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Regulatory T cells in the control of immune pathology

Kevin J. Maloy and Fiona Powrie
2001 Nature Publishing Group http://immunol.nature.com

It is now well established that regulatory T (T R) cells can inhibit harmful immunopathological responses directed against self or foreign antigens. However, many key aspects of T R cell biology remain unresolved, especially with regard to their antigen specificities and the cellular and molecular pathways involved in their development and mechanisms of action. We will review here recent findings in these areas, outline a model for how T R cells may inhibit the development of immune pathology and discuss potential therapeutic benefits that may arise from the manipulation of T R cell function.
The existence of a subpopulation of T cells that specialized in the suppression of immune responses was originally postulated in the early 1970s1. However, the cellular and molecular mechanisms responsible for these suppressive phenomena were never clearly characterized, with the result that interest in the field of suppressor T cells gradually dwindled. Nevertheless, a few groups doggedly persevered with the study of T cellmediated suppression; their efforts, together with advances in the definition of subpopulations of CD4+ T cells that control different types of immune responses, have led to a renaissance in the field. Suppressor T cells have been reborn as regulatory T (TR) cells. This is partly because there still exists something of a stigma towards suppressor cells, but mainly because this is a more accurate definition of their function. Several reviews have thoroughly covered the historical background and the spectrum of T cell populations to which regulatory functions have been attributed25, thus, we will mainly concentrate on naturally occurring CD4+CD25+ TR cells. We will review some of the key properties of these cells, describing what is known about their ontogeny and mechanisms of action as well as highlighting some of the key unresolved issues in their control of immune pathology. Although it is clear that clonal deletion in the thymus is of central importance, autoimmune diseases do occur and the presence of autoreactive T cells in normal healthy individuals indicates that potentially pathogenic, self-reactive T cells form part of the normal T cell repertoire68. Given that the recognition of peptide ligands by the T cell receptor (TCR) appears to be extremely degenerate9, it may be that the presence of autoreactive T cells is a consequence of the intrinsic TCR cross-reactivity required to ensure responsiveness to the vast array of foreign peptides that may be encountered10. Indeed, some T cells are reactive with both foreign microbial peptides and self-antigen peptides9,11. Nevertheless, autoimmunity occurs relatively rarely and even

the severe autoimmunity that is elicited by immunization with self-antigen in adjuvants is generally self-limiting in normal animals4. A number of mechanisms have been proposed to account for the maintenance of peripheral self-tolerance, including induction of T cell anergy12, T cell deletion13 and immunological ignorance14. Although these passive mechanisms certainly contribute, they do not explain the fact that, in many systems, tolerance can be adoptively transferred by T cells15,16. In addition, dominant extrathymic immune regulation by TR cells has the conceptual advantage that, as well as preventing pathological responses to self-antigens, such a mechanism may also serve to limit the development of harmful pathology during normal immune responses.

Naturally occurring TR cells inhibit immune pathology

Studies in a number of experimental models of organ-specific autoimmune disease provide convincing evidence that specialized TR cells capable of controlling autoimmunity are an integral part of the T cell repertoire in normal animals. These models have common characteristics: they usually involve manipulation of lymphocyte homeostasis, particularly influencing the thymus or peripheral T cells, and autoimmunity can be inhibited by adoptive transfer of CD4+ T cells from normal animals35. For example, neonatal thymectomy of mice (d3Tx) leads to the development of a wide spectrum of organ-specific autoimmune manifestations that include gastritis, oophoritis, orchitis and thyroiditis, all of which can be inhibited by the transfer of small numbers of CD4+ T cells from normal mice3,4. Similar autoimmune disease can be induced by reconstituting immunodeficient nude mice with CD4+ T cells from normal adult mice that have been depleted of CD4+CD25+ T cells8. CD4+CD25+ T cells prevent the development of autoimmunity induced by their CD4+CD25 counterparts, which confirms that TR cells are predominantly present in the small CD4+CD25+ T cell population8,17. Similarly, the development of autoimmune thyroiditis or diabetes in rats subjected to adult thymectomy followed by fractionated doses of sublethal irradiation (TxX) is inhibited by the transfer of CD4+ T cells from normal rats; in addition, the TR cells are located within the CD4+CD45RC T cell population6,18. CD4+ T cells from normal animals can inhibit the spontaneous development of autoimmune experimental allergic encephalitis (EAE) in mice transgenic for a myelin basic protein (MBP)-specific TCR19,20 and the development of spontaneous autoimmune diabetes in a genetically susceptible strain21. This indicates that immune suppression by TR cells is not restricted to experimentally induced disease. In an experimental model of inflammatory bowel disease (IBD), reconstitution of immunodeficient severe-combined immunodeficient (SCID) recipients with CD4+ CD45RBhi T cells from normal mice leads to the development of colitis, whereas cotransfer of the reciprocal CD4+CD45RBlo population from normal mice inhibits disease development22. The TR cells capable of inhibiting the development of IBD are predominantly found within the CD4+CD45RBloCD25+ population23. Although alternative interpretations

