Guarding the immune system: Suppression of autoimmunity by CD4 1 CD25 1 immunoregulatory T cells

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1 Immunology and Cell Biology (2006) 84, doi: /j x Review Article Guarding the immune system: Suppression of autoimmunity by CD4 1 CD25 1 immunoregulatory T cells TRICIA D ZWAR, IAN R VAN DRIEL and PAUL A GLEESON Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria, Australia Summary CD4 1 CD25 1 Foxp3 1 T cells (CD25 1 T regulatory [Treg] cells) are a naturally occurring suppressor T-cell population that regulates a wide variety of immune responses. A major function of CD25 1 Treg cells is to inhibit the activity of self-reactive T cells that can potentially cause autoimmune disease. This review examines the recent advances in CD25 1 Treg cell biology, with particular focus on the thymic and peripheral development of CD25 1 Treg cells, the signals that promote their expansion and maintenance in the periphery and the mechanism by which they mediate their suppressor activity in peripheral lymphoid tissues. An understanding of these issues is likely to facilitate the development of CD25 1 Treg-cell-based therapies for the treatment of autoimmune disease. Key words: autoimmunity, rodent, suppression, T cell, T regulatory cell. Introduction In recent years, active suppression of autoreactive T cells by immunoregulatory T cells has emerged as an important theme in the maintenance of self-tolerance. Suppressor T cells rose to prominence as a result of a series of experiments initially carried out in the late 1960s and early 1970s. Some of this work, pioneered by Robert Gershon, led to an explosion of work on CD8 1 suppressor T cells, which was spectacularly refuted after analyses using molecular technology between the late 1970s and early 1980s. 1 This high-profile fall from grace meant that other more reliable work on the immunoregulatory T cells was overlooked by most immunologists for many years. Some of the foundations of current day immunoregulatory T-cell research were laid by the work of Nishizuka and Sakakura who found that female mice that were thymectomized on day three of life developed oophoritis, 2 which, it was subsequently discovered, could be prevented by grafting of an intact thymus or by the transfer of thymocytes or peripheral splenocytes from adult mice. 3 In short, these cell populations appeared to suppress autoimmune disease. This work led to considerable research into suppressor cells in autoimmunity. Transplantation immunologists also continued to champion dominant or infectious suppression mediated by T cells as a means of preventing graft rejection. 4 However, much of this data was later reinterpreted in light of new discoveries in the late 1980s. 5 In 1995, the discovery of the high affinity IL-2 receptor a-subunit (CD25) as a reliable marker of immunoregulatory CD4 1 T cells, by Sakaguchi et al. (Sakaguchi, an early collaborator of Nishizuka) 6 rekindled widespread interest in Correspondence: Professor Paul A Gleeson, Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Vic. 3010, Australia. pgleeson@unimelb.edu.au Received 5 May 2006; accepted 3 July immunoregulation and facilitated the characterization of this lymphocyte population, although CD25 as marker of immunoregulatory T cells in transplantation was described by Hall et al. some years earlier. 7 Several different lymphocyte populations can show regulatory activity, including naturally occurring gd T cells, NKT cells and CD8 1 CD25 1 T cells, as well as conditionally induced Th3 and Tr1 (Tregulatory type 1) cells. However, these cell types are distinct from CD4 1 CD25 1 immunoregulatory T cells with respect to their mode of development, mechanism of suppression and sites of action and are not discussed in this review. From several studies published during the 1980s and early 1990s, it became apparent that CD4 1 T cells could be divided into functionally distinct subpopulations based on differential expressions of particular cell surface markers In 1995, Sakaguchi et al. showed that the immunoregulatory activity of CD4 1 T cells could be specifically attributed to the 5 10% that coexpress CD25. 6 Although CD25 is transiently expressed on all recently activated CD4 1 T cells and on less than 1% of CD8 1 T cells, the majority of CD4 1 CD25 1 T cells in the periphery are a unique lineage of immunoregulatory cells. The CD4 1 CD25 1 immunoregulatory T-cell (CD25 1 Treg) population does not express several markers of activated lymphocytes, stressing differences between the CD25 1 Treg population and activated conventional T cells Subsequent efforts to phenotype the CD25 1 Treg population showed that CD25 1 Treg cells express high levels of glucocorticoid-induced TNF receptor-related gene (GITR), 17,18 and low levels of cytotoxic T-lymphocyte antigen (CTLA)-4, 19 in contrast to naive conventional CD4 1 T cells. However, neither of these markers exclusively delineate immunoregulatory T cells. Therefore, a major breakthrough was the finding that the forkhead box (Fox)p3 transcription factor is expressed exclusively in CD25 1 Treg cells, but not in resting or activated conventional CD4 1 T cells and furthermore is required for CD25 1 Treg cell development Ó 2006 The Authors Journal compilation Ó 2006 Australasian Society for Immunology Inc.

