GENERATION ANDEFFECTOR FUNCTIONS

OF REGULATORYLYMPHOCYTES

Novartis Foundation Symposium 252

2003

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GENERATION ANDEFFECTOR FUNCTIONS

OF REGULATORYLYMPHOCYTES

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GENERATION ANDEFFECTOR FUNCTIONS

OF REGULATORYLYMPHOCYTES

Novartis Foundation Symposium 252

2003

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Generation and e¡ector functions of regulatory lymphocytes / [editors, Gregory Bock andJamie Goode].

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Contents

Symposium onGeneration and e¡ector functions of regulatory lymphocytes, held attheNovartisFoundation, London, 9^11July 2002

Editors: Gregory Bock (Organizer) and Jamie Goode

This symposiumwas based on a proposalmade byMatthias vonHerrath and Je¡rey Bluestone

Jean-Fran�ccois Bach Chair’s introduction 1

Shimon Sakaguchi, Shohei Hori,Yoshinori Fukui,Takehiko Sasazuki,Noriko Sakaguchi andTakeshiTakahashi Thymic generation and selection ofCD25+CD4+ regulatory T cells: implications of their broad repertoire and highself-reactivity for the maintenance of immunological self-tolerance 6Discussion 16

EthanM. Shevach, CiriacoA. Piccirillo, Angela M.Thornton andRebecca S. McHugh Control of T cell activation by CD4+CD25+ suppressorT cells 24Discussion 36

Adam P. Kohm, Pamela A. Carpentier and Stephen D. Miller Regulation ofexperimental autoimmune encephalomyelitis (EAE) by CD4+CD25+ regulatoryT cells 45Discussion 52

Elisa Boden, QizhiTang, Helene Bour-Jordan and Je¡reyA. Bluestone Therole of CD28 and CTLA4 in the function and homeostasis of CD4+CD25+

regulatory T cells 55Discussion 63

Clare Baecher-Allan, Julia A. Brown, GordonJ. Freeman andDavid A. Ha£erCD4+CD25+ regulatory cells from human peripheral blood express very high levelsof CD25 ex vivo 67Discussion 88

v

Fiona Powrie, Simon Read, Christian Mottet, HolmUhlig and Kevin MaloyControl of immune pathology by regulatory T cells 92Discussion 98

General discussion I TGFb 106

Maria Grazia Roncarolo, Silvia Gregori andMegan Levings Type 1Tregulatory cells and their relationship with CD4+CD25+ Tregulatory cells 115Discussion 127

Leonard C. Harrison, Natasha R. Solly andNathan R.Martinez (PRO)insulin-speci¢c regulatory T cells 132Discussion 141

Qing-ShengMi, Craig Meagher andTerry L. Delovitch CD1d-restricted NKTregulatory cells: functional genomic analyses provide new insights into themechanisms of protection againstType 1diabetes 146Discussion 160

Eli Sercarz, Emanual Maverakis, Peter van den Elzen, Loui Madakamutil andVipin Kumar Seven surprises in theTCR-centred regulation of immuneresponsiveness in an autoimmune system 165Discussion 171

KathrynJ.Wood, Hidetake Ushigome, Mahzuz Karim, Andrew Bushell,Shohei Hori and Shimon Sakaguchi Regulatory cells in transplantation 177Discussion 188

KimJ. Hasenkrug CD4+ regulatory T cells in chronic viral infection 194Discussion 199

General discussion II 203

MarcaH.M.Wauben, EstherN.M.’tHoen andLeonie S.Taams Modulation ofT cell responses after cross-talk between antigen presenting cells and T cells: a give-and-take relationship 211Discussion 220

Jacques Banchereau, Joseph Fay,Virginia Pascual and A. Karolina PaluckaDendritic cells: controllers of the immune system and a new promise forimmunotherapy 226Discussion 235

vi CONTENTS

Chrystelle Asseman andMatthias von Herrath Regulation of viral andautoimmune responses 239Discussion 253

General discussion III Active immune regulation 257

MargaretJ. Dallman, Brian Champion andJonathanR. Lamb Notch signallingin the peripheral immune system 268Discussion 276

Lucienne Chatenoud CD3 antibody treatment stimulates the functional capabilityof regulatory T cells 279Discussion 286

Allan McI Mowat, AnneM. Donachie, LucyA. Parker, Neil C. Robson,Helen Beacock-Sharp, Lindsay J. McIntyre, Owain Millington andFernando Chirdo The role of dendritic cells in regulating mucosal immunityand tolerance 291Discussion 302

Index of contributors 306

Subject index 308

vii CONTENTS

Participants

Abul K. Abbas Department of Pathology, University of California SanFrancisco, RoomM 590, 513 Parnassus Avenue, San Francisco, CA 94143, USA

Chrystelle Asseman (Novartis Foundation Bursar) LaJolla Institute for Allergyand Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA

Jean-Fran�ccois Bach (Chair) Laboratoire d’Immunologie, Ho“ pital Necker,161 rue de Se' vres, 75743 Paris Cedex 15, France

Jacques Banchereau Baylor Institute for Immunology Research, 3434 LiveOak, Suite 205, Dallas,TX 75204, USA

Je¡rey Bluestone UCSF Diabetes Center, 513 Parnassus Ave Box 0540,San Francisco, CA 94143-0540, USA

Lucienne Chatenoud Laboratoire d’Immunologie, Ho“ pital Necker, 161 rue deSe' vres, F-75743 Paris Cedex 15, France

Cristina Cuturi INSERM U437, CHUde Nantes, 30 solJ. Monnet, 44093,Nantes, France

Margaret Dallman Department of Biology, Sir Alexander Fleming Building,Imperial College of Science,Technology and Medicine, South Kensington,London SW7 2AZ, UK

Terry Delovitch TheJohn P. Robarts Research Institute, 1400 Western Road,London, Ontario, Canada N6G 2V4

Richard Flavell Department of Immunobiology,Yale University School ofMedicine, HHMI, 310 Cedar Street, FMB412, Box 208011, New Haven,CT 06520-8011, USA

David Ha£er Center for Neurologic Diseases, Harvard Medical School, 77Avenue Louis Pasteur, Boston, MA 02115, USA

viii

Leonard Harrison Autoimmunity andTransplantation Division,TheWalterand Eliza Hall Institute of Medical Research,The Royal Melbourne HospitalPO, Parkville, 3050 Victoria, Australia

KimHasenkrug Laboratory of PersistentViral Diseases, Rocky MountainLaboratories, National Institute of Allergy and Infectious Diseases, Hamilton,MT 59840, USA