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Correspondence should be addressed to F. P. (fiona.powrie@path.ox.ac.uk).
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moderate amounts of transforming growth factor- (TGF-)51. TR1 clones with a similar phenotype have also been generated by repetitive stimulation of murine TCR-transgenic CD4+ T cells with their cognate peptide plus IL-1051. TR1 clones can suppress the immune responses of other T cells in vitro and in vivo, including inhibiting the development of colitis in vivo by antigen-driven bystander suppression51,52. Similarly, T helper type 3 (TH3) cells that arise after oral administration of antiPhenotypic characteristics of naturally occurring TR cells gen produce high concentrations of TGF- and can inhibit the develCD4+CD25+ T cells, which constitute 10% of peripheral murine CD4+ opment of immune pathology in several animal models5355. Thus, anerT cells, possess potent regulatory activity both in vitro25,26 and in gic cells, TR1 cells and TH3 cells may be derived from the same popuvivo8,17,23,27. Murine CD4+CD25+ T cells express little CD45RB and a lation: they have a similar phenotype and usually mediate their supsignificant proportion express cytolytic T lymphocyte-associated anti- pressive activities via the release of the cytokines TGF- and IL-10. CD4+ T cells with regulatory activity appear to be key to the suppresgen 4 (CTLA-4)23,28,29. They show a partially anergic phenotype, in that they proliferate poorly upon TCR stimulation in vitro and their growth sion of graft rejection in a number of transplantation models56,57. Mice is dependent on exogenous interleukin 2 (IL-2)30. CD4+CD25+ T cells treated with nondepleting monoclonal antibodies (mAbs) to CD4 tolerate can inhibit autoimmune diabetes in mice28 and rats31, induce tolerance allografts and CD4+ T cells isolated from tolerant mice suppress the rejecto alloantigens3234, impede anti-tumor immunity35 and regulate the tion of grafts upon adoptive transfer into nave mice. These TR cells can expansion of other peripheral CD4+ T cells36. The mature CD4+CD8 induce nave CD4+ T cells to themselves differentiate into TR cells, a phesingle-positive (SP) thymocyte fraction from normal rodents also con- nomenon termed infectious tolerance58. The phenotypic characteristics tains a small proportion (510%) of the TR cells responsible for of CD25+ TR cells31,37. CD4+CD25+ infectious tolerance and the molecular mechanisms by which they T cells with regulatory functions mediate suppression remain to be similar to those described in roestablished. However, CD4+CD25+ dents have now been isolated from human thymus and peripheral cells from tolerant donors can blood3842. However, a number of adoptively transfer transplantation tolerance to nave recipients32 and caveats preclude the use of CD25 expression as a unique marker for CD4+CD45RBlo T cells from unTR cells. manipulated mice can prevent graft rejection in an adoptive transCD25 is transiently expressed Figure 1. TR cells are a normal product of thymic selection. TR cells may arise from relatively high-avidity interactions with self-peptideMHC complexfer model59. This suggests that TR upon activation of nave CD4+ T es, just below the threshold for negative selection (green area).This narrow avid+ cells and its expression by CD4 cells generated as a result of tolerity selection window ensures that Tr cells will constitute only a small fraction of ance-inducing protocols may have T cells is highly dynamic in the mature T cell pool and have a greater sensitivity to self-peptideMHC than emerged from naturally occurring vivo36. Importantly, the peripheral potentially pathogenic autoreactive T cells. TR cells. Future studies, particularCD4 +CD25 T cell population also possesses some regulatory ly those designed to provide comactivity, albeit less potent than that of their CD4+CD25+ counter- prehensive genetic and molecular expression profiles of highly purified parts23,31,36. Similarly, protection from EAE in the MBP-specific TCR- CD4+ TR cell populations, should help define the nature and extent of the transgenic model can be mediated by normal T cell populations that interrelationships of different TR cell populations. have been depleted of CD25+ cells43. Thus, the CD4+CD25+ T cell population is heterogenous and although a relatively high proportion Thymic generation of TR cells of these cells may be TR cells, it is unwise to assume that this is true Accumulating evidence that the thymus is important in the generation for the entire population or that all TR cells express CD25. of TR cells has led to the theory that the production of TR cells represents a key function of the thymus60. CD4+CD8 SP thymocytes are a potent source of TR cells that adoptively transfer protection from Other subsets of regulatory T cells A number of CD4+ T cell subpopulations capable of inhibiting the autoimmune diabetes in rodent models61,62. CD25 is expressed by response of other T cells have been described25. A complete character- 510% of CD4+CD8 SP thymocytes in humans38, rats31 and mice30,37; ization of all the different CD4+ TR cell populations is still lacking, but these cells can inhibit T cell proliferation in vitro and can prevent develsome of them share characteristics with the naturally occurring opment of gastritis37, diabetes31 and colitis63 in vivo. CD4+CD25+ TR cells. One such feature is that CD4+CD25+ T cells genStudies of transplantation tolerance support a key role for thymic erally show a low proliferative capacity in vitro25,26. Similarly, anergic T epithelium (TE) in the differentiation of TR cells. Grafting of xenogeneic cells are classically defined as T cells that do not proliferate or produce or allogeneic TE induces tolerance to a variety of peripheral tissues of IL-2 upon antigenic restimulation44. Some anergic T cell clones can donor origin that is mediated by, and transferable with, CD4+ T cells; this suppress immune responses in vivoincluding the prevention of allo- suggests that the selection of T cells on foreign TE generates TR cells that graft rejection via a mechanism that involves antigen-presenting cells provide dominant tolerance to donor tissues16,64. One pathway of (APCs)4547and T cells that have been anergized in vivo are strong intrathymic selection of TR cells has been delineated by elegant studies in producers of IL-1048,49. Interestingly, allogeneic stimulation of human which mice expressing a transgenic TCR that recognizes an influenza CD4+T cells in vitro in the presence of IL-10 also induces T cell aner- hemagglutinin (HA) peptide were crossed to a lineage expressing the HA gy50 and, after repetitive stimulation, a population of T cell clones (des- peptide65,66. In double-transgenic mice, the HA-specific CD4+T cells are ignated TR1 cells) emerges that secretes high amounts of IL-10 and not deleted: instead, 50% of them are CD25+ and function as TR cells65.
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have been proposed to account for TR cell phenomena24, the experimental evidence outlined above emphasizes three important points. TR cells are present in the T cell repertoire of normal animals, they may suppress harmful immunopathological responses to self or foreign antigens and they reside mainly within a minor subpopulation of CD4+ T cells that express the CD25 marker.