2 488 TD Zwar et al. The role of CD25 1 Treg cells in preventing organ-specific autoimmune disease has been shown in adoptive transfer studies. Sakaguchi et al. first showed that depletion of CD25 1 cells from BALB/c splenic cells, followed by the transfer of the residual CD25 population into syngeneic adult athymic nude mice, resulted in the development of organ-specific autoimmune disease affecting multiple organs. 6 Reconstitution of recipient mice with CD4 1 CD25 1 T cells within 10 days of transferring CD25 cells abrogated disease development in a dose-dependent manner, supporting the view that CD4 1 CD25 1 T cells have immunosuppressive qualities. 6 CD4 1 CD25 1 T cells were also found to be capable of inhibiting the initiation of organ-specific autoimmune disease that occurs following neonatal thymectomy. 23 CD25 1 Treg cells have been implicated in suppression of organ-specific autoimmunity in several other animal disease models. For example, CD25 1 Treg cells can suppress autoimmune diabetes in non-obese diabetic (NOD) mice 24,25 and in a murine inflammation-induced diabetes model 26 and CD25 1 Treg cells were shown to be protective in a mouse model of experimental autoimmune encephalomyelitis (EAE). CD25 1 Treg cells have also been shown to be suppressive in a murine model of inflammatory bowel disease, 27 which results from a pathological immune response to commensal gut bacteria. 28 Research in the past 10 years has implicated a role for CD25 1 Treg cells in suppression of a wide range of immune responses targeting various microbes, intracellular parasites, allergens, allo-antigens and tumours. 29 However, here we will focus primarily on the role of CD25 1 Treg cells in suppression of autoimmunity. Regulation by CD25 1 Treg cells In early studies of CD25 1 Treg cells, in vitro proliferation assays were used to identify and characterize the immunosuppressive effects of these cells. In such assays, TCR stimulation of CD25 1 Treg cells failed to induce proliferation and based on this in vitro behaviour were considered to be naturally anergic. In coculture conditions, TCR-stimulated CD25 1 Treg cells suppressed the proliferation of CD4 1 CD25 and CD8 1 T cells in a dose-dependent manner. 15,30 In particular, CD25 1 Treg cells induced cell cycle arrest in target cells on day two of in vitro culture 16 and this was associated with the inhibition of IL-2 production in target cells at the level of mrna production. 15,30 In addition, CD25 1 Treg cells were found to inhibit IFN-g production by CD8 1 T cells in vitro. 31 Importantly, stimulation of the TCR complex on CD25 1 Treg cells was required for the induction of suppressor activity, either by specific antigen, anti-cd3 mab or other mitogens. Similar to their murine counterparts, human CD4 1 CD25 1 T cells are also anergic to TCR stimulation in vitro and are capable of suppressing proliferation and cytokine production by responder CD4 1 CD25 T cells in vitro. 32 However, compared to mice, CD25 expression levels on human CD4 1 CD25 1 T cells are relatively low and this population is rather heterogeneous, rendering isolation of human CD25 1 Treg cells more difficult. 32 Addition of exogenous IL-2 and/or IL-4 to in vitro cultures of CD25 1 Treg cells breaks their anergic state and under such conditions, CD25 1 Treg cells fail to suppress proliferation of CD4 1 and CD8 1 T cells in coculture experiments. 15,30 More recently, it was shown that under these conditions IL-2 mrna levels in responder cells were inhibited by CD25 1 Treg cells despite responder cell proliferation. 33 This finding indicates that the proliferation of responder cells was driven by exogenous IL-2 rather than autocrine IL-2 produced by the responder cells themselves. These data also indicate that CD25 1 Treg cells may be able to control the effector function of CD4 1 T cells, even under conditions that promote their proliferation. CD25 1 Treg cells have been shown to be capable of regulating a range of in vivo immune responses mediated by CD4 1 T cells and CD8 1 T cells, as well as B cells in several cases. From in vivo studies, it is evident that CD25 1 Treg cells can regulate immune responses at various levels. CD25 1 Treg cells are capable of suppressing in vivo cell proliferation 34,35 and clonal expansion 36 that is associated with activation of naive T cells following TCR stimulation. However, in other studies, CD25 1 Treg cells had no effect or only modest effects on effector T-cell proliferation CD25 1 Treg cells have been found to inhibit events associated with gain of effector function such as the induction of cytokine production and chemokine receptor expression. 35,37,40 In addition, CD25 1 Treg cells can suppress well-established immune responses. For example, CD25 1 Treg cells were shown to cure established colitis, 41 and to revert autoimmune diabetes soon after disease onset. 42,43 The stage at which CD25 1 Treg cells suppress immune responses may depend on factors such as the strength and type of the immune response, the relative ratio of the effector T cells to CD25 1 Treg cells and the nature of the target antigen. Mechanism of suppression of CD25 1 Treg cells An important issue is the mechanism, or mechanisms, by which CD25 1 T cells mediate their suppression. A key observation in early studies was that CD25 1 Treg cells were unable to suppress the in vitro proliferation of responder CD4 1 T cells when the two populations were separated by a cytokinepermeable membrane. 15,30 This indicated that direct contact between CD25 1 Treg cells and responder CD4 1 T cells was required for suppression. Although these studies did not rule out a role for soluble immunosuppressive cytokines in CD25 1 Treg-cell-mediated suppression, they indicated that cytokines alone do not mediate suppression in vitro. An alternative interpretation of these data is that direct contact is not required per se, but rather that suppression is mediated by a soluble factor that operates over a very short distance, thus requiring close proximity between CD25 1 Treg cells and their T-cell targets. Contact or close proximity between CD25 1 Treg cells and responder T cells may be required either for the induction of CD25 1 Treg cell suppressor function, for the effector phase of suppression or for both. Role of immunosuppressive cytokines The role of immunosuppressive cytokines in CD25 1 Treg cellmediated suppression in vitro is somewhat controversial. Whereas CD25 1 Treg populations have been found to transcribe IL-10, IL-4 and TGF-b mrna, there is little evidence that these cytokines play a role in CD25 1 Treg-cell-mediated suppression in vitro. 15,30,44 For example, in early studies, the addition of neutralizing anti-il-10, anti-il-4 and anti-tgf-b