StephenMiller Immunobiology Center, NorthwesternUniversity School ofMedicine, 303 E. ChicagoAvenue, Chicago, IL 60611-3072, USA

AvMitchison Department of Immunology,Windeyer Institute of MedicalScience, University College London Medical School, 46 Cleveland Street,LondonW1P 6DB, UK

AllanMowat Department of Immunology and Bacteriology,Western In¢rmary,Glasgow G11 6NT, UK

Virginia Pascual Baylor Institute for Immunology Research, 3434 Live Oak,Suite 205, Dallas,TX 75204, USA

Fiona Powrie University of Oxford, SirWilliam Dunn School of Pathology,South Parks Road, Oxford OX13RE, UK

Maria Grazia Roncarolo San Ra¡aeleTelethon Institute for GeneTherapy(HSR-TIGET),Via Olgettina 58, I-20132 Milano, Italy

Shimon Sakaguchi Department of Experimental Pathology, Institute forFrontier Medical Sciences, Kyoto University, 53 Shogoin Kawahara-cho,Sakyo-ku, Kyoto 606-8507, Japan

Eli Sercarz The LaJolla Institute for Allergy and Immunology, 10355 ScienceCenter Drive, San Diego, CA 92121, USA

Ethan Shevach Cellular Immunology Section, NIAIDLaboratory ofImmunology, Building10, Room11N315,10 CenterDrive,MSC1892, Bethesda,MD 20892-1892, USA

Matthias von Herrath Department of Immune Regulation, LaJolla InstituteforAllergy and Immunology,10355 Science CenterDrive, SanDiego, CA 92121,USA

ix PARTICIPANTS

MarcaWauben Department of InfectiousDiseases and Immunology, FacultyofVeterinary Medicine, Utrecht University, POBox 80.165, NL-3508 TDUtrecht,The Netherlands

ChristophWalker Novartis PharmaUK, Novartis Horsham Research Centre,Wimblehurst Road, Horsham RH12 5AB, UK

KathrynWood The Nu⁄eld Department of Surgery, University of Oxford,John Radcli¡e Hospital, HeadleyWay, Headington, Oxford OX3 9DU, UK

x PARTICIPANTS

Chair’s introductionJean-Fran �ccois Bach

Laboratoire d’Immunologie, Ho“ pitalNecker, 161 Rue de Se' vres, 75743 Paris, Cedex 15, France

The notion of T cell-mediated suppression/regulation is not new. In 1971, RichardGershon published a classical paper describing the capacity of T cells from miceimmunized by sheep red blood cells (SRBCs) to down-regulate the production ofanti-SRBC antibodies in na|« ve recipients (Gershon&Kondo 1970). This so-calledinfectious tolerance opened the ¢eld of suppressor T cells which gave rise to anunusually abundant future in the following decade. It is not necessary to reviewhere all the claims that were made in this period and all the problems that werethen raised both at the experimental and the interpretational levels. In any event,these problems ineluctably led to the discredit of the whole ¢eld, whichdisappeared from the scene in the early 1980s. It was only a few years later thatnew experiments were performed and eventually published indicating that T cell-mediated regulation was a real phenomenon, probably important in the control ofthe development of autoimmune and alloimmune responses. This resurrectionrelied on three sets of observations. The ¢rst one was undoubtedly the proposalby Mosmann & Co¡man (1989) of the Th1/Th2 paradigm. The demonstrationthat Th1 cytokine could down-regulate Th2 cell di¡erentiation and function, andreciprocally Th2 cytokines could down-regulate Th1 cells through a non-antigen-speci¢c cytokine-mediated mechanism provided a new explanation for thesuppressor T cells which had been described 10 years earlier. Fifteen years later,the Th1/Th2 paradigm is still very vivid, even if its generality is not as apparentas it was initially thought.A second observation which proved to be crucial for the emergence of the

suppressor cell concept was that of bystander suppression. Oral administration ofan autoantigen can induce tolerance to that antigen. Surprisingly enough, thistolerance extends to antigens other than the tolerogen in as much as this antigenis expressed at the same site (organ or cell) as the tolerogen (Al Sabbagh et al 1994).Here again, cytokines were implicated in the phenomenon. It was assumed that theinitial regulatory reaction produced by a tolerogen led to the local production ofcytokines that show suppressive activity against immune responses directed atother antigens at the lesion site.The third set of observations dealt with the reappraisal of the thymectomy

experiments performed in the late 1960s. It had been shown at that time that

1

thymectomy at day 2^5 induced polyautoimmune syndrome (Nishizuka &Sakakura 1969). It was shown in the early 1990s that such polyautoimmunesyndrome could be prevented by the administration of CD4+CD25+ T cells(Asano et al 1996), excluding other explanations of the thymectomy inducedautoimmunity (e.g. absence of selection in the thymus). One should lastlymention experiments performed in various laboratories including oursshowing the presence in healthy mice of regulatory cells capable ofpreventing disease onset in various autoimmune models. We indeed showed innon-obese diabetic (NOD) mice that CD4+ T cells from pre-diabetic NOD couldprevent the transfer of diabetes a¡orded by diabetogenic T cells (derived fromdiabetic NODs) when injected into immunoincompetent recipients (Boitard et al1989).

Still unanswered questions

If there is a general although not absolute consensus about the existence ofregulatory T cells today, many questions remain unanswered. CD4+ T cells maybe regulatory. How many distinct T cell subsets comprise the CD4+ regulatoryT cells? The study of phenotypes is helpful, looking either at membrane markersor at cytokine production, pro¢le and dependency. It must be admitted, however,that study of phenotypes and cytokine production has not yielded convergentresults. As far as markers are concerned, CD25 is probably the most reliablemarker, although CD45RB (Powrie et al 1993) and CD62L (Herbelin et al 1998)are also useful and do not de¢ne exactly the same cells.Maybe othermarkers such asCTLA4 and glucocorticoid-induced tumour necrosis factor (TNF) receptorGITR(Zelenika et al 2002, McHugh et al 2002, Shimizu et al 2002) will prove useful,perhaps in a complementary fashion to CD25 and CD62L.Themode of action of regulatory T cells is very uncertain, and probably variable

from one subset to another. Cytokines are logical candidates. They probably play acentral role for Th2 cells and Treg1 cells (Groux et al 1997). Their role for CD25+ Tcells is more dubious, although a number of data have recently incriminatedtransforming growth factor (TGF)b (Nakamura et al 2001). A special role couldbe played by interleukin (IL)10, at least in certainmodels, such as colitis. It could beof crucial importance to determine the respective role of cytokines for the variousT cell subsets, both in terms of their mode of action but also of their growth anddi¡erentiation.Antigen speci¢city is a very open question, except of course for Th2 cells and

Treg1 cells, the de¢nition ofwhich is based on stimulation by antigen.Morework isneeded to determine the speci¢city of CD25+ T cells. Do CD25+ T cells act in aspeci¢c fashion particularly with regard to organ-speci¢c antigens? Can theantigen-speci¢c CD25+ T cells recently described in allograft and tumour

2 BACH

immunity be assimilated to the CD25+ T cells controlling the expression ofphysiological autoimmunity?