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Radioresistant thymic elements mediate selection of the CD4+CD25+ CD4+ T cells obtained from athyroid rats cannot inhibit the developcells, which arise first in the thymus, before gradually accumulating in ment of thyroiditis in susceptible TxX rats, although their ability to secondary lymphoid organs66. These observations are consistent with the inhibit the development of diabetes is unimpaired70. Strikingly, thymoappearance of CD4+CD25+ cells in fetal thymic organ cultures37 and show cytes from athyroid rats can inhibit the development of thyroiditis, that the CD4+CD25+ TR cells found in the thymus are not derived from which suggests that the failure to maintain this TR cell function in the recirculating activated peripheral T cells. Consistent with published stud- periphery is due to the absence of the specific autoantigen70. Similarly, ies30, CD25 expression is acquired relatively late in thymocyte develop- the ability of TR cells to suppress the rejection of allografts in the infecment, as the cells progress from the CD4+CD8+ double positive stage to tious tolerance transplantation model is highly dependent on the conthe CD4+CD8 SP stage. Strikingly, thymocytes bearing a lower affinity tinuous presence of antigen56. HA-specific transgenic TCR do not develop into CD4+CD25+ thymoOn the other hand, there is evidence that regulation of harmful T cell cytes in double-transgenic mice. This prompted the suggestion that those responses towards nonself-antigens may be mediated by TR cells that thymocytes that develop into CD4+CD25+ SP cells may express a TCR have never been exposed to the target antigens. Thus, although the IBD with a relatively high affinity for self-peptides present in the thymus66. induced in SCID mice reconstituted with nave CD4+CD45RBhi T cells Nevertheless, these results are also is driven by T cell reactivity compatible with an avidity-depentowards antigens present in the dent selection process, similar to intestinal flora63, peripheral CD4+ the model originally proposed64. CD45RBlo or CD4+CD25+ T cells In this scheme, TR cell developisolated from immunocompetent germ-free mice can still inhibit ment in the thymus would be the development of colitis63. It directed by relatively high avidity interactions between the TCR and should be noted, however, that self-peptide ligands expressed on CD4+ TR cells are naturally generthymic APCs (Fig. 1). Lower ated that maintain tolerance avidity interactions would pretowards components of the comdominantly promote the developmensal flora and a breakdown in ment of conventional CD4+CD25 this tolerance is observed in patients with IBD71,72. Presently, SP thymocytes, as observed when the affinity of the TCR was very little is known about the reduced in the double transgenic specificities of naturally occurmice. Higher avidity interactions ring TR cells and the existing eviwould lead to clonal deletion, as dence is supportive of the hypothfound when the high affinity HAesis that TR cells are a heterogespecific TCRtransgenic mice neous population that can react were crossed with other transwith peptides derived from both genic lines that expressed higher self-tissues and foreign antigens. Figure 2.TR cells can mediate their effector function via multiple mechamounts of the HA peptide66. A anisms. The inhibitory functions of TR cells on autoreactive T cells that are potennarrow avidity window for the Peripheral de novo develtially pathogenic (TPATH cells) may be mediated through the actions of cytokines selection of TR cells would ensure opment of TR? and/or via a cell contactdependent mechanism. Although triggering through the that they represented only a small The question of whether TR cells TCR is required to induce TR cell function, the precise role of CTLA-4 is not clear. Similarly, the existence of putative molecules which may be involved in direct T proportion of positively selected represent a unique lineage of cellT cell interactions also remains to be established. thymocytes, perhaps just enough CD4+ T cells imprinted with this to maintain self-tolerance in the function in the thymus or periphery. In addition, those T cells in the peripheral repertoire with the whether TR cell function may be acquired by nave T cells in the highest intrinsic affinity for self-peptidemajor histocompatibility com- periphery is still open. Evidence for the induction of tissue-specific plex (MHC) would be TR cells. TR cells in the periphery comes from studies with male mice that have been orchiectomized at birth and thymectomized as adults. Treatment of these mice with dihydrotestosterone induces the de novo developPeripheral maintenance of TR cells Little is known about how TR cells are incorporated into and maintained ment of a mature prostate that is not subjected to autoimmune attack. within the peripheral T cell pool, although their numbers appear to be In fact, such mice concommitantly develop TR cells that can inhibit relatively stable in the periphery throughout adult life67. Cytokines and the development of autoimmune prostatitis after transfer to post-d3Tx costimulatory molecules are important in the homeostasis of CD25+ TR mice73. Models of transplantation tolerance provide further support cells, as this population is absent in IL-2/ mice30 and severely dimin- for peripheral differentiation of TR cells. In addition to suppressing ished in CD28/ or B7/ nonobese diabetic (NOD) mice28. The activa- allograft rejection by nontolerant T cells, TR cells induced by infection status of APCs may also influence the accumulation of TR cells, as tious tolerance protocols, or by engraftment of allogeneic fetal thymic CD40-deficient and CD40 liganddeficient mice have reduced num- epithelium, can also educate nave CD4+ T cells to differentiate into bers of CD4+CD25+ cells63,68. These findings, together with the fact that TR cells in the periphery56,58,74. TR cells maintain a partially activated phenotype, suggests accumulaProtocols that induce T cell tolerance by mucosal administration of tion of these cells depends on continued peripheral stimulation69. antigen are also supportive of the existence of peripheral pathways of TR Evidence that maintenance of TR cells is driven by the presence of the cell induction55. In an ovalbumin (OVA)-specific transgenic CD4+ T cell target organs that they regulate comes from the finding that peripheral adoptive transfer model, in vivo tolerization of CD4+ T cellseither by
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a Steady state: LN
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TPATH TPATH mDC TPATH TPATH TM