3 CD4 1 CD25 1 immunoregulatory T cells 489 antibodies to in vitro cultures failed to abrogate the suppressive effects of CD25 1 Treg cells. 15,30 In a more recent study, CD25 1 Treg cells stimulated by a combination of anti-cd3 mabs and anti-cd28 mabs/il-2 were found to secrete IL-10 and TGF-b in vitro. 45 In this study, inhibition of in vitro suppression was observed in the presence of high concentrations of a neutralizing anti-tgf-b mab, which was dependent on cell cell contact but not on APC. 45 The authors proposed that suppression resulted from the contact-dependent delivery of latent cell surface-bound TGF-b on CD25 1 Treg cells to responder CD4 1 T cells. However, subsequent analyses have shown that CD4 1 T cells from Smad3-deficient mice or mice expressing a dominant negative form of the TGF-b receptor are fully susceptible to suppression by CD25 1 Treg cells in vitro, 46 despite being unresponsive to TGF-b-mediated signalling. 47,48 Finally, CD25 1 Treg cells from IL-4-deficient and IL-10-deficient mice and from young TGF-b-deficient mice are fully functional suppressors in vitro, confirming that these cytokines are not critical to the immunosuppression by CD25 1 Treg cells in vitro. 15,46 In contrast to the in vitro studies, there is evidence that immunosuppressive cytokines are required for CD25 1 Tregmediated suppression in some disease models. For example, administration of a blocking anti-il-10-receptor mababrogated suppression of colitis by CD4 1 CD45RB low T cells 49 and CD25 1 Treg cells from IL-10-deficient mice fail to suppress colitis, although they suppress autoimmune gastritis efficiently. 50 Suppression of colitis by CD4 1 CD25 1 CD45RB low T cells is also dependent on TGF-b, 27,51 as shown, for example, by the fact that colitogenic CD4 1 CD45RB high cells from mice expressing a dominant negative form of the TNF-b receptor II are refractory to suppression by CD25 1 Treg cells in vivo. 51 In this model, production of TGF-b1 by the CD25 1 Treg cells is dispensable for suppression of colitis, suggesting that CD25 1 Treg cells induce other cell types to express TGFb1. 51 However, other studies have failed to identify a role for TGF-b in CD25 1 Treg-mediated suppression in other autoimmune disease settings. 52,53 In a rare consensus in this area, in vivo CD25 1 Treg-mediated suppression of CD8 1 T-cell cytolytic activity appears to be dependent on TGF-b signalling in CD8 1 T cells. 54 One complication in the interpretation of many in vivo studies is that it is often difficult to exclude the possibility that cytokines are required for the development, maintenance and/or activation of CD25 1 Treg cells, rather than for the effector phase of suppression. Nevertheless, from these studies, it appears that there is a requirement for immunosuppressive cytokines in CD25 1 Treg suppressor function in some in vivo settings. Role of IL-2 The role of IL-2 as a T-cell growth factor was established many years ago, 55 although more recent analyses of IL-2-deficient mice have shown the redundancy of IL-2 in this role. 56,57 CD25 1 Treg cells themselves do not produce IL-2, but express all components of the high-affinity IL-2 receptor. Therefore, it was reasoned that CD25 1 Treg cells may be highly efficient consumers of IL-2 and may suppress proliferation of target cells by competing for IL-2 in the local environment. However, this proposal is not supported by the finding that CD4 1 Foxp3 1 cells present in IL-2Ra-deficient mice display similar in vitro suppressor activity to their wildtype counterparts. 58,59 In addition, the analysis of transgenic mice (Foxp3 gfp ) expressing a Foxp3 GFP fusion protein, thus allowing direct observation of Foxp3 expression, 60 showed a minor population of Foxp3-expressing CD4 1 CD25 T cells, which were immunosuppressive in vitro. 60 Therefore, IL-2 responsiveness is not a prerequisite for CD25 1 Treg suppressor function. However, IL-2 is crucial for the maintenance of CD25 1 Treg cells. Effects on APC Suppression may result from competition between CD25 1 Treg cells and conventional T cells for costimulatory molecules expressed on APC, or modulation of APC costimulatory function by CD25 1 Treg cells. However, neither proposal is supported by the available data. For example, in one study, activation of CD25 1 Treg suppressor function apparently did not require costimulation by CTLA-4 or CD28 interactions with CD80/CD86 expressed on APC. 61 Therefore, competition for costimulation is unlikely to be a major mechanism of suppression. In another study, suppression of CD8 1 T-cell proliferation by pre-activated CD25 1 Treg cells was observed in an APC-free system in vitro, in which soluble peptide : MHC class I tetramer was used to stimulate responder cells. 31 This latter work shows that APC are not required for suppression and suggests that suppression instead involves direct interaction between CD25 1 Treg cells and effector T cells. However, the use of two-photon laser scanning microscopy to examine in vivo priming of diabetogenic T cells in the presence of CD25 1 Treg cells failed to detect any sustained associations between these two T-cell populations. 35 Instead, the CD25 1 Treg cells appeared to form stable associations with dendritic cells (DC), resulting in reduced arrest of diabetogenic T cells. Specific effects of CD25 1 Treg cells on DC remain to be determined. Role of CTLA-4 CTLA-4 is generally not expressed on naive conventional CD4 1 T cells, but is upregulated following T-cell activation. On binding to CD80/CD86 ligands on APC, CTLA-4 transduces a negative signal that results in dampening of T-cell activation. 62,63 In contrast to conventional T cells, CD25 1 Treg cells constitutively express CTLA-4, suggesting an important role for this molecule in CD25 1 Treg biology. In support of this proposal, Takahashi et al. reported that CD25 1 Treg-cellmediated suppression of responder CD4 1 T-cell proliferation in vitro was inhibited by the addition of a blocking anti- CTLA-4 mab or its Fab fragment to in vitro cocultures. 19 By using responder cells from CTLA-4-deficient mice, this group confirmed that blockade of CTLA-4 signalling in CD25 1 Treg cells was responsible for this effect and therefore that CTLA-4 signalling is required for suppression. However, it should be noted that other groups have been unable to reproduce these results. 15 A role for CTLA-4 in CD25 1 Treg function is further supported by in vivo data. For example, in vivo blockade of CTLA-4 for a limited period in BALB/c mice by treatment with an anti-ctla-4 mab was reported to result in the induction of autoimmune gastritis. 19 In another study, CD25 1

