeficient hosts rather than protect them from the disease. J Exp Med 1997; 186:307-312. 63. Pakala SV, Kurrer MO, Katz JD. T helper 2 (Th2) T cells induce acute pancreatitis and diabetes in immune compromised nonobese diabetic (NOD) mice. J Exp Med 1997; 186:299-306.

CHAPTER 4

Genetic Engineering of Brain-Specific T Cells for Treatment of Diseases in the Central Nervous System A. Flügel and H. Wekerle

T

he concept of gene therapy developed rapidly from the originally somewhat futuristic idea to create “the” perfect causal therapy to correct genetically encoded defects in the organism.1-3 Over the years, the initial focus on the treatment of monogenic disorders has been significantly extended. At the present time, neoplastic, inflammatory and degenerative diseases are considered additional targets of gene therapy; in fact, they have become the paramount targets of current gene therapeutic protocols.4-8 Two principal gene transfer approaches can be distinguished. The ex vivo approach involves transfer of genes in culture using cells isolated from an individuum genetic manipulation in culture, followed by reintroduction of these genetically engineered cells back into the pathological lesion. The in vivo approach involves the direct application of gene vectors or genes into the target tissue without cell manipulation in vitro.

General Aspects of Gene Therapeutic Approaches in CNS Diseases Recent insights into physiology and pathophysiology of the central nervous system (CNS) have laid a firm base for the development of the research tools mentioned above. For example, genetic defects of several inherited CNS diseases, e.g., Huntington’s disease,9,10 neurofibromatosis,11 fragile X syndrome,12 and hereditary motor and sensory neuropathies such as Charcot-Marie-Tooth and Déjérine-Sottas disease,13 have been identified and now are prime targets for gene therapy. Then, the rapidly developing field of neurotrophic factors has provided candidate genes for the treatment of degenerative CNS diseases.7,14-18 Further, immunological research has led to the identification of inflammatory networks in neoplastic,19-21 infectious22-26 and autoimmune27-29 CNS processes, mechanisms with direct relevance for gene therapeutic strategies. Initially, gene therapy of the CNS seemed to be burdened with special, discouraging problems. The function and anatomy of the CNS was considered to be too complex for the gene therapeutic tools available. Genetic manipulation of CNS cells is indeed complicated by the fact that cells of neuronal and glial origin cannot be readily removed from the brain and reinserted in their anatomical location without causing serious functional damage. Furthermore, most CNS cells are hard to culture in vitro, and in the case of nondividing differentiated neurons, stable and safe gene transfer has not been achieved until recently. Targeting of DNA material in CNS diseases creates a number of additional problems. For example, some functional units of the CNS, such as the motor neuronal systems, are T Cell Autoimmunity and Multiple Sclerosis, edited by Marco Londei. ©1999 R.G. Landes Company.

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T-Cell Autoimmunity and Multiple Sclerosis

spread over immensely widespread areas. In these cases, CNS lesions are not concentrated in single circumscript locations. Second, even “monosystemic” disorders, such as Parkinson’s disease, usually involve distinct neuronal units that are often scattered throughout the brain. Probably the most important obstacle against targeted gene delivery in the CNS is created by its particular anatomical structure. The CNS is embedded almost entirely in bone structures that require neurosurgical intervention for external gene application. Perhaps even more important, internal gene application, via blood circulation, is hindered by the endothelial blood brain barrier (BBB), an anatomical structure impermeable to most macromolecules and cells.30,31 Numerous strategies have been developed to overcome these barriers. These include pharmacological, biological, and mechanical approaches.32-36 Some therapeutic strategies have concentrated on increasing the hydrophobicity of therapeutic agents to let them pass through the BBB, or on involving specific receptor -mediated transport mechanisms. Then, reversible opening of the BBB has been attempted using hyperosmotic agents like mannitol. Purely mechanical strategies include intrathecal administration of drugs via injection, implants, or pumps. All these strategies, effective as they may be in overcoming the BBB, have been limited with regard to precise targeting of genes or gene vectors to disease affected brain locations. Most gene therapy studies of CNS disorders use the ex vivo approach. 37 Gene manipulated cells derived from different tissues were transplanted stereotactically into lesioned areas of the brain.38 The engineered cellular transplants had been taken either from the recipient him/herself,39-43 or came from distinct (embryonic) human donors, or were even of nonhuman species origin.44,45 The latter were firmly encapsulated to protect the host against potential damage coming from the transplant (e.g., animal viruses) and to protect the implanted cells against host-derived immunological attack. Alternatively, for direct in vivo therapy, gene vectors such as adenoviruses46,47 or pure DNA48 similarly were applied stereotactically to affected areas. Suitable neurotropic vectors such as members of the Herpes virus family are still in development.49-52 As mentioned, ultrastructural and functional analyses established that the BBB is impermeable to most macromolecules and cells circulating through the blood. However, in recent years it has become clear that the barrier function is not absolute. Thus, upon activation, T lymphocytes can cross the BBB,53-55 a property of fundamental importance for the effective immune surveillance of the brain. The unique ability of activated T lymphocytes to patrol through virtually any type of tissue renders them attractive as vehicles for gene targeting. Indeed, recently improved gene transfer techniques allowed experimental testing of genetically engineered, brain-specific T lymphocytes as therapeutic tools. At least in theory, genetically manipulated T lymphocytes of known receptor specificity would deliver the transgene of interest specifically to the tissue expressing the antigen expressing the specific antigen that the T cells recognize. Gene delivery might be controlled by the life span of the T cells and by the respective gene regulating elements. Hence this strategy would not only elegantly overcome physical barriers encapsulating the CNS, but in addition, with the optimally suited target antigen chosen, it would allow further targeting of therapeutic transgenes to the specific, damaged CNS areas. Antigen-specific T lymphocytes were first used for gene therapy of cancer due to the natural function of T cells to kill tumor cells and to their specific homing behavior. The broad applicability of antigen-specific, gene engineered T lymphocytes as a potential therapeutic principle in CNS disease has been demonstrated in recent animal studies of inflammatory CNS and PNS disorders, and will be discussed later in this chapter. Application of T cell-mediated gene therapy to CNS diseases was possible only after careful studies of lymphocyte behavior in animal models of experimental allergic encephalitis (EAE) and after

GeneticEngineeringofBrain-SpecificTCellsforTreatmentofDiseasesintheCNS

39

development of effective gene transfer techniques for T lymphocytes. Thus, before coming to studies pursuing this therapeutic approach, T-cell trafficking in EAE and gene transfer techniques into T cells are discussed.

Brain-Specific T Cells as a Possible Therapeutic Tool EAE as a Model for T Lymphocyte Infiltration Into the CNS As mentioned above, traditionally the CNS was considered to be an immune privileged organ, its tissues being exempt from immune surveillance. Thus, the CNS lacks a fully organized lymphatic system and immune cells. There is no expression of molecules for antigen presentation, MHC I and MHC II in healthy brain tissue.56,57 Furthermore, most cells and molecules from the peripheral blood are excluded from the nervous milieu by the tight endothelial BBB.58-60 In fact, early transplantation studies which demonstrated unusually long survival of tissue grafts in the brain61-64 supported this concept. Under diverse pathological conditions, however, inflammatory processes with marked cellular infiltrations can be observed in the CNS. Some of these diseases, such as viral or sterile infiltrations, e.g., multiple sclerosis, are characterized by immune cell infiltration of the CNS through the intact BBB.55 Better understanding of immune cell passage through the BBB required a model system allowing study of its development over time and manipulation of individual cellular components involved in the individual phase of the inflammatory process. Such studies became possible thanks to the diverse versions of experimental allergic encephalomyelitis (EAE), which for the first time paved the way for a systematic analysis of lymphocyte traffic into the CNS tissues. Historically, the first time EAE-like symptoms were observed as sequelae of a therapeutic vaccination. Patients treated with Pasteur vaccine, fixed rabies virus grown in rabbit brain, developed in 0.1% adverse effects characterized by acute cerebral dysfunction.65 Clinically, the recipients suffered from a monophasic paralytic disease, which was reflected histopathologically by perivascular infiltrates and focal demyelination. The cause of the disease was correctly attributed to contamination of the vaccine with brain matter. Indeed, a similar disease was produced in monkeys, rodents, and other species by injection of uninfected brain homogenates.65 Subsequent work demonstrated that EAE can be caused not only by immunization of animals with whole CNS homogenates but also with individual CNS proteins, most prominently with myelin basic protein (MBP).66 Secondly, it turned out that T lymphocytes are the crucial agents mediating EAE.67-69 EAE could be triggered via transfer of CD4+ MBP-specific T lymphocytes into healthy syngeneic recipient animals.70,71 The most popular EAE model of passively transferred EAE and still one of the most intensively analyzed EAE models is the T-cell line-mediated EAE of the Lewis rat. Transfer of encephalitogenic T cells into naive Lewis rats results in a monophasic disease characterized by caudo-cranially ascendent paralysis. Surviving animals recover completely and often become resistant to later attempts to induce EAE. Histologically, EAE lesions are characterized by mononuclear cell infiltrates72 and marked activation of local microglial and astroglial cells, the latter reflected by enhanced formation of cytoskeletal glial fibrillary acidic protein (GFAP) 73,74 and MHC class I and class II antigens in microglia.75 The mononuclear infiltrates consist predominantly of monocytes and of CD4+ T lymphocytes,76 while B cells play a minor role.77,78 CD8 T lymphocytes were observed in the recovery phase.77,78 Apart from leptomeningeal inflammation,77,78 infiltrates are concentrated in the white matter of the CNS, typically around postcapillary microvessels, but there is also marked invasion of the perivascular parenchyma.72,79 In the active phase of the disease the BBB is ruptured, causing

40

T-Cell Autoimmunity and Multiple Sclerosis

local vasculogenic edema and fibrin deposition.80 However, it should be noted that primary demyelination is minimal in T cell-mediated Lewis rat EAE. Large scale demyelination is produced by the combination of encephalitogenic T cells with myelin-specific antibody.81 The lack of demyelination illustrates that Lewis rat EAE cannot be considered “the” model of human MS. However, EAE may well represent early inflammatory changes seen in the MS plaque.66,76 The adoptive transfer model using T-cell lines is highly versatile and productive. With its use, an ever growing number of diverse CNS proteins have turned out to qualify as potential encephalitogenic autoantigens. Furthermore, functional insights into interactions between brain-reactive T cells and brain cells were gained, which eventually provided a basis for evaluation of T lymphocytes as potential therapeutic transgene carriers. These include questions like: 1. Which of the T lymphocytes are able to enter the brain and cause disease? 2. Which factors promote, which inhibit these processes? 3. How do antigen-specific T lymphocytes produce pathogenic changes within the CNS? and 4. What fate awaits these lymphocytes after infiltrating the brain? The following section gives a brief survey of present knowledge on these issues.

T Lymphocytes in the Lewis Rat EAE Most if not all transfer models of Lewis rat EAE are based on the autoreactive CD4+CD8–,αβ+ T lymphocytes which recognize their target peptide epitopes in the context of MHC class II determinants. Analysis of MBP-specific T lymphocytes in Lewis rats revealed some unusual features, which are also seen in MBP-reactive T-cell responses of H-2u mice (PL/J, B10.PL) but not in other strains and species. MBP-specific T-cell lines raised from Lewis rats show epitope specificity dominated almost exclusively by one peptide segment (MBP p68-88). Further, almost all of these cells use Vβ8.2 genes for their receptor, with a preferred combination with certain Vαs.54,76 The cytokines released by practically all established encephalitogenic T lymphocyte lines have a typical T helper 1 subtype pattern, with release of interferon γ and IL-2, but no IL-4.82,83 What Enables T Lymphocytes to Penetrate the BBB? Quite soon after establishment of the adoptive transfer EAE model, it became clear that infiltration into the brain requires activated lymphocyte blasts.53,55,84-86 Resting lymphocytes are unable to induce EAE or to infiltrate the brain. Differential circulation behavior of resting compared to activated cells has been analyzed in detail.87,88 To date, the crucial difference which enables activated T cells exclusively to enter the brain and recruit other peripheral blood cells to the CNS remains unknown. It is possible that cytokines or membrane surface molecules, transiently expressed primarily in the activated phase of the T-lymphocyte restimulation cycle, play the pivotal role in this respect.89,90 One candidate for such a surface molecule is CD40L, which recently has been shown to induce endothelial expression of CD40, E-selectin and ICAM-1.91 No difference in the expression of adhesion molecules involved in CNS homing from that of those used in the periphery could be defined, suggesting that similar steps of lymphocyte extravasation via tethering, adhesion and transmigration take place in the CNS as in the periphery.87,92 This assumption is supported by CNS inflammation studies using transgenic animals and blocking antibodies, which show participation of all commonly used members of adhesion molecule classes for lymphocyte trafficking: the selectins,93-96 integrins,97-102 and adhesion molecules from the immunoglobulin superfamily.99,100,103-105 Increasingly, EAE research is focusing on the importance of chemokines, either in directly attracting inflammatory cells to the CNS or

GeneticEngineeringofBrain-SpecificTCellsforTreatmentofDiseasesintheCNS

41

via indirect activation of vascular and cerebral tissue.87,106-108 Despite some promising early results, it will likely be a while until the whole set of homing participants can be defined and their complex functional and temporal interplay will be understood. Analysis of specificity of T lymphocytes able to cause CNS infiltration gave surprising results. Several studies could show that T-lymphocyte infiltration of the brain is not antigen dependent.53-55,109 Injection of T-lymphocyte blasts specific for a CNS-irrelevant antigen (ovalbumin) resulted in brain infiltration of these cells. Even allogeneic lymphocytes were found to infiltrate the CNS.55 This infiltration of lymphocyte blasts takes place within a few hours after injection into healthy recipients. The difference between irrelevant lymphocyte blasts and brain antigen-specific lymphocyte blasts concerning CNS infiltration is that the former disappear rapidly, within two to three days after the first wave of infiltration. Brain-specific lymphocytes, however, persist and a second wave of infiltration can be observed immediately before onset of clinical symptoms. These results clearly demonstrate that the BBB is not an impermeable barrier for T lymphocytes and that the CNS is under surveillance of the immune system. Furthermore, the above mentioned findings, that homing to CNS tissue is in principle dependent on common adhesion molecules, have been corroborated, at least concerning T-lymphocyte migration. A crucial but still insufficiently answered question remains: How many of the injected lymphocytes can actually be found in CNS infiltrates? Previous studies trying to trace T lymphocytes in inflammatory brain lesions or to isolate T lymphocytes from them suggest a surprisingly low number of specific cells.110-113 Analyses of EAE lesions with transgene-carrying lymphocytes reveal a respectable representation (Fig. 4.1.) of the injected cells in the lesions. What Causes Pathogenicity of Lymphocytes and Where do They Infiltrate? No in vitro criteria have been identified which define the pathogenic potential of a T-cell line. However, testing of further potentially encephalitogenic antigens other than MBP in the Lewis rat lead to quite interesting observations. Not unexpectedly, other myelin proteins such as proteolipid protein (PLP) are also able to evoke an EAE.66,114 Others, such as myelin oligodendrocyte glycoprotein (MOG), surprisingly showed an encephalitis with almost no clinical signs of disease.115 The same held true for a non-myelin CNS protein, the calcium binding protein S100β.116 Here, EAE inducibility was very surprising, since S100β was shown to be expressed in several other organs, such as in Müller cells of the retina,117 Schwann cells of the PNS,118 and even the thymus.119 Similarly to MOG, the massive perivascular and parenchymal infiltration of the CNS with inflammatory cells would lead one to expect a severe clinical disease. Instead, the animals are almost completely healthy with slight weight loss.116 More careful histological analysis revealed a distinct relation of certain inflammatory cell populations in this type of EAE compared to the MBP-driven EAE.116 Whereas the T-lymphocyte number was comparable, the percentage of infiltrating monocytes in MOG and S100β EAE is much less. The explanation for this minor recruitment of monocytes is not known. S100β cells in vitro do not differ substantially from MBP T lymphocytes concerning testable properties such as the trimmings of membrane molecules and profile of cytokine release. A further striking, but better explained, difference in the appearance of S100β to MBP EAE is the different location of the lesions. As mentioned above, MBP lesions usually start in the lumbo-sacral spinal cord and only in very severe cases of EAE affect the forebrain. S100β EAE lesions are distributed throughout the CNS, affecting, according to their antigen specificity, also the uvea and retina of the eye and, to a lesser extent, the PNS.116 T lymphocyte lines directed against the peripheral P2 myelin cause a severe inflammation of the PNS, completely sparing CNS tissue.120,121 This regularity of migration to areas of antigen expression opens the possibility of designing T-lymphocyte lines specific for certain brain

42

T-Cell Autoimmunity and Multiple Sclerosis

Fig. 4.1. Transgene-carrying T lymphocytes in an EAE lesion. (a) In situ hybridization against transgenic GDNF (glial derived neuro-trophic factor)-positive, MBP-specific T lymphocytes infiltrating the sacral spinal cord in an adoptive transfer EAE; (b) W3/13 immunohistochemistry of the same animal, (c) in situ hybridization and simultaneous W3/13 immunohistochemistry; (d, e, f ) Higher magnification of an area of the upper picture indicated by arrow heads. Sections and staining were performed by H. Lassmann and R. Birnbacher, Vienna. regions, on condition that brain-specific antigens for the respective region are known. A different targeting mechanism for CNS-specific lymphocytes with important implications for gene therapy via brain-specific lymphocytes was observed by Meahlen et al.122 In their study they analyzed lymphocyte migration behavior of actively immunized Lewis rats treated with facial nerve crush. They found massive concentrations of inflammatory cells in the nuclear region of the injured nerve. Similar results were observed by Hickey, analyzing T-cell migration after actively induced EAE in a transection model of the optical nerve.123 Konno et al124 showed targeting of adoptively transferred MBP lymphocytes in injured CNS tissue. Exclusively CNS specific lymphocytes were shown to migrate into the lesions. The exact mechanism tracing the T lymphocytes into lesioned areas has still to be defined. Interestingly, lesioned areas can attract the lymphocytes even if this location usually is not favored by their “normal” CNS infiltration, e.g., MBP-specific T lymphocytes infiltrate the lesioned optical tract they usually spare.

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43

What Fate Awaits T Lymphocytes in the CNS? The question of what happens to the T lymphocytes after infiltrating the CNS is not definitely solved. From the concept of immune surveillance, one would postulate that T lymphocytes should be able to again leave the CNS, return to peripheral lymphatic organs and restart their patrol through the body to seek for further potential exposure to their antigen. Although there are some data supporting such a “lucky” end for CNS-infiltrating T lymphocytes,125-128 the majority of recent publications favor the brain as a kind of “dead end”, for at least most of the T lymphocytes, from which they can not escape. Schmied et al129 were the first who were able to attribute the apoptosis observed in brains of EAE in previous studies 130,131 to degenerating lymphocytes. Meanwhile, these results were corroborated by several other studies.132-134 In spite of many EAE studies, the knowledge about the exact inflammatory processes and T-lymphocyte biology in EAE is still incomplete. However, what could be learned from a T lymphocyte-driven gene therapeutic approach to CNS diseases? 1. Brain-specific T lymphocyte blasts applied systemically are able to migrate into the CNS to areas where their antigen is expressed. Irrelevant lymphocytes also transmigrate the BBB. Their stay in the CNS is, however, of very short duration. Therefore, transgene transport of lymphocytes into the brain should be possible, provided the lymphocytes are activated. 2. Migration of brain-specific lymphocytes is determined by the antigen specificity of the lymphocyte and the state of the target tissue. Damaged brain tissue attracts brain-specific lymphocytes. This could have important implications for targeting of transgene-carrying T lymphocytes. Searches for antigens specific for certain brain regions, and establishment of lymphocytes against these antigens, could possibly be avoided. 3. Brain-specific T lymphocytes capable of infiltrating the CNS do not necessarily evoke clinical disease, as demonstrated by MOG- and S100β-specific lymphocyte lines. These non-pathogenic T lymphocytes are ideal candidates for therapeutic use. Indeed, a beneficial effect of gene manipulated, S100β-specific lymphocytes could be demonstrated. In an adoptive transfer EAE study of the Lewis rat mediated by MBP-specific T lymphocytes, coinjection with S100β-specific lymphocytes expressing IL-4 led to a distinct amelioration of the disease (Flügel A, Willem M, Ritter Th, Klinkert WEF, and Wekerle H, unpublished data). 4. After CNS infiltration and a certain span of activity, brain-specific T-lymphocyte lines most likely perish in their target tissue. Apoptosis of brain-specific lymphocytes after infiltration and release of transgene products would greatly support the therapeutic usage of such lymphocytes, providing a local and time limited, focused therapeutic effect.

Gene Delivery Techniques for T Lymphocytes Introduction of Gene Transfer Systems Several gene delivery techniques for eukaryontic cells are available. Techniques used for therapy require a high standard of transfer efficiency, reliable transgene expression, and safety for the host.135 Choice of a certain technique depends on the susceptibility of the target cell type for the respective transfer technique, the method chosen for delivery, and the envisioned duration of transgene expression. Principally, either non-viral or viral gene delivery techniques can be applied. The general properties of gene transfer systems, with their respective positive and negative aspects, are listed in Table 4.1.

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T-Cell Autoimmunity and Multiple Sclerosis

Non-viral gene delivery techniques136 allow DNA transfer without major safety constraints. These methods include calcium phosphate precipitation, electroporation, cationic lipids, liposomes, virosomes, ligand-mediated gene delivery, polymers, or direct insertion of pure DNA into cells or tissues via (micro)injection. Successful transfer requires uptake of the transgene and bypass of cellular degradation processes. Stable transgene expression requires entry of the transgene DNA into the nucleus and integration into the host genome. The size of transferred DNA is not as tightly limited as in viral systems. Therefore, endogenous gene-specific regulatory sequences can more easily be included in the transgene construct. Targeting of DNA is achieved via local application. Very encouraging results, with long lasting in vivo expression of transgenes after injection of naked plasmid DNA into muscle or neural tissue, have been reported.137-142 In general, however, efficiency of non -viral gene transfer compared to viral techniques is low, and stable gene expression is hard to achieve. Thus, most gene therapeutic strategies engage viral gene transfer techniques. Viruses infect susceptible cells very efficiently. On the other hand, they often exert a cytotoxic effect on the host cells and may target the infected cell through the immune system. Therefore, viral gene transfer systems usually involve attenuated viruses. Parts of the viral genome are removed in order to avoid viral pathogenicity and to provide space for therapeutic gene sequences, since viruses from nature are extremely economically structured particles. Genes necessary for viral infectibility are provided in trans by so-called complementation or packaging cells. These cells usually are fibroblasts genetically designed to express the requisite viral proteins. Some viruses, such as retroviruses143,144 or adeno-associated viruses,145 guarantee a stable transformation of the target cell via integration into the host genome. In most gene therapeutic studies a Moloney murine leukemia virus (Mo MuLV)-based retroviral system is applied.143,144,146 The main advantages of the retroviral system are the effective and stable gene transfer into a broad spectrum of replicating cells, and low immunogenicity due to the loss of practically all endogenous viral genes. Furthermore, retroviruses, followed by the adenoviruses, represent the best characterized gene transfer vehicles, with practical experience in many in vitro applications and therapeutical in vivo studies. However, the retroviral approach also contains negative aspects: 1. Stable gene transfer is achieved exclusively in replicating cells; 2. The packaging capacity of the virus allows a transgene size limited to maximally 8 kb; and 3. There exists the theoretical possibility of damage to important host genes and insertional mutagenesis due to random integration of the viral vector into the host genome, although until now clinical studies have not found adverse side effects pointing in this direction. Adenovirus gene transfer systems usually are derived from the human pathogenic adenovirus type 2 or 5.145,147 Attenuation is achieved by deletion in an adenoviral early gene locus (usually E3, 4). Similarly to the retroviral system, missing genes necessary for adenoviral infectivity and virally mediated transgene expression are provided in trans by complementation or packaging lines. Due to the higher stability of the viruses, viral titers after harvesting of adenovirus packaging lines and concentration reach maximally 1011 colony forming units (cfu) compared to 106-108 cfu of retroviral preparation. Together with the high infectivity of the virus (at least for certain cell types such as epithelial cells), the impressive titer enables a very effective gene transfer to dividing as well as quiescent target cells. Adenoviral DNA does not integrate into the host genome. Transgene expression is therefore transient. The adenovirus gene transfer vector still contains many viral genes, causing a high immunogenicity.148,149 Strategies have been developed to overcome immunogenicity, which is most likely responsible for rapid clearance of adenoviruses by the host-derived

45

GeneticEngineeringofBrain-SpecificTCellsforTreatmentofDiseasesintheCNS

Table 4.1. Comparison of the most commonly used gene transfer techniques in clinical gene therapy Non-Viral

Retroviral

Adenoviral

Adeno-Associated

Effectivity

+

+

++

++

Safety

++

-+

+-

+

no limit

7kb

7-8kb

4-5kb

Infectibility of quiescent cells

-+

-

+

+

Infectibility of replicating cells

-+

+

+

+

Immunogenicity

-

-+

++

+

Handling

++

+

+

+

Expression

transient

stable

transient

stable

Cloning capacity

immune system, through suppression of the host immune response150-154 or through reduction of immunogenicity by further removal of viral genes.155 The effectiveness and applicability of such strategies in vivo waits for further approval. Parvovirus-derived adeno-associated viruses (AAVs)145 are dependent on, as the name implicates, viral components of adenoviral origin for replication. The RNA virus is able to integrate into the genome of the host. Using wild type AAV vectors, integration is targeted to the long arm of human chromosome 19q13.4. Packaging capacity is 4.7 kb, less in comparison to retroviral and adenoviral (both 7-8 kb) vectors.145 Until now experience in gene therapeutic approaches of this gene transfer technique has been very limited. The same is true for the Herpes virus. Applicability of this DNA virus, which is interesting in CNS gene therapy due to its neurotropism and persistence in neurons, is currently being tested.49-52

Gene Transfer Into T Lymphocytes

Gene transfer into lymphocytes has been studied for several diseases.156 In congenital disorders such as adenosine deaminase (ADA) or mucopolysaccharidosis type II, deficiency strategies aim to replace the inborn metabolic defect.157-162 In neoplastic diseases, genetically-engineered tumor infiltrating lymphocytes are established which are resistant against shutdown via the tumor,163-166 or hematopoetic stem cells are tolerized against chemotherapeutics via introduction of, for example, a multidrug resistance gene.167-169 In HIV, CD4 -positive lymphocytes are designed to render them resistant against infection.170-174 Recently, in autoimmune diseases, antigen -specific lymphocytes have been used as carriers for immune deviating factors.175-179 The appropriate gene delivery technique for lymphocytes depends strongly on the susceptibility and respective application.

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Lymphocytes are quite resistant to gene transfer in general. This is due to several biological properties of this cell type. Although lymphocytes can easily be isolated from peripheral blood, cultivation and propagation for longer periods remain difficult. Non-viral gene delivery techniques usually are very inefficient. This might be due to apoptosis induction.180 Retroviral gene delivery is the most often and successfully used transfer technique for lymphocytes.146,181-186 Other techniques applied in gene transfer into lymphocytes include AAVs,145,187 ballistic systems,188 and anti-CD3 receptor antibody-mediated189 and polybrene/ DMSO-assisted177,190,191 gene uptake. The applicability of adenoviral mediated gene transfer in lymphocytes is inconsistently discussed.192-196 Although promising transfer rates into lymphocytes were shown, there is practically no in vivo application of this technique. Initial studies of retroviral gene transfer into primary lymphocytes report transduction rates of around 1-10%.156,183,197-200 Cocultivation with packaging lines increased the rate to 15-40%.183,201 More recent studies developed optimized transduction conditions with transfer rates of up to 50%.146,185 Basically, the longer the in vitro cultivation can be performed, the higher is the probability of gaining a high percentage of stably transduced lymphocytes, due to the possibility of positively selecting-transgene carrying lymphocytes. Cloning greatly facilitates gene engineering of antigen-specific T lymphocytes, because longer in vitro culturing, including several restimulations, is necessary. For this approach we developed in the Lewis rat a gene delivery technique combining limiting dilution cultivation with retroviral gene delivery. Simultaneous cocultivation of T lymphocytes with packaging lines resulted in many different.206 clonoids of stably transduced lymphocytes, which could easily be amplified in one to two restimulations up to several hundred millions of lymphocytes. Separation of packaging cells is not necessary, due to the very effective cytotoxicity exerted by the T lymphocytes. The heterogeneity of the isolated lines is much lower compared to transduced lines obtained after transduction of bulk cultures. Via this technique, we generated MBP-specific GFP (GFP) (Fig. 4.2) or neurotrophic factorexpressing lymphocytes which showed stable transgene expression in vitro during an observation period of over six restimulations. They did not differ from their wild type colleagues concerning surface markers and functional criteria. Furthermore, the transgene could be traced in the brain, associated to infiltrating T lymphocytes, via in situ hybridization (Fig. 4.1). Therefore, in our opinion this technique represents an ideal delivery system for rapid establishment of antigen-specific T lymphocytes for in vivo studies.

Therapeutical Achievements with Genetically Engineered T Cells Gene delivery studies for T lymphocytes aim mainly in the following directions: 1. Modification of functional effects exerted by the lymphocytes in order to enhance or suppress lymphocyte-mediated inflammatory processes;202 2. Introduction of marker genes to trace lymphocyte traffic;197,203,204 3. Transfer of metabolic genes in order to reconstitute a genetic loss;157-162 and 4. Provision of lymphocytes with genes prohibiting infection by HIV.170-174 Recent studies have extended these approaches in so far as antigen-specific lymphocytes being employed as specific carriers of genes that are not necessarily immunologically derived, but which bring a potential therapeutic benefit to the diseased target tissue. The concept of engineering antigen-specific T lymphocytes was first implemented with tumor-infiltrating lymphocytes (TILs).165 TNF-α was retrovirally transferred to cytotoxic T lymphocytes to render them more aggressive against tumors.166 Other strategies to increase the cytolytic activity of the TILs against the tumor include introduction of a chimeric zeta chain/anti-tumor antigen antibody construct,163 or of the tyrosine kinase Fyn.164 Until now the attractive oncological approach has been limited by the ability of tumors to escape TIL attack through rapid growth and functional inactivation of the TILs.

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Fig. 4.2. Retrovirally transduced MBP-specific rat T lymphocytes. (a) MBP-specific rat T lymphocytes viewed in phase contrast microscopy. (b) Green fluorescence protein (GFP) transgene expression of the upper cells visualized by fluorescence microscopic analysis (emission data were obtained with excitation by UV light, 360-400 nm). (c) Anti-CD4 (W3/25) staining (phycoerythrin-labeled secondary antibody) of the upper cells.

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The first study exploiting site-specific delivery of therapeutic transgenes by T lymphocytes in the nervous system was performed by Kramer et al in a Lewis rat model of peripheral neuritis.205 They could demonstrate that a transgene can be delivered specifically to their target tissue by genetically engineered, antigen-specific T lymphocytes and produce a beneficial effect. In their study, NGF was released in the peripheral nervous system (PNS) by T lymphocytes specific for peripheral myelin P2. NGF release in the target tissue exerted a positive effect on neuronal myelin sheath integrity, and modified dramatically the histological picture of the experimental auto-allergic neuritis (EAN) lesions. Clinically, the course of EAN was also ameliorated. Recently, two EAE studies were published which applied a similar approach with therapeutic transgene delivery via antigen-specific T lymphocytes in animal models of chronic relapsing experimental allergic encephalomyelitis (CREAE). In the first study, MBP-specific hybridoma cells were retrovirally transduced with an IL-4 construct.179 These modified T lymphocytes were able to delay the onset and reduce the severity of actively induced EAE in (SJL/J x PL/J) F1 mice when injected before the onset of disease. In the second study,177 lymph node cells of (SWR x SJL) F1 mice immunized with proteolipid protein (PLP) peptide p139-151 were transfected by the DMSO/polybrene technique with an IL-10 construct. In a similar way to that of the previous study, these transfected T lymphocytes were injected into animals with induced active EAE. In addition to the previous study, however, the authors could also show an ameliorating effect of IL-10-transfected lymphocytes on already established disease by transferring the lymphocytes after onset of disease. Meanwhile, the application of T lymphocytes as therapeutic gene carriers has widened to disease models of peripheral organs such as arthritis175,176 and diabetes.178 Here also, inflammation-modifying cytokines or cytokine inhibitors have been transduced into joint-or pancreatic island-specific lymphocytes. These lymphocytes suppressed or ameliorated the respective disease.