Candidate regulatory T cells may beused with bene¢t for immunotherapy

Soluble autoantigens and altered peptide ligands apparently act by stimulatingTh2cells (Bach & Chatenoud 2001). CD3 antibody appears to act by stimulatingCD25+ T cells and, more precisely, by enhancing TGFb production by such cells(Chatenoud 2003, this volume). a galactosyl ceramide acts by stimulating NKTcells and has been shown to protect from diabetes onset in NOD mice (Sharif etal 2001, Hong et al 2001). It would be important to search for other methodsleading to the stimulation of the various regulatory T cell subsets and to analysethe feasibility of their clinical applications.

Conclusions: working hypothesis

More than 10 types of regulatory T cells have been described so far (Table 1). Onemay assume that some of these T cell types represent the variable expression of asingle T cell lineage, although this has not been proven. Theoretically one maybelieve that totally di¡erent lines of suppressor T cells exist. If this is the case, itwould be important to determine the conditions of their di¡erentiation. At ¢rstglance, one is tempted to separate cells which appear spontaneously in ontogenywithout deliberate intervention, such as CD25+ T cells or NKT cells. It isinteresting to note that depletion of these cells or genetic prevention of theirappearance leads to an increase of autoimmune disease in mice genetically proneto develop the disease. Thus, depletion of CD25+ T cells in NOD miceaccelerates diabetes onset and absence of NKT cells as a¡orded by geneticinvalidation of CD1d (Wang et al 2001) leads to accelerated diabetes onset in

CHAIR’S INTRODUCTION 3

TABLE 1 Classi¢cation of regulatory cells

Natural/innate Adaptive

CD25+ T cells Th1 cells

NKT cells Th2 cells

NK cells Tr1 cells

g/d T cells CD45RBlow T cells

Veto cells CD8+ T cellsAnti-idiotypic T cells

NOD mice. On the other hand, as mentioned above, Th2 cells, Treg1 cells andmaybe g/d CD8+ T cells (Harrison et al 1996) appear after administration ofantigens either as immunogens as in the case of experimentally inducedautoimmune disease, or as tolerogens as is the case when tolerance is induced inspontaneously autoimmune mice by administration of soluble autoantigens.This distinction is reminiscent of the opposition classicallymade between innate

and adaptive immunity. The word innate in the context of immunoregulationmaybe misleading since it is usually reserved to TCR-mediated immunity. Maybe itwould be more adequate to use the word ‘natural’ which relates to the appearanceof the cells in question independently of any contact with foreignness.

References

Al Sabbagh A, Miller A, Santos LM, Weiner HL 1994 Antigen-driven tissue-speci¢csuppression following oral tolerance: orally administered myelin basic protein suppressesproteolipid protein-induced experimental autoimmune encephalomyelitis in the SJL mouse.Eur J Immunol 24:2104^2109

Asano M, Toda M, Sakaguchi N, Sakaguchi S 1996 Autoimmune disease as a consequence ofdevelopmental abnormality of a T cell subpopulation. J Exp Med 184:387^396

Bach JF, Chatenoud L 2001 Tolerance to islet autoantigens and type I diabetes. Annu RevImmunol 19:131^161

Boitard C, Yasunami R, DardenneM, Bach JF 1989 T cell-mediated inhibition of the transfer ofautoimmune diabetes in NODmice. J Exp Med 169:1669^1680

Chatenoud L 2003 CD3 antibody treatment stimulates the functional capability of regulatory Tcells. In: Generation and e¡ector functions of regulatory lymphocytes. Wiley, Chichester(Novartis Found Symp 252) p 279^290

Gershon RK, Kondo K 1970 Cell interactions in the induction of tolerance: the role of thymiclymphocytes. Immunology 18:723^737

Groux H, O’Garra A, Bigler M et al 1997 A CD4+ T-cell subset inhibits antigen-speci¢c T-cellresponses and prevents colitis. Nature 389:737^742

Harrison LC, Dempsey-Collier M, Kramer DR, Takahashi K 1996 Aerosol insulin inducesregulatory CD8 gamma delta T cells that prevent murine insulin-dependent diabetes. J ExpMed 184:2167^2174

Herbelin A, Gombert JM, Lepault F, Bach JF, Chatenoud L 1998 Mature mainstream TCRab+CD4+ thymocytes expressing L-selectin mediate ‘active tolerance’ in the nonobesediabetic mouse. J Immunol 161:2620^2628

Hong S, Wilson MT, Serizawa I et al 2001 The natural killer T-cell ligand a-galactosylceramideprevents autoimmune diabetes in non-obese diabetic mice. Nat Med 7:1052^1056

McHugh RS, Whitters MJ, Piccirillo CA et al 2002 CD4+CD25+ immunoregulatory T cells:gene expression analysis reveals a functional role for the glucocorticoid-induced TNFreceptor. Immunity 16:311^323

Mosmann TR, Co¡man RL 1989 TH1 and TH2 cells: di¡erent patterns of lymphokine secretionlead to di¡erent functional properties. Annu Rev Immunol 7:145^173

Nakamura K, Kitani A, Strober W 2001 Cell contact-dependent immunosuppression byCD4+CD25+ regulatory T cells is mediated by cell surface-bound transforming growthfactor b. J Exp Med 194:629^644

Nishizuka Y, Sakakura T 1969 Thymus and reproduction: sex-linked dysgenesia of the gonadafter neonatal thymectomy in mice. Science 166:753^755

4 BACH

Powrie F, LeachMW,Mauze S, Caddle LB, Co¡man RL 1993 Phenotypically distinct subsets ofCD4+ T cells induce or protect from chronic intestinal in£ammation in C. B-17 scid mice. IntImmunol 5:1461^1471

Sharif S, Arreaza GA, Zucker P et al 2001 Activation of natural killer T cells by alpha-galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1diabetes. Nat Med 7:1057^1062

Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S 2002 Stimulation of CD25+CD4+

regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol 3:135^142

Wang B, Geng YB, Wang CR 2001 CD1-restricted NK T cells protect nonobese diabetic micefrom developing diabetes. J Exp Med 194:313^320