4 490 TD Zwar et al. Treg-mediated suppression of inflammatory bowel disease that is induced by transfer of CD4 1 CD45RB high cells to severe combined immunodeficiency (SCID) recipients, was inhibited by administration of anti-ctla-4 mabs. 27 Although these findings suggest that CTLA-4 signalling is required for CD25 1 Treg suppressor function, the cellular site of action of the anti-ctla-4 mab was not determined in these in vivo studies and therefore it remains possible that the antibody targeted the effector CD4 1 T cells rather than the CD25 1 Treg cells. Role of GITR In addition to CTLA-4, CD25 1 Treg cells also express constitutively high levels of GITR, a member of the TNF receptor superfamily, and upregulate GITR expression on activation. 17,18 The ligand for GITR, GITR ligand (GITR-L), is expressed on endothelial cells 64,65 as well as on APC such as B cells and mature and immature DC. 66 GITR is also expressed at low levels on naive CD4 1 CD25 T cells and is upregulated following T-cell activation. 18 GITR triggering on conventional CD4 1 and CD8 1 T cells has a costimulatory effect, augmenting in vitro proliferation in response to TCR stimulation. 67 In two early studies, it was reported that the ability of CD25 1 Treg cells to suppress the in vitro proliferation of CD4 1 CD25 T cells was abrogated in the presence of an agonistic anti-gitr mabs, suggesting that GITR stimulation on CD25 1 Treg cells inhibits their suppressor function. 17,18 The site of action of the anti-gitr mabs in these studies has been further investigated using CD25 1 Treg and responder T-cell populations from wild-type and GITR-deficient mice. In a study by Stephens et al., GITR engagement on CD25 T cells, either by anti-gitr mabs or by its natural ligand GITR-L, rendered those cells resistant to suppression by CD25 1 Treg cells. 66 However, GITR engagement on CD25 1 Treg cells had no effect on their ability to mediate suppression in vitro. 66 Thus, although CD25 1 Treg cells constitutively express high levels of GITR, signalling through GITR does not affect the suppressor activity of CD25 1 Treg cells themselves. Foxp3 Although the identification of CD25 as a specific marker of immunoregulatory CD4 1 T cells was a major discovery that allowed considerable advances in the field, there are problems with the use of the CD25 marker to define the CD25 1 Treg population. Most notably, CD25 is not a unique marker for CD25 1 Treg cells, as it is transiently upregulated to a similar level on conventional CD4 1 and CD8 1 T cells following activation. Therefore, a more specific marker of CD25 1 Treg cells, ideally with a role directly linked to CD25 1 Treg function, was required to better define this cell population. A major advance in the field was the identification of Foxp3 as a highly specific marker of CD25 1 Treg cells with an indispensable role in CD25 1 Treg cell development. The first clue to a link between Foxp3 and CD25 1 Treg cells was provided by the observation of an autoimmune and lymphoproliferative syndrome in mice of the scurfy mutant strain in which the Foxp3 gene is mutated. The syndrome closely resembles that which occurs in mice deficient in CD25 1 Treg cells as a result of knocking out genes that are required for CD25 1 Treg genesis or maintenance. Examples of such mice are mice deficient in CTLA-4, TGF-b, IL-2, and CD The scurfy mutant mouse strain contains a 2-base pair frameshift mutation in the X-chromosome gene, Foxp3, encoding a forkhead/winged-helix transcription factor. This mutation results in the production of a functionally inactive truncated gene product lacking the C terminal DNA binding domain. 69 In scurfy mice, the X-linked mutation is inherited recessively and therefore affects 100% of hemizygous male mice that typically die at days after birth. The disease observed in scurfy mice is mediated by CD4 1 T cells and is characterized by excessive lymphoproliferation, multiorgan lymphocytic infiltrates and wasting disease. 70 A similar disease occurring in humans, named immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome (IPEX) was subsequently mapped to a number of different mutations in the human FOXP3 analogue gene. 71,72 Most commonly, mutations result in either a truncated gene product or non-truncated products with altered DNA binding. IPEX is an aggressive and fatal disease that affects male children, typically manifesting as neonatal diabetes mellitus, thyroiditis, inflammatory bowel disease, severe allergy, autoimmune haemolytic anaemia, thrombocytopenia and severe infections. 70 Because Foxp3 mutations in both mice and humans result in a phenotype of immune dysregulation, the status of CD25 1 Treg cells in scurfy mice was investigated. Scurfy mice were found to lack CD25 1 Treg cells and adoptive transfer of CD25 1 Treg cells into neonatal scurfy mice prevented the onset of the characteristic lymphoproliferative disorder. 20 These findings suggested that the scurfy phenotype arises as a direct consequence of a lack of CD25 1 Treg cells and furthermore that Foxp3 is required for the genesis and/or maintenance of CD25 1 Treg cells. The role of Foxp3 in CD25 1 Treg development was confirmed by the generation and analysis of Foxp3-deficient mice. Such mice also lack CD25 1 Treg cells and develop a severe autoimmune syndrome similar to that of scurfy mice, which can be prevented by reconstitution with CD25 1 Treg cells early in life. 20 Consistent with a role in CD25 1 Treg development and/or maintenance, Foxp3 expression in lymphocyte populations of normal mice was found to be limited to CD4 1 CD25 1 peripheral T cells and CD4 1 CD8 CD25 1 thymocytes Importantly, unlike other markers of CD25 1 Treg cells, Foxp3 expression is not induced in conventional CD4 1 T cells on activation. There is now compelling evidence that Foxp3 is directly involved in the development of CD25 1 Treg cells. Transgenic Foxp3 overexpression converts CD4 1 CD25 and CD8 1 T cells to a suppressor phenotype, as such cells inhibited the proliferation of responder CD4 1 T cells in vitro. 22 Further, CD4 1 CD25 T cells, which ectopically expressed Foxp3 were found to show suppressor function in several in vivo and in vitro assays. 20,21 For example, Foxp3-transduced CD4 1 CD25 CD45RB high T cells suppressed the induction of inflammatory bowel disease and autoimmune gastritis that is normally induced in lymphopenic SCID mice following adoptive transfer of CD4 1 CD25 CD45RB high T cells. 21 Combined, these data suggest that expression of Foxp3 in otherwise non-regulatory CD4 1 T cells induces a programme of conversion to a CD25 1 Treg phenotype and indicate that Foxp3 is a master regulatory gene for the development of CD25 1 Treg cells.