Conclusion The pace of current advances strongly suggest that gene therapy will soon become an important tool for treatment of CNS diseases. In addition to the general problems of gene therapy, e.g., safe and effective gene delivery techniques together with adequately regulated gene expression, the complexity and importance of the CNS requires particularly clever strategies to render this elegant concept a useful tool. In this respect, the approach of using T lymphocytes as carriers for therapeutic agents represents an intriguing and, in part, already proven strategy. The first steps in this direction using different well established animal models have convincingly demonstrated very positive effects. No pharmacological design is likely to surpass the inborn biological specificity of the immune recognition machinery linked to a cell for which there exist almost no borders within the organism. Even for the brain, where T lymphocytes until recently were mainly known as harmful agents, this strategy has developed from a pure idea to an increasingly realistic therapeutic course. Progress toward answering the serious questions concerning, for example, efficiency and safety of T lymphocyte delivery techniques, T-lymphocyte migration, pathogenicity and activity and further fate of T lymphocytes after infiltration of the CNS, may allow a rational and powerful use of this tool against CNS diseases.

Acknowledgments We thank Dr. M. Willem for substantial support in the establishment of retroviral vectors, Prof. Dr. H. Lassmann, Dr. R. Birnbacher and I. Haarmann for the histological analysis and Dr. D. R. Johnson for critical reading of the manuscript.

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122. Maehlen J, Olsson T, Zachau A et al. Local enhancement of major histocompatibility complex (MHC) class I and class II expression and cell infiltrationin experimental allergic encephalomyelitis around axotomized motor neurons.J Neuroimmunol 1989; 23:125-132. 123. Hickey WF. Migration of hematogenous cells through the blood-brain barrier and the initiation of CNS inflammation. Brain Pathol 1991; 1:97-106. 124. Konno H, Yamamoto T, Suzuki H et al. Targeting of adoptively transferred experimental allergic encephalomyelitis lesion at the site of Wallerian degeneration. Acta Neuropathol 1990; 80:521-526. 125. Cserr HF, Knopf PM. Cervical lymphatics, the blood-brain barrier and the immunoreactivity of the brain: A new view. Immunol Today 1992; 13:507-512. 126. Gordon LB, Knopf PM, Cserr HF. Ovalbumin is more immunogenic when introduced into brain or cerebrospinal fluid than into extracerebral sites. J Neuroimmunol 1992; 40:81-88. 127. Krisch B, Leonhardt H, Oksche A. Compartments and perivascular arrangement of the meninges covering the cerebral cortex of the rat. Cell Tiss Res 1984; 238:459-474. 128. Schönrich G, Kalinke U, Momburg F et al. Down-regulation of T-cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction. Cell 1991; 65:293-304. 129. Schmied M, Breitschopf H, Gold R et al. Apoptosis of T lymphocytes-A mechanism to control inflammation in the brain. Am J Pathol 1993; 143:446-452. 130. Pender MP, McCombe PA, Yoong G et al. Apoptosis of αβ T lymphocytes in the nervous system in experimental autoimmune encephalomyelitis: Its possible implications for recovery and acquired tolerance. J Autoimmun 1992; 5:401-410. 131. Pender MP, Nguyen KB, McCombe PA et al. Apoptosis in the nervous system in experimental allergic encephalomyelitis. J Neurol Sci 1991; 104:81-87. 132. McCombe PA, Nickson I, Tabi Z et al. Apoptosis of Vβ8.2+ T lymphocytes in the spinal cord during recovery from experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein. J Neurol Sci 1996; 139:1-6. 133. Smith T, Schmied M, Hewson AK et al. Apoptosis of T cells and macrophages in the central nervous system of intact and adrenalectomized Lewis rats during experimental allergic encephalomyelitis. J Autoimmun 1996; 9:167-174. 134. Zettl UK, Gold R, Toyka KV et al. In situ demonstration of T-cell activation and elimination in the peripheral nervous system during experimental autoimmune neuritis in the Lewis rat. Acta Neuropathol 1996; 91:360-367. 135. Smith KT, Shepherd AJ, Boyd JE et al. Gene delivery systems for use in gene therapy: An overview of quality assurance and safety issues. Gene Ther 1996; 3:190-200. 136. Ledley FD. Non-viral gene therapy. Curr Op Biotechnology 1994; 5:626-636. 137. Danko I, Wolff JA. Direct gene transfer into muscle. Vaccine 1994; 12:1499-1502. 138. Davis HL, Whalen RG, Demeneix BA. Direct gene transfer into skeletal muscle in vivo: Factors affecting efficiency of transfer and stability of expression. Hum Gene Ther 1993; 4:151-159. 139. Davis HL, Demeneix BA, Quantin B et al. Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. Hum Gene Ther 1993; 4:733-740. 140. Hartikka J, Sawdey M, Cornefert-Jensen F et al. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum Gene Ther 1996; 7:1205-1217. 141. Manthorpe M, Cornefert-Jensen F, Hartikka J et al. Gene therapy by intramuscular injection of plasmid DNA: Studies on firefly luciferase gene expression in mice. Hum Gene Ther 1993; 4:419-431. 142. Partridge TA, Davies KE. Myoblast-based gene therapies. Br Med B 1995; 51:123-137. 143. Gordon EM, Anderson WF. Gene therapy using retroviral vectors. Curr Op Biotech 1994; 5:611-616. 144. Vile RG, Russell SJ. Retroviruses as vectors. Br Med B 1995; 51:12-30. 145. Kremer EJ, Perricaudet M. Adenovirus and adeno-associated virus mediated gene transfer. Br Med B 1995; 51:31-44.

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146. Bunnell BA, Muul LM, Donahue RE et al. High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes. Proc Natl Acad Sci USA 1995; 92:7739-7743. 147. Bramson JL, Graham FL, Gauldie J. The use of adenoviral vectors for gene therapy and gene transfer in vivo. Curr Op Biotech 1995; 6:590-595. 148. Kajiwara K, Byrnes AP, Charlton HM et al. Immune responses to adenoviral vectors during gene transfer in the brain. Hum Gene Ther 1997; 8:253-265. 149. Wood MJ, Charlton HM, Wood KJ et al. Immune responses to adenovirus vectors in the nervous system. Trends Neur 1996; 19:497-501. 150. DeMatteo RP, Markmann JF, Kozarsky KF et al. Prolongation of adenoviral transgene expression in mouse liver by T lymphocyte subset depletion. Gene Ther 1996; 3:4-12. 151. Kass-Eisler A, Leinwand L, Gall J et al. Circumventing the immune response to adenovirus-mediated gene therapy. Gene Ther 1996; 3:154-162. 152. Kolls JK, Lei D, Odom G et al. Use of transient CD4 lymphocyte depletion to prolong transgene expression of E1-deleted adenoviral vectors. Hum Gene Ther 1996; 7:489-497. 153. Kuzmin AI, Finegold MJ, Eisensmith RC. Macrophage depletion increases the safety, efficacy and persistence of adenovirus-mediated gene transfer in vivo. Gene Ther 1997; 4:309-316. 154. Scaria A, St GJA, Gregory RJ et al. Antibody to CD40 ligand inhibits both humoral and cellular immune responses to adenoviral vectors and facilitates repeated administration to mouse airway. Gene Ther 1997; 4:611-617. 155. Amalfitano A, Chamberlain JS. Isolation and characterization of packaging cell lines that coexpress the adenovirus E1, DNA polymerase, and preterminal proteins: Implications for gene therapy. Gene Ther 1997; 4:258-263. 156. Culver K, Cornetta K, Morgan R et al. Lymphocytes as cellular vehicles for gene therapy in mouse and man. Proc Natl Acad Sci USA 1991; 88:3155-3159. 157. Bordignon C, Notarangelo LD, Nobili N et al. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients. Science 1995; 270:470-475. 158. Braun SE, Pan D, Aronovich EL et al. Preclinical studies of lymphocyte gene therapy for mild Hunter syndrome (mucopolysaccharidosis type II). Hum Gene Ther 1996; 7:283-290. 159. Culver KW, Osborne WR, Miller AD et al. Correction of ADA deficiency in human T lymphocytes using retroviral-mediated gene transfer. Transplan P 1991; 23:170-171. 160. Ferrari G, Rossini S, Nobili N et al. Transfer of the ADA gene into human ADA-deficient T lymphocytes reconstitutes specific immune functions. Blood 1992; 80:1120-1124. 161. Hoogerbrugge PM, von BVW, Kaptein LC et al. Gene therapy for adenosine deaminase deficiency. Br Med B 1995; 51:72-81. 162. Williams DA, Orkin SH, Mulligan RC. Retrovirus-mediated transfer of human adenosine deaminase gene sequences into cells in culture and into murine hematopoietic cells in vivo. Proc Natl Acad Sci USA 1986; 83:2566-2570. 163. Altenschmidt U, Moritz D, Groner B. Specific cytotoxic T-lymphocytes in gene therapy. J Mol Med 1997; 75:259-266. 164. Fujita K, Ikarashi H, Takakuwa K et al. Enhancement of T-cell receptor signaling of tumor-infiltrating lymphocytes by retrovirally mediated fyn gene transduction. Japanese J Can Res 1994; 85:1073-1079. 165. Hwu P, Rosenberg SA. The use of gene-modified tumor-infiltrating lymphocytes for cancertherapy. Ann NY ACAD 1994; 716:188-97 [discussion#197-203]. 166. Hwu P, Yannelli J, Kriegler M et al. Functional and molecular characterization of tumor-infiltrating lymphocytes transduced with tumor necrosis factor-α cDNA for the gene therapy of cancer in humans. J Immunol 1993; 150:4104-4115. 167. Eckert HG, Stockschlader M, Just U et al. High-dose multidrug resistance in primary human hematopoietic progenitor cells transduced with optimized retroviral vectors. Blood 1996; 88:3407-3415. 168. Hesdorffer C, Antman K, Bank A et al. Human MDR gene transfer in patients with advanced cancer. Hum Gene Ther 1994; 5:1151-1160.

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169. Richardson C, Bank A. Preselection of transduced murine hematopoietic stem cell populations leads to increased long-term stability and expression of the human multiple drug resistance gene. Blood 1995; 86:2579-2589. 170. Chen JD, Yang Q, Yang AG et al. Intra- and extracellular immunization against HIV-1 infection with lymphocytes transduced with an AAV vector expressing a human anti-gp120 antibody. Hum Gene Ther 1996; 7:1515-1525. 171. Leavitt MC, Yu M, Yamada O et al. Transfer of an anti-HIV-1 ribozyme gene into primary human lymphocytes. Hum Gene Ther 1994; 5:1115-1120. 172. Lever AM. Gene therapy for HIV infection. v` 1995; 51:149-166. 173. Plavec I, Agarwal M, Ho KE et al. High transdominant RevM10 protein levels are required to inhibit HIV-1 replication in cell lines and primary T cells: Implication for gene therapy of AIDS. Gene Ther 1997; 4:128-139. 174. Weerasinghe M, Liem SE, Asad S et al. Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme. J Virol 1991; 65:5531-5534. 175. Chernajovsky Y, Adams G, Triantaphyllopoulos K et al. Pathogenic lymphoid cells engineered to express TGF beta 1 ameliorate disease in a collagen-induced arthritis model. Gene Ther 1997; 4:553-559. 176. Chernajovsky Y, Adams G, Podhajcer OL et al. Inhibition of transfer of collagen-induced arthritis into SCID mice by ex vivo infection of spleen cells with retroviruses expressing soluble tumor necrosis factor receptor. Gene Ther 1995; 2:731-735. 177. Mathisen PM, Yu M, Johnson JM et al. Treatment of experimental autoimmune encephalomyelitis with genetically modified memory T cells. J Exp Med 1997; 186:159-164. 178. Moritani M, Yoshimoto K, Ii S et al. Prevention of adoptively transferred diabetes in nonobese diabetic mice with IL-10-transduced islet-specific Th1 lymphocytes. A gene therapy model for autoimmune diabetes. J Clin Invest 1996; 98:1851-1859. 179. Shaw MK, Lorens JB, Dhawan A et al. Local delivery of interleukin 4 by retrovirus-transduced T lymphocytes ameliorates experimental autoimmune encephalomyelitis. J Exp Med 1997; 185:1711-1714. 180. Ebert O, Finke S, Salahi A et al. Lymphocyte apoptosis: Induction by gene transfer techniques. Gene Ther 1997; 4:296-302. 181. Imbert AM, Costello R, Imbert J et al. Highly efficient retroviral gene transfer into human primary T lymphocytes derived from peripheral blood. Canc Gene Ther 1994; 1:259-265. 182. Lam JS, Reeves ME, Cowherd R et al. Improved gene transfer into human lymphocytes using retroviruses with the gibbon ape leukemia virus envelope. Hum Gene Ther 1996; 7:1415-1422. 183. Mavilio F, Ferrari G, Rossini S et al. Peripheral blood lymphocytes as target cells of retroviral vector -mediated gene transfer. Blood 1994; 83:1988-1997. 184. Medin JA, Karlsson S. Viral vectors for gene therapy of hematopoietic cells. Immunotech 1997; 3:3-19. 185. Rudoll T, Phillips K, Lee SW et al. High-efficiency retroviral vector mediated gene transfer into human peripheral blood CD4+ T lymphocytes. Gene Ther 1996; 3:695-705. 186. Sharma S, Cantwell M, Kipps TJ et al. Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector. Proc Natl Acad Sci USA 1996; 93:11842-11847. 187. Muro-Cacho CA, Samulski RJ, Kaplan D. Gene transfer in human lymphocytes using a vector based on adeno-associated virus. J Immunother 1992; 11:231-237. 188. Burkholder JK, Decker J, Yang NS. Rapid transgene expression in lymphocyte and macrophage primary cultures after particle bombardment-mediated gene transfer. J Immunol Meth 1993; 165:149-156. 189. Buschle M, Cotten M, Kirlappos H et al. Receptor-mediated gene transfer into human T lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex. Hum Gene Ther 1995; 6:753-761.

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190. Aubin RA, Weinfeld M, Taghavi M et al. Highly effective delivery of foreign DNA to adherent cells via polybrene/DMSO-assisted gene transfer. Meth Mol Biol 1997; 62:319-342. 191. Chisholm O, Symonds G. Transfection of myeloid cell lines using polybrene/DMSO. Nucl Acid R 1988; 16:2352. 192. Horvath J, Weber JM. Nonpermissivity of human peripheral blood lymphocytes to adenovirus type 2 infection. J Virol 1988; 62:341-345. 193. Horvath J, Kulcsar G, Ugryumov JP et al. Effect of adenovirus infection on human peripheral lymphocytes. Acta Microbiol Hung 1983; 30:203-209. 194. Lavery D, Fu SM, Lufkin T et al. Productive infection of cultured human lymphoid cells by adenovirus. J Virol 1987; 61:1466-1472. 195. Mentel R, Dopping G, Wegner U et al. Adenovirus-receptor interaction with human lymphocytes. J Med Virol 1997; 51:252-257. 196. Silver L, Anderson CW. Interaction of human adenovirus serotype 2 with human lymphoid cells. Virology 1988; 165:377-387. 197. Kasid A, Morecki S, Aebersold P et al. Human gene transfer: Characterization of human tumor -infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man. Proc Natl Acad Sci USA 1990; 87:473-477. 198. Morgan RA, Baler-Bitterlich G, Ragheb JA et al. Further evaluation of soluble CD4 as an anti-HIV type 1 gene therapy: Demonstration of protection of primary human peripheral blood lymphocytes from infection by HIV type 1. AIDS Restt 1994; 10:1507-1515. 199. Wilson JM, Ping AJ, Krauss JC et al. Correction of CD18-deficient lymphocytes by retrovirus-mediated gene transfer. Science 1990; 248:1413-1416. 200. Woffendin C, Yang ZY, Udaykumar et al. Nonviral and viral delivery of a human immunodeficiency virus protective gene into primary human T cells. Proc Natl Acad Sci USA 1994; 91:11581-11585. 201. Finer MH, Dull TJ, Qin L et al. kat: A high-efficiency retroviral transduction system for primary human T lymphocytes. Blood 1994; 83:43-50. 202. Doughty LA, Patrene KD, Evans CH et al. Constitutive systemic expression of IL-1Ra or soluble TNF receptor by genetically modified hematopoietic cells suppresses LPS induction of IL-6 and IL-10. Gene Ther 1997; 4:252-257. 203. Cai Q, Rubin JT, Lotze MT. Genetically marking human cells—Results of the first clinical gene transfer studies. Canc Gene Ther 1995; 2:125-136. 204. Merrouche Y, Negrier S, Bain C et al. Clinical application of retroviral gene transfer in oncology: Results of a French study with tumor-infiltrating lymphocytes transduced with the gene of resistance to neomycin. J Clin Onc 1995; 13:410-418. 205. Kramer R, Zhang Y, Gehrmann J et al. Gene transfer through the blood-nerve barrier: Nerve growth factor engineered neuritogenic T lymphocytes attenuate experimental autoimmune neuritis. Nature Med 1995; 1:1162-1166. 206. Flügel A, William M, Berkowicz T, Wekerle H. Gene transfer into CD4+ T lymphocytes: Green fluorescent protein-engineered, encephalitogenic T cells illuminate brain autoimmune responses. Nat Med 1999; 5: 843-847.

CHAPTER 5

T Cells and Cytokines Enrico Maggi

A

ll infectious agents, although capable of eliciting an immunological response, have evolved numerous ways of evading the consequences of immune attack. In humans and other higher organisms, the mechanisms used by the immune system to defend against infection have been continuously shaped and refined. Viral antigens are synthesized within infected cells and presented on the surface of the cells in association with class I major histocompatibility complex (MHC) molecules, leading to the stimulation of CD8+ class I MHC-restricted cytotoxic T lymphocytes. The majority of microbial antigens, however, are endocytosed by APC, processed and presented preferentially in association with class II MHC molecules to CD4+ class II MHC-restricted T helper (Th) cells. CD4+ T cells collaborate with B lymphocytes for the production of antibodies which can challenge microbes living outside cells or neutralize their soluble toxic products (exotoxins). This branch of the specific Th cell-mediated immune response is known as ‘humoral immunity.’ Other microbes, such as mycobacteria, can survive and multiply within macrophages. CD4+ Th cells, once activated by mycobacterial soluble antigens, can activate macrophages, whose products (oxygen intermediates, reactive nitrogen intermediates, and TNF-α) then lead to destruction of the microbe. This branch of the specific Th cell-mediated immune response is known as ‘cell-mediated’ immunity. The occurrence of this response is revealed by an indurative skin reaction, commonly defined as delayed type hypersensitivity (DTH). Using chemically modified Salmonella flagellin as an antigen, Parish and Liew1 showed that antibody production and DTH could be reciprocally expressed (“split tolerance” or “immune deviation”). Very low concentrations of antigen yielded responses in which DTH reactions were dominant; when the antigen dose was increased, antibody responses increased and DTH responses diminished. Finally, at high doses of antigen, DTH again dominated. The mechanisms by which CD4+ Th cells may be responsible for this dichotomy remained unclear until 1986, when Mosmann and his coworkers2 provided evidence that repeated stimulation with given antigens results in the development of restricted patterns of lymphokine production (type 1 or Th1 and type 2 or Th2) accounting for effector reactions characterized by prevalent DTH or antibody response, respectively.

Type 1 and Type 2 T Helper Cells: Two Polarized Forms of the Specific Immune Response In mouse, Th1 cells produce IL-2, IFN-γ and TNF-β, and induce the production of IgG2a opsonizing and complement-fixing antibodies, macrophage activation, specific and antibody-dependent cell cytotoxicity and DTH.3 Thus, Th1 cells can be considered as responsible for the phagocyte-dependent host response.4,5 By contrast, Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 and provide optimal help for humoral immune T-Cell Autoimmunity and Multiple Sclerosis, edited by Marco Londei. ©1999 R.G. Landes Company.

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responses, especially at the mucosal level, through recruitment and growth of mast cells and eosinophils and differentiation to IgA synthesis. Some Th2-derived cytokines, such as IL-4, IL-10 and IL-13, inhibit several macrophage functions, thus leading to a phagocyteindependent host response.4,5 T cells expressing cytokines of both patterns have been designated Th0; they usually mediate intermediate effects depending upon the ratio of lymphokines produced and the nature of the responding cells.6-8 T cells producing high amounts of transforming growth factor (TGF)-β have been termed Th3,9 and additional patterns have been described among long term clones.10 Evidence for the existence of Th1 and Th2 cells in humans was provided by establishing CD4+ T-cell clones specific for peculiar antigens. CD4+ T lymphocytes that produce IL-4, but low or no IFN-γ, were found to occur in high frequencies in the allergen-specific repertoires of atopic donors.11,12 In addition, T-cell clones specific for Toxocara canis excretory/secretory (TES) antigens exhibited a Th2-like profile of cytokine secretion, whereas the great majority of PPD-specific T-cell clones derived from the same donors showed a clear-cut Th1 profile.13,14 Human T-cell clones exhibit a less restricted cytokine profile than murine T cells. IL-2, IL-6, IL-10, and IL-13 tend to segregate less clearly among human CD4+ subsets than in the mouse.15,16 Moreover, human Th1 and Th2 cells clearly differ for their cytolytic potential and mode of help for B-cell antibody synthesis. In fact, Th2 clones, which usually had no cytolytic potential, induced IgM, IgG, IgA, and IgE synthesis by autologous B cells in the presence of the specific antigen, with a response proportional to the number of Th2 cells added to B cells.17 In contrast, Th1 clones (of which the majority were cytolytic) provided B-cell help for IgM, IgG, IgA (but not IgE) synthesis at low T cell:B cell ratios. At T cell:B cell ratios higher than 1:1, there was a decline in B-cell help, related to their lytic activity against autologous antigen-presenting B cell targets.17 This may represent an important mechanism for the downregulation of antibody responses in vivo.14,17 Finally, human Th1 and Th2 cells exhibit different ability to activate monocytic cells.18 Th1, but not Th2, cells can help tissue factor (TF) production and procoagulant activity by monocytes. Indeed, both cell to cell contact with activated T cells and Th1 cytokines, in particular IFN-γ, were required for optimal TF synthesis, whereas Th2-derived cytokines (IL-4, IL-10 and IL-13) were inhibitory.18 Regardless of whether the variation in T-cell cytokine synthesis represents a continuum or discrete subsets, there is no doubt that many T-cell clones and in vivo immune responses show a dramatic Th1 or Th2 polarization. Thus, although Th1 and Th2 cells are certainly not the result of a preexisting functional dichotomy of CD4+ T cells, they can be regarded as polarized forms of the specific immune response that frequently occur under the combined action of genetic and environmental conditions. We currently speak about CD4+ T cells producing Th1 or Th2 type cytokines without considering the other set of cytokines. Bearing this in mind, the Th1/Th2 paradigm may provide an useful model for understanding the pathogenesis of several pathophysiologic conditions and possibly for the development for novel immunotherapeutic strategies.

Surface Molecules Preferentially Associated with Human TH1 or TH2 Cells Some molecules preferentially associated with the Th1 or the Th2 phenotype have also been described in human T cells. When expressed on T cells, CD26, an ectopeptidase present in a wide range of tissues, has been reported to correlate with Th1-like reactions in granulomatous diseases.19 In contrast, L-selectin (CD62L)-positive memory CD4+ T cells produce mainly Th2 type cytokines.20 Moreover, it has been shown that detection of surface membrane IFN-γ (IFN-γ is expressed on the cell surface before secretion) may be a useful

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marker for Th1 cells in both mouse and man.21 Recently the β2 chain of IL-12R has been associated with the Th1 profile of cytokine secretion.22 Other molecules that have been shown to be preferentially associated with human Th2 and Th1 clones are CD30 and lymphocyte activation gene (LAG)-3, respectively. CD30, a member of the TNF receptor family,47 was found to be consistently expressed, and its soluble form (sCD30) released, by Th2 and Th0 clones, whereas Th1 clones usually showed poor and transient or no CD30 expression.23 CD30 expression by Th0/Th2 clones was not constitutive, but required T-cell activation and the presence in culture of IL-2. Additional in vitro and in vivo observations support the view that CD30 expression and production of Th2 cytokines are significantly associated. First, costimulation of antigen-specific Th0 or Th2, but not Th1, clones with an agonistic anti-CD30 monoclonal antibody resulted in increased proliferation and cytokine production.23 Accordingly, CD30 ligation induced activation of NFκB transcription factors in Th0 and Th2, but not in Th1, clones.24 The preferential association of CD30 with T cells producing Th2 cytokines was also supported by some findings in vivo. Very small numbers of CD4+CD30+ T cells were detected in the blood of atopic pollen-sensitive donors during the seasonal exposure to grass pollens. When sorted, these cells developed into T-cell lines able to proliferate in response to grass allergens and to produce IL-4 and IL-5, but not IFN-γ and TNF-β. In the lymph node and skin biopsies of three children with Omenn’s syndrome (OS) and in the lymph node and peripheral blood of one child with Omenn’s-like syndrome a higher proportion of CD4+CD30+T cells was found.25 When circulating CD30+ T cells from this child were sorted from the CD30– T cells and cloned, most CD30+ T cells developed intoTh0/Th2 clones, whereas the majority of CD30– T cells differentiated into Th1-like clones. More recently, high proportions of CD30+ (CD4+) T cells were found in the skin biopsy specimens of patients with systemic sclerosis (SSc), another Th2-dominated disorder. It is of note, however, that neither CD30 mRNA expression nor accumulation of CD30+ T cells was found in biopsy specimens of gastric antrum mucosa from Helicobacter piloriinfected patients or in the gut from patients with Crohn’s disease, characterized by the presence of activated T cells producing IFN-γ, but not IL-4. Recently the association of CD30 expression with production of Th2 cytokines has also been observed in a model of TCR transgenic mice.26 IL-4–/– mice are unable to express the molecule on activatedT cells, and exogenous IL-4 can induce CD30 expression in these animals. By contrast, LAG-3, a member of the immunoglobulin superfamily,27 showed a different behavior in comparison with CD30. Surface LAG-3 expression correlated with IFN-γ but not IL-4, production in antigen-stimulated T cells. Secondly, LAG-3 expression was strongly upregulated by IL-12,28 a powerful Th1-inducing agent. Finally, following activation with PHA and IL-2, most CD4+ T-cell clones with an established Th1 profile of cytokine secretion expressed LAG-3 (but not CD30) on their surface, whereas the great majority of Th2 clones showed CD30 expression, but neither surface LAG-3 nor LAG-3 mRNA.28 Soluble LAG-3-related peptide(s) (sLAG-3) is released by T cells, and its concentrations in their supernatants correlated positively with the concentrations of IFN-γ, but inversely with those of IL-4.28 Finally, the opposite role of IL-4 and IFN-γ on the expression of CD30 and LAG3 by activated T cells from cord blood lymphocytes has been recently demonstrated.29 Thus, CD30 and LAG-3 expression by activated naive and memory CD4+ human T cells appear to be preferentially associated with the differentiation/activation pathway, leading to the prevalent production of type 2 or type 1 cytokines, respectively.

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Tc1 and Tc2 Cells and Their Functions

Although the great majority of CD8+ T cells produce IFN-γ, but no IL-4,30 CD8+ T-cell clones producing IL-4 can be obtained by stimulation of murine CD8+ T cells with anti-CD3 antibody,31 mitogen or allostimulation32 and antigen33 in the presence of IL-4. Based on these findings, the names Tc1 and Tc2 for cytotoxic CD8+ T cells secreting Th1-like and Th2-like cytokines were proposed.32 Tc2 cell clones have also been generated from the skin of immunologically unresponsive individuals with leprosy,34 the Kaposi’s sarcoma skin lesions35 and the peripheral blood of HIV-infected patients.36,37 It is of note that generation of Tc2 clones from HIV-infected patients did not require T-cell conditioning with exogenous IL-4, suggesting that in these patients there is an in vivo operating condition which favors the conversion of CD8+ T cells from the Tc1 to the Tc2 functional phenotype. More recently, we have found that when CD8+ T cells from HIV-infected individuals are cloned in the presence of autologous feeder cells, most of them develop into clear-cut Tc2 clones and that such a tendency can be corrected by addition in bulk culture of IL-12 and/or anti-IL-4 antibody.38 Finally, we have been able to develop Tc2 cells in vitro by incubating CD8+ T cells from healthy subjects with anti-CD3 Ab plus exogenous anti-IL-12 Ab and IL-4.38 While the functional role of Tc1 cells is well established, the in vivo functional meaning of Tc2 cells is still unclear. Murine Tc2 clones exhibited normal cytolytic potential and failed to provide cognate help for B-cell antibody production.32 However, Tc2 clones generated from HIV-infected individuals showed reduced anti-HIV cytolytic activity, expressed the CD40 ligand (CD40L) and provided optimal polyclonal B-cell helper activity for production of immunoglobulin, including IgE.36,37 It is also of interest that IL-4 in the absence of antigenic stimulation induces an anergy-like state in differentiated Tc1 cells (loss of IL-2 synthesis and autonomous proliferation).39 Thus, one possible explanation is that Tc2 cells act as suppressor or anti-inflammatory cells through the production of “helper” cytokines.40 It is of note that, as for CD4+ T-cell clones, CD30 and sCD30 are usually not detectable in CD8+ Tc1 clones, but they were consistently found in CD8+ Tc2 clones generated from HIV-infected individuals.41

TH1/ TH2-Polarizing Signals There is a general consensus that a single precursor can differentiate to either a Th1 or Th2 phenotype.42-45 Both genetic and environmental factors are responsible for the Th1 or Th2 differentiation, although the mechanisms by which the genetic background controls the type of Th cell differentiation still remain elusive.46,47 With regard to the environmental factors, a role for the site of antigen presentation, the physical form of immunogen, the type of adjuvant, the dose of antigen and microenvironmental factors has been demonstrated.

Site of Antigen Entry The site of antigen entry may play some role in determining the type of effector response. Inhaled, but not parenterally injected, ovalbumin (OVA) stimulates a Th2 response.48 When OVA is administered with anti-IL-4 antibody, a shift to the Th1 phenotype occurs, suggesting that the dominance of the Th2 cell phenotype in oral immunity is strongly influenced by the production and presence of IL-4.49 Moreover, when administered orally, antigens that stimulate a strong systemic antibody response can induce tolerance that is associated with T cell production of TGF-β.50 In particular, low-dose feeding induced prominent secretion of IL-4, IL-10, and TGF-β, whereas minimal secretion was observed with high dose feeding.51

Type of Antigen-Presenting Cells and Their Costimulatory Factors Murine hepatic accessory cells were found to support the proliferation of Th1 but not Th2 clones.52 Moreover, adherent cells stimulated optimal proliferation of Th1 clones,

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whereas purified B cells stimulated optimal proliferation of Th2 clones.53 Accordingly, when antigen was presented by macrophages, normal resting Th cells differentiated into Th1 cells, whereas presentation by B cells generated Th cells producing IL-2, which could differentiate into Th2 cells upon restimulation.54 However, no independent relationship between type of APC and cytokine response has been definitely established.55 Several costimulatory molecules present on the surface of APC are involved in T-cell activation and function. It was initially suggested that, at least, in vitro CD28 signaling may be required for Th1 but not for Th2 cells. Indeed, only Th1 clones need CD28 signaling in addition to TCR signaling for activation and IL-2 production.56 The effect of soluble recombinant CTLA-4, the CD28 homologue, with high affinity for B7 antigen, was studied. Injection of soluble CTLA-4 also abrogated progressive disease in L. major- infected susceptible BALB/c mice, without having any effect on the protective immune response developed by resistant C57BL/6 mice.57 These data suggest that interaction of B7 with its ligands is required for elevated Th2 cytokine gene expression and secretion during primary Th2 responses. Other findings, however, indicate that the regulation of costimulation mediated by the B7-CD28/CTLA-4 system is far more complex than previously appreciated.Among the three different molecules (B7-1; B7-2; B7-3), at least two can act as costimulatory molecules for CD28. B7-1 appeared to be a neutral differentiative signal, whereas B7-2 provided an initial signal to induce naive T cells to become IL-4 producers.58 In the experimental allergic encephalomyelitis (EAE) model, a classic Th1-mediated disorder, neutralization of B7-1 reduced disease severity and increased the production of IL-4, whereas neutralization of B7-2 increased both disease severity and IFN-γ production,59 suggesting that these interactions can influence the committment of precursors to the Th1 or the Th2 pathway. Other findings do not fit, however, with this possibility: B7-2 neutralization blocks the development of insulin-dependent diabetes mellitus in nonobese mice (a Th1 disorder)60 and stimulation of murine and human T cells with B7-1 or B7-2 transfectants induce similar levels of Th1 type and Th2 type cytokines.61 In the hapten (2,4,6-trinitrobenzene sulfonic acid)-induced colitis, the administration of anti-CD40L antibodies during the induction phase of the Th1 response prevented IFN-γ production by lamina propria CD4+ T cells, whereas the secretion of IL-4 was increased. As demonstrated by immunohistochemistry, the prevention of clinical and histological evidence of disease was caused by an inhibition of IL-12 secretion.62 IL-12 production by murine macrophages is indeed critically dependent on CD40L expression by activated T cells.63 Thus, CD40L-CD40 interaction seems to be crucial for the in vivo priming of Th1 cells via the stimulation of IL-12 secretion by APC. Finally, we have recently shown that CD30 triggering of activated Th cells by CD30L-expressing APCs may represent an important costimulatory signaling for the development of Th2 type responses. In fact costimulation of human PBMC with an agonistic anti-CD30 antibody resulted in the preferential development of antigen-specific T-cell lines and clones showing a Th2-like profile of cytokine secretion. Conversely, early blockade of CD30L/CD30 interaction by an anti-CD30L antagonistic antibody shifted the development of T cells towards a Th1-like phenotype.23

Properties of the Immunogen In mouse, corpuscolate immunogens more easily promote Th1 responses than soluble antigens and this is probably related to their greater ability to induce IL-12 production by macrophages that are responsible for phagocytosis. Antigen polymerization also preferentially evokes Th1 type responses in comparison to the unmodified antigen. Recent studies suggest that differentiated effector CD4+ T cells may produce different cytokines depending on the dose of antigen used. Exposure of CD4+ T cells from naive mice transgenic for a TCR

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recognizing cytochrome c bound to Ebb:Eak to high antigen doses led to differentiation into Th1-like cells producing abundant IFN-γ while low doses of the same peptide induced cells with the same TCR to differentiate into Th2-like cells producing abundant IL-4.64 On the other hand, by using naive CD4+ T cells from the D011.10 TCR transgenic mice, at doses of antigen that induced seemingly similar levels of T-cell proliferation, low to medium doses of antigen promoted the development of Th1 cells producing IFN-γ and undetectable levels of IL-4, whereas increasing the dose of antigen resulted in the disappearence of IFN-γ and the development of IL-4-producing cells.65 Direct sequencing and analysis of mRNA and genomic TCR expression of antigen-specific Th1 and Th2 clones demonstrated usage of the same receptors by both subsets of CD4+ cells, suggesting that the same peptide can underlie both responses.66 Nevertheless, it also appears that certain epitopes can preferentially induce one of the two subsets of Th cells (a repetitive peptide of L. major selectively activated Th2 cells) . More recently, it was reported that varying either the antigenic peptide or the MHC class II molecules could determine whether Th1-like or Th2-like responses are obtained. High MHC class II-peptide density on the APC surface favored Th1-like responses, while low ligand densities favored Th2-like responses.67 Likewise, by using a set of ligands with various class binding affinities, but unchanged T-cell specificity, it was shown that the highest affinity ligands induced IFN-γ production, whereas ligands with lower affinity induced only IL-4 secretion.68 A single MHC polymorphism may dictate Th1/Th2 selection by determining the level of peptide presented to a given TCR on APC.69 These findings suggest that the MHC binding affinity of antigenic determinants can be crucial for the differential development of cytokine patterns in T cells.