Zelenika D, Adams E, Humm S et al 2002 Regulatory T cells overexpress a subset of Th2 genetranscripts. J Immunol 168:1069^1079

CHAIR’S INTRODUCTION 5

Thymic generation and selectionof CD25+CD4+ regulatory T cells:implications of their broad repertoireand high self-reactivity for themaintenance of immunologicalself-toleranceShimon Sakaguchi*{, Shohei Hori*{, Yoshinori Fukui{, Takehiko Sasazuki{,Noriko Sakaguchi*{ and Takeshi Takahashi*

*Department of Experimental Pathology, Institute for Frontier Medical Sciences, KyotoUniversity, Kyoto 606-8507, {Laboratory of Immunopathology, Research Center for Allergyand Immunology, The Institute for Physical and Chemical Research (RIKEN), Yokohama230-0045, and {Division of Immunogenetics, Department of Immunobiology and Neuroscience,Medical Institute of Bioregulation, Kyushu University, and CREST, Japan Science andTechnology Corporation, Fukuoka 812-8582, Japan

Abstract. Besides positive andnegative selectionofTcells, another functionof the thymusinmaintaining immunological self-tolerance is the production ofCD25+CD4+ regulatoryT cells capable of preventing autoimmune disease. They acquire the regulatory activitythrough the thymic selection process, and are released to the periphery as a functionallyand phenotypically mature population. Our recent study with transgenic mice in whichevery class II MHCmolecule covalently binds the same single peptide has revealed that aparticular self-peptide/MHC ligand in the thymus can positively select a broad repertoireof functionally mature CD25+CD4+ regulatory T cells as well as na|« ve T cells.Interestingly, the regulatory T cells bear higher reactivity than other T cells to theselecting ligand in the thymus even after negative selection by the ligand. This broadrepertoire and high self-reactivity of CD25+CD4+ regulatory T cells, together with theirhigh level expression of various accessory molecules, may guarantee their prompt ande⁄cient activation upon encounter with a diverse range of self peptide/MHC complexesin the periphery, ensuring dominant control of self-reactive T cells.

2003 Generation and e¡ector functions of regulatory lymphocytes. Wiley, Chichester (NovartisFoundation Symposium 252) p 6^23

One aspect of peripheral self-tolerance is maintained by regulatory CD4+ cellsnaturally occurring in normal na|« ve animals (Sakaguchi 2000, Shevach 2000,Maloy & Powrie 2001). Direct evidence for the key contribution of these

6

naturally arising regulatory T cells to self-tolerance is that removal of asubpopulation of CD4+ T cells from the normal immune system leads tospontaneous development of various autoimmune diseases in geneticallysusceptible animals; and reconstitution of the removed population prevents thedevelopment of autoimmunity (Sakaguchi et al 1985, Powrie & Mason 1990).The normal thymus seems to be continuously producing this autoimmune-preventive regulatory T cell population (Itoh et al 1999, Seddon &Mason 2000).Here we discuss how the thymus produces the regulatory T cells and how theirsensitivity and repertoire in recognizing self-antigens contribute to their functionof controlling self-reactive T cells.

Self-tolerance maintained by CD25+CD4+ regulatory T cells

Autoimmune diseases can be produced in normal rodents simply by removing aT cell subpopulation,without immunizationwith self-antigens in potent adjuvant.For example, when splenic cell suspensions from normal mice or rats are depletedof CD5high, CD45RClow, or CD25+CD4+ T cells and the remaining cells aretransferred to syngeneic T cell-de¢cient animals, autoimmune diseasespontaneously develops in multiple organs of the recipients within a few months;co-transfer of the removed population inhibits the development of autoimmunity(Sakaguchi et al 1985, 1995, Powrie & Mason 1990) (Fig. 1). Expression of theCD25 molecule is so far most speci¢c for such an autoimmune-preventive T cellpopulation present in normal na|« ve animals. CD25+ T cells, which constitute5^10% of peripheral CD4+ T cells and less than 1% of peripheral CD8+ T cells inmice and humans, are CD5high and CD45RBlow. Removal of these CD25+ T cellsfrom normal mice produced autoimmune disease in a wider spectrum of organsand with higher incidences than removal of CD5high cells or CD45RBlow T cells(Sakaguchi et al 1995). The autoimmune diseases thus induced areimmunopathologically similar to the human counterparts, e.g. Hashimoto’sthyroiditis, type A autoimmune gastritis with pernicious anaemia, insulin-dependent diabetes mellitus, Addison’s disease (autoimmune adrenalitis),premature ovarian failure with autoimmune oophoritis or male infertility withautoimmune orchitis. These ¢ndings indicate that the normal immune systemharbours self-reactive T cells su⁄ciently pathogenic in speci¢city and a⁄nity toinitiate autoimmune diseases, that their activation and expansion is held incheck in the normal periphery by a regulatory CD4+ T cell subpopulation,and that elimination or reduction of this population su⁄ces to break naturalself-tolerance, leading to spontaneous activation and expansion of self-reactive Tcells which mediate chronic and destructive autoimmune disease (Fig. 1).The CD25+CD4+ T cells in the periphery of normal na|« ve mice have the

following immunological characteristics (Takahashi et al 1998, Thornton &

THYMIC SELECTION OF CD25+CD4+ REGULATORY T CELLS 7

Shevach 1998, Itoh et al 1999). First, they potently suppress the activation/proliferation of other T cells in vitro when the two populations are co-culturedwith antigen-presenting cells (APCs) and stimulated with antigen. They needstimulation through TCR to exert the suppressive activity; and, upon TCRstimulation, they suppress the proliferation of not only T cells with the sameantigen speci¢city but also other T cells speci¢c for other antigens; i.e.CD25+CD4+ regulatory T cells stimulated by a speci¢c antigen exerts antigen-non-speci¢c suppression (Takahashi et al 1998). Second, although CD25+CD4+

regulatory T cells require antigenic stimulation for their functional activation,they themselves are non-proliferative (i.e. anti-proliferative or anergic) to in vitro

8 SAKAGUCHI ET AL

FIG. 1. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells. While thenormal thymus deletes T cells highly reactive with self-antigens expressed in the thymus, itcontinuously produces potentially pathogenic self-reactive CD4+ T cells, which persist in theperiphery at CD25� quiescent state. The normal thymus also continuously produces anergicand suppressive CD25+CD4+ T cells. On APCs, they suppress the activation and expansion ofCD4+ self-reactive T cells (and CD8+ self-reactive T cells) from CD25� dormant state. They areunique in requiring a signal through CTLA4 for their activation. Signal through GITR, on theother hand, attenuates their suppressive activity. When regulatory CD25+CD4+ T cells areeliminated or substantially reduced, or their regulatory function is impaired (for example, byblockade of CTLA4 or signal transduction through GITR), CD25� self-reactive T cellsbecome activated, expand and di¡erentiate to autoimmune e¡ector T cells.