5 CD4 1 CD25 1 immunoregulatory T cells 491 The analysis of transgenic mice (Foxp3 gfp ) expressing a Foxp3 GFP fusion protein has provided pivotal insights into the biology of Foxp3 expression. 60 Within the lymphoid compartment of Foxp3 gfp mice, Foxp3 expression was limited to ab T cells, and was not detected in NK cells, NKT cells, B cells, macrophages or DC. Notably, the lymphoid compartment of recombination-activating gene (RAG)-1-deficient Foxp3 gfp mice, which lack B cells and T cells, was completely devoid of Foxp3 gfp -expressing cells. Foxp3 gfp expression was also undetectable in a range of other non-haematopoietic cell types and tissues examined, including thymic epithelium. Within ab T cells in the periphery, the vast majority (>97%) of Foxp3 gfp -expressing cells were CD4 1 (CD8 ) T cells. An analysis of Foxp3 and CD25 expression by CD4 1 T cells showed a discrete population of CD25 1 Foxp3 gfp1 cells, as expected, but also populations of CD25 1 Foxp3 gfp, CD25 Foxp3 gfp1 and CD25 Foxp3 gfp CD4 1 T cells. Analyses of these cell populations showed that in vitro suppressor function correlated with Foxp3 expression rather than CD25 expression. Therefore, the CD25 molecule, although expressed on the majority of Foxp3 1 Treg cells, is apparently dispensable for suppressor function. Consistent with its predicted role as a transcription factor, Foxp3 gfp fluorescence was limited almost exclusively to the nucleus of Foxp3 gfp1 cells. The precise gene targets of Foxp3 are unknown; however, consensus forkhead-binding domains have been identified in the promoters of several key cytokine genes, including IL-2, IL-4 and TNF. 70 Further investigation of the gene targets of Foxp3 should provide important insights into the mechanism of action of CD25 1 Treg cells. Thymic development of CD25 1 Treg cells More than 30 years ago, it was observed that thymectomy of mice at day three after birth induces organ-specific autoimmune disease. 2 In 1996, Asano et al. showed that day three thymectomy-induced autoimmunity can be prevented in BALB/c mice by the transfer of CD25 1 Treg cells isolated from normal adult mice, before day 10 of life. 23 This finding suggested that day three thymectomized mice intrinsically lack CD25 1 Treg cells and provided an early clue that CD25 1 Treg cells may originate in the thymus. Subsequently, it was discovered that CD25 1 Treg cells populate the periphery from the thymus with delayed kinetics relative to conventional T cells, with very few CD25 1 Treg cells present in the periphery at day three after birth. 23 It was, therefore, proposed that day three thymectomy generates a quantitative imbalance of CD25 1 Treg cells and conventional T cells in the periphery, resulting in dysregulated immune responses to self-antigens and consequently development of organ-specific autoimmune disease. 68 That CD25 1 Treg cells develop and acquire their suppressive phenotype in the thymus was shown more directly by Itoh et al., who found that CD25 1 CD4 1 CD8 thymocytes showed the same in vitro functional characteristics as their peripheral counterparts; namely they were naturally anergic and suppressed proliferative responses of other thymocytes and peripheral T cells. 73 In addition, CD25 1 CD4 1 CD8 thymocytes displayed a similar pattern of expression of CTLA-4, GITR and OX40, when compared with peripheral CD25 1 Treg cells. 18,73 Because most CD25 1 Treg cells have a thymic origin, it is clearly important to determine the nature of the signals driving CD25 1 Treg development in the thymus. Given that CD25 1 Treg cells express MHC class II-restricted ab TCR, like conventional CD4 1 T cells, it is likely that CD25 1 Treg cells or their precursors interact with self-antigen/mhc class II complexes on thymic APC. One possibility is that the strength of interaction of TCR with self-antigen/ MHC class II complexes is a critical factor in the differentiation of immature thymocytes into the CD25 1 Treg lineage. Alternatively, or in addition to TCR-derived signalling, signalling from other receptors may be required for CD25 1 Treg generation. There is evidence to support both hypotheses. Role of TCR signalling It is commonly observed that MHC class II-restricted TCR transgenic mice have a decreased number of CD25 1 Treg cells, when compared with wild-type mice. The relatively few CD25 1 Treg cells found in TCR transgenic mice typically express endogenous TCR-a chains at a higher frequency than the CD4 1 CD25 T-cell population. These observations suggest that signalling through the TCR formed by endogenous TCR-a and transgenic TCR-b chains is essential for CD25 1 Treg generation. This proposal is supported by the finding that TCR transgenic mice on RAG-deficient or TCR-adeficient backgrounds are often nearly devoid of CD25 1 Treg cells For example, mice transgenic for a TCR recognizing a determinant of myelin basic protein (MBP) contain a population of CD25 1 Treg cells that is enriched for cells expressing endogenous TCR-a chains. These mice remain healthy with no evidence of autoimmune disease. However, when crossed onto a RAG-1-deficient background, MBP-specific TCR transgenic mice no longer develop CD25 1 Treg cells and develop spontaneous EAE at an early age, probably due to reduced regulation of the encephalogenic MBP-specific CD4 1 CD25 T-cell population. 76 The requirement for TCR signalling in thymic CD25 1 Treg generation was first shown directly using TCR transgenic mice that recognized a major determinant of influenza haemagglutinin (HA), and expressed HA as a neo-self-antigen in thymic APC. 77 Peripheral CD4 1 T cells and mature CD4 1 thymocytes were found to contain a high proportion of clonotypic CD25 1 Treg cells in these double transgenic mice, when compared with single TCR transgenic mice. Development of the clonotypic CD25 1 Treg population was dependent on expression of the HA transgene in radioresistant thymic stromal cells and did not require expression of endogenous TCR-a chains. 77 These findings were suggestive of HA-induced positive selection of HA-specific CD25 1 Treg cells. In a different line of HA-specific TCR transgenic mice in which the TCR had a lower intrinsic affinity for the HA determinant, the expression of transgenic HA by thymic APC did not result in the development of a large CD25 1 Treg population. 77 This further suggested that TCR avidity was a critical factor in the generation of CD25 1 Treg cells, with relatively high-avidity TCR ligand interactions promoting the development of CD25 1 Treg cells. Several groups have now reported similar findings in different TCR transgenic systems. In TCR transgenic mice that express the cognate antigen in thymic epithelial cells, significant populations of CD25 1 Treg cells are positively selected with concomitant deletion of conventional CD4 1 T cells. 74,78,79 From these TCR transgenic