Cytokines It is largely accepted that cytokines themselves are the major factors determining the differentiation of naive, and probably even memory, Th cells into the polarized Th1 or Th2 phenotype. So far, the clearest examples of factors affecting the differentiation pathways of Th cells are cytokines released by APC and/or other cell types at the time of antigen presentation. It is generally accepted that the early presence of IL-12 or IL-4 is critical in determining the development of the naive Th cell into the Th1 or Th2 pathway, respectively.45 IL-12, a cytokine produced by the cells of the “natural immunity” set, macrophages and dendritic cells, is the dominant factor in inducing Th1 development.70,71 In spite of some controversy, IL-12-driven development of murine Th1 cells from naive Th cells appears to be dependent on IFN-γ, for which requirement it can be replaced by IFN-γ.72 Other cytokines, such as TNF-α and TGFβ, may also contribute to the development of Th1 cells.47 In mouse, IL-10 appears to act as a Th2-promoting factor, whereas the role of IL-1 is controversial. However, both IL-1 and IL-10 certainly have a minor effect in Th cell development in comparison to IL-4 and IL-12. Even more critical is the role of IL-4 in determining the development of Th2 cells,73,74 since the presence of IL-4 during primary stimulation of naive Th cells usually overcomes the Th1-promoting effect of IL-12.45 The IL-4 source which is required for priming naive T cells to develop into Th2 cells is still unclear. Three major candidates have been suggested. They include Fcεε receptor 1-positive (Fcεε R1+) non-T cells, the CD4+NK1+ subset and the naive CD4+ T cell itself. FcεεR1+ non-T cells may represent the “natural immunity” analogue for the development of Th2 cells.75 More importantly, in IL-4 gene-targeted mice, only those mice which are reconstituted with IL-4-producing T cells, but not with IL-4-producing non-T cells, produce antigen-specific IgE.76 Thus, IL-4 production by FceR1+ non-T cells triggered by antigen-IgE antibody immune complexes can play a role in amplifying secondary Th2 responses to

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parasites. The CD4+NK1+ T cell subset is a specialized population of T cells showing both T cell and NK cell surface receptors, very limited TCR repertoire and CD1 restriction.77 Several findings suggest that CD4+NK1+ T cells may represent an important source for IL-4 required by naive CD4+ T cells to develop into Th2 cells.78,79 Therefore, it is possible that immunogens having associated superantigens able to interact with a sufficiently large fraction of CD4+NK1+ cells may promote a pulse of IL-4 at the time of the primary response by naive CD4+ T cells to the antigen. It is unlikely, however, that all antigens able to promote the differentiation of naive Th cells into the Th2 pathway would necessarily activate CD4+NK1+, CD1-restricted T cells. A more likely possibility is that the source of IL-4 in the primary response is the naive CD4+ T cells themselves. First, low intensity signalling of TCR, such as that mediated by low peptide doses or by mutant peptides, leads to secretion of low levels of IL-4 by murine naive T cells.80 Moreover, naive T cells, activated in the presence of fibroblasts that express costimulatory molecules (i.e., in the absence of outside influences from other cells), can develop into cells producing IL-4 and IL-5 (Th2 cells). Such development was blocked by inhibiting IL-4 action, suggesting it was due to endogenous IL-4 produced by the naive T cells themselves.81 Interestingly, data obtained in humans by using in vitro models also suggest a critical regulatory role of cytokines in determining the development of Th cells into one or another cytokine profile.82 The addition in PBMC bulk culture, before cloning, of IFN-γ plus anti-IL-4 antibody, IFN-α, TGF-β or polynosinic-polycyitidylic acid (poly I:C) promotes the differentiation of T cells that are specific for Toxocara canis excretory/secretory antigen (TES) or allergen into Th0/Th1 instead of Th2 clones.83-86 IL-12 addition in bulk culture also shifts the development of allergen-specific T cells from the Th0/Th2 to the Th1 profile, whereas the neutralization in bulk culture of endogenously produced IL-12 by an anti-IL-12 antibody shifts the differentiation of PPD-specific T cells from the Th1 to the Th0/Th2 phenotype. 87,88 Taken together, these data suggest that IL-12 and even IFN-γ, IFN-α and TGF-β favor the development of human Th1 cells. IL-12 directly affects at single cell level the differentiation of both CD4+ and CD8+ human T cells, by inducing a stable priming effect for high IFN-γ production and is also able to promote transient IFN-γ mRNA expression and small but detectable IFNγ production by already established Th2 clones.88 Moreover, the Th1-promoting effect of poly I:C was recently ascribed to the combined activity of IL-12 and IFN-α released by macrophages.85 Therefore, it is reasonable to suggest that, given the capacity of intracellular bacteria and some viruses to stimulate macrophages to the production of IL-12 and IFN-α(that in turn induce IFN-γ production by both T and NK cells), human Th cells responding to these pathogens may be simultaneously presented with processed antigen plus cytokines that induce them to differentiate towards a Th1 phenotype.89 Recently, a new cytokine named as IFNγ-inducing factor (IGIF), which induces IFN-γ production by both NK and T cells more potently than does IL-12, has been cloned.90 IGIF may be involved in the development of Th1 cells, as well as in mechanisms of tissue injury in inflammatory reaction. A promoting role of IL-4 in the development of human Th2 cells was suggested by the demonstration that addition of recombinant IL-4 in bulk cultures before cloning shifted the differentiation of PPD-specific T cells from the Th1 to the Th0, or even to the Th2 phenotype.93 The interaction between CD30L and CD30 may also play some role in the development of human Th2 cells. In fact, the addition in bulk culture before cloning of an agonistic anti-CD30 antibody favored the development of tetanus toxoid-specific T cells into Th2-like clones.23 However, whether CD30L-CD30 interaction acts by favoring early IL-4 production by some cell type present in bulk culture or via a different mechanism is unclear.

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The source of IL-4 required by human T cells to develop into Th2 cells has also been investigated. IL-4 can be released by human bone marrow non-T, non-B cells belonging to the mast cell/basophil lineage,91 mast cells,92,93 basophils94 and eosinophils;95 however, for the reasons discussed above, it is unlikely that these cells are responsible for the development of Th2 cells. The recent demonstration that under certain experimental conditions both murine and human CD8+ T cells can develop into Tc2 cells34 raises the possibility that IL-4 produced by Tc2 cells may influence the development of Th cells. However, even if it is possible that IL-4-producing CD8+ T cells can have some effect in some pathological conditions (severe atopy, lepromatous leprosy, AIDS), it is highly unlikely that they play a physiologic regulatory role in the development of Th2 cells. On the other hand, human CD45RA+ (naive) adult peripheral blood T cells, as well as human neonatal T cells, have been found to develop into IL-4-producing cells in the absence of any preexisting source of IL-4 and despite the presence of anti-IL-4 antibodies.96,97 In addition, high proportions of T-cell clones showing a clear-cut Th2 profile of cytokine production could be generated from single thymus-derived CD4+ T cells, which required exogenous IL-12 (IFN-γ was not effective) to be primed to IFN-γ production.98 Thus, evidence is accumulating to suggest that the maturation of naive human T cells into the Th2 pathway is potentially the basic type of specific effector response, depending mainly on the levels and the kinetics of autocrine IL-4 production at priming. Such a Th2 maturation can occur without exposure to IL-4 produced by accessory cells and may likely be determined by 1) The genetic background of the individual and 2) the nature and the intensity of TCR signaling by the peptide ligand. Priming of naive T cells to IFN-γ production probably results from the stimulation of “natural immunity” and consequent release of IL-12 and IFNs (γ and α) by different pathogens. Since most studies performed with human Th cells reflect in vitro secondary responses, their meaning was initially questioned, the dogma being that only primary, but not secondary, immune responses can be influenced by exogenous cytokines. Recently, however, it has been demonstrated that even murine memory CD4+ T cells, regardless of prior commitment, may retain the capacity to be further influenced by IL-4, IFN-γ or IL-12 during effector cell development to become subsets that are at least temporarily polarized to express a particular pattern of cytokine secretion.99-101

Hormones The role of hormones in promoting the differentiation of Th cells or in favoring the shifting of differentiated Th cells from one to another cytokine profile has also been suggested. Glucocorticoids enhance Th2 activity and synergize with IL-4, whereas dehydroepiandrosterone sulphate enhances Th1 activity.102 Another major prohormone, 25-hydroxy cholecalciferol (25-(OH) vitamin D3) may have a reverse effect on the Th1/Th2 balance. The intense conversion of 25 (OH) vitamin D3 to 1,25-(OH)2 vitamin D3 (calcitriol) decreases secretion of IL-2 and IFN-γ and increases a Th2 pattern of response. Calcitriol analogues can also antagonize cyclosporin A in their ability to prolong survival of skin grafts by inhibiting Th1 activity.102 Recently, we found that progesterone favors the in vitro development of human Th cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established human Th1 clones.103 This may represent one of the mechanisms involved in the Th1/Th2 switch which has been hypothesized to occur at the maternal-fetal interface in order to improve fetal survival and promote successful pregnancy.104 In more recent experiments, relaxin (another corpus luteum-derived hormone) was found to favor the development of IFN-γ and TNF-β producing cells, without having any influence on IL-4 and IL-5 production, thus showing an opposite effect compared to

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progesterone.105 Therefore, increasing evidence is accumulating to suggest that hormones and peripherally activated prohormones may regulate the Th1/Th2 balance.

Other Factors Many other substances capable of exerting some influence in the development of Th1 and Th2 cells have been described. Prostaglandin E2 (PGE2) and forskolin differentially modulate cytokine secretion profiles of adult human Th lymphocytes by inducing a higher elevation of intracellular cAMP levels in Th0 and Th1 than in Th2 clones.106 However, in cord blood naive T cells, PGE2 inhibits acquisition of the ability to produce IFN-γ and IL-2 but not IL-4 and IL-5, thus showing a clear promoting effect towards the Th2 cell development.107 Nitric oxide (NO), which is produced by Th1 but not Th2 cells, selectively inhibits the proliferation and production of IL-2 and IFN-γ by Th1 cells, whereas it has no effect on IL-4 production by Th2 cells.108 Mercuric chloride (HgCl2), a chemical responsible for autoimmune manifestations in Brown Norway rats, induces early and high expression of IL-4 mRNA in T cells from these animals.109 Likewise, gold salts promote autoimmune manifestations and the development of Th0/Th2 cells in the same animals.110 Naturally occurring flavonoid compounds such as rutin (a polyphenol-containing glycoprotein from tobacco), preferentially activate Th2 cells.111 Vitamin A deficiency results in a strong regulatory T-cell imbalance with excessive Th1 response and insufficient Th2 cell development and function. At least three vitamin A activities that balance Th1 and Th2 functions, of Th1 cells directly downregulating IFNγ secretion, decreasing activated APC function, and promoting Th2 cell growth and/or differentiation, were identified.112

Genetic Background in the Th Development From the above mentioned results, it appears clear that there are striking differences in Th outcome depending on the genetic background of the host. The clearest example of genetic control of the Th1/Th2 responses is provided by murine L. major infection. Transgenic T cells from both the B10.D2 and BALB/c backgrounds showed development toward either the Th1 or Th2 phenotype under the strong directing influence of IL-12 and IL-4, respectively. However, when T cells were activated in vitro under neutral conditions in which exogenous cytokines were not added, B10.D2-derived T cells acquired a significantly stronger Th1 phenotype than T cells from the BALB/c background, correspondent with in vivo Th responses to L. major in these strains.113 Another interesting example is provided by the different effect exerted by HgCl2. In Brown Norway rats, HgCl2 induces early and strong IL-4 expression and autoimmunity, whereas it does not affect IL-4 expression in Lewis rats who do not develop autoimmunity.109 Well characterized examples of genetic differences in Th cell development include murine candidiasis114 and malaria,115 atopic disorders,116 autoimmune diabetes,117 intestinal helminth infections118 and Lyme disease.119 Recently, it has been found that T cells from B10.D2 mice (a murine strain easily inducible to Th1 responses) have an intrinsically greater capacity to maintain IL-12 responsiveness under neutral conditions in vitro compared to T cells from BALB/c mice (a strain inducible to Th2 responses).120 The locus controlling this genetic effect was localized to a region of chromosome 11 containing a cluster of genes important for T-cell differentiation, including IL-4, IL-5, IL-3, and IRF-1,121 similar to the homologous gene cluster on human chromosome 5 linked to several phenotypic markers of atopy.

Intracellular Signaling Involved in the TH1/TH2 Cell Development Relatively little is known about the biochemical basis for the differential production of cytokines by Th1 and Th2 cell types. Likewise, the intracellular signalings involved in the development of one or the other phenotype are still poorly understood.

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Cytokines exert their effects on cells by interacting with specific receptors expressed at the cell surface. This results in receptor homo- or hetero-dimerization, and triggering of intracellular signals. One of the earliest signaling events is the activation of protein tyrosine kinases (PTKs) which are physically associated with the receptor,122 which phosphorylate several substrates critical for signal transduction. PTKs utilized by cytokine/cytokine receptor complexes for signal transduction comprise two families of essential elements which are known as the JAK-STAT pathway.123 The Janus family of kinases (JAK) contains the members JAK1, JAK2, JAK3, and TYK2. They constitutively associate with the intracellular domains of cytokine receptors and become activated following ligand-induced assembly of receptor subunits at the cell surface. The second family is comprised of Src homology 2 (SH2) domain-containing, latent cytosolic transcription factors known as signal transducers and activators of transcription (STATs). Typically, stimulation by a particular cytokine results in the activation of a distinct pair of two of the four known JAK kinases. JAK kinases are required for tyrosine phosphorylation and activation of STATs, although it is not yet clear whether JAKs phosphorylate STATs directly. It has clearly been shown that IL-12 directly influences T cells for the induction of Th1 differentiation.87,88 Treatment of murine TCR-transgenic T cells with IL-12 produced an electromobility shift assay (EMSA) complex containing STAT3 and STAT4, whose mobility was distinct from that of complexes induced by IL-2 and IL-4, which contain STAT5 and STAT6, respectively.124 Interestingly, activation of EMSA complexes are evident in early Th1 cells. Th2 cells respond to IL-2, IL-4, IFN-γ involving activity of JAK1 and JAK2) and IFN-α (involving activity of JAK1 and TYK2), but not to IL-12. The lack of STAT4 phosphorylation in Th2 cells was not due to a lack of STAT4 protein, since Th2 cells expressed similar levels of STAT3 and STAT4 compared to Th1 cells, but in the failure to phosphorylate JAK2, STAT3, and STAT4.125 In human T cells, IL-12 induces tyrosine phosphorylation of JAK2 and TYK2, whereas JAK1 and JAK3 (phosphorylated in response to IL-2) are not phosphorylated after IL-12 treatment.126 More importantly, IL-12 induces tyrosine phosphorylation and activation of STAT4-, but not of STAT- or STAT2-containing complexes, which bind to the GRR DNA sequence.127 The recognition that IL-4 expression is critical for determining the development of Th2 cells has intensified interest in the molecular basis of its regulation. It is now clear that, following the interaction between IL-4 and its receptor on a given cell, the IL-4 induced a STAT protein termed STAT6. The essential role of STAT6 in IL-4 signalling has clearly been demonstrated in STAT6-deficient mice, where T cells are unable to develop into Th2 cells and production of IgE and IgG1 is virtually abolished.128 The mechanisms governing the IL-4 gene expression in T cells, however, are very complex and still unclear. An analysis of the IL-4 promoter has revealed functionally important binding sites for several transcription factors, including NF-AT, CCAAT box binding protein NF-Y, Oct 1, HMGI(Y), AP-1 members, NFkB, and an as yet unpurified factor termed PCC.129-135 Silencer elements further upstream that bind factors termed NRE have also been described.136 Nucleotide substitutions have been identified in the proximal IL-4 promoter that influence promoter activity in transfected cells and affect the interaction of DNA binding proteins with the polymorphic site. In another study, the nuclear extracts from individuals with atopic dermatitis were found to exhibit a higher affinity for a consensus P-element, suggesting that polymorphic residues in NF-AT family members themselves or in their posttranslationally modified versions may be involved.137 None of these factors, however, is expressed in a Th-selective manner. More recently, it has been shown that the protooncogene c-maf , a basic region/leucine zipper transcription factor, controls tissue-specific expression of IL-4. c-Maf is expressed in Th2 but not Th1 clones and is induced during normal precursor cell differentiation along a Th2 but not Th1 lineage. c-Maf binds to a c-Maf response element

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(MARE) in the proximal IL-4 promoter adjacent to a site footprinted by extracts from Th2 but not Th1 clones.138

Role of the Th1/Th2 cells in Autoimmune Disorders Autoimmune diseases develop as a result of abnormalities in the immune response mediated by activated T cells and T cell-derived cytokines. Strong evidence deriving from both studies in animal models and in human disease suggests that Th1 type cytokines are involved in the genesis of organ-specific autoimmune diseases. In contrast, a less restricted lymphokine pattern is emerging from experimental studies on systemic autoimmune diseases.

Systemic Lupus Erythematosus Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by over-production of a wide range of autoantibodies and several abnormalities involving the kidney, skin, brain, lungs, and other organs.139 In theory, a prevalent production of Th2 cytokines may be critical to disease induction by contributing to the increased B cell activation and to disease maintenance of SLE patients.140 Experimental Models It is well known that autoimmune alterations observed in the context of allogeneic reactions and following exposure to some chemicals such as gold salts and mercurials are Th2-mediated disorders. 141 Brown Norway rats treated with HgCl 2, gold salts or D-penicillamine also exhibit CD4+ T cells recognizing MHC class II molecules and produce Th2 cytokines.141 These chemicals are able to directly trigger IL-4 production by a population of T cells in genetically susceptible animals.109 Results presently available in lupus-prone mice suggest that both Th1 type and Th2 type cytokines or cells showing no restricted cytokine profile may play a pathogenic role. For example, administration of anti-IFN-γ antibody attenuates the severity of the disease in (NZB x NZW),142 but continuous administration of anti-IL-10 antibodies also delays onset of autoimmunity, whereas IL-10 accelerates the onset of autoimmunity in (NZB x NZW) F1 mice.143 Autoreactive T-cell lines producing both IL-4 and IFN-γ have been derived from MRL lpr/lpr mice, which manifest an autoimmune disease state. These lines trigger a nephritis which is prevented by administration of anti-IFN-γ antibody.144 Analysis of cytokine gene expression by RT-PCR revealed that IL-1, IL-5, IL-6, IFN-γ, TNF-α TNF-β, and TGF-β were transcribed by various T-cell subsets.145 IFN-γ was most markedly augmented in MRL/l mice, IL-1 was most severely overexpressed in BXSBm mice, while IL-10 was equally increased in both strains.146 IL-12 and NO play also an important role in the development of spontaneous autoimmune disease in MRL/MP-lpr/lpr mice.147 Human SLE SLE patients have significantly fewer cells producing Th1 cytokines, IFN-γ and IL-2, but significantly more cells secreting Th2 cytokines, IL-6 and IL-10, than normal controls.148 SLE patients also have excess IL-4 production and increased numbers of IL-4-secreting cells,149,150 as well as increased serum levels of sCD30, which correlate with disease activity.151 However, the major abnormalities seem to involve IL-6 secretion and IL-6/IL-6 receptor interactions.152 Moreover, abnormal production of both IL-6 and IL-10 in SLE seems to be due to macrophages and B cells148,149 rather than to T lymphocytes. A general defect in suppressor activity, as shown in TGF-β1 knockout mice which exhibit high levels of IL-2, IL-6, and IL-10, as well as of anti-nuclear autoantibodies, rather than the occurrence of a Th1- or Th2-dominated response, may be suggested.

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Systemic Sclerosis Systemic sclerosis (SSc) is a disorder characterized by inflammatory, vascular, and fibrotic changes of the skin (scleroderma) and a variety of internal organs, such as the gastrointestinal tract, lungs, heart, and kidney. Increased numbers of T cells are present in skin lesions, as well as in other organs in the early stages of the disease.153 Several soluble factors secreted by T cells or other cells of the immune system may modulate fibrosis or promote vascular damage, such as IL-1α/β IL-6, TNF-β, and TGF-β. All these factors are able to alter various fibroblast activities such as growth, production of extracellular matrix components, production of collagenase or prostaglandins.154 IL-4 also induces human fibroblasts to synthesize elevated levels of extracellular matrix proteins, as well as to stimulate the growth of subconfluent fibroblasts and induce chemotaxis of these cells.155 PBMC from patients with SSc produce higher amounts of IL-2 and IL-4 than controls156 and IL-2, IL-4, and IL-6, but not IFN-γ, were found in the sera from SSc patients.157 More recently, analysis by RT-PCR of spontaneous cytokine and CD30 gene expression by PBMC from SSc patients showed spontaneous IL-4, IL-5, and CD30, but no IFN-γ or IL-10, mRNA expression. T-cell clones generated from the skin cellular infiltrates were in great majority Th2. Accordingly, large numbers of CD4+ T cells present in the perivascular infiltrates of skin biopsy specimens were CD30+ and high levels of sCD30 were found in the serum of most SSc patients, especially those showing active disease.157 These data suggest a predominant activation of Th2 cells in SSc and support the view that abnormal and persistent IL-4 production may play an important role in the genesis of fibrosis.

Rheumatoid Arthritis Rheumatoid arthritis (RA) is an autoimmune chronic synovitis which often leads to joint destruction. Experimental Models: Collagen-Induced Arthritis A murine collagen (antigen type II collagen—CII—emulsified with CFA)-induced arthritis has been used as a major model for RA. By using this model, it has been clearly shown that CD4+ T cells are involved in the induction phase, whereas their role in the effector phase of the inflammatory reaction is still unclear.158 The proinflammatory cytokines mainly produced by macrophages are also heavily involved in the pathogenesis of arthritis. In fact, anti-TNF-α, anti-IL-1α and anti-IL-1β antibodies or recombinant soluble TNF receptor after the onset of symptoms reduced the clinical and histological severity of arthritis.159 Upon stimulation with CII in vitro, spleen cells from immunized mice synthesize IFN-γ. Moreover, IL-12 in combination with CII can replace Mycobacteria in causing severe arthritis of DBA/1 mice and this effect correlates with a 3-10 fold increase in the production of IFNγ by antigen-stimulated T cells.160 More importantly, neutralization of IFN-γ in vivo prevented the development of arthritis in collagen-immunized and IL-12-treated mice.161 Finally, oral administration of an immunodominant human collagen peptide modulates collageninduced arthritis.162 These data suggest that a Th1 type autoimmune reaction is responsible for the collagen-induced arthritis in DBA/1 mice, even though other findings do not fit with this interpretation.163 Human Disease There is a general consensus that proinflammatory cytokines (TNF-α, IL-1, GM-CSF, IL-6),164,165 and chemokines (IL-8, RANTES, and MIP-1)166 highly produced by the synovial membrane play an important role in the pathogenesis of RA. These factors do indeed induce bone resorption and cartilage destruction, and can stimulate PGE2 release and collagenase production. In contrast T cell-derived cytokines are not easily detectable in RA synovium,167

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even though synovial membrane infiltrating T cells appear phenotypically activated. An alternative view is that T-cell cytokines may be important, but are expressed at levels too low for detection by conventional methods.168 In favor of this possibility is the observation that mRNA for IFN-γ and IL-2 is present in synovial tissue of RA patients, whereas IL-4 mRNA is detectable only in a few cases; moreover, most T-cell clones derived from the RA synovium are of the Th1 type,169 even if some rheumatoid inflammatory T-cell clones exhibit a Th0-like profile.170 On the other hand, high levels of IL-1RA, soluble TNF receptors (of both the 55 and 75 kDa receptor), TGF-β, and IL-10 were found in the RA synovium,171 which can be interpreted as attempts to contain inflammation and limit joint destruction. Indeed, blocking IL-10 in synovial membrane cultures resulted in increase of both TNF-α and IL-1β.171 Due to its powerful inhibitory activity on Th1 cytokine production, it is reasonable to suggest that IL-10 represents the (or one of the) reason(s) of the “elusiveness” of Th1-derived cytokines in RA. However, the question of whether increased production of proinflammatory cytokines in RA is secondary to Th1 cell activation or represents a primary phenomenon still remains unclear.

Sjogren Syndrome Sjogren syndrome (SS) is a autoimmune disease clinically defined by the presence of keratoconjunctivitis sicca and xerostomia, and immunologically by a hypergammaglobulinemia, multiple autoantibodies and lymphocytic infiltration of the glands, in patients not fulfilling criteria for any other chronic inflammatory connective tissue disease. The results of different studies suggest a predominant activation of Th1 cells in patients with SS. First, the cytokines IL-1, IL-6, TNF-α and IFN-γ were identified in labial salivary gland specimens from patients with SS.172 Second, increased levels of IL-6 and IFN-γ have been found in the serum of SS patients.173 Finally, spontaneous IFN-γ mRNA in freshly isolated unstimulated T cells from SS patients has been observed.174 Increased production of IL-10 by stimulated, and spontaneous IL-10 mRNA expression by freshly isolated, PBMC of SS patients174 may reflect a mechanism downregulating the inflammatory reaction induced by the Th1 type cytokines. Recently, however, a SS-like lymphoproliferation associated with high levels of anti-nuclear autoantibodies has been observed in TGF-β1 knockout mice.175 These mice produced high amounts of IL-2, IL-6 and IL-10, suggesting that in this disease a general defect of suppressor mechanisms rather than a prevalence of Th1 or Th2 responses may play the major pathogenic role.

Autoimmune Thyroid Diseases Hashimoto’s thyroiditis (HT) is an organ-specific autoimmune disease characterized by massive infiltration of lymphoid cells in the thyroid gland and parenchymal destruction leading to hypothyroidism. Grave’s disease (GD) is an autoimmune disorder of the thyroid gland with a histologic picture similar to HT, but distinguishable by associated ophthalmopathy and production of thyroid-stimulating antibodies leading to hyperthyroidism. Experimental Autoimmune Thyroiditis An experimental autoimmune thyroiditis (EAT) can be induced by immunization of susceptible mice with thyroglobulin in CFA, which results in strong activation of thyroidal antigen-specific T cells.176 Autoreactive T-cell clones can indeed transfer a disease very similar to that induced by immunization.177 Transfer of the disease can be achieved with both CD4+ and CD8+ specific T-cell lines and clones specific for thyroid epithelial cells that exhibit a clear-cut Th1 pattern of cytokine production.178 Moreover, IL-12 and IFN-γ play a major role in the expression of thyroiditis in the BioBreeding (BB) rat, in whose inflammatory lesions Th1 lymphocytes predominate.179

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Human Autoimmune Thyroiditis Although the precise etiology of these diseases remains largely unknown, there is a general consensus that infiltrating T lymphocytes play an essential role in their pathogenesis. T cells from thyroid infiltrates of HT or GD patients exhibit a restricted Th1 lymphokine profile, with production of high TNF-α and IFN-γ concentrations, and strong cytolytic potential.180 A prevalent Th1-like cytokine profile was also observed by RT-PCR in the thyroid gland of patients with HT and from retroorbital infiltrates of patients with Graves’ ophthalmopathy.181 In contrast, by using RT-PCR, other authors found a more heterogenous cytokine profile in both the thryoid gland and retroorbital infiltrates of patients with GD.182,183 These discrepancies may be explained by the different cytokine profiles of thyroid antigen-specific T cells; thyroid peroxidase (TPO)-specific clones showed a Th1 profile, whereas the cytokine profile of clones specific for thyroid-stimulating hormone receptor (TSHR) were of Th0 or Th2 type,184 which are more active in promoting the synthesis of pathogenic autoantibodies.

Autoimmune Uveites Experimental Autoimmune Uveoretinitis Immunization with retinal antigens (S antigen) in CFA induces an experimental autoimmune uveoretinitis (EAU) in susceptible rat strains LEW and (LEW x BN) F1 hybrids.185 This disease is mediated by T lymphocytes and is probably a Th1-mediated disease, inasmuch as it can be transferred by S antigen-specific Th1-like T-cell lines.186 Furthermore, administration of HgCl2 (which triggers IL-4 production) protects from the development of EAU.187 Other findings, however, argue against the possibility that EAU is a Th1-mediated disorder. For example, it is surprising that administration of IFN-γ given at the initiation of the disease is capable of preventing the induction of EAU.188 Behçet’s Disease Behçet’s disease (BD), is a chronic, relapsing-remitting systemic vasculitis, whose manifestations include orogenital ulcers, synovitis, thrombophlebitis, uveitis, and other symptoms related to the CNS. The uveitis in BD may be a human equivalent of the EAU. In fact, T-cell clones specific for the bovine S-antigen have been generated from PBMC of patients with BD.189 Histopathological studies have revealed cellular infiltration consisting of lymphocytes, plasma cells and polymorphonuclear neutrophils. Increased serum levels of IL-1β and IFN-γ and spontaneous production of TNF-α, IL-6, IL-8 and IFN-γ from cultured T lymphocytes have been observed.190 Recently, several Th0-cell clones, generated from the CSF of patients with neuro-BD, showed the ability to produce both IFN-γ and IL-4 in response to stimulation with PMA plus anti-CD3 antibody; however, the amounts of IFN-γ produced by the majority of these clones were exceptionally high (Parronchi P, unpublished results).