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Figure 3. Model for the control of pathogenic immune responses by TR cells. (a) In the steadystate, low numbers of immature DC traffic to the draining lymph node (LN) from uninflamed tissues and present self-peptides (yellow) to both TR and TPATH cells. The relatively high ratio of TR:TPATH cells, together with the intrinsically higher affinity of the TR cells, leads to low-level activation of the TR cells (red arrow) and inhibition of TPATH cells (green arrow). iDC, immature DC. (b) During an immune response high numbers of activated mature DCs traffic to the LN where they present peptides derived from both self (yellow) and foreign antigens (red). This strong stimulus (red arrows) leads to activation not only of T cells specific for the infectious agent (TE cells), but also to full activation of the TR cells. This results in their proliferation and a transient loss of suppressive function, which, in turn, allows activation and expansion of TPATH cells. mDC, mature DC. (c) After the infectious agent is cleared, in the absence of foreign peptides, TE cells either die or become memory T cells (TM). The regulatory phase is characterized by presentation of self-peptides, again leading to competition between TR and TPATH cells.As both have proliferated, the ratio of TR:TPATH cells is maintained so that regulatory activity dominates; this leads to the inhibition of TPATH cells and down-regulation of APCs. To prevent a chronic pathogenic inflammatory response, TR cells may also migrate to the inflammatory site.