antigenic stimulation, and this anergic state is closely linked with suppression.Importantly, the anergic/suppressive state of CD25+CD4+ T cells appears to betheir basal and default condition. When CD25+CD4+ T cells are TCR-stimulatedand treated with interleukin (IL)2 (or anti-CD28), anergy/suppression is broken,but they spontaneously revert to their original anergic state and re-acquire thesuppressive activity when IL2 (or anti-CD28 antibody) is removed (Takahashi etal 1998). Third, the roles of accessorymolecules are di¡erent betweenCD25+CD4+

regulatory T cells and other T cells. For example, the majority of CD25+ CD4+ Tcells in normal na|« ve mice constitutively express CTLA4 (CD152) and GITR(glucocorticoid-induced tumour necrosis factor receptor family gene) at highlevels. (Takahashi et al 2000, Read et al 2000, Solomon et al 2000, Shimizu et al2002, McHugh et al 2002). Blockade of CTLA4 or active signal transductionthrough GITR abrogates CD25+CD4+ T cell-mediated suppression, therebyproducing autoimmune diseases similar to those induced by elimination ofCD25+CD4+ T cells. The expression pattern of other accessory molecules onCD25+ CD4+ regulatory T cells (e.g. CD45RBlow, CD44high, CD5high, CD54[ICAM1]high, CD11a/CD18 [LFA1]high, partly CD62Llow) is in part similar to‘primed’, ‘activated’, or ‘memory’ T cells (Sakaguchi et al 1995, Itoh et al 1999),suggesting that the regulatory T cells may be primed and continuously stimulatedby self-antigens in the normal internal environment.

Thymic production of CD25+CD4+ regulatory T cells: another keyfunction of the thymus in self-tolerance

How are such autoimmune-preventive regulatory T cells produced in the immunesystem? The following ¢ndings indicate that the normal thymus produces themajority, if not all, of the CD25+CD4+ regulatory T cells in a functionallymature form. First, transfer of CD4+CD8+ mature thymocyte suspensionsdepleted of CD25+ thymocytes produces various autoimmune diseases insyngeneic nude mice, as shown with the transfer of CD25�CD4+ spleen cells(Itoh et al 1999) (Fig. 1). Neonatal thymectomy can also produce similarautoimmune diseases presumably by blocking the thymic production ofCD25+CD4+ regulatory T cells from the beginning of their production (Asano etal 1996, Suri-Payer et al 1998). Second, CD25+CD4+CD8� thymocytes in normalna|« ve mice are naturally anergic and exhibit equivalent in vitro suppressive activityto that of CD25+CD4+ T cells in the periphery (Itoh et al 1999). Third, theexpression pattern of cell surface accessory molecules is similar betweenCD25+CD4+ CD8� thymocytes and CD25+CD4+ T cells in the periphery; forexample, both constitutively express CTLA4 and GITR, being CD45RBlow,CD44high, and CD5high (Itoh et al 1999, Takahashi et al 2000, Shimizu et al 2002)and are characteristically resistant to superantigen-induced clonal deletion

THYMIC SELECTION OF CD25+CD4+ REGULATORY T CELLS 9

(Papiernik et al 1998). Furthermore, in TCR-transgenic mice, RAG2-de¢ciencyabrogated both CD25+CD4+ thymocytes and T cells, which predominantlyexpress endogenous TCRa chains (Itoh et al 1999). These ¢ndings collectivelyindicate that the normal thymus is continuously producing not only pathogenicself-reactive CD4+ T cells but also functionally mature regulatory CD25+CD4+ Tcells that control them, and releasing both to the periphery. The ¢ndings alsosuggest that CD25+CD4+ regulatory thymocytes and T cells may have adevelopmental continuity as a common T cell lineage and constitute a T cellsubpopulation functionally distinct from other T cells or thymocytes.

Thymic selection of CD25+CD4+ regulatory T cells by self-peptide/MHC

Several ¢ndings reported so far suggest that thymic generation of phenotypicallyand functionally mature CD25+CD4+ regulatory T cells may require uniqueselection events. For example, in mice expressing transgenic TCR speci¢c fornon-self antigens, a large fraction of CD25+CD4+ T cells expressed endogenousTCR a chains whereas other CD4+ T cells mainly expressed transgenic TCR aand b chains. Furthermore, RAG2 de¢ciency, which blocks the generearrangement of the endogenous TCR a-chain locus, abrogated the thymicdevelopment of CD25+CD4+ T cells in TCR transgenic mice (Itoh et al 1999). Ina double-transgenic strain that expressed a transgene-encoded speci¢c peptide inthe thymic stromal cells at a certain high level, the majority of T cells expressingtransgenic TCR a and b chains speci¢c for the peptide di¡erentiated intoCD25+CD4+ regulatory T cells (Jordan et al 2001, Kawahata et al 2002). Theregulatory T cells failed to develop, however, when double-transgenic miceexpressed either low-a⁄nity transgenic TCR for the same peptide or a highconcentration of the peptide in the thymic stromal cells because of insu⁄cientpositive selection or strong negative selection, respectively. Furthermore, H2-DMa-de¢cient mice, in which class II MHC molecules display a limited array ofself-peptides, developed CD25+CD4+ regulatory T cells, whereas MHC class II-de¢cient mice, which can generate a small number of CD4+ T cells restricted toclassical or non-classical MHC class I antigens, did not (Bensinger et al 2001).These ¢ndings altogether suggest that the thymic generation of CD25+CD4+

regulatory T cells may require rather high avidity interactions between TCRs andself-peptide/class II MHC ligands on the thymic stromal cells.To assess more directly this possible high self-reactivity of CD25+CD4+

regulatory T cells, we analysed B2L-TKO mice, a transgenic strain in whichevery class II MHC molecule covalently binds the same single peptide Ea52-68(Fukui et al 1997). We addressed whether a particular single peptide/MHC ligandcan positively and negatively select CD25+CD4+ regulatory T cells that arephenotypically and functionally similar to those found in normal mice, and, if