Type 1 or Insulin-Dependent Diabetes Mellitus The development of insulin-dependent diabetes mellitus (IDDM) in humans and in the spontaneous animal models is the result of an autoimmune process (insulitis) that selectively destroys the pancreatic islet beta cells, due to T lymphocytes and, in a lesser degree, to macrophages around the islets.191 IDDM in BB and NOD Mice In BB mice, both the IL-12 p40 chain and IFN-γ mRNA are present in the inflammatory lesions, whereas mRNA for IL-2 and IL-4 is usually undetectable.192 All NOD mice spontaneously develop insulitis early in life, but later it progresses to diabetes mainly in the

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females. There is extensive infiltration of the islets by CD4+ and CD8+ T cells, B cells and macrophages, as occurs in human IDDM. IFN-γ-producing Th1-like clones cause IDDM after transfer into neonatal NOD mice,193 and transfer of T-cell clones that secrete IFN-γ and IL-2 is able to precipitate diabetes in recipient NOD mice.194 By contrast, Th2 clones are unable to transfer diabetes. NOD mice produce large amounts of IFN-γ in response to glutamic acid decarboxylase (GAD), a key β-cell antigen recognized by both T cells and B cells.195 Administration of anti-IFN-γ antibodies can prevent the development of diabetes induced in NOD mice as well as the injection of IL-12 induces massive Th1-cell infiltration in the pancreatic islets and accelerates IDDM development in NOD mice.196 Moreover, IL-12 p40 knockout mice on a NOD background showed no insulitis, and their spontaneous IDDM incidence was very low. Finally, systemic administration of IL-4 prevents diabetes in NOD females.197 This latter finding suggests a possible protective role of Th2 cells in the development of IDDM in NOD mice. Human Type 1 Diabetes There are very few studies on the cells infiltrating the insulitis lesion in human type 1 diabetes. They were largely confined to autopsies on patients who died of recent onset diabetes where the pancreatic tissue had been formalin fixed. The cell infiltrate is composed of both lymphocytes and macrophages, and high proportions of lymphocytes contain IFN-γ.198 Autoreactive T-cell clones generated from newly onset patients with IDDM exhibited a predominant Th1 profile, whereas those derived from a prediabetic patient were prevalently Th2.199

Crohn’s Disease. Crohn’s disease (CD) belongs to the idiopathic inflammatory bowel diseases (IBD), which include disorders marked by the presence of chronic inflammation of the gastrointestinal tract not ascribed to a specific pathogen. At one end of the spectrum is ulcerative colitis (UC), a disease that affects the large bowel exclusively; at the other end is CD, which most commonly involves the terminal ileum and the ascending colon. Experimental Models of IBD An IBD-like disease develops in mice with alterations in T-cell subpopulations and T cell selection (TCR-α chain-deficient mice, TCR-β chain-deficient mice, MHC class II-deficient mice),200 in knockout mice for IL-2, IL-10, and TGF-β,201 in mice lacking signaling proteins (G protein subunit Gα2-deficient mice) or subject to rectal application of TNBS.202,203 In all these models T cells play a critical role in the normal regulation of intestinal immune responses. The Th1-derived cytokines IFN-γ and TNF-α appear to be responsible for the pathogenesis of colitis in SCID mice restored with memory CD4+ T cells, as disease was prevented by the administration of anti-IFN-γ antibody and significantly reduced in severity by anti-TNF antibody.204 Moreover, antibodies to IL-12 abrogated established experimental colitis induced in mice by rectal application of the hapten reagent TNBS.203 These data suggest a pivotal role of IL-12 and Th1 cytokines in the induction of murine chronic intestinal inflammation. On the other hand, IL-10 and TGF-β seem to play an important role in the regulation of pathogenic inflammatory responses, as TGF-βand IL-10-deficient mice develop colitis.205 Crohn’s Disease Increased levels of IL-1, IL-6, IL-8, TNF-α as well as of IL-2 and IL-2 receptor, have been found in the intestinal mucosa and/or serum of patients with IBD,206,207 suggesting activation of macrophages and perhaps of Th1-like cells. Moreover, increased expression of

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mRNA for IL-2, IFN-γ and IL-10 are present in the mucosa of patients with active CD, whereas IL-4 mRNA expression was frequently below the detection limits. Recently we found that T-cell clones generated from the mucosa of patients with CD produce higher levels of IFN-γ, and lower levels of IL-4 and IL-5, in comparison with T-cell clones derived from the colonic mucosa of control patients.208 High numbers of activated CD4+ T cells showing CD26, LAG-3, and IFN-γ reactivity, as well as of IL-12-containing macrophages, were found to infiltrate the muscularis mucosa of gut from patients with CD, but not of controls. In addition, culturing IL-2-conditioned T cells from the mucosa of CD patients in the presence of anti-IL-12 antibody had an inhibitory effect on the development of IFN-γ producing T cells, suggesting that constitutive IL-12 production plays a critical role in the development of Th1 cells at intestinal level.208

Inbalance of Th1/Th2 cells in Multiple Sclerosis Multiple sclerosis (MS) is considered as a prototypic autoimmune disorder of the CNS. Active lesions in the brains of MS patients are characterized by lymphocytes (mainly CD4+ T cells) and macrophage infiltration associated with a progressive demyelinizating process. Although the etiology of the disease is still unclear, the pathogenesis clearly involves an autoimmune reaction against myelin antigens such as MBP, PLP, and possibly other proteins.209 Experimental Allergic Encephalomyelitis A classical experimental model for MS showing clinical and pathological similarities to the human disease is experimental allergic encephalomyelitis (EAE). EAE can be induced by immunizing several species of animals with different CNS-related proteins emulsified in CFA, including MBP and PLP. This results in the activation of peptide-specific CD4+ T cells, which in turn can adoptively transfer the disease in naive animals. The majority of T-cell clones derived from mice immunized with MBP or PLP peptides exhibit a restricted Th1 phenotype.210,211 Production of TNF-α and TNF-β, which are known to kill oligodendrocytes in vitro,212 also strongly associates with EAE induction. Moreover, at the peak of disease, perivascular infiltrates within the CNS stain positive for the macrophage-derived cytokines IL-1, IL-6, IL-8, TNF-α, and the Th1-derived cytokines IL-2 and IFN-γ. By contrast, disease recovery associates with the appearence of TGF-β1 and IL-4.213 Finally, treatment of EAE with IL-4 or IL-4-inducing chemicals results in the induction of MBP-specific Th2 cells, diminished demyelinization, inhibition of the synthesis of inflammatory cytokines in the CNS, and improvement of clinical manifestations.214 TGF-β or IL-10 also prevents EAE relapses, whereas anti-IL-10 antibody increases both the incidence and the severity of such relapses. The expression of IL-4 in brains of animals recovering from EAE suggests that remission of disease might be related to the presence of antigen-specific Th2 cells. This hypothesis is also supported by the following findings: 1. Transfer of Th2 cells does not induce EAE, but rather ameliorates the disease; 2. Most MBP-specific CD4+ T-cell clones generated from SJL/J mice fed and immunized with MBP secrete TGF-β1 and/or IL-4 and IL-10 and are protective; 3. PLP-specific Th2 clones, producing high amounts of IL-4 and IL-10, suppress the induction of EAE if transferred at the time of active immunization, or at the onset of clinical symptoms.59 Thus, it is likely that myelin peptide-specific Th1 cells are involved in the pathogenesis of EAE, whereas Th2 cells play a protective role in this disease. The occurrence of a Th1 response in mice affected by EAE is probably due to both the genetic background and the emulsification of the immunizing peptide with CFA. Susceptibility to EAE varies according to the mouse strain, although the genes controlling this process have not yet been identified.

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Several data suggest that autoantigenic stimulation requires high affinity binding to MHC class II molecules.215 However, EAE-resistant BALB/c mice may have highly encephalitogenic MBP-specific T-cell clones that recognize a MBP peptide with high affinity for MHC class II.216 It is notable that among SJL mice males are significantly more resistant to EAE induction than age-matched female mice, suggesting a possible influence of hormonal regulation.217 Furthermore, adoptive transfer of macrophages from EAE-susceptible female mice followed by immunization with self-antigen, results in the induction of EAE in the disease-resistant male mice, suggesting that macrophage APC populations in EAE-resistant mice are deficient in either a costimulatory molecule or secretion of cytokine(s) required for Th1-cell priming.217 The role for costimulatory molecules in the differential activation of Th1/Th2 development pathways has been demonstrated using anti-B7 antibodies: anti-B7-1 antibodies reduced the incidence of disease, while anti-B7-2 increased disease severity by altering the T-cell cytokine profile.59,214 The contemporaneous administration of anti-B7-1 antibody at immunization promoted the prevalent generation of Th2 clones, whose transfer not only prevented induction of EAE, but also abolished established disease. Moreover, cotreatment with anti-IL-4 antibody prevented disease improvement, clearly suggesting that the protective effects of anti-B7-1 antibody result from activation of the Th2 pathway.59 In this respect, it is also of note that blockade of CD28-B7 interactions by injecting soluble CTLA4-Ig prevented the appearence of EAE by inducing a shift in the response toward the Th2 function. 218 On the other side, induction of active EAE as a consequence of emulsification of myelin antigen with CFA probably reflects the stimulation of macrophage IL-12 production by components of the mycobacterial cell wall, with subsequent increase in IFN-γ production. Indeed, in vitro stimulation of antigen-primed lymph node cells with PLP-primed mice and recombinant IL-12 enhances their subsequent encephalitogenicity, whereas inhibition of endogenous IL-12 in vivo after lymph node cell transfer prevented paralysis, suggesting that endogenous IL-12 plays a pivotal role in the pathogenesis of EAE.219 Furthermore, administration of anti-IFN-γ antibody at the time of immunization inhibits EAE development.214 Other important factors are the route of antigen administration, as well as the dose and the structure of antigen. The role of antigen route is exemplified by studies of oral tolerance to myelin antigens. EAE can be suppressed by the oral administration of MBP, which also induces a profound decrease in MBP-reactive, IL-2 and IFN-γ secreting lymphocytes relative to control animals. Such a reduction is probably related to the induction of a state of clonal anergy.220 At high doses, deletion by apoptosis may indeed be the predominant tolerizing mechanism, but at lower antigen doses the induction of T cells which produce Th2 cytokines IL-4 and IL-10 (or TGF-β and protect SJL/J mice from the induction of EAE by MBP and PLP, are demonstrable.9 The mechanisms by which cells producing IL-4, IL-10, and TGF-β are induced have not yet been clearly defined. Low dose feeding appears capable of inducing prominent secretion of IL-4, IL-10 and TGF-β, whereas minimal secretion of these cytokines was observed with high dose feeding.51 However, oral MBP partially suppresses serum antibody responses, especially at high doses, and can be associated with increased salivary IgA,221 thus suggesting an overproduction of switching factors for IgA such as IL-2 and TGF-β. EAE can be suppressed in MBP-TCR transgenic mice orally tolerized with MBP,222 and the nasal administration of MBP peptides also suppresses EAE.223 It is also of note that epitopes of MBP that trigger TGF-β release after oral tolerization are distinct from encephalitogenic epitopes and mediate epitope-driven bystander suppression.224 Studies performed in EAE induced with PLP 139-151, by altering single amino acids, also suggest an important role for the structure of the ligand. These altered peptides are able to protect

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from disease induced with the native peptide, because they activate antigen-specific T cells which are crossreactive but produce Th2-type cytokines on activation. Interestingly, this change in phenotype occurs despite immunization being carried out in the presence of CFA.225 In vivo tolerization of a T-cell clone specific for the MBP epitope p87-99 and capable of inducing EAE with an analogue of p87-99 resulted in reversion of established paralysis, with regression of brain inflammatory infiltrates. It is of note that an antibody raised against IL-4 reversed the tolerance induced by the altered peptide ligand.226 It should also be remembered that some studies are not consistent with a critical role for IFN-γ in the pathogenesis of EAE, and with the protective function of Th2 cells in Th1-induced EAE. First, mice with a disrupted IFN-γ gene are still susceptible to the induction of EAE227 Moreover, neuroantigen-specific Th2 cells were inefficient suppressors of EAE induced by effector Th1 cells,228 whereas induction of EAE was shown by transferring a Th2 T-cell clone in immunodeficient mice.229 Finally, very recently, Th2 immune deviation during EAE in marmoset led to a lethal form of the disease associated with increased antibody responses.230 These findings suggest that the cytokine network may be more complicated than previously suggested in autoimmune diseases, and may account for the difficulties in defining a clearcut Th1/Th2 paradigm in MS. Multiple Sclerosis Several findings in human disease suggest a role for TNF-α and IFN-γ in the pathogenesis of MS. High levels of TNF-α are present in the cerebrospinal fluid (CSF) of patients with chronic progressive MS.231 High levels of TNF-α in both plasma and CSF may predict relapses in MS patients. By using RT-PCR, mRNA of TNF-α was also found in PBMC from MS patients232 and increased TNF-α expression appeared to precede relapses by 4 weeks in patients with relapsing-remitting MS, whereas IL-10 and TGF-β expression was decreased at the same time. Most clones derived from both PBMC and CSF of patients with MS showed a Th1 profile;233 increased numbers of IFN-γ-secreting T cells, which produced IFN-γ upon activation by several myelin antigens and several MBP peptides, were found in the CSF of MS patients.234 T-cell clones specific for PLP peptides generated from MS during an acute attack showed a clear-cut Th1 profile, whereas during remission in the same patients a more heterogenous cytokine profile was found. These clones produced levels of both IL-10 and TGF-β significantly higher than those of clones isolated during acute attacks.235 High levels of LAG-3 expression and sLAG-3 production by T-cell clones generated from the CSF were also found. Moreover, high levels of sLAG-3 were detected in the serum of patients with relapsing remmitting MS.28 By immunohistochemistry, both TNF-α and TNF-β were identified in acute and chronic active MS lesions, but not in spleens or PBMCs from MS patients. TNF-β expression was associated with T cells, whereas TNF-α was associated with astrocytes in all areas of the lesion.236 By using semiquantitative RT-PCR and immunohistochemistry, the costimulatory molecules B7-1, B7-2 and the cytokine IL-12 p40 were found to be upregulated in acute MS plaques from early disease cases. The differences in mRNA expression were specific for IL-12 p40, whereas no differences were observed for other cytokines, suggesting that an early event in the initiation of MS involved upregulation of costimulatory molecules and IL-12.237 These probably represent the conditions that maximally stimulate Th0-cell activation and induce Th1 type immune responses. Moreover, the antagonistic effects of treatments with IFN-γ and IFN-β on the course of the clinical outcome of the disease provide further indirect support for a pathogenic role of Th1 cells in MS. In some clinical trials, it has been shown that IFN-γ administration induces relapses of MS,238 probably because it upregulates MHC class II expression by microglial cells and APC-like macrophages and stimulates the secretion

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of myelinotoxic mediators (TNF-α). By contrast, IFN-β, which inhibits IFN-γ-induced MHC class II expression, has been shown to reduce the rate of relapse in MS.239 Finally, MBP- and PLP-specific TGF-β-secreting Th3 type cells have been shown in PBMC of MS patients treated with oral bovine myelin preparation, but not in the control group.240 In these patients, no increase of IFN-γ-producing cells was seen. However, a placebocontrolled double blind phase III trial of single dose bovine myelin in a large number of MS patients and controls did not show any difference between placebo and treated groups in the number of relapses; analysis of magnetic resonance imaging data showed, however, significant changes favoring oral myelin in certain patients.241 In conclusion, although in vitro and in vivo studies of the T-cell immune response in MS are not yet so clear as studies of EAE, there is convincing evidence to suggest that Th1 cells play a critical pathogenic role in this disease.

Conclusion There is now emerging evidence that the Th cell population contains functionally distinct subsets that are characterized by the patterns of cytokines they produce in response to different types of antigenic stimulation. Strong evidence now exists that Th1 and Th2 cells exist in mice, rats and humans. These two extremely polarized forms of the specific cellular immune response, evoked by intracellular parasites and gastrointestinal nematodes, respectively, provide a useful model for explaining not only the different types of protection but also the pathogenic mechanisms of several diseases. The development of polarized Th1 or Th2 in vitro is regulated by either environmental factors, including dose of antigen and nature of immunogen, or other undefined factors in the genetic background, operating mainly at the level of so-called “natural immunity” (costimulatory molecules expressed by APC, APC-released cytokines, etc.). Th1-oriented responses are effective in eradicating infectious agents, including those hidden within the cell; however, if the Th1 response is not effective or excessively prolonged, it may become dangerous for the host, due to both the activity of cytotoxic cytokines and the strong activation of phagocytic cells. By contrast, Th2 responses are not sufficiently protective against the majority of infectious agents, but they provide protection against some gastrointestinal nematodes. Th2 cells are indeed able to make the life of these complex microrganisms in the body unpleasant and, at the same time, they inhibit phagocyte activity, thus limiting attempts to destroy large parasites through Th1-mediated responses that may be harmful to the host itself. Thus, Th2 responses should also be regarded as an important downregulatory mechanism for exaggerated and/or excessively prolonged Th1 responses. Autoimmunity has been treated as being synonymous with the development of clinical disease. It is now evident that this is not the case and that it is possible to draw a clear distinction between what can be described as either destructive or non-destructive autoimmunity. Destructive autoimmunity is associated with clinical disease, whereas non-destructive autoimmune responses remain asymptomatic. The study of autoimmune disorders in the context of Th1 and Th2 T-cell responses indicates that the relative contribution of either T-cell type to the development of a particular autoimmune response can influence whether or not this response leads to clinical disease. The majority of autoimmune diseases studied, especially organ-specific autoimmune diseases, appear to be mediated by Th1 cells, the two clearest examples being EAE and its presumed human equivalent MS, and IDDM. Interestingly, in these diseases, switching from Th1 to a Th2 response can prevent Th1-mediated destruction of tissue. In other autoimmune diseases a Th1/Th2 polarization is not so clear, but there are also autoimmune disorders in which Th2 responses predominate. Th2 cells are indeed involved in SLE induced by chemicals and in

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PSS. It is of note that the chronic autoimmune diseases related to a prevalent Th2 response can be prevented by switching from a Th2 to a Th1 response. Taken together, these findings suggest that, by modulating the relative contribution of Th1/Th2 T cells to an autoimmune response, it is possible to regulate the development of clinical autoimmune disease, thus opening the way for new therapeutic strategies. These include the induction of nonresponsiveness in antigen-specific Th1/Th2 cells by antigen peptides or redirection of antigen-specific Th1/Th2 responses by exogeneous cytokines, altered peptide ligands, antigens bound to appropriate adjuvants, and plasmid DNA vaccination. Oral tolerance of autoantigens and the induction of autoreactive Th2 cells by immune deviation with selected autoantigens are the most promising approaches for redirecting Th1 responses in inflammatory autoimmune diseases such as multiple sclerosis.

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191. Foulis AK, Farquharson MA. Aberrant expression of HLA-DR antigens by insulin containing beta cells in recent onset type 1 (insulin-dependent) diabetes mellitus. Diabetes 1986; 35:1215-1224. 192. Wong FS, Visintin I, Wen L et al. CD8 T-cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells. J Exp Med 1996; 183:67-76. 193. Haskins K, Portas M, Bergman B et al. Pancreatic islet-specific T-cell clones from non-obese diabetic mice. Proc Natl Acad Sci USA 1989; 86:8000-8004. 194. Healey D, Ozegbe P, Arden S et al. In vivo activity and in vitro specificity of CD4+ Th1 and Th2 cells derived from the spleens of diabetic NOD mice. J Clin Invest. 1995; 95:2979-2985. 195. Kaufmann DL, Clare-Salzler M, Tian J et al: Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 1993; 366:69-72. 196. Trembleau S, Penna G, Bosi E et al. Interleukin-12 administration induces T helper type 1 cells and accelerates autoimmune diabetes in NOD mice. J Exp Med 1995; 181:817-821. 197. Rapoport MJ, Jeramillo A, Zipris D et al. Interleukin-4 reverses T-cell proliferative unresponsiveness and prevents the onset of diabetes in nonobese diabetic mice. J Exp Med 1993; 178:87-99. 198. Foulis AK, McGill M, Farquharson, MA. Insulitis in type 1 (insulin-dependent) diabetes mellitus in man. Macrophages, lymphocytes, and interferon-gamma containing cells. J Pathol 1991; 165:97-103. 199. Chang JC, Linarelli LG, Laxer JA et al. Insulin-secretory-granule specific T-cell clones in human insulin dependent diabetes mellitus. J Autoimm 1995; 8:221-234. 200. Mombaerts P, Mizoguchi E, Grusby MJ et al. Spontaneous development of inflammatory bowel disease in T-cell receptor mutant mice. Cell 1993; 75:275-282. 201. Sadlack B, Merz H, Schorle H et al. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 1993; 75:253-261. 202. Rudolph U, Finegold MJ, Rich SS et al. Ulcerative colitis and adenocarcinoma of the colon in G alpha i2-deficient mice. Nature Genet. 1995; 10:143-150. 203. Neurath MF, Fuss I, Kelsall BL et al. Antibodies to interleukin-12 abrogate esablished experimental colitis in mice. J Exp Med 1995; 182:1281-1290. 204. Powrie F, Correa-Oliveira R, Mauze S et al. Regulatory interactions between CD45RBhi and CD45RBlo CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity J Exp Med 1994; 179:589-600. 205. Kuhn R, Lohler J, Rennick D et al. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 1993; 75:263-274. 206. Mueller Ch, Knoflach P, Zielinski CC. T-cell activation in Crohn’s disease. Increased levels of soluble interleukin-2 receptor in serum and in supernatants of stimulated peripheral blood mononuclear cells. Gastroenterology 1990; 98:639-646. 207. Mullin E, Lazenby AJ, Harris ML et al. Increased interleukin-2 messenger RNA in the intestinal mucosa lesions of Crohn’s disease but not ulcerative colitis. Gastroenterology 1992; 102:1620-1627. 208. Parronchi P, Romagnani P, Annunziato F et al. Type 1 T helper (Th1)-predominance and IL-12 expression in the gut of patients with Crohn’s disease. Amer. J Pathol 1997; 150:823832. 209. Windhagen A, Nicholson LB, Weiner HL et al. Role of Th1 and Th2 cells in neurologic disorders. Chem. Immunol 1996; 63:181-186. 210. Renno T, Krakowski M, Piccirillo C et al. TNF-alpha expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis. Regulation by Th1 cytokines. J Immunol 1995; 154:944-953. 211. Kuchroo VK, Sobel RA, Laning JC et al. Experimental allergic encephalomyelitis mediated by cloned T cells specific for a synthetic peptide of myelin proteolipid protein. J Immunol 1992; 148:3776-3782 212. Ruddle NH, Bergman CM, McGrath KM et al. An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis. J Exp Med 1990; 172:1193-1200.

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213. Khoury SJ, Akalin E, Chandraker A et al. CD28-B7 costimulatory blockade bt CTLA4Ig prevents actively induced experimental autoimmune enecphalomyelitis and inhibits Th1 but spares Th2 cytokines in the central nervous system. J Immunol 1995; 155:4521-4524. 214. Racke MK, Scott DE, Quigley L et al. Distinct role for B7-1 (CD-80) and B7-2 (CD-86) in the initiation of experimental allergic encephalomyelitis. J Clin. Invest. 1995; 96:2195-2203. 215. Acha-Orbea H, Mitchell DJ, Timmerman L et al. Limited heterogeneity of T-cell receptors from lymphocytes mediating autoimmune enecphalomyelitis allows specific immune intervention. Cell 1988; 54:263-273. 216. Abromson-Leeman S, Alexander J, Bronson R et al. Experimental autoimmune encephalomyelitis-resistant mice have highly encephalitogenic myelin basic protein (MBP)-specific T-cell clones that recognize a MBP peptide with high affinity for MHC class II. J Immunol 1995; 154:388-398. 217. Cua DJ, Hinton DR, Stohlman SA. Self-antigen-induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice. J Immunol 1995; 155:4052-4059 218. Perrin PJ, Scott D, Quigley L et al. Role of B7:CD28/CTLA-4 in the induction of chronic relapsing experimental allergic encephalomyelitis. J Immunol 1995; 154:1481-1490. 219. Leonard JP, Waldburger KE, Goldman SJ Prevention of experimental auotimmune encephalomyelitis by antibodies against interleukin-12. J Exp Med 1995; 181:381-386. 220. Whitacre CC, Gienapp IE, Orosz CG, Bitar DM. Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy. J Immunol 1991; 147:2155-2163. 221. Fuller KA, Pearl D, Whitacre CC. Oral tolerance in experimental autoimmune encephalomyelitis: Serum and salivary antibody responses. J Neuroimmunology 1990; 28:15-26. 222. Chen Y, Inobe J-I, Kuchroo VK et al. Oral tolerance in myelic basic protein T-cell receptor transgenic mice: Suppression of automimmune encephalomyelitis and dose-dependent induction of regulatory cells. Proc Natl Acad Sci USA 1996; 93:388-391. 223. Metzler B, Wraith DC. Inhibition of experimental autoimmune encephalomyelitis by inhalation but not oral administration of the encephalitogenic peptide: Influence of MHC binding affinity. Int Immunol 1993; 5:1159-1165. 224. Miller A, Al-Sabbagh A, Santos LMB et al. Epitopes of myelin basic protein that trigger TGF-β release after oral tolerization are distinct from encephalitogenic epitopes and mediate epitope-driven bystander suppression. J Immunol 1993; 151:7307-7315. 225. Kuchroo VK, Greer JM, Kaul D et al. A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis mediated by a diverse T-cell repertoire. J Immunol 1994; 153:3326-3336. 226. Brocke S, Gijbels K, Allegretta M et al. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. Nature 1996; 379:343-346 227. Ferber IA., Brocke S, Taylor-Edwards C et al. Mice with a disrupted IFN-γ gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J Immunol 1996; 156:5-7. 228. Khoruts A, Miller SD, Jenkins MK. Neuroantigen-specific Th2 cells are inefficient suppressors of experimental autoimmune encephalomyelitis induced by effector Th1 cells. J Immunol 1995; 155: 5011-5017. 229. Lafaille JJ, Van de Keere F, Hsu AL et al. Myelin basic protein-specific type 2 T helper cells cause experimental autoimmune encephalomyelytis in immunodeficient hosts rather than protect them from the disease. J Exp Med 1997; 186:307-312. 230. Genain CP, Abel K, Belmar N et al. Late complications of immune deviation therapy in a nonhuman primate. Science. 1996; 274:2054-2057. 231. Sharief MK, Hentges R. Association between tumor necrosis factor alpha and disease progression in patients with multiple sclerosis. N. Engl. J Med 1991; 325:467-472. 232. Rieckmann P, Albrecht M, Kitze B et al. Cytokine mRNA levels in mononuclear blood cells from patients with multiple sclerosis. Neurology 1994; 44:1523-1526. 233. Brod S, Benjamin D, Hafler DA. Restricted T-cell expression of IL2/IFN-γ mRNA in human inflammatory disease. J Immunol 1991; 147:810-815. 234. Olsson T. Cytokines in neuroinflammatory disease: Role of myelin-autoreactive T-cell production of interferon-gamma. J NeuroImmunol 1993; 40:211-218.

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235. Correale J, Gilmore W, McMillan M et al. Patterns of cytokine secretion by autoreactive proteolipid protein-specific T-cell clones during the course of multiple sclerosis. J Immunol 1995; 154:2959-2968. 236. Selmaj K, Raine CS, Cannella B et al. Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions. J Clin Invest 1991; 87:949-954. 237. Windhagen A, Newcombe J, Dangond F, et al. Expression of costimulatory molecules B7-1 (CD80), B7-2 (CD86), and interleukin-12 cytokine in multiple sclerosis lesions. J Exp Med 1995; 182:1985-1996. 238. Panitch HS, Hirsch RL, Schindler J et al. Treatment of multiple sclerosis with gammainterferon: Exacerbations associated with activation of the immune system. Neurology 1987; 37:1097-1102. 239. The IFNB Multiple Sclerosis Study Group. Interferon beta-1b is effective in relapsingremitting multiple sclerosis. I. Clinical results of a multicenter, randomized, double-blind, placebo-controlled trial. Neurology 1993; 43:655-661. 240. FukauraH, Kent SC, Pietrusewicz MJ et al. Induction of circulation of myelin basic protein-specific transforming growth factor b1 secreting TH3 T cells by oral administration of myelin in Multiple Sclerosis patients. J Clin Invest 1996; 98:70-77. 241. Weiner HL. Oral tolerance: Immune mechanisms and treatment of autoimmune diseases. Immunol Today 1997; 18:335-343.

CHAPTER 6

Cytokines in Multiple Sclerosis and Its Experimental Models Tomas Olsson

I

n our present understanding of MS, to a large extent obtained through studies of its models in experimental autoimmune encephalomyelitis (EAE), activated immunocompetent cells in the systemic circulation enter the CNS where they provoke a further recruitment of inflammatory cells. Immune-mediated mechanisms then lead to damage to myelin sheaths and also, as recently emphasized, damage to neurons/axons, with ensuing neurological defects. Cytokines are by definition important for the outcome of MS and EAE, since they are the low molecular weight proteins responsible both for effector functions of immunocompetent cells for and enabling communication between them.1 Thus, studies of the regulation of cytokine production and effects thereof in MS/EAE is very important for future progress in understanding of the natural course of the disease, the effects of more or less selective immunomodulatory treatments and the genetics of the disease. This chapter will review selected aspects of these matters.

General Features of Cytokines, with Implications for Their Study and Interpretation Cytokines are low molecular weight proteins enabling communication between mononuclear cells in the immune system and are usually produced as the result of antigen stimulated cellular activation.1 A broad spectrum of target cells have high affinity receptors for cytokines, and responding cells are profoundly affected with respect to growth and differentiation. A number of basic features of cytokines neccessitate caution and thought when studying their expression in relation to disease and when conducting experimental manipulations. I will in the following section consider their redundancy, character as local hormones, mutual interactions, as well as time-, site- and dose-dependent effects.

Redundancy Certain cellular reactions can be mediated by multiple cytokines signaling through the same or different receptors. For example, tumor necrosis factor α TNF-α and lymphotoxin (LT) are distinct cytokines acting on the same receptor2 as do interferons α and β. In addition, interferon γ (IFN- γ) acts on a distinct receptor, but activates a set of genes that overlap with that of interferon α/β stimulation.3 This type of redundancy an has impact on the interpretation of results from studies on genomically deleted mice, as discussed further below. Lack of effect on a certain disease upon genomic deletion of a particular cytokine does not T-Cell Autoimmunity and Multiple Sclerosis, edited by Marco Londei. ©1999 R.G. Landes Company.

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exclude that the cytokine is indeed having important effects in the wild type mouse. Ontogenetic adaptation to the deletion may have further enforced a role of a similarly acting, partly redundant cytokine.

Local Actions A second important point is the character of cytokines as local hormones. Most of them act autocrinely or paracrinely. Therefore, to estimate the role of a cytokine it is seldom sufficient to measure free levels in body fluids such as serum or cerebrospinal fluid, and sometimes even in cell culture supernatants. Data obtained this way represent levels after local receptor binding in tissues or in the cell culture, and cytokines often break down rapidly in body fluids. Thus, it is imperative to study cellular production of cytokines in tissues or cell suspensions obtained from local sites of immune reactions. There are a series of ways to do this, each having certain advantages and disadvantages: 1. Measurement of mRNA for the produced cytokine is commonly performed by polymerase chain reaction (PCR) or in situ hybridization. Advantages of these methodologies are that any cytokine with known sequence can be studied, and there is no need for specific antibody reagents. PCR can be applied to most materials, but is seldom quantitative. Furthermore, there is always a potential problem with signal to noise ratio. If very few cells in a sample express the cytokine, the mRNA may be difficult to detect.This is often the situation with immunocompetent cells in which very few cells expressing a particular cytokine can mediate biologically meaningful effects.This phenomenon is avoided with in situ hybridization, where even a single cell expressing a particular cytokine can be detected in a sample with numerous non-expressing cells. In our studies of MS and EAE, we have employed a methodology where synthetic oligonucleotide probes are constructed based on published sequences.4,5 Even minute deviations in numbers of cells expressing cytokines such as IL-4 and transforming growth factor β (TGF-β) appeared to be relevant for the EAE disease course in certain inbred rat strains.6,7,8An obvious disadvantage with detection of mRNA is that it is always uncertain if detected levels reflect finally produced and bioactive protein. For certain cytokines, such as IFN-γ, there is quite a good correlation between numbers of cells expressing mRNA and the amount of secreted product.5,6 On the other hand, TGF-β is an example of a cytokine with a very complex regulation.The bioactive protein is cleaved off from a precursor and further subjected to binding by other proteins,9-11 so that mRNA levels cannot be expected to reflect final bioactivity. 2. Measurements by immunostaining (fluorescence detection or enzyme histochemistry gives actual numbers of cells expressing particular cytokines, and is applicable both to cell suspensions and sectioned tissues. An important point as to this methodology is that only some antibodies against cytokines function properly, and this necessitates extensive testing.12,13 This is at the same time an important limitation of the technique. It depends on the existence of proper anti-cytokine reagents.This is often a problem when working with the rat, but less so with the mouse and humans. 3. Cytokines secreted by cultured cells can be detected either in supernatants or captured to the culture well by anti-cytokine antibody. In this last case, single cells secreting the cytokine can be enumerated by applying the so-called Elispot system.14-17 These methodologies also depend on properly tested antibodies, and can be applied to cell suspensions from various body compartments. Such suspensions can also be subjected to a variety of stimuli, including specific antigens15,16 or polyclonal stimulants. I have noted that it is common to apply polyclonal stimulants such as Con A to such cultures and draw quite far reaching conclusions from this on the in

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vivo production of cytokines. An obvious drawback with cultured cells is that by necessity in vivo conditions may not be accurately reflected during in vitro culture.