administration of intravenous OVA peptide or by feeding intact OVA led to the emergence of CD4+CD25+ transgenic T cells with immunoregulatory properties75. Thus, additional pathways may exist whereby nave CD4+ T cells can be induced to differentiate into TR cells in the periphery and education of nave CD4+T cells provides a potential mechanism to extend the functional repertoire of TR cells in the periphery. The differentiation of nave CD4+ T cells is crucially determined by two interrelated factors: the APC that stimulates them and the cytokine environment in which the activation occurs76,77. As noted above, IL-10 proved an excellent adjunct for inducing the development of TR1 cells or anergic T cells in vitro, which suggests that it may function as a growth or differentiation factor for TR cells. IL-10 may mediate its function via dendritic cells (DCs) because IL-10 prevents the maturation of DCs78 and stimulation of T cells with immature DCs leads to the development of T cells with regulatory activity79,80. The development of CD4+CD25+ TR cells from human nave T cells has been induced ex vivo by culturing the T cells with TGF-81. However, prior depletion of the rare CD25+ cells within the nave T cell population markedly reduces the efficacy of TR cell development, which suggests that TGF- may preferentially expand precommitted TR cells, rather than inducing their de novo differentiation81. Stimulation in vivo with APCs that are overexpressing Serrate-1 (a Notch ligand), also results in the differentiation of CD4+ TR cells82; however, the cellular and molecular interactions that normally direct TR cell development in vivo remain to be determined.