6 492 TD Zwar et al. systems, it has been proposed that in the absence of cognate ligand, the pairing of endogenous TCR-a chains with transgenic TCR-b chains is required to generate TCR that interact with self-peptide/mhc class II complexes with sufficiently high avidity to promote CD25 1 Treg positive selection. In the absence of endogenous TCR-a gene rearrangement, such as in RAG-deficient or TCR-a-deficient mice, highly selfreactive TCR are not generated and therefore such mice lack CD25 1 Treg cells. However, in the presence of cognate ligand, the transgenic ab TCR interacts with cognate ligand MHC complexes on thymic APC with high avidity, thus recruiting clonotypic T cells into the CD25 1 Treg lineage without any involvement of endogenous TCR-a chains. 29 The finding that CD25 1 Treg generation is dependent on TCR MHC interactions is consistent with the results of an earlier study by Bensinger et al. in which CD25 1 Treg cells were lacking from the periphery of MHC class II-deficient mice. 80 However, in MHC class II-deficient mice that expressed an MHC class II transgene exclusively in cortical thymic epithelial cells, CD25 1 Treg cells were restored to near-normal levels. Collectively, these studies suggest that strength of signalling through the TCR complex is the major factor directing developing thymocytes into the CD25 1 Treg lineage (Fig. 1). More recently, an alternative model of CD25 1 Treg generation was proposed by van Santen et al. 81 This group analysed TCR transgenic mice recognizing an epitope of moth cytochrome C (MCC), which expressed the MCC epitope in thymic stromal cells in a gene expression system that can be regulated. Groups of double transgenic mice expressing different levels of the MCC epitope were generated and the status of CD25 1 Treg cells in each group was examined. Consistent with previous studies, increasing antigen dose over the midrange (and thus increasing avidity of TCR ligand interactions) did correlate with increased frequency of CD25 1 Treg cells in the thymus and periphery. However, the total number of CD25 1 Treg cells did not increase and, in fact, the CD25 1 Treg population only appeared to increase in size as conventional T cells declined in number because of agonist-induced deletion. Furthermore, at the highest antigen dose used in this study, CD25 1 Treg cells were eliminated. From these data, the authors concluded that CD25 1 Treg cells are more resistant to clonal deletion induced by agonist peptide/mhc class II complexes than conventional CD4 1 CD25 T cells. Based on this conclusion, the authors proposed an alternative model of thymic CD25 1 Treg generation, in which differentiation of thymocytes into the CD25 1 Treg lineage is not driven by TCR signalling in the thymic medulla, but rather by unknown signalling at an earlier stage in T-cell development. In this model, the signal or signals directing immature thymocytes into the CD25 1 Treg lineage would alter the balance of pro-apoptotic and anti-apoptotic factors. It was suggested that GITR may mediate the increased resistance of CD25 1 Treg cells to clonal deletion observed in this study, since GITR has been implicated in T-cell apoptosis and is constitutively expressed by CD25 1 Treg cells. 81 This model is consistent with results of an earlier study in which CD25 1 Treg cells were shown to be resistant to viral superantigen-mediated clonal deletion. 44 Recently, an alternative approach was used to track the development of a polyclonal population of CD25 1 Treg cells, using Foxp3 gfp mice. 60 In these mice, most Foxp3-expressing cells in the thymus are localized to the medullary region, which harbours the most mature thymocyte population. A smaller population of Foxp3-expressing cells is found at the corticomedullary junction. Corresponding with its spatial distribution, Foxp3 expression is mainly limited to single-positive (i.e. mature) CD4 1 thymocytes, with small populations of Foxp3-expressing CD8 1 single-positive, double-positive and double-negative thymocytes also present. Strikingly, Foxp3 expression was not detectable in thymocyte populations of MHC-deficient Foxp3 gfp mice. The finding that Foxp3 expression is biased toward mature thymocyte populations and is Increasing TCR affinity Thymic repertoire Deletion CD25 + Treg cells Conventional CD4 + T cells Death by neglect Early thymic emigrants Selection Final T-cell repertoire CD25 + Treg cells Conventional CD4 + T cells Figure 1 A model of thymic and peripheral CD25 1 T regulatory (Treg) cell selection. In the thymus, differentiation of immature thymocytes into the CD25 1 Treg lineage is dependent on TCR interaction with self-peptide/mhc class II complexes within a moderate-to-high range of avidity. In the periphery, TCR interaction with self-antigen is required for survival of CD25 1 Treg cells; therefore, the final repertoire is also likely to be influenced by the set of peripheral self-antigens. For conventional naive CD4 1 T cells, however, contact with self-antigen in the periphery is also required for survival, but will result in clonal deletion if the avidity of TCR interaction is high.