Mutual Cytokine Interactions A third issue complicating assessment of the role of cytokines in a certain situation is their mutual interactions. They often either induce or downregulate each other. In descriptive studies, it is thus often difficult to decide on primary or secondary events with regard to particular cytokines. For example, expression of IFN-γ, TNF-α and IL-1 is simultaneous using certain stimuli. By use of IFN-γ receptor deleted mice, induction of IL-1 and TNF was abrogated, strongly suggesting that these appeared secondarily to induction by IFN-γ.18 When using cytokine production as outread for detection of antigen-specific T cells or studying lectin-induced cytokine production, similar complications arise. For example, a certain culture may contain cells capable of producing cytokines such as TGF-β and IL-4, both of which may dampen the production of IFN-γ. If this last-mentioned cytokine is used alone as outread for the T-cell recognition, the result will be detection of an artificially low reactivity. This type of argument is valid for any bulk culture detection of T cells and can be termed intracultural immunoregulation. Concordantly, it is important to be aware of the in vitro conditions when studying cytokine production. The additions of, for example, IL-2, IL-419 or low molecular weight substances such as glutathione,20 will strongly direct differentiation into certain cytokine profiles.

Time-, Dose- and Site-Dependency A fourth issue to consider is that: 1. The effect of a single particular cytokine may strongly depend on the timepoint during an immune response it appears, or is deliberately administered; 2. The dose given or level of endogenous production may strongly affect the outcome, opposing effects being achieved at low doses as compared to high, as has been observed for IFN-γ;21,22 3. Presence of the cytokine systemically or at the local site of an immune response drastically affects the outcome, as has been observed for TGF-β. These matters mainly complicate conclusions from studies in which cytokines are given to experimental animals. Furthermore, a possible scenario is that a cytokine given systemically to an animal may shut off the production at the local inflammatory site, with a paradoxical outcome. Accordingly, the cytokine may induce production of antibodies against the cytokine, thereby neutralizing its effects. Treatment with anti-cytokine antibodies or soluble receptor constructs may give similar paradoxical complications. Neutralization of a cytokine systemically might alleviate feedback inhibition and lead to a higher cytokine production at the local site. Treatment with anti-IL-6 has led to an increased in vivo production of the cytokine.23 A further possible complication with antibody blocking, or blocking with soluble receptor constructs, is that binding of the cytokine may protect it from breakdown, with increased sustained release of the cytokine, as was recently suggested to be a potential risk when blocking TNF.24 A series of conclusions can be drawn from even these basic features of cytokines: 1. Cytokines are difficult to study. Preferably, cellular production should be studied. When evaluating reported work one should scrutinize methods for detection, types of samples, in vitro conditions and possibilities for paradoxical effects. 2. Cytokines or anti-cytokine reagents may turn out to be difficult to apply as therapeutic agents themselves. It will be difficult to predict outcomes from in vitro effects, and effects of cytokines may differ considerably between acute monophasic models and the often chronic human

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diseases.This does not exclude a variety of therapeutic modalities aiming at changing the endogenous cytokine production, which by definition is important for the outcome of an inflammatory disease.

Polarization of the Cytokine Profile T cells have been subtyped based on their spectrum of cytokine expression with striking correlation to functional T cell abilities.25,26 This appears to be true both for CD4+ T helper cells and for CD8+ T cells.27 T1 cells function as T-effector cells mainly in delayed hypersensitivity-like reactions. This subtype produces lymphotoxin or tumor necrosis factor beta (TNF-β ) and IFN-γ, while T2 cells produce IL-4, -5, -6, -10 and -13. This subset mainly provides help for B-cell proliferation and differentiation. A third subset producing high levels of relatively isolated TGF-β has been denoted T3. The polarized T1 and T 2 subtypes have been characterized mainly using cloned long-term cultured cells in vitro. Under shortterm culture conditions, the cytokine production patterns may not be as polarized.Thus, the term T0 is used, reflecting a mixture of produced T1 and T2 cytokines. In general, the T1 and T2 subsets mutually interact, and cytokines secreted from one of them often counteract the effects or activation of the other. Preferential recruitment or bias in profile into any of the two patterns strikingly influences the course and outcome of an inflammatory disease, perhaps best illustrated in infectious diseases. Host defense against intracellular microorganisms depends on a strong T cell-mediated immunity with production of IFN-γ. The intracellular parasite Leishmania is eliminated in mouse strains mounting a strong T1-biased response, while mouse strains having a T2-dominated response succumb due to infection.28 A similar scenario is present in leprosy.29 Host defense against extracellular microorganisms such as Trypanosoma brucei depend on antibodies. Consistent with this, we have shown that mouse strains able to produce IL-4 during infection have strikingly lower levels of parasitemia than mouse strains having no such IL-4 production.30 Thus, cytokine profile bias strongly affects the outcome of certain infectious diseases and the bias is affected both by genetic and environmental factors. Some studies have addressed the same questions in autoimmune neuroinflammation, as is discussed below.

Role of Cytokines in EAE Since observations in human MS are by necessity in most cases descriptive rather than experimental, much of our current knowledge on cytokines stems from various models of EAE. In the following section, I will discuss data obtained from studies of: 1. Expression in tissues; 2. Effects on cytokine expression by immunomodulatory treatments; 3. T-cell transfer studies; 4. Selective in vivo manipulations of cytokines; and finally 5. MHC and non-MHC genetics of cytokine differentiation.

Dynamics of Cytokine Tissue Expression While a chronic disease such as MS can be expected to have both ongoing disease up and downregulatory events simultaneously with poor correlation to cytokine patterns, an acute monophasic disease such as actively induced EAE in the Lewis rat can be expected to have better correlations in this respect. We have therefore examined a number of putative disease up and downregulatory cytokines in the target tissue at distinct phases of the disease, using mRNA in situ hybridization.31-33 Shortly before clinical onset, at the time of appearance of the first inflammatory cells in the CNS, cells expressing lymphotoxin and IL-12 appear.32 Both cytokines are proinflammatory and IL-12 is an important inducer of IFN-γ production.34 In parallel with clinical signs, TNF-α and IFN-γ are abundantly expressed,

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both at the mRNA level31,32 and at the protein level.35,36 Between height of disease and recovery, TGF-β peaks, followed in the recovery period by an increasing expression of IL-10.31,32 This type of expression does not necessarily correlate with data from PCR based studies, since numbers of cells expressing cytokines above a certain threshold are detected, while PCR detects the total message in the tissue. However, increased levels of IL-10 are present in the recovery phase of mouse EAE as also detected by PCR.37 Next, we examined the expression of these cytokines in a rat EAE model with a protracted relapsing course, in DA rats immunized with the same protocol. Interestingly, target tissue expression of IL-10 and TGF-β was deficient in these rats, consistent with a causal relation between a strain-dependent deficient expression of putative disease downregulatory cytokines and chronicity.33 This is obviously a genetically regulated event which may map both to MHC and non-MHC genes, as discussed below. Thus, the kinetics of the expression of particular cytokines obviously makes sense if comparing previously made in vitro observations of cytokine function.

Effects on Cytokine Expression by Immunomodulatory Treatments Certain experimental immunomodulatory treatments also provide data consistent with expected cytokine functions. Treatment with sulfosalazine or cyclosporine A induces chronicity in otherwise monophasic Lewis rat EAE, and is paralleled by increased production of IFN-γ in the target.38,39 The anti-depressant drug rollipram, which inhibits the production of the proinflammatory cytokines IFN-γ and TNF, also ameliorates EAE.40,41 Treatment of rats with recombinant IFN-β curtails the intra-CNS production of IFN-γ. 42 These observations encourage similar studies of cytokine production during therapeutic studies with immunomodulatory drugs in humans, in order to understand their mode of action. Cytokine expression has also been studied in the context of tolerogenic treatments. Neonatal tolerance with myelin basic protein correlates with induction of a T2 biased immune response.43 Peroral tolerance with MBP induces a myelin antigen-specific immune response, with production of TGF-β and IL-4,44 consistent with a disease-promoting role of T1 biased immunity and a downregulatory function of T2- or T3-myelin-specific T cells. In agreement, therapy of mouse EAE with altered peptide ligands, either the MBP 87-9945 or PLP 139-151,46 has been reported to generate antigen-specific T cells secreting T2 cytokines rather than T1 cytokine producing pathogenic T cells. It has also been reported that CD4+ “suppressor” T cells recognizing encephalitogenic TCR produce IL-4.47 Even coimmunization with a CNS irrelevant antigen known to give rise to a T2-biased immunity, namely KLH, switched the myelin-specific T1-biased encephalitogenic response to a T2 response, and disease was abrogated.48 DNA vaccination with a TCR construct for a dominantly used and encephalitogenic TCR induced a T2-biased response linked to protection.49 However, other tolerogenic protocols using either DNA vaccination with a construct for an encephalitogenic peptide in rats (Lobell A, Weissert R, Storch M et al, unpublished data), or soluble MBP peptide,50 abrogated disease by inducing anergy. Thus, a shift to T2 is not a universal rule for tolerance induction in EAE. As discussed further below, however, autoantibody mediated attack is also a possible pathogenetic pathway. A T2-supported stimulation of such autoantibody production with ensuing myelin damage is consistent with data from MOG-induced EAE in the marmoset, in which a tolerogenic protocol induced expression of T2 cytokines and exacerbated disease.51

Transfer of Myelin-Specific T Cells with Particular Cytokine Expression Patterns T-cell transfer of myelin antigen-specific T cells with certain profiles of cytokine expression has to some extent yielded informative data. Here it is firmly established that

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myelin antigen-specific T1 cells transfer acute monophasic EAE,52-55 while T2 cells do not mediate EAE if transferred into immunocompetent hosts.54,55 However, T2 cell transfer has not unequivocally been proven to protect against EAE , while IL-4 treatment does.56 A form of “allergic” EAE was recently demonstrated after transfer of MBP-specific T2 cells into immunocompromised hosts.57 It may not be surprising, however, that such cells do what they are programmed to do, and the relevance for human disease is unclear.

Selective In Vivo Manipulations of Cytokines Attempts to understand the role of particular cytokines in EAE by a variety of other “more selective” experimental manipulations have been less rewarding and in part paradoxical, most probably due to the above discussed basic features of cytokines being redundant, acting as local hormones and having time-, site- and dose-dependent effects. In the following section, I will discuss IFN-γ, TNF, IL-4 ,TGF-β and IL-10 with respect to cytokine treatments, antibody blocking, genomic deletions and transgenic expressions. Cytokine and anti-cytokine treatments have in part given counterintuitive results. Interferon gamma is of particular interest. A brief glimpse at Table 6.1, summarizing a series of proinflammatory actions of this cytokine relevant to the central nervous system, would suggest an important disease-promoting role of IFN-γ. For example, expression of MHC antigens, activation of macrophages/microglial cells, T-cell homing, increased NO production, astrogliosis and oligodendrocyte death are all features of MS and EAE lesions. Furthermore, deliberate treatment of MS patients with IFN-γ worsened the disease course.58 In contrast to this, mouse EAE data have demonstrated that treatment with IFN-γ protected against EAE, while antibody treatment worsened disease. In accordance, EAE is inducable in both IFN-γ receptor and in IFN-γ knockout mice.59-63 These last experiments clearly demonstrate that IFN-γ is not the only cytokine that can mediate symptomatic neuroinflammatory disease, and may be an example of the redundancy in the cytokine system, especially evident in genomically deleted mice, subject to ontogenetic adaptation to the defect. If hypothesizing that IFN-γ would indeed be disease promoting, the data from antibody blocking and direct cytokine treatments are more difficult to accept. However, as discussed above, time, dose and site dependency might play a role, as well as artifactual disturbances of a variety of feedback regulations. Recently, transgenic CNS expression of IFN-γ was reported to induce hypomyelination and reactive gliosis,64 and a similar transgenic approach implicating the expression of IFN-γ in the CNS at older ages of the mice revealed a number of hallmarks of immunemediated CNS disease, with upregulation of MHC molecules, gliosis and lymphocytic infiltration.65 Thus, despite divergent experimental data, IFN-γ is still a putative diseasepromoting cytokine for MS and EAE. So far, IL-12 has performed as expected from in vitro data after its in vivo manipulation in EAE. IL-12 is a potent inducer of IFN-γ production, and its in vivo blocking with an antbody ameliorated EAE.66 Systemic injection of the cytokine promoted disease in otherwise EAE resistant mice.67 Similar scenarios to that of IFN-γ are evident when scrutinizing a variety of data on TNF and LT. Their expression patterns in the CNS during acute EAE,32, 36, 68-70 and the ability of TNF/LT-producing myelin antigen-specific cells to transfer EAE71 and oligodendrocyte/ myelin damaging effects,72,73 are consistent with a disease-promoting role of these cytokines. Systemic in vivo blocking of the cytokines either with monoclonal antibodies or soluble receptor has ameliorated EAE.74-77 Injection of TNF causes EAE relapse.78,79 Furthermore, transgenic expression of TNF leads to spontaneous demyelinating inflammatory disease.80 In spite of this, mice deleted of both LT and TNF developed full blown EAE after immunization with whole spinal cord.81 However, a knockout for LT immunized with a

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Table 6.1. Selected reported effects of IFN-γ with impact on autoimmune neuroinflammation Effect

Reference(s)

Activates macrophages/microglial cells

159,160

Induces MHC expression on glial, endothelial cells and neurons

121,126,127, 161-165

Induces IL-1 and TNF-α production

166

Induces recognition molecules involved in T-cell homing

167

Promotes B-cell differentiation into Ig secretion

168

Activates astrocytes

169

Kills oligodendrocytes

170

Induces nitric oxide production

171-173

Affects behavior

174

Increases the spinal nociceptive reflex

175

MOG peptide was protected.82 Two MS patients were treated with a TNF neutralizing antibody and clinically deteriorated.83 Thus, despite intense efforts, TNF or LT cannot be categorized as having overall harmful or beneficial effects on EAE or MS and, as with other cytokines, their disease-inducing potentials may depend on time, site and level of production as well as interplay with other cytokines. I now switch emphasis to potential disease downregulatory cytokines, with respect to their in vivo manipulations, and will subsequently discuss IL-4,TGF-β , IL-10 and IL-13. With regard to IL-4, it has been reported that direct treatment with the cytokine, simultanous with transfer of encephalitogenic T cells and MBP, led to disease abrogation.56 IL-13, a closely related cytokine, acting on the same receptor, abrogates EAE in rats.84 However, attempts to obtain protection against disease by transfer of IL-4 producing myelin antigen-specific T cells has failed.85 As discussed above, transfer of IL-4 producing myelinspecific cells to immunocompetent hosts fail to induce disease,54,85 while if transferred to immunocompromised hosts they even induce some form of allergic EAE.57 Apart from abrogation of protection in APL treatment with anti-IL-4 experiments,45 I am not aware of any study attempting to interfere with EAE using such antibodies. Nor have knockout or transgenic expression studies been reported for IL-4.

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IL-6 has been less explored. In a very delicate study, Gijbels et al86 treated mice with anti-IL-6 and determined that the treatment reduced EAE. However, this was associated with increased bioactivity in the CNS of the cytokine, suggesting the possibility that the antibody treatment caused a decreased feedback inhibition of IL-6 in the CNS. A series of potent “immunosuppressive” abilities of IL-10 makes data from selective manipulations of this cytokine intriguing. Of particular note are that, in view of its increased expression in the CNS during EAE recovery (see above), it represents the immunosuppressive activity in the CSF after Listeria infection,87 that IL-10 is more potent than IL-4 in inhibiting encephalitogenic T1 cells88 and its ability to induce long term anergy.89 While systemic IL-10 treatment prevents EAE in rats,90 it even worsened disease in sjl/j mice.91 A variety of reasons, as discussed above, may lie behind these discrepancies. I am am not aware of any studies using anti-IL-10 antibody blocking, gene deletion or transgenic expression. TGF-β stands out as a potentially very important “immunosuppressive” cytokine, especially in view of data reported on its genomic deletion, which resulted in a widespread inflammatory disease.92 Furthermore,genetically regulated differences in TGF-β expression either in the CNS or by immunocompetent cells after myelin antigen recognition (see below) correlate to differences in susceptibility to EAE. With this cytokine, direct treatment or antibody blocking has given the expected suppression or enhancement of EAE, respectively.93-95 I am not aware of any experiments with its transgenic expression in the context of EAE.

Genetics of Cytokine Expression in EAE There is a strong genetic influence on both MS and EAE, perhaps best illustrated in the human disease by twin studies, for which there is a concordance rate of 25-30% for monozygotic, as opposed to 2-4% for dizygotic twins.96 The nature of the genetic influences as well as their positional clonings are very important for definition of relevant therapeutic targets. If one accepts that MS and EAE are diseases mediated by pathogenic immune responses and that cytokines are key regulators in these responses, it is likely that polymorphic genes which determine disease susceptibility and severity act by affecting the magnitude and quality of cytokine production, or by action on target cells. This might occur either by polymorphisms in the cytokine or cytokine receptor genes themselves, or in any step that regulates cytokine interactions. Alternatively, these features may well be secondarily affected. In either case, evaluation of cytokines in the genetics of neuroinflammatory disease may give important clues on how a genetic influence is mediated and be helpful in phenotypic evaluation in the context of attempts for positional cloning. In general, the genetics of both MS and EAE can be divided into that of the MHC complex and that of non-MHC background genes. In both the human disease97-100 and its experimental models101,102 there is firm evidence for a strong influence on disease susceptibility from genes in both locations. In the following section, I will discuss cytokine expression in relation to these in EAE (for MS see further below).

MHC Influences on Cytokine Differentiation and Disease There are data emerging with regard to the MHC complex and cytokine expression in EAE. The MHC complex contains numerous immunoregulatory genes with possible impact on both disease and cytokine expression. So far, influences have not been mapped precisely to single genes within this complex, though there has been mapping to some extent to subregions, e.g., the regions containing the genes for the antigen-presenting class I and II molecules. We recently analyzed the contribution of the MHC complex to relapsing disease in the DA rat which develops after immunization with whole spinal cord in this strain, but not in the more commonly used Lewis rat.33,103 By varying the non-MHC background genes on

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the DA rat MHC complex (RT1av1), and comparing with the same backgrounds on other MHC haplotypes using MHC congenic rats, it was demonstrated that relapsing disease correlated to the RT1av1 haplotype. Furthermore, while strains displaying no chronicity had a conspicuous expression of TGF-β and IL-10 in the CNS, relapsing RT1av1-bearing strains lacked this expression of putatively immuno-downmodulatory cytokines.104 One can thus conclude that polymorphisms of genes within the MHC complex affect cytokine expression. However, at present the RT1av1 regulatory influence has not been mapped within the MHC; neither is it clear if regulation primarily affects cytokine regulation or if the different expression patterns are secondary to some other event. In another set of experiments, using MHC congenic rats on the EAE permissive Lewis background, we have analyzed MHC disease restriction patterns in response to immunization with encephalitogenic MBP peptides in relation to ex vivo responses of primed lymphoid cells to the peptide in the form of elicited cytokine responses.6,7,8 From experiments using immunization with MBP 63-88, it became clear that the MHC haplotype influences could be categorized into three different forms: 1. Disease permissive haplotypes in which ex vivo T-cell responses were characterized by a pure T1 type cytokine response, with production of IFN-γ in response to the peptide; 2. Disease resistant haplotypes with absence of T-cell responses to the peptide ex vivo. In this case this may be due either to absence of proper MHC class II molecule peptide binding or to an efficient thymic deletion of any putative peptide autoreactive T cell. 3. The third category is especially interesting. Certain rat MHC haplotypes did not allow disease to develop, but displayed an autoreactive response to the peptide ex vivo. This response was characterized by a production of TGF-β and T2 cytokines such as IL-4, in addition to IFN-γ production. It is thus suggested that not only the peptide response per se, but in addition the MHC-regulated cytokine pattern in response to a potentially encephalitogenic peptide, determines the outcome of an autoimmune response. Use of MHC congenic rat strains with recombinations between the MHC class I and II genes allowed mapping of the TGF-b response to the class I region and to a particular class I allele.The protective inluence conferred by this allele could also be ameliorated by depletion of CD8+ cells with antibodies, suggesting that there are allele-specific class I-restricted CD8+ “suppressor” cells.7 In addition to the allele class I-mediated protection, there was a class II region protective influence, explaining the T2 T-cell response. Interestingly, it was recently demonstrated that varying class I alleles in humans changes the risk for MS.105 Interestingly, these influences are peptide and MHC haplotype-specific, since similar protective influences gave a different MHC restriction pattern using another MBP peptide (89-101).8 Although the basic molecular mechanistic reason for the MHC influence on cytokine differentiation and disease outcome is yet not clear, the latter finding suggests that it may be determined by the peptide-MHC-TCR interaction and not by neighboring genes. Studies have reported a correlation between peptide binding strength and T1/T2 cytokine differentiation, so that strong peptide binders provoke a pure T1 response, while weak binders provoke a T2 response.106 One could argue that the altered peptide ligand therapy represents a means by which one could utilize the peptide-MHC-TCR interaction regulating cytokine differentiation for therapeutic purposes, since at least some of the basic studies on this suggest that the therapeutic effect is due to production of immuno-downmodulatory cytokines in response to the APL.45,46

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It will be of interest to explore whether parts of the MHC influence on human MS can be explained by regulation of the cytokine spectrum. Interestingly, one recent study demonstrated that T-cell lines obtained from DR2+ donors secreted significantly more TNF-α and lymphotoxin than T-cell lines from DR2– donors.107 Since the genes for TNF and LT are located in the MHC complex, it is speculated that the MHC influence on MS may be due to polymorphisms in these genes, but to date no influence has been determined that is independent from that of the class II genes.108,109

Non-MHC Background Gene Effects on Cytokine Differentiation and Disease The effects of background genes on inflammatory diseases was first explored in infections in which strain-dependent T1- or T2-biased immune responses were decisive for the outcome of the disease (see above). One recent example of non-MHC background genetic influences on T1/T2 phenotype development is a genetically determined difference in maintenance of IL-12 receptor expression, partially localized to a chromosomal region.110,111 Data has also started to emerge in these respects with regard to EAE. Availability of microsatellite-based genetic mapping techniques for both mice and rats112 will allow the first steps in the molecular cloning of these influences. In mouse EAE it has been demonstrated that a strain-dependent unresponsiveness to EAE induction correlates to an inability of T cells to produce IFN-γ in vitro. Strikingly, treatment with IL-12, a potent inducer of T1 differentiation, allowed disease development in the resistant strain.67 In another study in mice, a genetic difference influencing the T1/T2 commitment correlated to ability of T-cell lines from the respective strains to transfer EAE, and this difference mapped outside the MHC.113 We have made observations in rat EAE. Using intercrosses between the EAE resistant E3 strain and the EAE susceptible DA strain, a non-MHC genetic resistance correlated with the ability of lymphoid cells to express mRNA for TGF-β in response to an encephalitogenic peptide ex vivo.114 In a F2 cross between EAE susceptible DA rats and resistant BN rats, we have demonstrated that one gene region outside the MHC has a dramatic disease-promoting influence, in turn correlating to increased expression of mRNA for IFN-γ (Dahlman I, unpublished data). In another set of experiments we have studied MOG-induced EAE in different rat strains. Immunization with MOG leads to a very MS-like disease both clinically and histologically. Here, non-MHC background gene-determined low susceptibility to disease correlates to inability of lymphoid cells to produce IFN-γ in response to antigen. (Weissert R, Wallstrom E, Storch M et al., unpublished data). In one F2 cross between DA and PVG1 av1 rats, disease susceptibility and a T1 bias as measured by anti-MOG isotype pattern were mapped to a particular gene region (Dahlman I, Lorentzen J, Graaf K et al, unpublished data). Thus , at present there are strong indications of non-MHC background influences on both EAE and cytokine differentiation, and it will be important to positionally clone these, as well as to determine any causal relationships between the two.

Cytokine Expression and Target Immune Reactivity Cytokines produced in the target during neuroinflammatory disease can originate principally from infiltrating lymphoid cells, but also from CNS resident cells such as microglia, astrocytes and even neurons.This will have an impact on the “target immune reactivity” and the regulation of this last-mentioned production and its “quality” may well affect the outcome of a CNS-directed autoimmune response, or any other insult to the CNS. In addition, primarily non-autoimmune conditions such as trauma and infection may hypothetically influence the target immune reactivity and susceptibility to autoimmune conditions by inducing a variety of cytokine cascades.

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Evidence for these types of regulation have been obtained to some extent in EAE combined either with viral infections115 or with nerve trauma.116 As to the latter types of experiments, peripheral nerve trauma outside the central nervous system may be employed, avoiding any direct penetration leading to disturbances of the blood brain barrier. Subjecting a peripheral nerve to axotomy, by a crush or cut injury, will lead to a retrograde response in the corresponding nerve cell bodies located within the CNS. The neurons readjust their transmitter related protein synthesis to production of growth related proteins.117-119 Some years ago it was observed that this nerve cell body response was also accompanied by induction of immunological recognition and effector molecules in neurons and surrounding glia. Thus , neurons and microglial cells start to express MHC class I molecules.120,121 MHC class II molecules only appear on glial cells.122 These molecules are classically induced by cytokines such as IFN-γ. Therefore, elicitation of a local cytokine cascade was suspected. If this experimental maneuver was combined with an active EAE induction, a much stronger infiltration of T cells and other inflammatory cells was observed in the vicinity of reacting nerve cell bodies than elsewhere.116 In a subsequent study, nerve trauma alone was determined to expand MBP-autoreactive T cells, which in the EAE susceptible Lewis strain led to a subclinical EAE, but not in the EAE resistant BN strain. In this study, infiltration of inflammatory cells was also more outspoken near injured nerve cell bodies.123 These studies demonstrate in principle that: 1. Neurons can regulate the degree of intra-CNS immune reactivity; and 2. Target immune reactivity is important to the degree of CNS inflammation. It will be important to exactly define the molecular playmates in these experimental paradigms and any potential genetically regulated bottlenecks in them. Some progress has been made in these respects. A first cytokine to consider in this context is IFN-γ in view of its potent MHC class I- and II-inducing capability and its generally potent proinflammatory abilities (see Table 6.1). We noted a strong upregulation of IFN-γ-like immunoreactivity in motorneurons after peripheral axotomy,122 detectable with two non-competetive monoclonal antibodies (unpublished data). Also, upregulation of NADPH diaphorase and IFN-γ like immunoreactivity in neurons correlate.124 Neuronal IFN-γ isolated from sensory ganglion cells, in which small sized neurons constitutively express the material, reveal IFN-γ bioactivity tested in different ways.125 An endogenous MHC class I upregulating capability of sensory neuron IFN-γ has been reported.126,127 However, detection of mRNA for classical IFN-γ with methods that readily detect lymphocyte-derived cytokine, has failed (ref. 128 and our own unpublished data), and therefore the nature of the neuronal IFN-γ-like material is still unclear. Other cytokines also appear in neurons and /or surrounding cells. Reportedly,IL-6 and GM-CSF are upregulated,129,130 as well as TGF-β.131 Precise experimental tools able to influence these cytokines will be needed to determine their significance for inflammatory CNS damage. There are certain indications that target cytokine production may be genetically influenced. Thus, certain cytokines are produced much more vividly by astrocytes from Lewis rats that from BN rats,132 and the macrophage reaction in response to CNS trauma is more intense in Lewis rats that in outbred Sprague Dawley rats.133 In our own still unpublished experiments, non-MHC background genes are decisive for a much stronger microglial cell activation and neuronal death following nerve avulsion in rats (Piehl F, Lundberg C, Olsson T, unpublished). Genetic mapping and molecular characterization of these target inherent cytokine/inflammation-related events will be important.

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Cytokines in MS Since MS arises due to an inflammatory attack on components of the CNS, and inflammation by definition is orchestrated by cytokines, an understanding of their function, interplay, genetic regulation and alteration by therapy is very important in this disease. However, as discussed above, their study is complicated by a series of factors already evident in experimental models. In human disease of the CNS, a series of additional complicating factors arise which should be taken into account when designing cytokine studies in MS and when evaluating published reports. The first matter to consider is the chronic nature of MS. Even if very clear deviations in cytokine patterns have occurred at early phases of disease, this may well be indiscernible at the late stages for which we have access to samples for study. Furthermore, in the chronic phase years after onset, disease up and downregulatory events may well occur simultaneously in different lesions, and perhaps even in single lesions. It seems likely that a kind of chaotic dysregulation may prevail, in which it will be difficult to discern clear-cut correlations to cytokines with defined experimental functions. This may well be the case even if the cytokines indeed perform their functions as suspected based on experimental systems. A second principal obstacle is the relative inaccessibility of the target tissue. The local character of cytokines prompts investigations of local productions. Autopsy and biopsy material is seldom accessible from the CNS, and sequential studies of such materials during disease courses or treatment regimens is of course not possible. Cytokine studies in MS are therefore in most cases restricted to peripheral blood specimens. Material from the cerebrospinal fluid (CSF), in which mononuclear cells to some degree may reflect events ongoing in the CNS parenchyma, is somewhat better. It is important to distinguish data obtained by analysis of fresh samples from those obtained after in vitro culture, either short term or long-term. The last-mentioned are subject to numerous in vitro regulatory events that may distort any pattern that may have occurred in vivo. In spite of these shortcomings, some data have also accumulated on cytokine expression and patterns in MS, which will be discussed in the following section regarding expression in fresh samples and antigen-induced conditions. Brief remarks will be made on relation to therapy and genetics. I will mainly discuss reports on cellular expression of cytokines, with the belief that this is more relevant than free levels detected in body fluids. Firstly, a number of putative disease-promoting cytokines have been measured in MS. TNF-α, LT and IFN-γ have all been localized to MS lesions with immunohistochemistry.134-136 Cells producing IFN-γ are detected at increased levels in the CSF of MS patients , as compared to controls.16 One study reports increased levels of TNF in the CSF, correlating to active disease.137 In addition, with PCR, increased levels of mRNA for TNF appeared to precede clinical relapses.138 In other settings, it has been difficult to detect any clear-cut correlations to independent disease parameters with any of these cytokines. A very consistent finding in our own laboratory from a few years back, using in situ hybridization with synthetic oligonucleotide probes for cytokines, is that MS patients display increased numbers of peripheral blood cells expressing mRNA for these proinflammatory cytokines. There is, furthermore, a strong enrichment of such cytokine-expressing cells among CSF cells.139-141 We have failed, however, to detect any clear relations to clinical variables. This does not exclude a disease-promoting role of these cytokines, however, due to reasons discussed above. The findings also encourage attempts to study cytokine expression in relation to therapeutic interventions and genetic variables. The in situ mRNA detection can be combined with surface phenotype staining using immunohistochemistry. This methodology allows study of activated cells in vivo for cytokine expression belonging to the CD4+ or CD8+ phenotype. We recently found that approximately

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30% of the IFN-γ mRNA-expressing cells belonged to the CD8+ phenotype, suggesting that MHC class I-restricted cells of this phenotype may be active during MS (Wallström E, Khademi M, Andersson A, Olsson T). This is interesting in view of the potential for both disease-promoting and -protective roles of CD8+ cells in experimental autoimmune disease,7,142,143 in certain cases also shown to be class I haplotype-specific,7 and in view of the recent description of risk-increasing class I alleles in human MS.105 There is less data on putative disease downregulatory cytokines analyzed in fresh MS samples. With the same in situ hybridization methodology, we have observed increased numbers of cells expressing mRNA for TGF-β, IL-4, IL-10 and IL-13.139-141,144,145 There are also higher frequencies of cells expressing these cytokines in the CSF. The only consistent correlation observed so far to clinical variables is an increased number of cells expressing TGF-β mRNA in patients with mild MS compared to severe MS.140 Once again, this paucity of correlations to clinical variables does not exlude important disease regulatory effects of the cytokines, but rather encourages further attempts for studies during therapy and in relation to genetic factors. Detection of cytokines as a consequence of antigen-induced activation in vitro represents a different approach. Here, the cytokine production can be regarded as a measure of T cells with a certain specificity. Since the cytokines produced are strongly affected by the in vitro conditions used, one cannot easily make conclusions about differences in cytokine profiles in the in vivo situation. However, cytokine production may be a good way to measure specific T cells. Traditionally, antigen-specific T cells have mainly been measured with assays of proliferation or frequency of T-cell clones reactive with a certain antigen. In MS, a lot of interest has of course been focused on myelin antigens, which have been proven to be encephalitogenic in animals. Measures of bulk culture proliferative responses have mainly failed to reveal any differences in myelin antigen-autoreactive T cells between MS patients and controls.146 This is also largely the case with T-cell cloning procedures.147-149 Using cytokine production as outread for myelin antigen responses, either with so-called Elispot analysis or in situ hybridization, a very consistent finding has been increased numbers of cells in MS patients compared to controls that recognize a series of myelin antigens such as MBP, PLP and MOG. 16,141,150-153 Both putative disease-promoting and immunodownmodulatory cytokines appear in response to these antigens. In this case, there is also a prominent enrichment of memory cells to the CSF. Myelin peptide-specific responses can also be studied in this way.151 We found that the response to several MOG peptides increased in DR2+ MS patients as compared to healthy DR2+controls. One particular peptide was immunodominant, MOG 63-87 (Wallström E, Khademi M, Andersson M et al., unpublished). Thus, even if immune responses are increased to several myelin antigens in MS, there are prospects for definition of a limited set of myelin antigen epitopes which might be approached with specific immunotherapy. In view of the data in MOG EAE, in which MOG-induced IFN-γ production but not MOG-induced proliferation, discriminated between disease susceptibility and resistance (see above), we think this might be a valid way to measure autoimmune responses in humans. It might also be a way to measure antigenspecfic T-cell responses during a variety of more or less selective immunotherapeutic interventions in humans. The only treatment so far with any proven effect on the natural course of MS is interferon-β. It therefore seems attractive to analyze how this treatment affects cytokine expression systemically and in the CNS. Data obtained so far are quite limited, and in most cases confined to analysis of the systemic compartment. Dayal et al154 reported an increased number of cells in the peripheral blood producing IFN-γ in response to con A. In contrast, rather soon after initiating therapy, Rudick et al155 recorded an induction of IL-10 production. When studying cytokine production at a later stage, 2-6 months after initiating therapy, at a

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timepoint when the therapeutic effects start to become evident, we instead recorded a decreased number of IL-10-expressing cells.156 It is still unclear if these cytokine deviations: 1. Have any casual relationships to the therapeutic effect; 2. Represent unwanted disease-promoting phenomena while the therapeutic effect targets another mechanism; or 3. Represent inert epiphenomena. Genetic influences on cytokine expression in MS have so far only been explored superficially. A strong indication that cytokine bias may play a role comes from an epidemiologic study in which IgE-mediated disease known to be T2-biased was strongly underrepresentated in patients with MS.157 With respect to HLA influences, T-cell lines from DR2+ subjects were demonstrated to produce increased amounts of TNF as compared to DR2– subjects.106 Accordingly, HLA DR1-, 2- and 6-positive individuals have been reported to respond in mixed lymphocyte reactions with higher IFN-γ production than DR3-, 4-, 5- and 7-positive ones.158 The relevance of these findings for disease is yet not clear, nor is the gene locus within the HLA complex. It can be anticipated that this area of cytokine research will expand, especially when loci affecting disease and cytokine expression will be defined in the animal models. In conclusion, the cytokine orchestration is by definition important for the outcome of neuroinflammatory disease. A number of basic features of cytokines makes them difficult to study, and also difficult to manipulate, at least if one wishes to obtain predictable effects, often necessary when going into therapeutic trials in humans. However, properly studied, very important information can be obtained in relation to T-cell specificities, genetics and trials of more or less selective immunomodulatory treatments.Thus, despite their complex nature, I expect continued intense studies of cytokines in both EAE and MS.