model, protection from colitis does not require IL-4, but is crucially dependent on TGF-83 and on IL-10 production by T cells84. TR1 cells that inhibit colitis via bystander suppression also produce high amounts of IL-10 and TGF-, but not IL-451. The TH3 cells induced in oral tolerance protocols have also been associated with the same set of suppressive cytokines55, which implicates these molecules as being essential for maintaining tolerance at mucosal surfaces. Similarly, in the rat thyroiditis model, inhibition of disease is dependent on TGF- and IL4, but the role of IL-10 remains to be determined18. The immune-suppressive properties of IL-10 and TGF- are most likely explained by the ability of these cytokines to inhibit APC function8588 and to mediate direct anti-proliferative effects on T cells8991. Indeed, one mechanism by which TR cells inhibit immune pathology may be by controlling the expansion of other T cell populations, via a process that requires IL-1036. Although the precise pathways operational in vivo are not known, the available data suggest that it is important that myeloid cells are able to respond to IL-10 because mice in which macrophages and neutrophils are rendered hyporesponsive to IL10 develop IBD92. Conversely, the action of TGF- on T cells is revealed in mice expressing a T cellspecific dominant-negative form of the TGF- receptor II, which develop inflammatory infiltrates in both the colon and lungs90.

Regulation through cell-contact

A defining feature of CD4+CD25+ TR cells in both mice and humans is their ability to inhibit the proliferation of other T cell populations in vitro25,26,3842. In vitro suppression requires activation of TR cells via their TCR, does not involve killing of the responder cells and is mediated through a cell contactdependent mechanism independent of IL-4, IL-10 and TGF-25,26,3842. Although the activation of CD4+CD25+ T cells is antigen-specific, once activated, these cells inhibit both CD4+ and CD8+ T cell responses in an antigen-nonspecific manner25,93. CD4+CD25+ T cells
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Regulation through suppressive cytokines

In keeping with their heterogeneous nature, it is becoming clear that TR cells may use multiple mechanisms to suppress immune responses and that the relative importance of these mechanisms depends on the experimental model. Studies in animal models provide strong evidence of a role for cytokines in the effector function of TR cells in vivo, but the cytokines involved vary depending on the model. In the SCID IBD
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do not prevent initial responder T cell activation, as up-regulation of early activation antigens is not affected; however the cells fail to proliferate and undergo cell cycle arrest at the G0/G1 stage93. Suppression is overcome by addition of exogenous IL-2 or anti-CD28 to the cultures, which suggests that limiting IL-2 may be responsible for the lack of a sustained T cell response25,26. This is not attributable to simple consumption of IL-2 by CD4+CD25+ T cells because TR cells prevent IL-2 production by normally responsive T cells25,26. In addition, activated CD4+CD25 T cells that are induced to express high amounts of CD25 do not inhibit T cell activation in vitro, which suggests unique immunosuppressive properties of naturally occurring CD4+CD25+ T cells26,41. There is conflicting data on whether the cell contactdependent inhibitory effects of TR cells are mediated via APCs. Coculture with activated CD4+CD25+ T cells led to reduced amounts of the costimulatory molecules CD80 and CD86 on DCs and B cells94 and similar observations were made with anergic T cells47. Conversely, others have found that CD4+CD25+ TR cells can inhibit responses induced by fixed APCs and that suppression occurs even when the antigens recognized by the TR cells and responder T cells are presented by separate APC populations. This suggests the involvement of a direct T cellT cell interaction that is independent of APCs25,93. Although these data are compatible with the view that antigen recognition by CD4+CD25+ T cells induces expression of a surface-bound molecule that can bind to receptors on target T cells and induce cell cycle arrest95, in the absence of molecular characterization of this interaction it is difficult to assess its contribution to the function of TR cells in vivo. It is also difficult to discount the possibility that short-range soluble factors may contribute to in vitro suppression. However this mechanism may explain the ability of CD4+ TR cells to inhibit gastritis and diabetes in an IL-4 and IL-10independent manner96,97, although whether this in vivo suppression of organ specific autoimmune disease is entirely cytokine independent is not known and it is notable that the contribution of TGF- has not been assessed.