7 CD4 1 CD25 1 immunoregulatory T cells 493 completely dependent on interactions with MHC molecules is consistent with a model of TCR ligand interactions inducing Foxp3 expression and thus CD25 1 Treg development. This TCR-signalling model is further supported by a recent study that analysed TCR usage of activated T cells in Foxp3- deficient mice. 82 Hsieh et al. shown that the TCR repertoire of activated T cells in Foxp3-deficient mice (which are likely to be pathogenic self-reactive T cells) was more similar to that of CD25 1 Treg cells than of conventional CD25 T cells from Foxp3-sufficient TCR-b transgenic mice. These findings suggest that in the wild-type thymus, highly self-reactive thymocytes are induced to express Foxp3 and thus differentiate into the CD25 1 Treg lineage. However, in Foxp3-deficient mice, a pool of these highly self-reactive thymocytes are not converted to CD25 1 Treg cells, but nonetheless escape thymic deletion, and subsequently contribute to autoimmune pathology in the periphery. APC involvement In the models discussed above the positive selection of antigenspecific CD25 1 Treg cells is dependent on the expression of cognate antigen by radioresistant thymic epithelial cells. One possibility is the existence of specific epithelial niches that support positive selection of CD25 1 Treg cells. In HA-specific TCR transgenic mice expressing transgenic HA in thymic epithelium, the use of different promoters to drive HA expression influenced the size of the antigen-specific CD25 1 Treg population generated. 83 Use of different promoters to drive antigen expression may result in different amounts of antigen being expressed (thus altering the density of TCR ligand interactions on T cells), and in different cell types, both of which may be important factors in the positive selection of CD25 1 Treg cells. A recent study has described a specific subpopulation of human thymic DC capable of generating de novo CD25 1 Treg cells in an in vitro system. 84 The authors of this study identified an IL-7-like cytokine, named thymic stomal lymphopoietin (TSL), as critical for the activation of immature thymic CD11c 1 DC. TSL is produced by thymic epithelial cells of Hassal s corpuscles and was shown to be capable of inducing upregulation of MHC class II as well as the costimulatory molecules CD80 and CD86 in this DC population. Such TSLactivated DC induced proliferation and differentiation of single-positive CD4 1 CD25 thymocytes into CD4 1 CD25 1 Foxp3 1 regulatory T cells in vitro. These results, combined with the observation that CD25 1 Treg cells are found closely associated with CD86 1 DCs and Hassal s corpuscles in the human thymus, suggest the presence of a dedicated epithelial niche for the positive selection of CD25 1 Treg cells. The promiscuous expression and presentation of peripheral self-antigens is a property of medullary thymic epithelial cells, allowing the induction of central tolerance to many self-antigens. The transcription factor Aire is required for thymic expression of many self-antigens. Aire-deficient mice display autoimmune pathology in range of organs, which has been attributed to a defect in central tolerance of conventional CD4 1 T cells. Aire-dependent expression of peripheral selfantigens may also be required for the positive selection of CD25 1 Treg cells that are necessary for the control of autoimmunity. However, an analysis of Aire-deficient mice showed a similar frequency and total number of CD25 1 Treg cells in the thymus and spleen, when compared with wild-type mice. 85 Importantly, autoimmune pathology observed in athymic mice grafted with Aire-deficient thymi was not prevented by simultaneously grafting wild-type (Aire-sufficient) thymi, showing that a CD25 1 Treg deficiency is not the primary defect of Aire-deficient mice. 85 It remains possible that a subset of CD25 1 Treg cells is positively selected in an Aire-dependent manner, but clearly this does not apply to the majority of CD25 1 Treg cells. Role of non-tcr signals Analysis of thymocyte populations in mice genetically deficient in molecules such as B7, CD28 and CD40 have shown deficits in CD4 1 CD8 CD25 1 T cells, indicating an important role for these costimulatory molecules in CD25 1 Treg generation. Signalling through such molecules may increase the strength of TCR signalling, which is likely to be a major factor determining the positive selection of CD25 1 Treg cells. 29 Mice which lack the functional genes for either IL-2, IL-2Ra (CD25) or IL-2Rb (CD122) contain significantly reduced numbers of thymic and peripheral CD4 1 CD25 1 T cells compared with their wild-type counterparts, 44,86,87 and are characterized by massive T-cell lymphoproliferation and autoimmune disease. 56,57,88,89 A lack of CD25 1 Treg cells is responsible for this phenotype as the pathology could be controlled by reconstitution of these mice with normal CD25 1 Treg cells. 90 The markedly reduced numbers of mature thymic CD4 1 CD25 1 T cells in IL-2-deficient and in IL-2R-deficient mice was initially taken as evidence that IL-2 signalling was required for thymic development of CD25 1 Treg cells. However, two recent studies have shown that IL-2-deficient and IL-2Ra-deficient mice do in fact contain a significant population of thymic CD4 1 Foxp3 1 T cells. 58,59 Single-positive CD4 1 Foxp3 1 T cells from IL-2-deficient mice expressed reduced levels of CD25 compared with their wild-type counterparts, presumably because IL-2 plays a role in regulating CD25 expression levels. 59 However, thymic single-positive CD4 1 Foxp3 1 T cells were not detected in mice deficient in the common cytokine receptor g chain (CD132), suggesting that signalling from other cytokines are necessary for thymic CD25 1 Treg development. Maintenance of CD25 1 Treg cells in the periphery Early studies of the in vitro behaviour of CD25 1 Treg cells showed that although they require stimulation through their TCR to become active suppressor cells, CD25 1 Treg cells do not proliferate in response to TCR stimulation alone. In vitro proliferation of CD25 1 Treg cells is observed when relatively large amounts of either exogenous IL-2 or anti- CD28 mab is added to culture medium; however, under such conditions, proliferating CD25 1 Treg cells lose their ability to suppress proliferation of responder CD4 1 T cells. 15,30 These early studies had a major influence on the field, as CD25 1 Treg cells were initially thought to be naturally anergic and CD25 1 Treg proliferation and suppressor function were thought to be mutually exclusive. Analysis of CD25 1 Treg proliferation and suppressor function in vivo has showed both of these assumptions to be