Acknowledgments Studies cited from the author´s laboratory have received grant support from the Swedish medical research council, the Swedish society for neurologically disabled, the AFA foundation, Petrus and Augusta Hedlunds foundation, Bibbi och Nils Jenssens foundation and the EC Biomed 2 program. I thank Bob Harris for linguistic advice and Robert Weissert for viewpoints on the manuscript.

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49. Waisman A, Ruiz PJ, Hirschberg DL et al. Suppressive vaccination with DNA encoding a variable region gene of the T-cell receptor prevents autoimmune encephalomyelitis and activates Th2 immunity. Nature Med 1996; 2:899-905. 50. Marusic S, Tonegawa S. Tolerance induction and autoimmune encephalomyelitis amelioration after administration of myelin basic protein-derived peptide. J Exp Med 1997; 186:507-515. 51. Genain CP, Abel K, Belmar N et al. Late complications of immune deviation therapy in a nonhuman primate. Science (Wash DC) 1996; 274:2054-2057. 52. Sedgwick JD, McPhee JHM and Puklawec M. Isolation of encephalitogenic CD4+ T-cell clones in the rat. Cloning methodology and IFN-γ secretion. J Immunol Meth 1989; 143:3492-3497. 53. Ando DG, Clayton J, Kono D et al. Encephalitogenic T cells in the B10. PL model of experimental allergic encephalomyelitis (EAE) are of the Th-1 lymphokine subtype. Cellular Immunol 1989; 124:132-143. 54. Baron JL, Madri JA, Ruddle NH et al. Surface expression of a VLA-4 integrin by CD4 T cells is required for their entry into brain parenchyma. J Exp Med 1993; 177:57-68. 55. Van der Veen R, Kapp JA, Trotter JL. Fine-specificity differences in the recognition of an encephalitogenic peptide by T helper 1 and 2 cells. J Neuroimmunol 1993; 48:221-226. 56. Röcken M, Racke M, Shevach EM. IL-4-induced immune deviation as antigen-specific therapy for inflammatory autoimmune disease. Immunol Today 1996; 17:225-231. 57. Lafaille JJ, Van de Keere F, Hsu AL et al. Myelin basic protein-specific T helper 2 (Th2) cells cause experimental autoimmune encephalomyelitis in immunodeficient hosts rather than protect them from disease. J Exp Med 1997; 186:307-312. 58. Panitch HS, Hirsch RL, Schindler J et al. Treatment of multiple sclerosis with gamma interferon: Exacerbations associated with activation of the immune system. Neurol 1987; 37:1097-1102. 59. Billiau A, Heremans H, Vandekerckhove F et al. Enhancement of experimental allergic encephalomyelitis in mice by antibodies against IFN-γγ. J Immunol 1988; 140:1506-1510. 60. Duong TT, St-Louis J, Gilbert JJ et al. Effect of anti-interferon-γγ and anti-interleukin 2 monoclonal antibody treatment on the development of actively and passively induced experimental allergic encephalomyelitis in the SJL/J mouse. J Neuroimmunol 1992; 36:105-115. 61. Voorthuis JAC, Uitdenhaag BMJ, de Groot CJA et al. Suppression of experimental allergic encephalomyelitis by intraventricular administration of interferon-γγ in Lewis rats. Clin Exp Immunol 1990; 81:183-188. 62. Krakowski M, Owens T. Interferon-γγ confers resistance to experimental allergic encephalomyelitis. Eur J immunol 1996; 26:1641-1646. 63. Willenborg DO, Fordham S, Bernard C et al. IFN-γ plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encepyhalomyelitis. J Immunol 1996; 157:3223-3227. 64. Corbin JG, Kelly D, Rath EM et al. Targeted CNS expression of interferon-γγ in transgenic mice leads to hypomyelination, reactive gliosis, and abnormal cerebellar development. Mol and Cellular Neuroscience 1996; 7:354-370. 65. Horwitz MS, Evans CF, McGavern DB et al. Primary demyelination in transgenic mice expressing interferon-γγ. Nature Medicine 1997; 3:1037-1041. 66. Leonard JP, Waldburger KE, Goldman SJ. Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12. J Exp Med 1995; 181:381-386. 67. Segal BM and Shevach EM. IL-12 unmasks latent autoimmune disease in resistant mice, J Exp Med 1996; 184:771-775. 68. Renno T, Krakowski M, Piccirillo C et al. TNF-α expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis. J Immunol 1995; 154:944-953. 69. Baker D, O´Neill JK, Turk JL. Cytokines in the central nervous system of mice during chronic relapsing experimental allergic encephalomyelitis. Cell Immunol 1991; 134:505-510.

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137. Sharief MK, Hentges R. Association between tumor necrosis factor-α and disease progression in patients with multiple sclerosis. New England J Med 1991; 325:467-472. 138. Rieckmann P, Albrecht M, Kitze B et al. Cytokine mRNA levels in mononuclear blood cells from patients with multiple sclerosis. Neurology 1994; 44:1523. 139. Link J, Söderström M, Kostulas V et al. Optic neuritis is associated with myelin basic protein and protwolipid protein reactive cells producing IFN-γ, IL-4 and TGF-β. J Neuroimmunol 1994; 49:9-18. 140. Link J, Söderström M, Olsson T et al. Increased TGF-β, IL-4 and IFN-γ in multiple sclerosis. Ann Neurol 1994; 36:379-386. 141. Navikas V, He B, Link J et al. Augmented expression of tumor necrosis factor α and lymphotoxin mRNA in mononuclear cells in multiple sclerosis and optic neuritis. Brain 1995; 119:213-223. 142. Zhang G, MaC, Xiao B, Bakhiet M et al. Depletion of CD8+ cells suppresses the development of experimental autoimmune myasthenia gravis in Lewis rats. Eur J Immunol 1995; 25:1191-98. 143. Zhang G, Xiao B, Bakhiet M et al. Both CD4+ and CD8+ T cells are essential to induce experimental autoimmune myasthenia gravis. J Exp Med 1996; 184:349-356. 144. Navikas V, Link J, Palasik W et al. Increased mRNA expression of IL-10 in mononuclear cells in multiple sclerosis and optic neuritis. Scand J Immunol 1995; 41:171-178. 145. Matusevicius D, Kivisäkk P, Navikas V et al. Autoantigen-induced IL-13 mRNA expression is increased in blood mononuclear cells in myasthenia gravis and multiple sclerosis. Eur J Neurol 1997; 4:468-475. 146. Johnson D, Hafler DA, Fallis RJ et al. Cell-mediated immunity to myelin-associated glycoprotein, proteolipid protein and myelin basic protein in multiple sclerosis. J Neuroimmunol 1986; 13:99-108. 147. Tournier-Lasserve E, Hashim GA, Bach MA. Human T-cell response to myelin basic protein in multiple sclerosis patients and healthy subjects. J Neurosci Res 1988; 19:146-156. 148. Ota K, Matsui M, Milford EL et al. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 1990; 346:183-187. 149. Pette M, Fujita K, Kritze B et al. Myelin basic protein-specific T-lymphocyte lines from MS patients and healthy individuals. Neurology 1990; 40:1770-1776. 150. Link J, Frederikson S, Söderström M et al. Organ-specific autoantigens induce transforming growth factor β mRNA expression in multiple sclerosis and myasthenia gravis. Ann Neurol 1994; 35:197. 151. Olsson T, Sun J, Hillert J et al. Increased numbers of T cells recognizing multiple myelin basic protein epitopes in multiple sclerosis. Eur J Immunol 1992; 22:1083-1087. 152. Sun J, Olsson T, Wang W et al. Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. Eur J Immunol 1991a; 21:1461. 153. Sun J, Link H, Xiao B et al. T and B cell responses to myelin-oligodendrocyte glycoprotein in multiple sclerosis. J Immunol 1991b; 146:1990. 154. Dayal AS, Jensen MA, Lledo A et al. Interferon-gamma-secreting cells in multiple sclerosis patients treated with interferon β-1b. Neurology 1995; 45:2173-2177. 155. Rudick RA, Ransohoff RM, Peppler R et al. Interferon beta induces interleukin-10 expression—relevance to multiple sclerosis. Ann Neurol 1996; 40:618-627. 156. Andersson M, Khademi M, Wallström E et al. Cytokine profile in interferon-beta treated multiple sclerosis patients: Reduction of interleukin-10 mRNA expressing cells in peripheral blood. Eur J neurol 1997; 4:567-571. 157. Oro AS, Guarino TJ, Driver R et al. Regulation of disease susceptibility: Decreased prevalence of IgE-mediated allergic disease in patients with multiple sclerosis. J Allergy Clin Immunol 1996; 97:1402-1408. 158. Petrovsky N, Harrison LC. HLA class II-associated polymorphism of interferon-γ production. Implications for HLA-disease association. Human Immunol 1997; 53:12-16.

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CHAPTER 7

Antigen-Specific T-Cell Responses in Autoimmune Demyelinating Disease Johannes M. van Noort and Sandra Amor

I

n MS, the target tissue of inflammatory damage is CNS white matter. With the exception perhaps of the eyes, other parts of the body such as the peripheral nervous system are affected only rarely. As with other organ-specific autoimmune diseases, it is not fully clear what determines this apparent tissue specificity of the inflammatory process in MS. Possible disease-contributing factors that could play a role in this respect include infection by a neurotropic pathogen, a selective homing pattern by autoreactive lymphocytes into the CNS, their selective recruitment by an activated white matter blood-brain barrier or the contribution by white matter-specific antibodies. At first sight, however, the most straightforward explanation for the tissue selectivity in MS appears to be restricted expression of the relevant target antigens to CNS white matter. Although this assumption has directed the search for target antigens in MS primarily to myelin, it may not be fully accurate. Recent advances in basic immunology have clarified that autoantigens which can trigger tissue-restricted inflammation do not necessarily have to be restricted in their expression to that particular tissue. Studies in mice have demonstrated that tissue-restricted expression of a target antigen under the control of a transgene in an appropriate background of potentially reactive T cells will not trigger an inflammatory response unless costimulatory molecules are coexpressed at the same site. 1 T-cell receptor transgenic mice with a monospecific T-cell repertoire directed against tissue-restricted antigens such as MBP usually fail to develop autoimmune disease unless additional costimulatory factors are present, such as those which accompany microbial infection.2 Thus, the development of inflammation depends on the combination of target antigen and costimulatory molecules and either one of these factors alone will not initiate reactivity. Given this dual signal requirement of T cells, autoantigens for tissue-restricted inflammation may well be expressed elsewhere in the body without causing problems at those other sites if the lack of costimulatory molecules renders them locally ‘invisible’ to potentially reactive T cells. Bearing these findings in mind, it still appears reasonable to assume that autoantigens relevant to the development of autoimmune demyelinating diseases are expressed in CNS white matter or, more specifically, that they are associated with myelin sheaths and glia cells. Yet, they need not be restricted in their expression to CNS white matter, since additional costimulatory factors will have to accompany the antigen in the target tissue to allow it to be recognized by the immune system.

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In this chapter, we will discuss current data and ideas on the role of antigen-specific T-cell responses in the development of MS as well as of animal models for MS. We will limit ourselves to reactivity of CD4+ helper T cells that carry the classical αβ T-cell receptor. Much remains to be established on the possible contribution by antigen-specific CD8+ T cells or γδ T cells. We will emphasise the dazzling complexity of myelin as a source of protein antigen, pointing out that myelin contains a dynamic, complex array of protein structures. We will review data on the relevance of selected antigen-specific T-cell responses to the development of inflammatory responses in animal models of MS, on the antigens to which these responses are directed and on how such responses may change during the course of chronic disease. We will summarize the data that have been reported on antigen-specific responses in MS patients as detected in peripheral blood or in the cerebrospinal fluid. Again, the issue of chronicity will be discussed, and how this may affect T-cell responses over time. Finally, we will make an attempt to summarize current data in order to give a perspective on how antigen-specific responses may be selectively targeted as an approach to therapy in MS.

Myelin Antigens CNS Myelin: A Dynamic Complexity of Potential Antigens As explained above, it appears reasonable to assume that autoantigens relevant to MS are to be found in CNS myelin, although they may well also be expressed elsewhere in the body. Myelin sheaths are composed of 75-80% lipids and 20-25% proteins. The two most abundant proteins in myelin are the hydrophobic transmembrane protein PLP that accounts for about 50% of the total protein mass in myelin and the strongly hydrophilic MBP that represents 10 to 15% of all protein. In addition to these abundant proteins, myriad other proteins are found associated with CNS myelin, each of which usually does not represent more than 1% of the total protein mass. These other proteins include structural proteins such as MAG and MOG that play an important role in the maintenance of the myelin structure and its interactions with other cellular components, as well as enzymes such as CNPase, transaldolase-H, kinases, acyltransferases and methylases that can modify the myelin structure upon receiving the appropriate signals. In addition, molecular chaperones such as the small stress protein αB-crystallin can also be found in purified CNS myelin. Apart from the fact that a multitude of different proteins can be found in CNS myelin, many of them still unidentified, two additional factors greatly contribute to the structural complexity of myelin proteins. The first is differential splicing of primary transcripts that encode myelin proteins.3 As a rule, the splicing process inside the oligodendrocyte generates multiple forms of RNA transcripts that encode the different myelin proteins. These transcripts differ in their exon composition, which results in the biosynthesis of protein variants that share some polypeptide segments but are different in others. In this way, the PLP gene, for example, gives rise to two different proteins designated PLP and DM-20, and the MBP gene can give rise to as many as four different versions of the MBP molecule. Also, gene transcripts encoding MOG, MAG or CNPase emerge as differentially spliced messenger RNAs which lead to the production of different molecules in each case. To further add to the complexity, patterns of RNA splicing will change during ontogeny and as a result of tissue damage or disease, which may therefore lead to different protein compositions of CNS myelin as a function of these external factors.4 A second factor that contributes to the structural complexity of CNS myelin is posttranslational modification. Again, as a rule, CNS myelin proteins are covalently modified following their biosynthesis.5 Glycosylation, acylation, acetylation, methylation, deamidation, deimination and phosphorylation are among the common modifications that CNS myelin proteins undergo. Patterns of such modification change as the individual develops or as

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Table 7.1. Post-translational modifications of MBP and their relevance to immune recognition Modification

Modified Residues

Immunological Relevance

acetylation

Ala-1

T-cell reactivity (ref. 8) antibody reactivity (ref. 9)

phosphorylation

Ser-7 Ser-12 Ser-56 Thr-98 Ser-102 Ser-115 Ser-136

antibody reactivity (ref. 10)

deimination

Arg-9 Arg-25 Arg-33 Arg-122 Arg-130 Arg-159 Arg-170

T-cell reactiviy (refs. 11,12) antibody reactivity (ref. 9)

methylation

Arg-107

glycosylation

Thr-95 Thr-98

oxidation

Met-21

ADP-ribosylation

Arg-9

damage or stress occurs and tissue regeneration takes place.6,7 In Table 8.1, a summary is given of modifications that can be found in MBP, but it should be stressed that in fact all individual myelin proteins known to date are posttranslationally modified in one or more ways. In the case of MBP, the special lipid-bound form of the protein is also currently investigated for potential relevance as an autoantigen in demyelinating disease.13 Data on the effects of posttranslational modifications upon myelin recognition by either T cells or antibodies are scarce, but they already clarify that changes in either the patterns of mRNA splicing or patterns of posttranslational modification of myelin proteins may have a significant impact on T -ell or antibody recognition of myelin, and may well generate altered self-determinants for which the immune system may not be tolerant (Table 8.1). Recent data obtained by us illustrate that phosphorylated amino acid side chains in the myelinassociated stress protein αB-crystallin, for example, can be directly recognized by T-cell receptors (Van Stipdonk et al, submitted). Thus, the possibility that altered myelin determinants play a role in the development or perpetuation of autoimmune responses in MS is a real one.

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Factors That Govern the Encephalogenicity of Myelin Antigens The potential of a myelin antigen to induce encephalitis, i.e., inflammation within the CNS and accompanying neurological disease, is dependent on a variety of both intrinsic and extrinsic factors. Environmental influences such as diet,14 stress and (prior) exposure to infective agents as well as multigenic factors which control, for example, the details of antigen processing and presentation of encephalitogenic antigens, are known to contribute to the development of disease. In addition, several gene loci, gender and hormonal influences, particularly those governing neuroendocrine interactions, influence the qualitative and quantitative nature of the T-cell response in the experimental situation and therefore ultimately influence the encephalitogenic nature of a myelin antigen. It is probable that the major factor influencing onset of disease is the ability of the antigen to induce the influx of sufficient numbers of autoreactive T cells and/or antibody into the CNS. Myelin antigens on their own, being sequestered antigens that are usually absent from secondary lymphoid organs, are probably unable to activate sufficient numbers of autoreactive T cells. Thus, environmental factors such as stress and infection (or in the case of experimental models, appropriate adjuvant) may provide the necessary trigger to break self-tolerance and induce significant levels of encephalitogenic T-cell precursors. One factor known to significantly exacerbate the encephalitogenic potential of myelin reactive T cells is prior exposure to superantigens,15 such as may be provided by viruses and bacterial infections. Localization, posttranslational modifications and levels of expression of the myelin antigen in the CNS may be equally important in determining the topography and histopathological features of the lesion. In the autoimmune model of MS which may be induced with a number of different encephalitogens, the severity of disease and localization of the lesion for example is dependent on the experimental encephalitogen used. In addition, antigen specificity in itself is known to codetermine either pro-or anti-inflammatory cellular responses, further demonstrating that, at least in EAE, the nature of the target antigen has a distinct impact on the location and histopathological features of the lesions that are induced.16 Another factor known to influence susceptibility to disease is the genetic variation in the stress response. Comparison between rat strains that differ in their basal corticosteroid levels has demonstrated significant differences in EAE susceptibility correlating with such levels.17 Whereas Lewis rats, characterized by low cortisone levels, are extremely susceptible to acute monophasic EAE, PVG rats that have high levels of corticosteroids are resistant. In line with this, PVG rats become susceptible to EAE following adrenalectomy and supplementation of low basal levels of corticosterone. Yet a further consideration—and one that may explain the gender differences in the susceptibility to MS as well—is the finding that young adult male SJL mice are resistant to EAE as induced by CNS homogenate, whereas female mice are susceptible. Furthermore, male mice immunized with the encephalitogenic PLP epitope 139-151 do not exhibit relapses; female mice do, and they appear to produce higher levels of Th2 cytokines.18 By far the most important factor contributing to the encephalitogenic potential of a myelin antigen, however, is the genetic background of the host. In particular, the molecules encoded by major histocompatibility complex (MHC) genes play a crucial role. For a peptide to be recognized by T cells, its presentation by the proper MHC class II molecules is a prerequisite. Induction and progression of EAE is thus dependent on the proper binding of a peptide sequence to the MHC class II molecule, which imposes certain structural requirements upon the peptide sequence. For some MHC molecules, amino acid motifs for encephalitogenic epitopes have been identified, suggesting that encephalitogenicity in that particular genetic background may be restricted to proteins that harbor such sequence motifs. However, MHC-binding motifs are usually degenerate,19 and to date it remains to be established how restricted the above requirement actually is.

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How exquisitely specific the effects of interactions within the trimolecular complex between peptide, MHC molecule and T-cell receptor upon the development of EAE can be, has been clearly demonstrated in SJL mice following induction of disease with PLP peptide 139-151. In these studies, the use of altered peptide ligands has shown that substitution of a single amino acid at position 144 (Q144) renders the PLP peptide non-encephalitogenic and also protects against disease induced with the original peptide.20 On the other hand, studies of the effects of complementary mutations in an encephalitogenic region of MBP on their recognition by human T cells has again shown remarkable plasticity of the peptideMHC-TCR complex.21 While suggesting that TCR are capable of recognizing many more peptide-MHC complexes than originally thought, these findings also suggest that many more exogenous antigens may be capable of stimulating self-reactive T cells. That particular peptide-MHC complexes can influence susceptibility as well as resistance to autoimmune disease is clearly demonstrated in non-obese diabetic (NOD) mice which express the H-2 A g7 molecule. While such mice are susceptible to spontaneous insulin-dependent diabetes mellitus, transgenic mice which carry Eαd and, thus, express an H-2 E molecule, are resistant to diabetes. In contrast, non-transgenic NOD mice immunized with PLP 56-70 have a low incidence of EAE,22 while EAE in transgenic mice expressing I-E exhibit exacerbated disease associated with increased IFN-γ production.23 Also, studies of congenic strains of Lewis rats expressing the different MHC class II molecules RT1n, RT1a or RT1u have demonstrated that EAE induction is strongly influenced by the MHC class II gene region, while background genes confer protection. Yet other factors known to influence the encephalitogenicity of myelin proteins are the expression of proinflammatory cytokines such as IL-12 and IFN-γ and upregulation of adhesion molecules such as VLA-4,24 which allow the migration of activated cells in to the CNS. In conclusion, a myriad of factors contribute to the encephalogenicity of myelin antigens, underscoring that encephalitogenicity of selected antigens must always be considered within the context of these different factors.

Experimental Autoimmune Encephalomyelitis in Rodents Although chronic relapsing EAE induced in strain 13 of guinea pigs is an excellent model of MS, exhibiting both the clinical characteristics and histological lesions of demyelination, the use of small rodent models has the advantage of the wealth of laboratory reagents such as antibodies and gene probes with which to investigate the disease. The use of mice in particular allows the use of specific knockout or transgenic animals with which to dissect pathogenic mechanisms. Also, many strains of mice are not only susceptible to autoimmune models of EAE but also to viral models of MS. This combination allows the study of how viruses may influence and precipitate autoimmune neurological disease. The autoimmune model of MS, EAE is an acute, chronic or chronic relapsing autoimmune disease of the CNS following immunization with myelin proteins and peptides containing encephalitogenic epitopes within the protein molecule in adjuvant. The use of such adjuvants is necessary for the induction of ‘active’ EAE as may be Bordetella pertussis bacteria or its toxin. Alternatively, EAE may be induced in naive recipients following transfer of MHC class II restricted CD+ T cells specific for myelin proteins or the peptides (‘passive’ EAE).25 In rats and mice, both active and passive EAE disease may be augmented by coinjection of myelin specific antibodies26,27 suggesting that antibodies—in addition to T lymphocytes— play an important copathogenic role in the recreation of clinical and histopathological features of MS. Spontaneous EAE is unknown in normal animals despite the presence of myelin-reactive T cells. Even transgenic mice that have large numbers of T cells carrying a T cell receptor specific for the encephalitogenic MBP peptide Ac1-11 develop EAE in the absence of adjuvant only in an infectious environment, again stressing the requirement for infectious agents in precipitating autoimmune disease.2

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The clinical course of EAE is variable and dependent on the antigen, strain of animal and immunization protocol employed, and hyperacute, acute, chronic or chronic relapsing forms of EAE have been described. Neuropathologically, EAE is characterized by lesions of mononuclear cellular inflammation and varying degrees of demyelination within the CNS. Acute EAE lesions in rodents are characterized by subpial lesions of inflammation within the spinal cord and, if demyelination is present, it is generally the result of axonal damage, i.e., of secondary or Wallarian degeneration. While such models may be useful to study mechanisms of axonal repair and early pathological events, EAE models that involve protracted and chronic relapsing disease with inflammation and foci of primary demyelination followed by remyelination and gliosis may be more suitable to study mechanisms of demyelination. Consequently, these latter models may also be suitable for the evaluation of experimental therapeutic strategies. One example of such a model is the recently described chronic relapsing and/or protracted disease in DA rats.28 Furthermore, the use of neuroantigens such as MOG, in which demyelination in the CNS more closely resembles the lesions that are observed in relapsing-remitting MS,29 make it possible to investigate at least part of the wide spectrum of disease profiles as observed in MS. Thus, models which reflect such lesional variation are important to study. In the Biozzi ABH mouse, the influence of differing encephalitogenic myelin antigens on the clinical course and pathology within the CNS has been clearly demonstrated.22,29 In this mouse strain, native MBP does not induce disease, although an isolated short sequence of MBP does indeed induce mild acute disease.30 MOG-induced CREAE in Biozzi ABH mice has features typical of MS which are rarely documented with MBP or PLP. These include relapsing-remitting paralytic disease, with clinically silent lesions29 and marked demyelination induced by anti-MOG antibodies.27 Due to their availability, the abundant myelin antigens MBP and PLP have been studied most extensively in many animal strains. The availability of recombinant technology and synthetic peptides has recently enabled the evaluation of the encephalitogenic potential and definition of encephalitogenic epitopes of minor myelin proteins such as MOG, MAG and oligodendrocyte specific protein (OSP). Encephalogenicity of selected antigens, however, is not only restricted to myelin-specific proteins, since disease is also observed following immunization with the non-myelin-specific proteins S100β, glial fibrillary acidic protein (GFAP) and the small heat shock protein αB-crystallin,31 revealing that many different antigens of the central and peripheral nervous system can activate encephalitogenic T cells and, possibly, autoantibody responses. The use of inbred rodents has allowed investigation of the role of the peptide-MHC-TCR trimolecular complex in the development of EAE and on its use as a specific target for intervention in disease. These experiments have demonstrated a large variety of approaches via which the development of EAE can be influenced by manipulating the trimolecular complex. Blocking of class II molecules with specific antibodies prevents disease in experimental animals. Furthermore, peptides that bind with high affinity to MHC molecules may, by competing with the encephalitogenic peptide, inhibit disease. Likewise, such peptide therapy has also been aimed at blocking specific TCR interactions.32 An alternative to blocking peptide-MHC-TCR interactions is immune deviation of the pathogenic CD4+ Th1 cells to a Th2 phenotype. This has been achieved by blocking costimulation molecules,33 the use of peptide analogues34 and by administration of immunomodulatory lipids.35 Together, these data show the intriguing potential of antigen-specific therapy in autoimmune diseases and emphasize the importance of the definition of relevant autoantigens in human autoimmune diseases.

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Encephalitogens in Rodents As discussed above, major factors controlling susceptibility to EAE operate within the trimolecular complex between peptide, MHC molecule and TCR. In mice, different MHC haplotypes typically associate with different encephalitogenic regions of myelin antigens.36,37 Yet, different haplotypes may well associate with one and the same encephalitogenic epitope and, conversely, any single haplotype can usually accommodate more than one encephalitogenic epitope, as is illustrated in Table 8.2. The three commonly used mouse strains for EAE studies, PLJ (H-2 Au), Biozzi ABH (H-2 Ag7) and SJL (H-2 As), all respond to the encephalitogenic myelin proteins PLP, MBP and MOG. However, a hierarchical pattern of responses with respect to the severity and type of EAE are usually observed in these mice, and they reflect the differential response of different haplotypes to encephalitogenic antigens. As discussed above, one rule governing the encephalitogenic potential of a myelin antigen is the presence of a peptide sequence within the protein that can meet the peptide binding requirements of the MHC molecule it is facing. For some rodent MHC class II molecules, detailed studies have been performed to define these requirements, assisted by computer modeling of selected peptide-MHC complexes. Such studies suggest that, while many peptides may interact to at least some extent with the class II molecule, significantly fewer induce T-cell responses and fewer still are encephalitogenic. Still, many neuroantigens may be expected to harbor sequences fulfilling all the requirements for encephalitogens. For many years MBP was the antigen of choice for EAE studies but recently, the encephalitogenic potential of other myelin proteins has been examined more closely. As a result, encephalitogenic epitopes have been identified in the myelin proteins PLP, MOG, MAG, OSP and the non-myelin specific antigens, αB-crystallin, GFAP and s100β (Table 8.2). Together, these data suggest that perhaps any myelin antigen will be able to provide an encephalitogenic epitope for any given strain of experimental animal. Yet, it may be the interplay between cellular responses to each of these that will determine whether or not disease ensues. As explained above, the Biozzi ABH mouse is not susceptible to EAE following immunization with whole native MBP. Yet, the synthetic peptide 12-26 induces acute disease, albeit mild. Although it is not fully clear why the sequence 12-26 cannot induce disease when it is part of the whole protein, similar findings have been reported also for other model systems of autoimmunity. Apart from details of antigen processing that could perhaps explain part of this phenomenon, the type of MHC molecule involved may also be crucial, since it may allow competition for binding of the encephalitogenic sequence 12-26 by other non-encephalitogenic sequences derived from the same protein antigen, or it may have a general propensity to trigger protective Th2 type of responses when presenting peptides to passing T cells. Such possibilities must be seriously taken into account when studying responses to autoantigens in outbred populations where some class II molecules may confer protection against induction of disease.

General Features of Encephalitogenic T Cells Apart from their specificity, other features of myelin-directed T cells have been suggested to be relevant to their encephalitogenic potential. In this section, we briefly summarize these features. Characterization of encephalitogenic T cells has revealed that as a rule, they have the T helper 1 phenotype in secreting proinflammatory cytokines such as IFN-γ and TNF-α as well as IL-2 which recruits other T cells and macrophages that induce inflammation. In contrast, cells of the T helper 2 phenotype secrete regulatory cytokines IL-4 and IL-10 that suppress inflammation. A third T helper phenotype secreting TGF-β that would be preferentially activated following oral administration of antigen has been proposed,56 but this particular distinction is still controversial.