Where do TR cells act to suppress immune responses?

Another issue that remains to be addressed is identification of the anatomical sites where TR cells act in vivo. Inhibition of colitis by TR cells is associated with a decreased accumulation of activated DCs and T cells in the mesenteric lymph nodes (MLNs), as well as a marked decrease in the number of T cells present in the colon100. However, whether the TR cells act in the colon to inhibit the migration of activated DCs, mediate their suppressive effects on the DCs and pathogenic T cells in the MLNs or both remains to be established. Development of colitis is associated with markedly increased expression of a number of inflammatory chemokines and their receptors in the colon and this is inhibited by cotransfer of TR cells101. Presently, little is known about the homing characteristics and chemotactic migratory responses of TR cells and such information, when combined with in vivo tracking studies, should help identify their sites of action.

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A model for TR cell control of immune responses

An obvious paradox is how TR cells preferentially inhibit autoreactive T cell responses while simultaneously allowing responses to pathogens to proceed. However, it is possible to formulate a testable model by which this balancing act may be achieved (Fig. 3). Many important factors that contribute to the decision between immunity and tolerance have been outlined previously: the activation status of the innate immune system and of APCs102,103, the localization and distribution of antigen14 and the recirculation patterns of T cells104. In our quantitative model, the maintenance of self-tolerance is dependent on the ratio of TR cells to potentially pathogenic autoreactive T (TPATH) cells that respond to a given peripheral antigen. In a given lymph node, this ratio will be dynamic and fluctuate depending on the infectious status of the local tissue. In the steady-state, immature DCs may traffic through peripheral tissues105; here they can efficiently phagocytose proteins and apoptotic debris arising from normal cell turnover in the tissue without becoming activated106,107(Fig. 3a). Even in the absence of inflammation, a few of these immature DCs will migrate to the draining lymph nodes where they will present a panel of self-peptides to both TR cells and TPATH cells108110. However, autoimmunity will not occur, perhaps because the autoreactive T cells are insufficiently activated by immature DCs or, alternatively, because the immature DC preferentially stimulate TR cells79,80. Consistent with the latter hypothesis, TR cells can respond to much lower concentrations of cognate peptide ligands than conventional nave CD4+ T cells25 and immature DCs express relatively low amounts of MHC class II and costimulatory molecules79. In contrast, the presence of an infectious agent will induce DC activation and migration111 so that high numbers of mature DCs will arrive in the lymph node and present peptides derived from the pathogen and from self-antigen (Fig. 3b). In our model, this stimulus is potent enough to transiently override TR cell activity, permitting a huge clonal expansion of anti-pathogen T cells (TE), but also allowing expansion of TPATH cells. Importantly, this transient loss of TR cell activity will also be associated with extensive proliferation of the TR cells themselves, analogous to the in vitrohyperstimulation of CD4+CD25+ TR cells with anti-CD3 + IL-2 or anti-CD28, which results in proliferation of the TR cells accompanied by a transient loss of suppressive activity25,26. The factors that may provide a similar stimulus to the TR cells in vivo are not known, but they may be derived from the mature DCs or other proliferating T cells in the lymph node. As the infectious agent is eradicated, there will be fewer and fewer pathogen-derived antigenic peptides presented in the lymph node, which will lead to contraction of the anti-pathogen TE cell population. Thus, in the regulation phase of the response, there will again be again competition
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Role of CTLA-4
Ligation of CTLA-4 on the surface of activated T cells, by its ligands on APCs (CD80 and CD86), strongly inhibits T cell activation98. The fact that CTLA-4 expression is primarily restricted to CD4+CD25+ T cells23,28,29 suggests that it may also be functionally important. Indeed, antiCTLA-4 treatment abrogates the ability of CD4+CD25+ T cells to inhibit colitis23 and also induces the development of gastritis in normal mice29. Blockade of CTLA-4 inhibits the function of TR cells in vitro, even under circumstances in which CTLA-4 is present only on the TR cell population29; this suggests that CTLA-4 expression on CD4+CD25+ T cells is involved in their function. However, others have found that in vitro suppression cannot be abrogated by blockade of CTLA-426,41,42 and CD4+CD25+ T cells from CTLA-4deficient mice also exhibit some suppressive activity29. Precisely how CTLA-4 may be involved in the function of TR cells remains to be defined. One possibility is that it preferentially binds B7 molecules as a result of its higher affinity, thereby preventing CD28-mediated signals that might otherwise abrogate the suppressive function of TR cells29. Alternatively, cross-linking of CTLA-4 on activated T cells may induce TGF- secretion99. These findings show that TR cells may use multiple protective mechanisms to inhibit immune activation. At present there is no easy way to reconcile all the evidence to delineate precise molecular pathways involved in suppression, but an overview of the possible interactions is shown (Fig. 2). The relative roles of different suppressive mechanisms in protection from immune pathology in vivo may be highly dependent on the local environment of the target organ and also on the nature of the pathological immune response that must be inhibited.
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between TR cells and TPATH cells in the draining lymph nodes for self-peptides presented on arriving DCs (Fig. 3c). As both populations will have expanded, the ratio of TR:TPATH cells will remain high enough for the TR cells to become dominant. The expansion and activation of TPATH cells in the lymph nodes will gradually decrease and additional APCs will be rapidly deactivated as they arrive in the lymph nodes. Similarly, during a persistent low-level infection the relatively low ratio of foreign peptides to self-peptides presented by APCs in the lymph nodes may allow TR cell activation to prevent immune pathology mediated by chronic T cell responses to pathogens. This still leaves the problem of controlling any TPATH cells that have already migrated to the inflamed tissue. For this reason it would seem advantageous to have some TR cells migrate to the inflammatory site where they could act locally to down-regulate the response by acting on APCs and/or effector T cells.