10 SAKAGUCHI ET AL

this is the case, the degree of diversity of the TCR repertoire, in particular, whetherthey aremore reactive than otherT cells to the selecting ligand.B2L-TKOmice didindeed develop CD25+ T cells as 1.73+0.5% of CD4+CD8� thymocytes (n¼5) or3.26+8% of CD4+ splenic T cells (n¼11) (Fig. 2A). The majority of splenicCD25+ CD4+ T cells were CTLA4high, GITRhigh, CD5high, CD44high, CD54high,CD11a/CD18high and CD45RBlow, being similar to CD25+CD4+ T cells innormal na|« ve mice. Functionally, B2L-TKO CD25+CD4+ T cells were anergicand suppressive upon TCR stimulation whereas CD25�CD4+ T cells were not(Fig. 2B). Stimulation by B6 APCs elicited proliferative responses in B2L-TKOCD25-CD4+ T cells, but not in CD25+CD4+ T cells, whereas B6 APCs and IL2

THYMIC SELECTION OF CD25+CD4+ REGULATORY T CELLS 11

FIG. 2. The presence of CD25+CD4+ regulatory T cells in the thymus and periphery of B2L-TKO mice. (A) Thymocyte suspensions (top), which were depleted of CD4+CD8+ T cells byanti-CD8 and complement treatment, or lymph node and spleen cell suspensions (bottom)prepared from a 2-month-old B2L-TKO mice were stained with FITC-anti-CD25 and PE-anti-CD4. (B) B2L-TKO CD25+CD4+ T cells, CD25�CD4+ T cells, or the mixture of the twopopulations at various ratios were stimulated with anti-CD3 mAb along with irradiatedautologous APCs. They were also stimulated with B6 APCs. (C) B2L-TKO CD25+ orCD25�CD4+ T cells were stimulated for 3 days with B6 or autologous B2L-TKO spleen cellsin the presence or absence of exogenous IL2 (100U/ml) and the amount of incorporated[3H]TdR during last 12 h of the culture was shown. A representative of four independentexperiments.

induced strong proliferation of both populations, indicating that they recognizeddiverse natural self-peptides bound to I-Ab molecules as non-self (Fig. 2B,C). Bycontrast, stimulation with autologous B2L-TKO spleen cells and IL2 constantlyelicited signi¢cantly high proliferative responses in B2L-TKO CD25+CD4+ Tcells but not in CD25�CD4+ T cells, which showed higher responses than theformer to other TCR stimuli (such as anti-CD3 antibody stimulation) (Fig. 2C).The TCR repertoire of CD25+CD4+ T cells in B2L-TKO mice is as diverse as

that in normal mice, and similar to the repertoire of CD25�CD4+ T cells. Forexample, there is no signi¢cant di¡erence between the two populations in theproportion of T cells expressing particular Vb gene segments (T. Takahashi,unpublished data). Assessment by the immunoscope technique of the CDR3 sizeof TCR a or b chains utilizing a particular Va or Vb family also revealed repertoirediversity (S. Hori, unpublished data).Taken together, these results indicate that a particular peptide/MHC ligand in

the thymus can positively select (or fail to negatively select, or both) CD25+CD4+

regulatory T cells with a diverse TCR repertoire and that the selected CD25+CD4+

regulatory T cells have higher avidity for the ligand compared with that of other Tcells also selected by the same ligand. In normal animals, summation of each broadrepertoire selected by each self-peptide/MHC ligand may well form a broadrepertoire of CD25+CD4+ regulatory T cells, which is almost ‘duplicated’ in theCD25+ and CD25�CD4+ population, but with higher reactivity of the former intotal to the thymic self-peptide/MHC ligands.

Are CD25+CD4+ regulatory T cells highly self-reactive inthe normal periphery?

A critical question then is whether CD25+CD4+ regulatory T cells present in theperiphery of normal animals are more reactive with peripheral self-antigens. Thisindeed appears to be the case since CD25+CD4+ T cells from normal na|« ve miceshowed higher in vitro proliferative responses than CD25�CD4+ T cells toautologous APCs presenting diverse self-peptides when stimulated with theAPCs in the presence of IL2; and the responses could be signi¢cantly reducedby blocking class II MHC molecules with speci¢c antibody (Fig. 3).Furthermore, such self-stimulated CD25+CD4+ regulatory T cells can suppressthe activation/proliferation of other T cells. For example, in the co-culture ofCD25�CD4+ T cells from OVA-peptide-speci¢c TCR transgenic mice andCD25+CD4+ T cells from normal non-transgenic mice, the latter stimulated byautologous APCs signi¢cantly suppressed the peptide-speci¢c proliferation ofthe former when the concentration of the peptide was relatively low (Fig. 3).At high peptide concentration, CD25�CD4+ T cells overcame the suppression,exhibiting equivalent degrees of responses as CD25�CD4+ T cells alone. Thus,

12 SAKAGUCHI ET AL

the self-reactive speci¢city of CD25+CD4+ regulatory T cells, their high levelexpressions of various accessory molecules and their speci¢c mode ofintracellular signal transduction through TCR or accessory molecules (such asCTLA4) may make them highly sensitive in their ability to recognize variousself-antigens in the periphery and therefore easily activated by them. Theregulatory T cells continuously stimulated by self-antigens may exertsuppression not only on self-reactive T cells but also on T cells with otherantigen-speci¢cities as well as through antigen-nonspeci¢c suppression, leadingto some degree of general immunosuppression in normal animals. Thesuppression may be su⁄cient to control self-reactive T cells, which generallybear low-a⁄nity TCRs, but insu⁄cient to suppress T cells with high a⁄nityTCRs for non-self antigens.

Implications of high sensitivity of CD25+CD4+ regulatory T cells toself-antigens in maintaining self-tolerance

The diverse TCR repertoire of CD25+CD4+ regulatory T cells and their highsensitivity to self-antigens have the following implications for their role inmaintaining natural self-tolerance.First, given that the CD25�CD4+ T cell population in normal na|« ve animals has

a pathogenic self-reactive repertoire even after thymic negative selection (Fig. 1),

THYMIC SELECTION OF CD25+CD4+ REGULATORY T CELLS 13

FIG. 3. High self-reactivity of CD25+CD4+ regulatory T cells in the normal periphery andtheir suppression in the physiological state. (A) CD25+ or CD25�CD4+ T cells from a normalBALB/c spleen were stimulated with autologous APCs in the presence of IL2 (100 U/ml) for 3days. Anti-pan-class II MHC monoclonal antibody (CA4) or rat Ig was added to the wells. Arepresentative result of four independent experiments. (B) CD25�CD4+ T cells from DO11.10TCR transgenic mice expressing the transgenic TCRs speci¢c for OVA-peptide (323^339) wereco-cultured with an equal number of CD25+CD4+ T cells from the transgenic mice or normalnon-transgenic wild-type (WT) BALB/c mice in the presence of various concentrations of theOVA peptide.