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Table 7.2. Encephalitogenic antigens in rodents Protein

Animal/strain

MBP

SJL mouse PLJ mouse Biozzi ABH mouse SWR mouse A.CA mouse B10.RIII mouse C57/BL mouse Lewis rats

PLP

Class II

Epitope

Reference

s u g7 q f r b RT-1

17-27,89-101 Acl-11 12-26 89-101 89-101 89-101 67-76 69-86

38,39 40 30 41 42 43 44 45

SJL mouse PL/J mouse Biozzi ABH NOD mouse BALB/c mouse SWR mouse C3H/He mouse Lewis rats

s u g7 g7 d q k RT1

139-151,178-191 43-64 56-70 56-70 56-70 103-116 215-232 217-240

46,47 48 22 22 22 49 50 51

MOG

SJL mouse PLJ mouse Biozzi ABH mouse C3H.SW mouse C57/BL mouse Lewis rats

s u g7 b b RT1

92-106 36-45 8-22,43-57,134-148 40-48 40-48 35-55

29 52 29 53 53 54

MAG

Biozzi ABH mouse

g7

97-112

Lewis rats

RT1

20-34,124-137,354-377

Morris et al, unpublished 16

s100β

Lewis rats

RT1

76-91

55

GFAP

Lewis rats Biozzi ABH mouse

RT1 g7

63-85 whole protein

16 Morris et al, unpublished

αB-crystallin

Biozzi ABH mouse

g7

1-16

31

OSP

SJL mouse

s

57-72

Morris et al, unpublished

Curiously, the vast majority of encephalitogenic MBP-and PLP-specific rodent T cells express a limited number of TCR variable chains, in particular Vβ8.2, and for some specificities similarities in the junctional regions have also been described. Possibly, this phenomenon is the result of a largely monospecific and, hence, oligoclonal response to these antigens. That such may not always be the case is illustrated by MOG-specific

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encephalitogenic T cells that are characterized by diverse Vβ gene usage. Only 48% of these T cells have the Vβ8.2 chain, suggesting that the anti-MOG response is more polyclonal than that against, e.g., MBP.57 Thus, the TCR usage may be quite dependent on the encephalitogenic antigen, which suggests that therapies targeting specific TCR may not be the ideal approach when one is dealing with a multi-determinant response.

Myelin-Directed T Cells in Virus Models of MS

Many studies have implicated infectious agents in MS58 and, given that the majority of naturally occurring CNS demyelinating diseases of man and animals of known etiology are virus-induced, viruses are obvious candidate etiological agents in MS. In humans, panencephalitis including demyelination is observed following measles infection (subacute sclerosis panencephalitis), rubella infection, HTLV-1 and EBV, whereas natural demyelinating infections of animals include canine distemper and visna/maedi infection of sheep. There are a number of ways in which viruses may have a role in autoimmune demyelinating disease. One of these is that they induce demyelination via a direct cytopathic effect, as observed in progressive multifocal encephalopathy following JC virus infection and release of local antigens for immune recognition. Alternatively, infection may significantly increase the local level of expression of MHC molecules, costimulatory molecules and adhesion molecules, up to the threshold required for disease. Indeed, viral infection increases the incidence of EAE and can render otherwise resistant animals susceptible to EAE. Infection of EAE-resistant B6 mice with the neurotropic Semliki Forest virus (SFV) gives rise to acute EAE which coincides with an increased number of MBP-reactive T cells.59 Likewise, juvenile guinea pigs infected with SFV60 are more sensitive to induction of EAE, as are BALB/c mice. Also, in other ways viruses can lead to the activation of myelin-reactive T cells. These include superantigenic activation or activation via molecular mimicry. An intriguing mechanism by which viruses may activate myelin-reactive T cells is molecular mimicry, the condition that sequence similarities between viral determinants and myelin components may lead virus-activated T cells to become crossreactive against myelin. Examination of protein data bases has revealed several sequence similarities between viruses and, e.g., PLP and MBP. The possible functional implication of this has been elegantly demonstrated by the induction of EAE in rabbits following immunization with a peptide of hepatitis B virus polymerase containing a sequence that is identical to an epitope of myelin basic protein.61 Such structural similarities between viral proteins and myelin antigens may also have implications for human T cells. Indeed, MBP-specific T-cell clones from MS patients may be activated in the presence of viral peptides including sequences from EBV, influenza type A virus and HSV.62 Yet another mechanism to explain the apparent impact of viruses on the development of autoimmunity leading to demyelination (especially well documented for humans) has been proposed following the finding that αB-crystallin, a small heat shock protein, is not only expressed in the lesions of MS patients but also emerges as an MHC class II-presented endogenous antigen in peripheral blood B cells following viral infection. This is discussed in greater depth further on. Immune responses to myelin antigens are frequently observed in viral models of MS (Table 8.3). However, it is difficult to assess whether such responses are due to molecular mimicry, superantigen activity or induction of autoreactivity to myelin antigens released as a result of myelin damage. The concept that neurotropic viruses may give rise to an autoimmune response has been supported by the finding that animals infected with demyelinating neurotropic viruses TMEV, SFV, measles, rubella and canine distemper mount both cellular and humoral responses against CNS antigens. In particular, infection of SJL mice with TMEV gives rise to anti-viral T-cell responses early in infection, but T-cell proliferative and DTH responses appear to develop against the encephalitogenic PLP

SJL

Lewis rat

JHM

MOG8-22

Biozzi ABH

SFV

PLP139-151

(SWRxSJL) F1

JHM

SFV

measles virus

PLP139-151

SLJ/J

Lewis rat

MBP84-104

SLJ/J

Measles

MBPAc1-11

(SJLxB10.PL)F 1

Chronic EAE

Immunising antigen

Animal strain

Model

SFV

MOG8-22

PLP121-174

PLP139-151

MBP84-104

MBPAc1-11

acute phase

MBP

MBP

MBP

MOG8-22; PLP56-70 αB-crystallin, MAG

PLP173-198; 249-273; MBP87-99

PLP139-151; 178-191

MBP84-104; PLP139-151

MBPAc1-11;35-47; 81-100;121-140

relapse phase

T cell responses to myelin antigens

Table 7.3. Myelin antigen specific T-cell responses in experimental models of MS

67

59

66

Morris et al, unpublished

65

65

64

63

Reference

122 T-Cell Autoimmunity and Multiple Sclerosis

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139-151 later on. Likewise, cell-mediated immune responses to MBP are observed during coronavirus (JHM) infection of rats and adoptive transfer of these MBP-reactive T cells can induce EAE in naive recipients.67 Such responses have also been described following measles virus infection of Lewis rats,66 thus suggesting that immune recognition of myelin antigens, including those that can be pathogenic, can be a major consequence of experimental infection.

Determinant Spreading in Animal Models for MS Myelin damage as a final result of immunization with either myelin antigens, as in EAE, or as a consequence of a neurotropic virus infection, may lead to the development of immune responses to otherwise sequestered myelin antigens. Such responses, which include those directed to epitopes not recognized in the whole protein molecule (cryptic epitopes), are secondary to the initial insult and have been termed ‘determinant’ or ‘epitope spreading’.63,64 Description of determinant spreading has largely been confined to the chronic EAE model and—to a lesser extent—to viral models of MS (Table 8.3), but it has been suggested to be an important event in the perpetuation and chronicity of disease. As discussed above, it is difficult to determine whether true ‘determinant spreading’ occurs during the course of virus infection or if autoreactivity results from, for example, crossreactivity to viral proteins. Also, it is still unclear whether determinant spreading results from leakage of myelin components into secondary lymphoid organs in the periphery or whether naive T cells may be locally activated against myelin components. Given the limited antigenpresenting function of CNS-derived antigen-presenting cells or infiltrating macrophages, local activation of truly naive T cells appears to be an unlikely event. Whether determinant spreading actually occurs in chronic inflammatory diseases in humans is still under debate, but this possibility should be an important consideration in the development of antigenspecific immunotherapies aimed to control diseases such as MS. The relapsing-remitting and progressive nature of chronic relapsing EAE has allowed investigation of the broadening of the T-cell repertoire and the importance of determinant spreading in the progression, and in the subsequent control of, disease. As discussed above, immunization of SJL/J mice with the encephalitogenic peptide PLP 139-151 gives rise to chronic relapsing EAE. In the acute phase of disease, T-cell responses to PLP are restricted to the administered sequence 139-151. During the relapse phase, however, activated T cells can be detected in the periphery directed to other epitopes both within the PLP molecule itself (intramolecular spreading) and within other myelin antigens such as MBP (intermolecular spreading).65 The importance of the spreading of the immune response to other myelin epitopes in the progression of disease has been demonstrated by the ability of such secondary immune responses to induce disease in their own right. Thus, PLP 178-191-specific T cells recruited as a result of priming to PLP 139-151 are encephalitogenic in naive recipients following in vitro activation with the peptide 178-191.68 Determinants that may be cryptic or subdominant in the initial phase of disease can therefore become immunogenic and, indeed, encephalitogenic during subsequent phases of disease. We have observed similar responses in CR-EAE in Biozzi ABH (H-2 Ag7) mice following immunization with the immunodominant epitope MOG 8-22. During the relapse phase, spreading of T-cell proliferative responses occurs to other epitopes within MOG, i.e., 43-57, 134-148; to the other known encephalitogenic epitopes PLP 56-70 and MBP 12-26; and to sequences of the small heat shock protein αB-crystallin (Morris et al, unpublished). So far, the exact role of these ‘secondary’ immune responses in disease progression in Biozzi ABH mice is unknown and current studies are aimed at clarifying whether or not they can be encephalitogenic. In other models, determinant spreading during CREAE has been found to correlate with the degree of tissue damage and does not appear to be a result of crossreactive epitopes, since spreading of the immune response does not develop in mice tolerized to the

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initial immunizing peptide either prior to or following acute disease.69 That epitope spreading in EAE is probably important in induction of relapses during the chronic stages is further suggested by the finding that tolerance to MBP 87-99, the peptide associated with relapses in (SWR x SJL) F1 mice, blocks disease progression following immunization with PLP 139-151.

Antigen-Specific T-Cell Responses in MS Immunohistochemical analysis has generated compelling evidence for the presence of activated T cells within MS lesions. Local T cells can be found to express surface markers typical for activated T cells such as the CD40 ligand; these cells synthesize high levels of mRNA encoding cytokines and they secrete such cytokines. In sharp contrast to the clear evidence for locally activated T cells in MS-affected white matter is the lack of evidence for activated myelin-reactive T cells in the periphery of MS patients. For quite some time now, intense studies have been devoted to myelin-specific T-cell responses in peripheral blood. From the very beginning, an important objective has been the identification of specific cellular responses that could help in understanding the pathogenesis of MS. Thus, efforts were directed at identifying responses that could be found in MS patients but not in healthy control subjects, or responses that were detectable only during episodes of active disease. So far, these attempts have remained unsuccessful: All responses that have been detected in either peripheral blood or cerebrospinal fluid of MS patients have been found also in healthy controls. As evidence accumulated that myelinspecific T cells form part of a normal immune repertoire, attention was focused on precursor frequencies of such T cells, their HLA-restriction patterns, their state of activation, the nature of their T-cell receptor, the quality of the ensuing response and the fine specificity of the response rather than on overall proliferative responsiveness. Also, mechanisms have been investigated by which potentially autoreactive T cells could become inadvertently activated in some individuals. In this latter respect, the possible contribution by pathogens has gained much attention, fuelled by consistent epidemiological evidence that viral infection in particular could well explain patterns of both disease susceptibility and disease relapses. Superantigenic stimulation of potentially myelin-reactive T cells has been examined, as well as the possibility of molecular mimicry between myelin determinants and pathogen-derived determinants. Below, we will summarize studies on myelin antigen-specificity of human T cells. Studies on MBP as an autoantigen have been particularly popular because of the early demonstration of its encephalitogenicity in rodents and since MBP is readily available and suitable for in vitro experimenting. Only in more recent years has attention shifted towards other myelin antigens.

Myelin-Specific T Cells in Peripheral Blood and Cerebrospinal Fluid Over 10 years ago now, MBP-specific T cells were first isolated from the peripheral blood of MS patients, but it soon became apparent that such cells could also be readily isolated from the blood of perfectly healthy donors. 70,71 Precursor frequencies of MBP-specific T cells in populations of peripheral blood lymphocytes have been estimated at somewhere between 0.5 x 10-7 and 3 x 10-5, dependent on the read-out system employed. In samples of the cerebrospinal fluid of MS patients, higher frequencies of such cells have reportedly been found, up to almost 2 x 10-3. These data are reviewed in ref. 72. Yet, reports on precursor frequencies are often confusing; the use of different culture systems and readout assays apparently have a significant impact on the outcome. Some claims have been reported on significantly higher precursor frequencies of MBP-specific T cells in MS patients as compared to controls or on a larger fraction of such cells to exist in a pre-activated state.73,74 However, others have met with considerable difficulties when attempting to reproduce such

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differences. Data collected so far fail to provide convincing and consistent evidence that in vivo activation of MBP-specific T cells accompanies clinical symptoms of MS. Such activation could be either the cause or the consequence of the disease but, so far, neither appears to be the case, although improved assays in the future may very well reveal differences that have remained obscure so far. In recent years, studies on T cells recognizing other myelin proteins have received increasing attention. In handling individual myelin antigens other than MBP, however, the strongly hydrophobic character of many myelin proteins has been technically frustrating. Consequently, several groups have studied T-cell responses to peptides representing parts of myelin proteins rather than whole protein itself. This has greatly assisted the study of, for example, PLP-specific responses. Under normal culturing conditions, PLP-specific responses are frequently quite difficult to detect. This may perhaps be due to the fact that extremely hydrophobic proteins such as PLP (and MOG) can cause cultured lymphocytes to agglutinate, which leads to nonspecific proliferation within the first days of culture (Van Noort JM, unpublished data). This may conceivably suppress selective development and outgrowth of T cells that respond to the antigen in an MHC-restricted, specific way. Studies using peptide antigens revealed that T-cell reactivity to PLP sequences can again be readily detected in MS patients as well as in healthy controls.75,76 As in the case of MBP, with respect to T-cell reactivity against PLP, data have also been reported to suggest increased frequencies of reactive (preactivated) T cells in MS patients as compared to controls.74,77 These data, however, still await further substantiation and confirmation using other culture systems and read-out assays to verify possible differences between MS patients and healthy controls. A number of other, minor myelin antigens have been suggested as potential target antigens, and interest in such less abundant myelin targets is still increasing. Currently, the most advanced are studies on the minor protein MOG, a surface protein that is selectively expressed on oligodendrocytes in the CNS and which has excellent encephalitogenic qualities in experimental animals. A limited number of studies on T-cell reactivity against MOG have suggested increased reactivity to this protein by MS patients. One study showed higher frequencies of specific T cells in peripheral blood of MS patients, while another indicated a higher proportion of peripheral blood T-cell samples from MS patients as compared to controls to display significant proliferative responses to preparations of MOG.78,79 Again, however, the reported data are somewhat confusing, since they are not internally consistent and appear to be strongly influenced by the technical approaches taken. Also, MOG preparations derived from whole myelin may not be completely free from contaminants. Earlier studies have shown that even minute quantities of such myelin contaminations may trigger T-cell responses that are stronger than those directed against the predominant protein component of the preparation.80 Studies using recombinant myelin proteins are currently performed to circumvent this problem. Yet, studies with recombinant antigen will fail again to detect the possible influence of protein modification. Other myelin antigens under study include CNPase and transaldolase-H.72,81 As with the other antigens, T-cell reactivity to these minor myelin components can be found in both MS patients and controls, and responsive T cells are usually HLA-DR-restricted and frequently display cytotoxic activity. Further studies should clarify whether any of these antigens is preferentially recognized by MS patients versus controls or at certain stages of disease.

αB-crystallin as Candidate Autoantigen in MS Recently, an approach to the study of T-cell reactivity to myelin antigens was taken that circumvented the problem of both protein purification and protein modification.82 In this study, responses by human peripheral blood T cells were examined to complete protein

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extracts from human CNS myelin, thus including minor protein components along with abundant proteins, and differentially spliced proteins together with posttranslationally modified variants. In addition, responses were compared against CNS myelin proteins from either healthy control tissue or from MS-affected white matter regions, in view of the strong possibility that the biochemical features of CNS myelin from MS brains could somehow be different from healthy tissue with respect to patterns of modification or levels of individual components. In this study, predominant responses were recorded against a single protein component present in CNS myelin derived from MS brain only. This target protein was subsequently identified as αB-crystallin, a small stress protein. Examination of MS brains by immunohistochemical analysis (using an antibody preparation raised against αB-crystallin derived from MS brains) revealed strongly elevated expression of αB-crystallin in oligodendrocytes within early developing MS lesions, but not within inactive lesions or healthy white matter.82,83 These results indicate that αB-crystallin, despite being only a minor protein component in purified CNS myelin, triggers much stronger T-cell responses than any other myelin protein when it is present at stress-induced levels. As appears to be the case with several other myelin antigens, no gross differences have so far been detected between MS patients and healthy controls in their response to αB-crystallin. Further studies characterizing the human T-cell response to αB-crystallin and its encephalitogenic potential in rodents are currently underway. Data collected so far fail to provide evidence for the idea that already activated T cells against myelin antigens circulate in MS patients at levels that are substantially higher than in healthy control subjects. In fact, the presence of myelin-reactive T cells appears to be a general feature of any human T-Cell repertoire. This repertoire may well be involved in the development of MS when it becomes activated in the CNS, but the presence of the repertoire as such does not trigger disease. Since activated T cells are indisputably present within active MS lesions, this raises the question of how and where myelin-specific T cells could become activated during disease development. Local activation of myelin-specific memory T cells in the CNS as the result of infectious or inflammatory damage to myelin sheaths is a good possibility. Activation of myelin-specific T cells during the acute phase of infection in experimental models for MS has been well documented, especially following viral infection of mice. Other ways for pathogens to activate myelin-specific T cells include molecular mimicry or superantigen-mediated stimulation of autoreactive T cells. Recently, we collected evidence to indicate that viral infection of peripheral blood B lymphocytes can lead to endogenous expression of αB-crystallin and its subsequent presentation via HLA-DR molecules. Under normal conditions, no expression of αB-crystallin can be detected in peripheral blood lymphocytes. Thus, viral induction of class II MHC-restricted presentation by B cells of an otherwise undetectable self-protein represents yet another mechanisms via which microbial infection can activate T cells that are crossreactive to myelin (van Sechel AC et al, submitted). We currently take these findings as an explanation for the remarkably high precursor frequencies of αB-crystallin-specific T cells in adult human peripheral blood as compared to other myelin-reactive T cells. Figure 7.1 depicts a hypothetical pathway for the build up of anti-myelin responsiveness byT cells as the result of viral infection, based on αB-crystallin being an antigen shared between CNS myelin and virus-infected lymphocytes. Local events in the CNS could then lead to recruitment of the memory T-cell repertoire into a site where the same antigen will again be presented to T cells, triggering their local reactivation. More data, however, are required to establish whether or not any of the above mechanisms for activation of myelin-reactive T cells are indeed functional during the development of MS.

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Fig. 7.1. Simplified model depicting a role for αB-crystallin as an antigen shared between virally-infected peripheral blood B cells and CNS myelin. Recent data indicate that viral infection of peripheral blood B cells induces expression and MHC class II-associated presentation of endogenous αB-crystallin, a self antigen that is normally not detectable in peripheral blood lymphocytes. Circulation of virus-infected cells into secondary lymphoid organs could lead to priming of naive T cells, or reactivation of memory T cells directed against this small HSP. At a later stage, local stress events within the CNS could lead to activation of the blood-brain barrier and to nonspecific recruitment of leukocytes, while at the same time triggering the expression of αB-crystallin within oligodendrocytes (ODC) and/or astrocytes. Reencountering their specific antigen, the infiltrated proinflammatory, αB-crystallin-reactive T cells could become reactivated and contribute to development of local inflammatory responses.

Myelin-Specific Responses Over Time Only a limited number of studies have been performed to examine whether or not the specificity of peripheral T-cell responses to myelin antigens change over time in groups of MS patients or in individuals. Clearly, such changes would be highly relevant to the rational design of antigen-specific therapy. While signs of determinant spreading have been documented for chronic stages of EAE, no convincing data are available to suggest that in individual MS patients the specificity of cellular responses against myelin antigens would change over time in any predictable pattern. In fact, epitope-specific responses against, for example MBP, that can be detected in individuals frequently remain remarkably stable over periods as long as a few years.84,85 Continued research on antigen-specific responses in a longitudinal set up, or comparing responses during active disease with those during remissions, should further clarify this issue.

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Conclusion Many questions remain on the issue of antigen-specific T-cell responses in MS. Given the experience in animal models, several different myelin specificities or even non-myelin specificities can have a pathogenic demyelinating potential, dependent on the MHC background of the individual along with several other factors, as discussed in this chapter. The human adult T-cell repertoire does indeed harbor many such specificities, but so far no differences have been found between MS patients and healthy subjects that help explain why MS develops in some and not in others. It appears likely that a myelin-specific T-cell repertoire is just a normal part of an adult immune repertoire and that in MS patients, this repertoire is somehow recruited and activated, while in healthy humans such events are better regulated or do not occur at all. In this perspective, deletion or active suppression of appropriate myelin specificities in the adult T-cell repertoire would represent a useful approach to reduce the recruitable potential. For the rational design of antigen-specific therapy in MS, identification of dominant myelin specificities is a key step. It has become clear that T-cell reactivities can be detected against many different CNS myelin proteins, if not against all of them, including different protein isoforms or posttranslationally modified structures. Recent data obtained in vitro indicate that in MS brain, the small stress protein αB-crystallin acts as the immunodominant T cell trigger, but further studies are required to verify whether its immunodominant properties also hold under in vivo conditions. The fact that viral infection of B cells can lead to class II MHC-restricted presentation of this very same antigen, thus priming T cells against it, is intriguing. It points to the possibility that the microbial status of the host in which autoimmune T-cell responses are examined may have a distinct impact on the potential myelin-reactive memory repertoire one is dealing with. Humans and laboratory animals are clearly different in this respect. Finally, the phenomenon of determinant spreading will continue to be examined. While in experimental models for MS the recruitment of novel myelin specificities during chronic disease has been well documented, data on humans are still scarce. Delineating patterns of myelin reactivity as a function of disease duration or disease activity is clearly important for the design of therapeutic strategies. Currently, the experimental model employing Biozzi ABH mice appears to hold significant promise as a tool to investigate determinant spreading. For this particular strain of mice, encephalitogenic epitopes within a number of different myelin antigens have been identified, allowing detailed analysis of the recognition pattern for such epitopes during chronic disease.

Acknowledgments Our research is supported by the Netherlands Prevention Fund, The Netherlands Foundation for the Support of MS Research, the Multiple Sclerosis Society of Great Britain and Northern Ireland and the European Commission via the concerted action program “T-cell Autoimmunity in MS.”

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47. Greer JM, Kuchhroo VK, Sobel R.A et al Identification and characterization of a second encephalitogenic determinant of myelin proteolipid (residues 178-191) for SJL mice. J Immunol 1992; 149:783-788. 48. Whitman RH, Jones RE, Hashim GA et al. Location of a new encephalitogenic epitope (residues 43-64) in proteolipid protein that induces relapsing experimental autoimmune encephalomyelitis in PL/J and (SJL X PL) F1 mice. J Immunol 1991; 147:3803-3808. 49. Tuohy V K, Lu Z, Sobel RA et al. A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis. J Immunol 1988; 141:1126-1130. 50. Endoh M, Kunishita T, Neihei J et al. Susceptibility to proteolipid apoprotein and its encephalitogenic determinants in mice. Int Arch Allergy Appl Immunol 1990; 92:433-438. 51. Zhao W, Wegmann KW, Trotter JL et al. Identification of an N-terminally acetylated encephalitogenic epitope in myelin proteolipid apoprotein for the Lewis rat. J Immunol 1994; 153:901-909. 52. Kerlero de Rosbo N, Mendel I, Ben-Nun A. Chronic relapsing experimental autoimmune encephalomyelitis with a delayed onset and an atypical clinical course, induced in PL/J mice by myelin oligodendrocyte glycoprotein (MOG)-derived peptide: Preliminary analysis of MOG T-cell epitopes. Eur J Immunol 1995; 25:985- 993. 53. Mendel I, Kerlero de Rosbo N, Ben-Nun A. A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: Fine specificity and T-cell receptor V beta expression of encephalitogenic T cells. Eur J Immunol, 1995; 25:1951-1959. 54. Adelmann M, Wood J, Benzel I et al. The N-terminal domain of the myelin oligodendrocyte glycoprotein (MOG) induces acute demyelinating experimental autoimmune encephalomyelitis in the Lewis rat. J Neuroimmunol 1995; 63:17-27. 55. Kojima K, Berger T, Lassmann H et al. Experimental autoimmune panencephalitis and uveoretinitis transferred to the Lewis rat by T lymphocytes specific for the S100 beta molecule, a calcium binding protein of astroglia. J Exp Med 1994; 180:817-829. 56. Miller A, al-Sabbagh A, Santos LMB et al. Epitopes of myelin basic protein that trigger TGF-β release after oral tolerization are distinct from encephalitogenic epitopes and mediate epitope-driven bystander suppression. J Immunol 1993; 151:7307-7315. 57. Mendel I, Kerlo de Rosbo and Benn-Nun A. Delineation of the minimal encephalitogenic epitope within the immunodominant region of myelin oligodendrocyte glycoprotein: Diverse Vβ gene usage by T-cell recognizing the core epitope encephalitogenic for T-cell receptor Vβb and T cell receptor Vβa H-2b mice. Eur J Immunol 1996; 26: 2470-2479. 58. Russell WC. Viruses and MS. In: Russell W.C. eds. Molecular Biology of Multiple Sclerosis. Chichester, Wiley & Sons Ltd. 1997:243-254. 59. Mokhtarian F and Swoveland P. Predisposition to EAE induction in resistant mice by prior infection with Semliki Forest virus. J Immunol 1987, 138:3264-3268. 60. Suckling AJ, Wilson NR, Rumsby MG. Experimental allergic encephalomyelitis: Modification of optic nerve pathology by antecedent virus infection. Acta Neuropathol 1982; 58:101-106. 61. Fujinami RS and Oldstone MBA. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: A mechanism for autoimmunity. Science 1985; 230:1043-1045. 62. Wucherpfennig KW and Strominger JL. Molecular mimicry in T cell-mediated autoimmunity: Viral peptides activate human T-cell clones specific for myelin basic protein. Cell 1995; 80:695-705. 63. Lehmann PV, Forsthuber T, Miller A et al. Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 1992; 358:155-157. 64. McRae BL, Vanderlugt CL, Dal Canto MC et al Functional evidence for epitope spreading in the relapsing pathology of experimental allergic encephalomyelitis. J Exp Med 1995; 182:75-85. 65. Yu M, Johnson JM and Tuohy VK A predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: A basis for peptide-specific therapy after onset of clinical disease. J Exp Med 1996; 183:17771788.

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66. Liebert UG, Linington C, ter Meulen V. Induction of autoimmune reactions to myelin basic protein in measles virus encephalitis in Lewis rats. J Neuroimmunol 1988; 17:103-118. 67. Watanabe R, Wege H, ter Meulen V. Adoptive transfer of EAE-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis. Nature1983; 305;150-153. 68. Miller SD, McRae BL, Vanderlugt CL et al. Evolution of the T-cell repertoire during the course of experimental immune-mediated demyelinating diseases. Immunol Rev 1995; 144:225-244. 69. Miller SD and Karpus WJ. The immunopathogenesis and regulation of T cell-mediated demyelinating diseases. Immunol Today 1994; 15:356-361. 70. Burns J, Rosenzweig A, Zweiman B et al. Isolation of myelin basic protein-reactive T-cell lines from normal human blood. Cell Immunol 1983; 81:435-440. 71. Pette M, Fujita K, Wilkinson D et al. Myelin autoreactivity in multiple sclerosis: Recognition of myelin basic protein in the context of HLA-DR2 products by T lymphocytes from multiple sclerosis patients and healthy donors. Proc Natl Acad Sci USA 1990; 87:7968-7972. 72. Martin R and McFarland HF. Immunology of multiple sclerosis and experimental allergic encephalomyelitis. In: Raine CS, McFarland HF, Tourtelotte WW eds. Multiple sclerosis; clinical and pathogenic basis. London, Chapman & Hall 1997:221-242. 73. Allegretta M, Nicklas JA, Sriram S et al. T cells responsive to myelin basic protein in patients with multiple sclerosis. Science 1990; 247:718-721. 74. Zhang J, Markovic-Plese S, Lacet B et al. Increased frequency of interleukin-2 responsive T cells specific for myelin basic protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. J Exp Med 1994; 179:973-984. 75. Pelfrey CM, Trotter JL, Tranquil LR et al. Identification of a novel T-cell epitope of human proteolipid protein (residues 40-60) recognized by proliferative and cytolytic CD4+ T cells from multiple sclerosis patients. J Neuroimmunol 1993; 46:33-42. 76. Pelfrey CM, Trotter JL, Tranquil LR et al. Identification of a second T-cell epitope of human proteolipid protein (residues 89-106) recognized by proliferative and cytolytic CD4+ T cells from multiple sclerosis patients. J Neuroimmunol 1994; 53:153-161. 77. Sun JB, Olsson T, Wang WZ et al. Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. Eur J Immunol 1991; 21:1461-1468. 78. Sun JB, Link H, Olsson T et al. T- and B- cell responses to myelin-oligodendrocyte glycoprotein in multiple sclerosis. J Immunol 1991; 146:1490-1495 79. Kerlero de Rosbo N, Milo R, Lees MB et al. Reactivity to myelin antigens in multiple sclerosis. Peripheral blood lymphocytes respond predominantly to myelin oligodendrocyte glycoprotein. J Clin Invest 1993; 92:2602-2608. 80. Van Noort JM, van Sechel AC, Boon J et al. Minor myelin proteins can be major targets for peripheral blood T cells from both multiple sclerosis patients and healthy subjects. J Neuroimmunol 1993; 46:67-72. 81. Banki K, Colombo E, Sia F et al. Oligodendrocyte-specific expression and autoantigenicity of transaldolase in multiple sclerosis. J Exp Med 1994; 180:1649-1663 82. Van Noort JM, van Sechel AC, Bajramovic JJ et al. The small heat-shock protein αB-crystallin as candidate autoantigen in multiple sclerosis. Nature 1995; 375:798-801. 83. Bajramovic JJ, Lassmann H and van Noort JM. Expression of αB-crystallin in glia cells during lesional development in multiple sclerosis. J Neuroimmunol 1997; in press. 84. Salvetti M, Ristori G, D’Amato M et al. Predominant and stable T-cell responses to regions of myelin basic protein can be detected in individual patients with multiple sclerosis. Eur J Immunol 1993; 23:1232-1239. 85. Meinl E, Weber F, Drexler K et al. Myelin basic protein-specific T-lymphocyte repertoire in multiple sclerosis. Complexity of the response and dominance of nested epitopes due to recruitment of multiple T-cell clones. J Clin Invest 1993; 92:2633-2643.

CHAPTER 8

Immunopathogenesis of MS M. Vergelli, L. Massacesi, and H.F. McFarland

M

ultiple sclerosis (MS) is a disease of the central nervous system (CNS) characterized by destruction of myelin. Clinically the disease is extremely variable but is most often characterized, at least during the initial stages, by episodes of neurological dysfunction followed by spontaneous improvement. This course is known as the relapsing-remitting course of MS.1 Although most patients (about 90%) begin with a relapsing-remitting course, about 70% of these patients will go on to develop a progressive course over time. Progression can occur either as a result of incomplete recovery from an exacerbation or through slow progression independent of exacerbations, a course termed secondary progressive MS. In addition, patients will vary with respect to the rapidity of progression. Some patients will have an extremely mild course, often termed benign MS. Others will have a rapidly progressive course that can lead to death within a year. This form is termed malignant or Marburg type MS.2 Fortunately, the Marburg form is seen in less than 1% of patients. Pathologically, MS is characterized by destruction of myelin associated with perivascular inflammatory infiltrates.3 Although it is generally thought that axonal damage only occurs in old, chronic lesions and even then is limited, pathological studies now raise the possibility that axon damage may occur in some early lesions. As the lesion ages, demyelination becomes complete and a well defined edge of intact myelin can usually be seen. In addition, a reactive gliosis occurs in the area of destroyed myelin, leading to a lesion seen on gross examination as grey and firm to the touch. The pathological hallmark of MS is that lesions of different ages are present.3,4 The events involved in the evolution of the lesion in MS are examined in detail below.

The MS Lesion Pathology Although not known with certainty, the initial event in the MS lesion is thought to be disruption of the blood-brain barrier (BBB) by activated T cells which have entered the CNS nonspecifically and recognize antigen(s) presented within the nervous system.5 Support for BBB disruption as an initial event comes from longitudinal studies of patients using contrast enhanced Magnetic Resonance Imaging (MRI), which will be discussed in detail below. Pathological examination of very early lesions has been limited. In general, these lesions are characterized by substantial inflammation in a perivascular distribution.3,4 However, some lesions have been described that have prominent edema with only limited cellular infiltrate. These lesions may show prominent loss of oligodendrocytes. It has been suggested recently that different, distinct pathological processes may occur in different patients.6 T-Cell Autoimmunity and Multiple Sclerosis, edited by Marco Londei. ©1999 R.G. Landes Company.