Therapeutic benefits from manipulating TR function

The ability to induce or expand TR cells in vivo and in vitro could have important implications not only in the field of autoimmunity, but also in transplantation tolerance56. An important advantage is that because TR cells can exert bystander suppression in a nonantigen-specific manner, they need not necessarily recognize the target antigen(s) that are the subject of immune attack. Induction of TR cells that react to any local tissueexpressed molecule may be sufficient to inhibit immune pathology. Identification of the sites of action of TR cells and of their antigen reactivities will be paramount in applying this kind of strategy to the treatment of inflammatory diseases. One important area that remains to be addressed is whether TR cells can also down-regulate ongoing immunopathological reactions. If so, what manipulations are required to re-establish dominant TR cell activity in vivo? In addition, identification of downstream cellular targets and molecular mechanisms of TR cell action should further enhance the development of treatments that inhibit immune pathology. Manipulation of TR cells may also have important clinical benefits in the induction of protective immunity. Transient depletion of CD25+ TR cells can break immunological unresponsiveness to syngeneic tumors and lead to markedly enhanced protection35. However, such strategies should be approached cautiously, as long-term depletion of TR cells may predispose the host to the development of autoimmunity112. There may be a delicate balance between enhancing immune responses to tumors or infectious agents while avoiding autoimmunity.

Concluding remarks
The study of TR cells is progressing to a new level where the need to demonstrate their existence has been replaced by the need to understand their biology. Many key issues remain to be resolved, particularly concerning their specificities and mechanisms of action. As interest in the field broadens, it is important that stringent criteria be applied to the classification of TR cells, so that an accurate, focused body of knowledge can be developed that will facilitate a more rapid realization of their therapeutic potential.
Acknowledgements We thank O. Annacker, S. Read, D. Mason, L. Stephens and A. Gallimore for numerous helpful discussions and critical review of the manuscript. K. M. and F. P. are supported by the Wellcome Trust.
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