it is highly likely that CD25+CD4+ regulatory T cells also contain a similar self-reactive repertoire including the speci¢cities for the self-antigens to be targeted inautoimmune disease. Considering that suppression exerted by antigen-stimulatedCD25+CD4+ regulatory T cells is antigen-nonspeci¢c, CD25+CD4+ T cellsexpanded by stimulating them with target self-antigens may e¡ectively suppressautoimmune responses even if the antigens may not be the primary self-antigeninitiating the autoimmunity. The self-antigen-speci¢c regulatory T cells thusprepared can be used to treat or prevent autoimmune disease.Second, if CD25+CD4+ regulatory T cells, or at least a proportion of them, are

continuously stimulated by self-antigens in the normal physiological state andtherefore continuously exerting some degree of suppression in vivo (Fig. 3), theymay hamper immune responses bene¢cial for the hosts, for example, againstautologous tumour cells or invading infectious agents. Indeed, e¡ective tumourimmunity can be provoked in mice by removing CD25+CD4+ T cells prior toinoculation of tumour cells (Shimizu et al 1999). Removal of CD25+CD4+ T cellsalso elicited strong immune responses to infecting agents in chronically infectedanimals (Hori et al 2002).Third, the high sensitivity of CD25+CD4+ regulatory T cells to self-antigens

indicates that they may also be sensitive to self-mimicking non-self antigens,easily activated by them, hence able to suppress activation of other T cells by themimicking antigens. Considering high cross-reactivity of the TCR in peptide/MHC recognition and consequent promiscuity in self-non-self discrimination,exposure of self-reactive T cells to self-mimicking non-self peptides may not berare events (Ohno 1991, Mason 1998, Hemmer et al 1998). Similarly, T cellclones established by repeated stimulation with a particular self-antigen can beactivated in vitro by many structurally similar or dissimilar non-self peptides(Wucherpfennig & Strominger 1995). It is generally di⁄cult, however, to induceautoimmune disease in normal animals by immunizing with self-mimickingantigens or peptides. One reason for this di⁄culty may be the low threshold foractivation of regulatory T cells by self-mimicking antigens, and resultingdominant suppression on self-reactive T cells to be activated by molecularmimicry. The regulatory T cells may have evolved to prevent autoimmunity dueto molecular mimicry.

Acknowledgements

We thank Dr K. J. Wood for critically reading the manuscript. This work was supported bygrants-in-aid from the Ministry of Education, Science, Sports and Culture, the Ministry ofHuman Welfare and the Organization for Pharmaceutical Safety and Research of Japan.

14 SAKAGUCHI ET AL

References

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Bensinger SJ, Bandeira A, Jordan MS, Caton AJ, Laufer TM 2001 Major histocompatibilitycomplex class II-positive cortical epithelium mediates the selection of CD4+25+ immuno-regulatory T cells. J Exp Med 194:427^438

FukuiY, IshimotoT,UtsuyamaMet al 1997Positive and negative CD4+ thymocyte selection bya single MHC class II/peptide ligand a¡ected by its expression level in the thymus. Immunity6:401^410

Hemmer B, Vergelli M, Pinilla C, Houghten R, Martin R 1998 Probing degeneracy in T-cellrecognition using peptide combinatorial libraries. Immunol Today 19:163^168

Hori S, Carvalhi TL, Demengeot J 2002 CD25+CD4+ regulatory T cells suppress CD4+ T cell-mediated pulmonary hyperin£ammation driven by Pneumocystis carinii in immunode¢cientmice. Eur J Immunol 32:1282^1291

Itoh M, Takahashi T, Sakaguchi N et al 1999 Thymus and autoimmunity: production ofCD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus inmaintaining immunologic self-tolerance. J Immunol 162:5317^5326

JordanMS, BoesteanuA,ReedAJ et al 2001Thymic selection of CD4+CD25+ regulatory T cellsinduced by an agonist self-peptide. Nat Immunol 2:301^306

Kawahata K, Misaki Y, Yamauchi M et al 2002 Generation of CD4+CD25+ regulatory T cellsfrom autoreactive T cells simultaneously with their negative selection in the thymusand from nonautoreactive T cells by endogenous TCR expression. J Immunol 168:4399^4405

MaloyKJ, Powrie F 2001 Regulatory T cells in the control of immune pathology. Nat Immunol2:816^822

Mason D 1998 A very high level of crossreactivity is an essential feature of the T-cell receptor.Immunol Today 19:395^404

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Ohno S 1991 To be or not to be a responder in T-cell responses: ubiquitous oligopeptides in allproteins. Immunogenetics 34:215^221

Papiernik M, de Moraes ML, Pontoux C et al 1998 Regulatory CD4 T cells: expression ofIL-2R alpha chain, resistance to clonal deletion and IL-2 dependency. Int Immunol 10:371^378

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Read S, Malmstrom V, Powrie F 2000 Cytotoxic T lymphocyte-associated antigen 4 plays anessential role in the function of CD4+ CD25+ regulatory cells that control intestinalin£ammation. J Exp Med 192:295^302

Sakaguchi S 2000 Regulatory T cells: key controllers of immunologic self-tolerance. Cell101:455^458

Sakaguchi S, Fukuma K, Kuribayashi K, Masuda T 1985 Organ-speci¢c autoimmune diseasesinduced in mice by elimination of T-cell subset. I. Evidence for the active participation of Tcells in natural self-tolerance; de¢cit of a T-cell subset as a possible cause of autoimmunedisease. J Exp Med 161:72^87

Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M 1995 Immunologic self-tolerancemaintained by activated T cells expressing IL-2 receptor a-chains (CD25). Breakdown of asingle mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155:1151^1164

THYMIC SELECTION OF CD25+CD4+ REGULATORY T CELLS 15

Salomon B, Lenschow DJ, Rhee L et al 2000 B7/CD28 costimulation is essential for thehomeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmunediabetes. Immunity 12:431^440

Seddon B, Mason D 2000 The third function of the thymus. Immunol Today 21:95^99Shevach EM 2000 Regulatory T cells in autoimmunity. Annu Rev Immunol 18:423^449Shimizu J, Yamazaki S, Sakaguchi S 1999 Induction of tumor immunity by removingCD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity.J Immunol 163:5211^5218

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regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol 3:135^142