8 494 TD Zwar et al. incorrect. For example Malek et al. showed that following transfer into IL-2Rb-deficient mice, which lack endogenous CD25 1 Treg cells, donor polyclonal CD25 1 Treg cells expand in the hosts. 87 Furthermore, the autoimmune/lymphoproliferative syndrome that normally occurs in IL-2Rb-deficient mice is cured by such transfer of CD25 1 Treg cells, showing that CD25 1 Treg proliferation and suppression may occur concurrently. 87 Polyclonal CD25 1 Treg cells also expand following transfer to neonatal Foxp3-mutant mice and concurrently inhibit the onset of the characteristic lymphoproliferative syndrome in these recipients. 20 TCR signalling and CD25 1 Treg survival Given that TCR signalling is a major requirement for the thymic generation of CD25 1 Treg cells, it is likely that the majority of CD25 1 Treg cells exiting the thymus express self-reactive TCR and thus could potentially contact cognate antigen in the periphery. For naïve conventional CD4 1 T cells, TCR interactions with self-peptide/mhc complexes in the steady-state promote survival and thus homeostasis. 91 An important issue is whether such interactions also promote homeostasis of CD25 1 Treg cells. Several studies have examined homeostasis of CD25 1 Treg cells in normal (non-lymphopenic) mice. In one study, polyclonal CD25 1 Treg cells formed a small, but long-lived population following transfer to normal mice. 92 This population included both long-lived quiescent cells, and a subpopulation of rapidly dividing cells. Antigen-driven proliferation of CD25 1 Treg cells in normal hosts has also been shown in a number of cell transfer studies, in which antigen-specific CD25 1 Treg cells proliferated in response to the transgenic expression of cognate antigen in the pancreas 79,92 or ubiquitously in the periphery. 93 Similarly, following transfer to normal recipients, antigen-specific CD25 1 Treg cells proliferated in response to immunization with cognate peptide in IFA. 38,79 In contrast, in a study by Gavin et al., CD25 1 Treg cells expressing a transgenic TCR specific for hclip failed to proliferate following transfer to normal mice and immunization of those recipients with hclip peptide in CFA. 94 However, in this latter study, the failure of CD25 1 Treg cells to proliferate may be related to the pro-inflammatory effects of the adjuvant used. In lymphopenic environments, the requirements for survival and proliferation of conventional CD4 1 T cells are more complex than in normal non-lymphopenic mice. In addition to TCR engagement, factors such as cytokine-derived signalling are required. In particular, the cytokine IL-7 is critical for the so-called homeostatic proliferation of CD4 1 T cells in lymphopenic environments. 95 The requirements for homeostatic proliferation of memory T cells differ from those of naïve T cells, as various studies have shown that proliferation of memory CD4 1 and CD8 1 T cells in lymphopenic mice does not require interaction of their TCR with MHC molecules. 91 Several studies have analysed the role of TCR signalling in homeostasis of CD25 1 Treg cells in lymphopenic mice. In one study, polyclonal CD25 1 Treg cells proliferated extensively in lymphopenic RAG-1 / mice, but not in MHC class II I-Ab-deficient RAG-1 / mice, indicating that TCR interactions with MHC class II were required for homeostatic proliferation of CD25 1 Treg cells. 94 In another study, HAspecific CD25 1 Treg cells failed to proliferate following transfer to irradiated normal mice, but did proliferate in irradiated HA-transgenic mice, further showing the dependency of CD25 1 Treg homeostatic proliferation on TCR signalling. 93 Interestingly, the proliferation of HA-specific CD25 1 Treg cells was augmented in irradiated HA-transgenic mice compared with non-irradiated HA-transgenic mice, suggesting that irradiation-induced lymphopenia and/or inflammation may provide additional stimuli that promote proliferation of CD25 1 Treg cells. 93 In a recent study by Hsieh et al., the TCR repertoires of thymic and peripheral CD25 1 Treg cells were assessed and compared. 82 Considerable differences were observed in the repertoires of thymic and peripheral CD25 1 Treg populations from individual mice. These findings suggest that interactions with self-antigen/mhc class II complexes in the periphery play a role in shaping the TCR repertoire of CD25 1 Treg cells, most likely by providing signalling to CD25 1 Treg cells to proliferate and/or survive. IL-2 and CD25 1 Treg homeostasis In several studies discussed above, in vivo-expanded CD25 1 Treg populations were reisolated from recipient mice and found to be anergic to restimulation in vitro. This failure of TCR-stimulated CD25 1 Treg cells to proliferate in vitro may reflect the requirement for additional factors to drive proliferation, which are absent in the in vitro proliferation assay. Given that CD25 1 Treg anergy in vitro can be overcome by high concentrations of exogenous IL-2, high in vivo levels of IL-2 may permit CD25 1 Treg proliferation. 96 The requirement for IL-2 in CD25 1 Treg homeostasis was shown in a series of experiments examining engraftment of CD25 1 Treg cells in recipient mice in the presence or absence of IL In this study, transfer of CD25 1 Treg cells into neonatal IL-2Rbdeficient mice (that lack endogenous CD25 1 Treg cells) resulted in long-term engraftment of the donor cells and recipients were subsequently protected from autoimmunity. In contrast, when CD25 1 Treg cells were transferred into IL-2-deficient mice, donor CD25 1 Treg cells were undetectable in the recipients several weeks later. Consequently, these mice developed the autoimmune and lymphoproliferative disorder that is characteristic of IL-2 and IL-2R mice. These experiments suggest that expansion of CD25 1 Treg cells in IL-2Rb-deficient mice is dependent on production of IL-2 by the recipient. This proposal is supported by two more recent studies that reported that peripheral CD4 1 Foxp3 1 T cells are significantly reduced in frequency in IL-2-deficient and IL-2Ra-deficient mice, when compared with wild-type mice. 58,59 Role of costimulation in CD25 1 Treg survival In the NOD mouse model of spontaneous autoimmune diabetes, disease is exacerbated and accelerated in mice deficient in CD Salomon et al. showed that B7-1/B7-2-deficient and CD28-deficient NOD mice are deficient in CD25 1 Treg cells, implying that costimulation through the CD28 pathway may be critical for thymic development and/or peripheral homeostasis of CD25 1 Treg cells. 25 Short-term blockade of B7 signalling in the periphery of NOD mice resulted in a decrease of CD25 1 Treg cells and exacerbated disease in young mice, confirming a role for CD28 in CD25 1 Treg homeostasis in