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Most acute lesions are associated with inflammatory infiltrates3,7 and T cells and macrophages comprise the majority of infiltrating cells at the sites of active demyelination.8 Characterization of the T cells in the inflammatory infiltrates indicate that both CD4+ and CD8+ T cells are present, but that CD4+ T cells predominate at the edge of active demyelination.9,10 The T cells appear activated, as they express IL-2 receptor and HLA class II molecules.11,12 In early lesions, numerous activated macrophages or microglial cells are present and breakdown products of myelin can be demonstrated within these monocytes. Remyelination is also a common finding in early lesions, and oligodendrocytes are present.3 In contrast to acute or recent lesions, chronic lesions show a marked loss of oligodendrocytes and complete loss of myelin.13 Some microglia/macrophages persist, some with residual myelin breakdown products, and a substantial degree of astrogliosis is seen.14 The edge of the lesion is usually distinct and shows an increased number of macrophages.

MRI Following the initial applications of MRI in MS it became clear that MRI provided a powerful tool for studying the disease. For the first time the pathological changes in MS could be imaged. During the last ten years the use of MRI to assist in the diagnosis of MS, to examine the natural history of the disease and as an adjunct to monitor new therapies have led to substantial progress in MS research.15-17 With these advances have come additional questions. The relatively poor correlation between the extent of disease seen on MRI and disability as measured by rating scales such as the Expanded Disability Status Scale (EDSS) have raised questions pertaining to the significance of what is seen on MRI, and to what the actual cause of disability is.18,19 Although many questions remain unresolved, the relationship between changes on MRI and the pathological process in MS is becoming clearer. Initially, imaging was restricted to what is now considered standard or conventional imaging techniques, T2-weighted images in which tissue with increased proton concentration results in an increased signal and T1-weighted images in which tissue with increased protons appears dark. T2-weighted images, spin echo sequences, provide an excellent representation of areas of diseased white matter when postmortem brains are imaged. However, increased signal on T2-weighted images are not pathologically specific; they can represent edema, inflammation, demyelination and gliosis, since all of these processes result in increased free water. It is generally agreed that the initial stage in the evolution of most, if not all, MS lesions is disruption of the BBB. This event can be imaged on T1-weighted images following the administration of a paramagnetic contrast agent such as gadolinium-DTPA.20,21 Acute or new lesions which enhance are also seen on T2-weighted images reflecting the inflammation and edema associated with these lesions. As the acute phase of the lesion resolves and enhancement is no longer seen, hyperintensity continues to be seen on T2-weighted images, which at this stage probably reflects tissue damage. Nonetheless, recent data generated by brain histopathological evaluation of MRI-scanned non-human primates affected by a chronic relapsing form of Experimental Autoimmune Encephalomyelitis(EAE) closely resembling MS have shown that histologically active CNS lesions in some cases do not enhance (Massacesi et al, unpublished results). These data suggest that the BBB may heal behind inflammatory infiltrates that may persists in the CNS for a long time, inducing lesions that may became irreversible (Massacesi et al, unpublished results). The above described lack of pathological specificity probably accounts, in part, for the poor correlations between the extent of diseased tissue seen on MRI and disability. This problem is further compounded by the use of the EDSS to measure disability. Since the EDSS is heavily weighted towards ambulation, lesions in the spinal cord and brain stem will have a disproportionate impact on this measure. However, MRI measures of disease burden focus on the

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cerebrum, since measurements in the spinal cord are difficult to quantitate. Consistent with this hypothesis is the greater correlation between disability and the diameter of the cervical spinal cord; a reduced diameter reflecting more significant involvement of the spinal cord has shown a strong correlation with disability, especially in patients with progressive MS. Some additional pathological specificity may be achieved measuring areas of hypointensity on T1-weighted images. Since not all areas of hyperintensity on T2-weighted images appear hypointense on T1-weighted images, it has been suggested that these lesions may represent more extensive tissue destruction, and recent studies have described a substantially greater correlation with disability compared to lesion burden on T2-weighted images.22 More recently, attention has turned to three new techniques, magnetization transfer imaging, magnetic resonance spectroscopy, and diffusion imaging. To date there is insufficient data to know if these techniques will provide an improvement in imaging specific aspects of the MS lesion, which have unique pathological characteristics. Since the introduction of MRI as a means to study MS, several longitudinal studies examining serial changes have been done. The results of these studies have provided a new understanding of the natural history of the disease, especially during the early RR phase of the disease.23,24 In a population of patients with minimal disability and early MS, a substantial degree of new disease activity is seen, indicating that the disease in many patients is progressive even during the early stages. This finding has had important implications on therapeutic approaches.

MS Pathogenesis A leading hypothesis concerning the cause of MS is that self-reactive T lymphocytes specific for myelin antigens participate in the pathogenesis of the CNS lesions. This assumption is based on the following observations: 1. Perivenular and white matter MS lesions are characterized by the presence of inflammatory infiltrates that, at least in acute lesions, are mostly reminescent of other classical T cell-mediated inflammatory reactions; 2. Localization of the lesions mainly in perivenular white matter of the central nervous system (periventricular white matter, corpus callosum, optic nerve and chiasm, brain stem white matter), but not in the peripheral nervous system, strongly argues for a certain level of specificity of the immune-mediated attack to one or more antigens contained in this “tissue”; 3. Enrichment of activated and clonally expanded B and T lymphocytes in the cerebrospinal fluid (CSF) suggests that the repertoire of immune cells present in the CNS, is antigen selected;25-28 4. Morphology of the CNS lesions and clinical course of experimental autoimmune encephalomyelitis (EAE) induced by myelin antigens may mimic those of MS.29,30

Major Lessons from EAE Most of the current theories on the imunopathogenic mecanisms leading to inflammatory and demyelinating lesions of MS are based on data obtained in the animal model named EAE. EAE has been extensively investigated and the results have provided a number of new insights into our understanding of the pathogenesis of, as well as therapeutic strategies for, organ-specific autoimmune diseases. EAE has been discussed in depth elsewhere in this text. Here we summarize some of the findings in EAE that in our opinion are more relevant for understanding the immunopathogenesis of multiple sclerosis: 1. In susceptible animals many proteins of the CNS are potentially encephalitogenic.31-35 Indeed, different myelin antigens such as myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) can induce EAE

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under appropriate experimental conditions. Nonetheless, a single autoantigen induces different types of pathology and clinical courses in different species and strains. In addition, since each inbred strain represents a genetically distinct individual, the differences in pathology and clinical courses among the different animal strains and species in response to immunization with each CNS protein may reflect variability of the immune responses to the same protein. 2. In susceptible animals, a relapsing-remitting form of EAE can be induced by the passive transfer of myelin antigen-specific CD4+ T lymphocytes (but not of immune serum or B cells).36-39 3. Transgenic mice expressing myelin basic protein (MBP)-specific TCR virtually in the whole T-lymphocyte repertoire develop spontaneous EAE.40,41 4. T cells reactive with myelin antigens can be easily cultured from peripheral blood and lymphoid organs of naive experimental animals as well as from healthy humans. These autoreactive T cells originating from healthy individuals have pathogenic (encephalitogenic) potentiality when transferred upon activation into syngenic recipients, indicating that potentially autoaggressive T lymphocytes reactive for CNS antigens exist in the “normal” repertoire of T cells in any individual.42,43

Mechanisms of Activation of Self-Aggressive T Cells (Breaking the Tolerance) Since myelin reactive T cells can be easily demonstrated in the repertoire of normal individuals, it is unlikely that clonal deletion of potentially self-reactive T cells is the major mechanism for preventing autoimmunity. It is now clear that immmunological tolerance, even to nervous system specifc self-antigens present in relatively high amounts, is absent or at least incomplete. In EAE, the mechanism leading to the expansion and activation of autoreactive T cells is determined by the experimental conditions (either active immunization or passive transfer of activated T cells); in MS the mechanisms which could be involved in sensitization, expansion or activation of these cells are not understood. Similarly to other autoimmune diseases, it has been postulated that infections, particularly viral infections, in genetically susceptible individuals may represent the mechanism underlying the failure of immunological tolerance and the trigger of autoimmunity. In this context, a relationship between infections and the occurrence of clinical relapses in MS has been reported.44 There are several possible mechanisms that could account for an infection-induced loss of tolerance. Induction of Changes in the Antigenicity of Target Tissues Dramatic changes may occur in tissues during an infection: Genes that are not normally transcribed can be activated and normal gene products can be modified at the posttranscriptional level, resulting in the generation and release of “new” autoantigens that are not tolerated by the immune system. In addition, infectious agents, by themselves or through the induction of an inflammatory reaction, can induce the expression of MHC as well as costimulatory molecules in the target tissue, lowering the treshold for the presentation of self-antigens. Molecular Mimicry According to this intriguing hypothesis, exogenous antigens such as those from bacteria or viruses may trigger autoimmunity by cross-stimulation of autoantigen-specific T cells.45 Until recently, mimicry was thought possible only if the exogenous antigen and the self peptide shared the same or a closely related amino acid sequence.46,47 It is now clear that contiguous identity is not required for cross-stimulation. Cross-recognition may even occur

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under circumstances in which little or even no amino acid sequence identity exists.48 In this context, Wucherpfenning and Strominger used previously established structural criteria for the characterization of the immunodominant T-cell epitope of MBP p83-99 presented by the MS-associated MHC class II molecule HLA DR15 in order to search databases for potentially crossreactive exogenous peptides that matched the structural features (and not the sequence) of the predicted T-cell determinant motif.49 Several of the candidate peptides could actually stimulate MBP-specific T-cell clones in vitro, although their amino acid sequence differed substantially from the MBP epitope recognized by the autoreactive clones.49 Finally, data generated in our own laboratory have recently demonstrated that the likelihood of T-cell cross-recognition is probably much wider than previously thought. This assumption is derived by the observation that functional peptide recognition by at least some class II-restricted T cells is extremely degenerate. The extensive flexibility of antigen recognition was demonstrated either by using combinatorial peptide libraries or a panel of ligands with single and multiple amino acid substitutions.50,51a Using these approaches, we have identified a stimulatory ligand for a TCC not sharing a single amino acid in corresponding positions with the antigen used to establish that T-cell clone itself.51b These data raise questions about the models of antigen cross-recognition based on sequence homology or conserved TCR contact positions, and provide new leads to examine crossreactivity between antigens from any source during autoimmune responses. Superantigen Stimulation Potentially autoaggressive T cells can be activated during the course of an ongoing infection by stimulation with a viral or bacterial superantigen. Superantigens stimulate T cells by binding to the Vβ portion of the TCR beta chain.52,53 Since the superantigen binding site is shared by a large number of T-cell clones with different specificities, superantigen stimulation usually results in the activation of a large fraction of T cells. This fraction may comprise autoreactive T cells. Nonspecific Mechanisms Autoreactive T cells could also be stimulated by completely nonspecific mechanisms. High local concentrations of certain cytokines, as well as nonspecific recrutiment of bystander T cells due to increased expression of cell adhesion molecules, may induce the activation of T cells carrying TCR specific for self-antigen molecules at the site of an inflammatory reaction. It should be noted that these hypothetical mechanisms are not mutuallly exclusive and that even during the course of the same disease different mechanisms may be operating at different stages or overlap each other. Possibly, MS could occur when a threshold is reached, regardless of the mechanism or mechanisms involved in reaching the threshold of T-cell sensitization and activation. Further, different mechanisms may operate in different individuals, adding to the complexity of the pathogenesis of the disease.

Disruption of the BBB and Entry of Self-Reactive T Cells in the CNS Regardless of the mechanisms involved in the generation and activation of T cells which could initiate an autoimmune demyelinating disease, it is generally thought that these events occur outside the nervous system. Information gained from studies of various animal models indicate that activated T cells can cross an intact BBB as part of normal immune surveillance.5,54 A reasonable hypothesis is that T cells specific for an antigen and sensitized through the mechanisms discussed above enters the nervous system nonspecifically. If a T cell encounters its antigen after entering the nervous system, further activation occurs and the production of proinflammatory cytokines is initiated. It is still unknown which cells could act as antigen-presenting cells within the CNS. The most likely

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candidates are perivascular macrophages and microglial cells.55,56 A subset of perivascular macrophages seems to constitutively express MHC molecules irrespective of the activation stage. These few strategically located cells may be responsible for the presentation of myelin peptides released from adjacent oligodendrocytes during the physiological turnover of myelin proteins in the initial stage of the disease. Again, studies using the EAE model indicate that the number of antigen-specific T cells that enter the nervous system after passive transfer is small, and that these cells remain in the perivascular cuff.57 Most likely, the production of proinflammatory cytokines and chemokines serves to activate the endothelial cells, with upregulation of cell adhesion molecules, which results in the recruitment of an inflammatory infiltrate which is largely antigen nonspecific. The next step in this process is initiation of the actual effector mechanism(s) which lead to myelin damage. It is possible that in some cases an inflammatory response can be initiated without significant myelin damage, and this would suggest that two distinct mechanisms operate with respect to induction of inflammation and myelin damage. Since both processes are most likely under genetic control, this scenario would be consistent with the complex data evolving with regard to genetic susceptibility in MS.58 A feature of the MS lesion is that the inflammation resolves over time, a picture consistent with the relapsing-remitting nature of the disease. It is unclear what downregulates the response. Several possibilities exist, including the production of regulatory cytokines such as IL-4, IL-10 and TGF-β59 or apoptosis of the T cells driving the response.60

Effector Mechanisms of Myelin Damage A crucial question in understanding the immunopathogenesis of demyelinating diseases is whether the direct target of the autoimmune attack is represented by myelin or oligodendrocytes. The lack of expression of MHC class II molecules on oligodendrocytes61-63 indicates that oligodendrocytes themselves are not capable of presenting myelin antigens to the invading CD4 T cells. A number of possible, mainly indirect, effector mechanisms have ben hypothesized. Here we summarize some of these hypothetical mechanisms and briefly discuss the controversial points that have been raised for each of them. CD8-Mediated Cytotoxicity In vitro studies have demonstrated that under certain experimental conditions oligodendrocytes may express MHC class I molecules. This observation raised the possibility of a direct killing of the myelin-forming cells by myelin-specific CD8+ class I-restricted T lymphocytes. To date, however, there is no convincing evidence of MHC class I expression on human oligodendrocytes in situ.12 In addition, data generated in the EAE have clearly demonstrated that the disease is inducible in animals deficient of CD8 T cells.64,65 Cytokine-Mediated Bystander Demyelination Cytokines are considered the most likely mediators of the myelin damage in MS. It is well known that CD4 T lymphocytes can be divided into two different subpopulations according to their cytokine production pattern. 66-68 Th1 cells produce IFN-γ, IL-2 and TNFαβ and mediate delayed type inflamatory reactions, whereas Th2 T cells produce IL-4 and IL-5 and cooperate with B cells for antibody production.68 It has been established in EAE that Th1 cells activated by myelin antigens are necessary for the development of the disease.69 On the other hand, Th2 cytokines are thought to be protective and improve the clinical picture in EAE.70 This paradigm has recently been challenged by the observation that Th2 immune deviation induced during EAE in marmoset by administration of antigen in soluble form led to a lethal form of the disease associated with increased antibody

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production.71 In another study, induction of EAE was demonstrated by transferring a Th2 myelin-specific T-cell clone in immunodeficient mice.72 These findings suggest that the cytokine network in autoimmune disease may be more complicated than previously thought and may account for the difficulties of defining a clear-cut Th1/Th2 paradigm in MS.73 These findings also raises the issue of the potential importance of antibody to myelin in the evolution of the MS lesion. Nevertheless, Th1 cytokines such as IFN-γ and TNF-αβ are likely to play a key role in the development of MS lesions and TNF especially is probably directly involved in myelin damage. It is known from clinical data that the systemic administration of IFN-γ resulted in an increased relapse rate in MS patients.74 TNF-α and LT-αβ are expressed in the CNS during acute EAE 75,76 and the production of TNF has been associated with the encephalitogenic potential of T-cell clones.77 Strategies aimed at blocking TNF activity (monoclonal antibodies, soluble receptors, pharmacological inhibitors) lead to the prevention and reversal of EAE.78,79 In addition, it has been demonstrated that the susceptibility to develop EAE in different mouse strains is correlated to the amount of TNF production by glial cells.80 Finally, TNF-α and LT-α mediate direct oligodendrocyte and myelin damage in vitro81 and overexpression of TNF-α in the CNS causes demyelination.82 Role of Macrophages and Antibodies When early MS plaques are examined by electron microscopy, macrophages seem to be responsible for starting the initial step of myelin destruction by stripping myelin lamellae away from the axons.3,29 This process starts at the level of the nodes of Ranvier, where the myelin layers produced by adjacent oligodendrocytes meet each other. The removal of myelin lamellae itself is achieved through receptor-mediated endocytosis, a process that starts by attachment of the macrophage membrane to superficial layers of the myelin sheath.3,29 Parts of the myelin membrane are then engulfed into vesicles at the macrophage surface called coated pits. Since coated pits represent a concentration of surface receptors, the myelin-macrophage contact seems to be mediated by a receptor ligand interaction. It is well known that activated macrophages upregulate both Fc and complement receptors, and both of them could contribute to this process. According to findings reported in EAE, the transfer of anti-myelin antibodies (especially antibodies against MOG, a protein found on the outer surface of myelin lamellae) is a major determinant in the development of demyelination.83 The lesions of classical MBP-induced EAE in Lewis rats that is produced by the transfer of purified MBP-specific T cells are mainly inflamatory, not demyelinating. If, however, MOG-specific antibodies are transferred following induction of the disease, large demyelinating lesions develop.83,84 Therefore, it is conceivable that the attachment of specific immunoglobulin to membrane proteins embedded in the myelin sheath plays a major role in the macrophage-mediated myelin damage. In addition to their direct mechanism, antibody-driven macrophages may damage the myelin sheath by indirect mechanisms such as the directed release of complement and inflammatory mediators, including reactive oxygen species85 and eicosanoids. Finally, activated macrophages may secrete TNF-α also involved in myelin injury (see above). Gamma Delta T Cells A small number of T lymphocytes express a TCR comprising one γ and one δ chain.86 These cells are unable to recognize antigens presented by classical MHC class I and class II MHC molecules. They are speculated to be evolutionarily older than αβT cells and to recognize antigens in the context of non-classical MHC molecules as a first line of defense against bacterial infections. Heath shock proteins (HSP) are targets of γδ T cell recognition.86,87 T cells expressing γδ T cell receptors have been found in the brains of patients with MS,88

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although some disagreement exists as to their frequency and whether they are more commonly found in acute or chronic lesions.89 It is also still unclear whether or not these cells are clonally expanded in the lesions as well as in the CSF of MS patients.90,91 The interest in this T-cell subpopulation has been raised by the observation that γδ T cells are able to lyse isolated oligodendrocytes.92 In addition, it is known from histochemical studies that HSP60 and HSP70 are expressed in the brain of MS patients and that both are inducible on isolated oligodendrocytes. 93 Although the in vitro oligodendrocytolytic activity of γδ T cells is independent of the expression of HSP in the target cells, it has been suggested that γδ-expressing T cells may be responsible for the myelin damage in vivo. However, it has to be mentioned that if such a mechanism contributes to oligodendrocyte destruction, it is probably more important in the evolution of chronic lesions, as oligodendrocytes are not consistently lost in acute MS lesions. Fas-Fas Ligand System Fas is a cell surface receptor belonging to the superfamily of TNF receptors that transduce an apoptotic cell death signal upon engagement by its natural ligand (Fas ligand, FasL). T cells as well as macrophages (cells types identified in active MS lesions) express FasL94,95 and may mediate cell cytotoxicity in vitro by interacting with Fas-expressing target cells. Immunohistochemical studies have recently demonstrated in tissue samples derived from MS lesions an elevated expression of Fas on oligodendrocytes, compared with oligodendrocytes derived from control tissue collected from subjects with or without other neurologic disease.96 In these lesions microglia and infiltrating cells displayed intense immunoreactivity to FasL.97 In addition, cells undergoing apoptosis were present in MS lesions and showed a colocalization with FasL expression. Taken together these findings suggest that Fas-mediated signaling might contribute to a novel cytotoxicity mechanism for immune-mediated oligodendrocyte damage in MS.

Failure to Demonstrate Antigen-Specific Responses in MS In spite of a large number of studies carried out in recent years, the antigens and the cells involved in myelin-specific T-cell responses in a specific MS patient or group of patients are still poorly understood. Antigen-dependent approaches have so far failed to demonstrate a T-cell response to myelin determinants that is somehow specific for MS.98 Following the elegant studies carried out in EAE demonstrating that myelin-specific T cells can mediate autoimmune demyelination, T-cell response to various myelin antigens has been deeply investigated in MS patients. The main strategies were based on the search for antigen-specific T-cell expansions in peripheral blood, or the search for an enrichment in myelin antigen-specific T cells in the CSF compared with peripheral blood and, finally, on the characterization of myelin-reactive T cells in terms of phenotype, fine specificity and TCR usage. Although differences in the frequency and in the functional phenotype of myelin-reactive T cells were reported in MS patients compared to normal donors99-103 these findings were not convincingly reproduced by other groups.104,105 A number of theoretic and experimental problems may underlie this failure: 1. Different antigens in different patients. Available data are strongly biased toward MBP. The frequency and the characteristics of T-cell response to other myelin proteins are poorly investigated. MBP is easy to handle, but might not be the more relevant myelin autoantigen. It is well known that MBP is expressed in the peripheral nervous system, whereas in MS the inflammatory response is confined to the CNS. 2. Lack of stratification of patients and controls for their immunogenetic background. It is well established that the MHC phenotype plays a major role in shaping the

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individual T cell repertoire, as well as in determining which epitopes within a complex protein can elicit a T-cell response. 3. Lack of stratification for timing of sampling. In the various studies, T cells have been obtained from MS patients irrespective of their stage of disease. Very few studies have attempted to correlate myelin-specific T-cell responses to the stage of disease activity at the time of sampling. In addition, only recently it became obvious that clinical parameters are not a reliable measure of the inflammatory activity in the CNS. MRI will be an useful tool to directly measure disease activity in studies aimed at linking immunological parameters to the various phases of disease. 4. Epitope spreading. The identification of the target antigen and T-cell epitopes involved in MS may be further complicated by the possibility that the autoantigen triggering the autoimmune attack may change during the course of the disease. Intramolecular and intermolecular spreading have been elegantly demonstrated in EAE.106-108 T cells reactive to cryptic epitopes have encephalitogenic potential and are probably involved in myelin destruction during the relapses of the disease. No conclusive evidence for modification over time of the characteristics of T-cell response to myelin antigen has been reported in MS. It is has been suggested for MBP-reactive T cells that the number of epitopes recognized and the heterogeneity of TCR expressed by these cells is smaller during the first stages of the disease. However, in the few instances that T-cell response to MBP was serially evaluated in MS patients, it was found to be stable in terms of epitope specificity as well as TCR usage.109,110 5. No real data on CD8+ T-cell responses to myelin antigens.

Degeneracy of TCR Recognition, Molecular Mimicry and Autoreactivity As mentioned above, T cells specific for self myelin antigens can be isolated from the peripheral blood of patients with MS as well as from healthy individuals. The biological meaning of these autoreactive circulating T cells remains unknown. One surprising finding is the impressively high frequency of MBP-reactive T cells that can be isolated from both MS patients and normal individuals using high antigen concentrations in the primary cultures (Vergelli et al, manuscript submitted). When the functional features of these T cells generated with high antigen concentration were examined, it became obvious that they recognize a wider spectrum of MBP epitopes and require higher antigen concentrations to get activated compared to MBP-specific T-cells obtained using low antigen concentration (Vergelli et al, manuscript submitted). The affinity (antigen requirement) of MBP-specific T cells appears therefore to be strictly related to the concentration used in the primary culture: at low antigen concentrations a small number of high affinity T cells was isolated; on the other hand, the use of high antigen concentration in the primary culture yielded a high number of low affinity T cells. These findings indicate that a high number of low affinity autoreactive T cells is part of the “normal” T-cell repertoire. Recent studies from our group have shown a high degree of degeneracy in antigen recognition by MBP-reactive T cells.111 The identification of single amino acid substitutions of MBP peptides yielding increased T-cell responsiveness,51 as well as the proliferative response to combinatorial peptide libraries consisting of mixtures containing up to 1014 different peptides,50 suggested that the number of productive ligands for a single autoreactive T-cell clone is much higher than previously expected. These approaches enabled us to identify crossreactive peptides inducing T-cell response at lower antigen concentration compared to the native MBP peptide, suggesting that the “real” specificity of MBP-reactive T cells may be different from the antigen used for the selection and the expansion of T cells in culture. The MBP peptide could just happen to be a low affinity crossreactive ligand.

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The presence of high numbers of circulating low affinity MBP-reactive T cells fits with the hypothesis that, due to the large extent of degenracy in T-cell recognition, self-reactive T cells are commonly activated and expanded during the course of protective immune reactions to foreign agents. The low affinity antigen recognition may prevent these autoreactive T cells from triggering autoimmunity even if they should encounter the crossreactive self antigen within the target organ (i.e., the CNS) upon cross-activation in the periphery. However, if a concomitant inflammatory process is ongoing in the CNS, leading to an increased expression of MHC and costimulatory molecules, it is conceivable that the low affinity can be compensated by the elevated number of available MHC-peptide complexes on the surface of APC and by costimulatory factors raising the overall avidity beyond the threshold required for triggering an effector T-cell response. Once the inflammatory reaction is established within the CNS, the process can be further amplified by the release of self-antigens. In addition, successive cross-stimulation of low affinity self-reactive T cells in the periphery might result in exacerbations of the inflammatory reaction in the CNS due to the “susceptible” local environment. Therefore, both a systemic and an organ related factor are required to trigger autoimmunity in this scenario. Alternatively, a second but not mutually exclusive scenario can be envisioned in which foreign antigen may interact and cross-activate T cells with high affinity self-reactive TCR. This will lead to expansion and activation of self-reactive T cells that may trigger autoimmunity by themselves. This is probably similar to what happens in experimental autoimmunity when animals are immunized with a self-antigen, leading to the expansion of high affinity self-reactive T cells. However, it is known from experimental autoimmunity that the simple expansion of self-reactive T cells may not be sufficient, and several questions remain open: Why do we need adjuvant to induce an active experimental autoimmune disease? Why do we not need adjuvant in passive transfer autoimmune diseases? Do the in vitro activated autoreactive cells have the right repertoire of adhesion/costimulatory molecules? Why in the MBP-TCR-transgenic mice does the autoimmunity develop only in a germ-containing environment? Something more than cross-activation of high affinity self-reactive T cells probably has to happen before autoimmunity is triggered. In any case, in this second scenario everything leading to the autoimmune process may happen outside the target organ.

Conclusion Dynamic View of MS Pathogenesis and Natural History Both in MS and in EAE, the pathology of the CNS lesions indicates that myelin antigen-specific T lymphocytes are probably responsible of initiation of the inflammatory infiltrates passing the BBB as activated T cells and remaining in the perivascular cuffs. Non-antigen-specific T lymphocytes, B lymphocytes and macrophages are subsequently recruited nonspecifically by lymphocytokines and other inflammatory factors and activation of endothelial cells, leading to disruption of the BBB. It is probably this step that is seen in contrast enhanced MRI. Evidence from both pathological studies and MRI data indicate that some lesions, once they reach a chronic stage of progression may become independent of further BBB disruption or influx of additional inflammatory cells. This could be due to either an antibody-mediated process due to resident B cells or a process mediated by activated macrophages. Regardless, the suggestion that the lesion can be thought of as having several distinct steps has important implications both for our understanding of the disease mechanism and for planning therapeutic strategies. For example, treatments that target T cells such as those designed to block activation in the periphery may only be effective if used relatively early in the course of the disease. Similarly, interferon-β 1a and 1b have both

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been shown to have a substantial effect on reducing BBB disruption. Although the mechanism is not known, it is thought that the drugs may influence T-cell activation or events at the endothelial cell. In either case, this would again be a treatment that would be most effective in the early stage of the process. At present, additional attention needs to be given to strategies that will target the existing lesion and modify effector mechanisms leading to demyelination or even stimulate remyelination. Some studies in this direction are now underway.

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Index Symbols

E

αB-crystallin 114, 115, 118-123, 126-128, 132

Experimental allergic encephalitis (EAE) 12, 13, 19-25, 29, 33, 34, 38-43, 48, 63, 74-77, 88, 91, 92, 94-101, 103, 104, 116-119, 121, 123, 124, 127, 134-136, 138-142

A Altered peptide ligands 36, 78, 81, 95, 117 Antibodies 5, 6, 12, 19, 23-27, 30, 40, 51-53, 59, 63, 66, 69, 70-75, 84, 86-88, 92-94, 96, 97, 99, 101, 107, 108, 113, 115, 117, 118, 130, 139 Antigen-specific therapy 107, 118, 127, 128 Astrogliosis 96, 134 Autoimmune disease 5, 7, 9, 13, 19, 20, 31-34, 45, 69, 71, 76-78, 80, 84, 86, 89, 103, 107, 113, 117, 118, 135, 136, 139, 142 Autoimmunity 1, 19, 24, 26, 29, 31-35, 37, 59, 67, 69, 77, 83, 84, 86, 91, 105, 106, 110, 113, 133, 136, 142 Autoreactivity 121, 123 Axonal loss 1

B Blood brain barrier (BBB) 38-41, 43, 101, 113, 127, 133, 134, 137, 142, 143

C CD1 7, 8, 65 CD30 61-63, 65, 66, 69, 70, 79, 80, 82, 85 CD5 9 Central nervous system (CNS) 1, 9, 10, 12-14, 19, 22, 24, 26, 29, 31, 34, 37-43, 45, 48-54, 72, 74, 87, 91, 94-96, 98-103, 106-108, 110, 113, 114, 116-118, 121, 123, 125-128, 133-137, 139-142 Cytokines 6-8, 15, 24, 25, 27, 32, 33, 36, 40, 48, 49, 52, 53, 59-71, 73-82, 85-88, 91-106, 108, 109, 116, 117, 119, 124, 137-139, 142

D Demyelination 1, 13, 19, 22-24, 27, 39, 40, 133, 134, 139, 140, 143 Determinant spreading 123, 127, 128

G Gene therapy 37, 38, 42, 45, 48 Gene transfer 37-39, 43-46 Green flouresence protein (GFP) 46 Glycoprotein 8, 17, 19, 41, 52, 53, 67, 107, 111, 135

H Heat shock proteins 5

I IL-4 59-84, 92-99, 103, 105-107, 111, 119 Inflammatory infiltrates 24, 76, 133-135, 142 Interferon-γ (IFN-γ) 32,33, 59-83, 86, 88, 92-97, 99-105, 107, 110-112, 119 Isopentenylpyrophosphate (IPP) 11

M Macrophages 8, 24, 32, 34, 35, 54, 59, 63-65, 69, 70, 72-76, 81, 87, 96, 97, 106, 112, 119, 123, 133, 134, 137-140, 142 Molecular mimicry 34, 35, 121, 124, 126, 141 Moloney murine leukemia virus (Mo MuLV) 44 Magnetic Resonance Imaging (MRI) 22, 23, 27, 108, 133-135, 141, 142, 144 Myelin 1, 10, 11, 13, 14, 19, 20, 22-27, 29, 31, 34-36, 39-41, 48, 49, 51-54, 74-77, 80, 87-89, 91, 95-98, 103-119, 121-128, 130-143, 147 Myelin basic protein 19, 26, 27, 29, 31, 34-36, 39, 51, 52, 54, 88, 89, 95, 104-108, 111, 112, 121, 130, 135, 136 Myelin damage 95, 121, 123, 138-140 Myelin protein 24, 41, 114, 115, 117-119, 125, 126, 128, 138, 140

150

T-Cell Autoimmunity and Multiple Sclerosis

O

T

Oligodendrocyte 10, 11, 15, 18, 19, 41, 52, 53, 74, 96, 97, 107, 108, 111, 112, 114, 118, 125-127, 133-135, 138-140

T-cell clones 2, 6, 18, 24, 29-36, 53, 60-62, 66, 70-76, 78-81, 84-88, 103, 105, 107, 108, 121, 132, 137, 139 T-cell homing 96 TCR γδ 1, 2, 5-7, 10-13 Th1/Th2 cells 69, 74, 78 Transduction 46, 68

P Pathology 14, 17, 19, 22-24, 34, 51, 52, 118, 135, 136, 142 Phosphorylated nonpeptidic metabolites 2, 3, 11

R Retrovirus 44

V Vδ genes 2 Vγ genes 2