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DISCUSSION

Bach: I’d like to press you on antigen speci¢city. At the end of your talk youalluded to transplantation and tumour immunity, and also anti-infectiousimmunity. How would you see the speci¢city of CD25+ T cells in these models?Are they di¡erent fromordinary T cells speci¢c for awhole variety of antigens?Doyou think that the reaction is restricted to the antigen that is initially recognized, ordo you think that there is a phenomenon of bystander suppression, which wouldexpand their function to other cell types? I have some di⁄culty in putting togetherthe self-reactive CD25+T cells and the cells thatKathrynWoodwill speak about intransplantation (Wood et al 2003, this volume).Sakaguchi: They are as diverse as ordinary T cells in their usage of TCR Va/Vb

subfamilies as revealed with normal mice and single-peptide/MHC transgenicmice. On the other hand, their TCR repertoire seems to be more skewed torecognizing self antigens. They need antigenic or TCR stimulation to exertsuppression, which is antigen-non-speci¢c in its e¡ector phase and may mediatebystander suppression. Considering these properties of CD4+CD25+ regulatoryT cells, it is highly likely that they are continuously activated to certain degreesby recognizing self-antigens in the normal internal milieu as illustrated by theircell surface phenotype, and exerting a basal level of suppression in the

16 DISCUSSION

physiological state. This basal level of suppression may suppress immuneresponses in general to some degree. For example, it may hamper thedevelopment of e¡ective tumour immunity or antimicrobial immunity in chronicinfection. This means that if you remove these regulatory T cells, you can enhancetumour and microbial immunity and immune responses in general. They can alsosuppress immune responses to alloantigens as they can be strongly stimulated byalloantigens as ordinary T cells.Abbas: I want to come back to the question of why in the thymus some cells are

not deleted but turn into regulatory cells. I want to suggest an alternative idea andsee how you respond. The idea is that where T cells see self antigen in the cortex ofthe thymus, they will be deleted, because double-positive thymocytes are poisedfor deletion. But if they see self-antigens in the medulla, then they have passed thedeletion stage andwill turn into regulatory cells. One of the surprises that has comeout of work by Bruno Kyewski (Derbinski et al 2001) and others is that a hugenumber of self antigens are expressed in the medulla, which is not the site ofnegative selection. Perhaps the reason why self antigens are present in themedulla is to generate regulatory T cells: it is not avidity, it is all anatomy.Sakaguchi: For example, Bensinger et al (2001) showed that thymic cortical

epithelial cells alone can contribute to the generation of regulatory T cells. In themedulla, they also succumb to clonal deletion as ordinary T cells do. We don’tknow how they become anergic or acquire suppressive activity through theseselection processes.Bluestone: I’d propose an alternative explanation to that of AbulAbbas. Since the

repertoire looks like it is very broad, perhaps if these cells encounter self-antigenstoo early before they have matured their signalling complex appropriately todelete, this kind of partial signalling of T cells might divert them to the CD25+

regulatory cells.Sakaguchi: That’s possible.Harrison: Self-antigen-expressing cells referred to a moment ago are not just

present in the thymus, but also the periphery. We don’t know how these self-antigens are expressed. They may be expressed on class II MHC, but this isn’tknown for sure. There is also controversy about the nature of the cell type. Canyou tell us something about the generation of these regulatory cells in theperiphery? We have heard that they may be generated in response to oral antigen,but we don’t know much about what is happening in the periphery.Sakaguchi:It is a controversial issue.Whatwecan say at themoment is thatnormal

thymus is de¢nitely producing them. There are some reports showing thatregulatory T cells with similar phenotype and function as naturally arisingCD4+CD25+ regulatory T cells can develop in the periphery from na|« ve T cells inoral tolerance or upon exposure to low dose antigens (Thorstenson & Khoruts2001). There are also recent reports that when certain T cells are stimulated by

THYMIC SELECTION OF CD25+CD4+ REGULATORY T CELLS 17

immature DCs they can somehow become regulatory (Jonuleit et al 2000). It iscritically important to determine in these experiments whether CD4+CD25+

regulatory T cells already present in the periphery are somehow expanded orstrengthened in their suppressive activity, or whether na|« ve CD25� T cells candi¡erentiate into regulatory T cells upon antigen exposure. Even if the latter is thecase, there is a possibility that naturally occurring regulatory T cells may be presentalso in the CD25� fraction (Stephens &Mason 2000), and such a population maysomehow become CD4+CD25+ regulatory T cells upon antigenic stimulation.Bach: We can go further on this. You mentioned that in vitro they do not

proliferate much. What is the evidence that they proliferate in vitro in response toan autoantigen? Do you have any data suggesting the enhancement of the pool ofthese cells in certain settings? Another way to pose the question is what kind ofevidence do you have about the autoantigen speci¢city? If there was suchspeci¢city one might think that the autoantigen-speci¢c clones in the peripherywould be expanded. Don Mason has published a paper indicating that therecould be further education in the periphery (Seddon &Mason 1999).Sakaguchi: The direct demonstration of the autoantigen speci¢city of regulatory

T cells in the natural population hasn’t yet been done. As I mentioned, the TCRrepertoire is almost duplicated between CD25+ and CD4+CD25� cells withpossible a bit skewing of the former to self. If we accept that CD4+CD25� T cellscontain pathogenic self-reactive T cells causing gastritis, IDDM and otherautoimmune diseases, it is natural to think that the CD4+CD25+ populationcontains regulatory T cells speci¢c for self-antigens in the gastric mucosa orLangerhans islets. CD4+CD25+ regulatory T cells can be expanded in vitro whenthey are stimulated with antigen in the presence of high dose IL2 or along withstrong CD28 co-stimulation. This means that regulatory T cells recognizing self-antigens may expand in certain situations in vivo. We have evidence thatalloantigen-reactive CD4+CD25+ regulatory cells can expand in vivo whenstimulated with alloantigens. We haven’t directly demonstrated that self-antigen-speci¢c regulatory T cells can expand in vivo.Shevach: One way to determine the antigenic speci¢city of the CD4+CD25+ T

cells is to use an e¡ector cell of de¢ned speci¢city. In the gastritis model, we havedeveloped several transgenic mice that recognize a de¢ned peptide from theautoantigen, the H/K ATPase. Fortunately, this autoantigen has been knockedout by several groups, so the question will be, can we take CD25+ cells frommice that lack the autoantigen, and how e⁄ciently will they suppress theautoreactive T cell that recognizes an epitope on the a chain of the H/K ATPase.We are just waiting to get enoughmice backcrossed onto the right backgrounds todo these experiments.Bluestone: That may be problematic. There are recent data on the plasticity of

repertoire development, and the precise autoantigen may not be the only thing

18 DISCUSSION