More MS news articles for Nov 2001

Regulatory T cells in the control of immune pathology

http://www.nature.com/cgi-taf/DynaPage.taf?file=/ni/journal/v2/n9/special_full/ni0901-816_r.html

Nature Immunology 2, 816 - 822 (2001) © Nature America, Inc.
Kevin J. Maloy & Fiona Powrie
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Correspondence should be addresed to F Powrie http://www.nature.com/email_response/email.taf?address=fiona.powrie%40path.ox.ac.uk

It is now well established that regulatory T (TR) cells can inhibit harmful immunopathological responses directed against self or foreign antigens. However, many key aspects of TR 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 TR cells may inhibit the development of immune pathology and discuss potential therapeutic benefits that may arise from the manipulation of TR cell function.

The existence of a subpopulation of T cells that specialized in the suppression of immune responses was originally postulated in the early 1970s (1). 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 cell–mediated 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 attributed (2-5), 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 repertoire (6-8). Given that the recognition of peptide ligands by the T cell receptor (TCR) appears to be extremely degenerate (9), 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 encountered (10). Indeed, some T cells are reactive with both foreign microbial peptides and self-antigen peptides (9, 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 animals (4). A number of mechanisms have been proposed to account for the maintenance of peripheral self-tolerance, including induction of T cell anergy (12), T cell deletion (13) and immunological ignorance (14). Although these passive mechanisms certainly contribute, they do not explain the fact that, in many systems, tolerance can be adoptively transferred by T cells (15, 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 animals (3-5). 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 mice (3, 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 cells (8). 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 population (8, 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 population (6, 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 TCR (19, 20) and the development of spontaneous autoimmune diabetes in a genetically susceptible strain (21). 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 development (22). The TR cells capable of inhibiting the development of IBD are predominantly found within the CD4+CD45RBloCD25+ population (23). Although alternative interpretations have been proposed to account for TR cell phenomena (24), 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.

Phenotypic characteristics of naturally occurring TR cells

CD4+CD25+ T cells, which constitute approx 10% of peripheral murine CD4+ T cells, possess potent regulatory activity both in vitro (25, 26) and in vivo (8, 17, 23, 27). Murine CD4+CD25+ T cells express little CD45RB and a significant proportion express cytolytic T lymphocyte-associated antigen 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 is dependent on exogenous interleukin 2 (IL-2) (30). CD4+CD25+ T cells can inhibit autoimmune diabetes in mice (28) and rats (31), induce tolerance to alloantigens (32-34), impede anti-tumor immunity (35) and regulate the expansion of other peripheral CD4+ T cells (36). The mature CD4+CD8- single-positive (SP) thymocyte fraction from normal rodents also contains a small proportion (approx 5–10%) of CD25+ TR cells (31, 37). CD4+CD25+ T cells with regulatory functions similar to those described in rodents have now been isolated from human thymus and peripheral blood (38-42). However, a number of caveats preclude the use of CD25 expression as a unique marker for TR cells.

CD25 is transiently expressed upon activation of naïve CD4+ T cells and its expression by CD4+ T cells is highly dynamic in vivo (36). Importantly, the peripheral CD4+CD25- T cell population also possesses some regulatory activity, albeit less potent than that of their CD4+CD25+ counterparts (23, 31, 36). Similarly, protection from EAE in the MBP-specific TCR-transgenic model can be mediated by normal T cell populations that have been depleted of CD25+ cells (43). Thus, the CD4+CD25+ T cell population is heterogenous and although a relatively high proportion of these cells may be TR cells, it is unwise to assume that this is true for the entire population or that all TR cells express CD25.

Other subsets of regulatory T cells

A number of CD4+ T cell subpopulations capable of inhibiting the response of other T cells have been described (2-5). A complete characterization of all the different CD4+ TR cell populations is still lacking, but some of them share characteristics with the naturally occurring CD4+CD25+ TR cells. One such feature is that CD4+CD25+ T cells generally show a low proliferative capacity in vitro (25, 26). Similarly, anergic T cells are classically defined as T cells that do not proliferate or produce IL-2 upon antigenic restimulation (44). Some anergic T cell clones can suppress immune responses in vivo—including the prevention of allograft rejection via a mechanism that involves antigen-presenting cells (APCs) (45-47)—and T cells that have been anergized in vivo are strong producers of IL-10 (48, 49). Interestingly, allogeneic stimulation of human CD4+T cells in vitro in the presence of IL-10 also induces T cell anergy (50) and, after repetitive stimulation, a population of T cell clones (designated TR1 cells) emerges that secretes high amounts of IL-10 and moderate amounts of transforming growth factor-b (TGF-b) (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-10 (51). 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 suppression (51, 52). Similarly, T helper type 3 (TH3) cells that arise after oral administration of antigen produce high concentrations of TGF-b and can inhibit the development of immune pathology in several animal models (53-55). Thus, anergic cells, TR1 cells and TH3 cells may be derived from the same population: they have a similar phenotype and usually mediate their suppressive activities via the release of the cytokines TGF-b and IL-10.

CD4+ T cells with regulatory activity appear to be key to the suppression of graft rejection in a number of transplantation models (56, 57). Mice treated with nondepleting monoclonal antibodies (mAbs) to CD4 tolerate allografts and CD4+ T cells isolated from tolerant mice suppress the rejection of grafts upon adoptive transfer into naïve mice. These TR cells can induce naïve CD4+ T cells to themselves differentiate into TR cells, a phenomenon termed "infectious tolerance" (58). The phenotypic characteristics of the TR cells responsible for infectious tolerance and the molecular mechanisms by which they mediate suppression remain to be established. However, CD4+CD25+ cells from tolerant donors can adoptively transfer transplantation tolerance to naïve recipients (32) and CD4+CD45RBlo T cells from unmanipulated mice can prevent graft rejection in an adoptive transfer model (59). This suggests that TR cells generated as a result of tolerance-inducing protocols may have emerged from naturally occurring TR cells. Future studies, particularly those designed to provide comprehensive genetic and molecular expression profiles of highly purified CD4+ TR cell populations, should help define the nature and extent of the interrelationships of different TR cell populations.

Thymic generation of TR cells

Accumulating evidence that the thymus is important in the generation of TR cells has led to the theory that the production of TR cells represents a key function of the thymus (60). CD4+CD8- SP thymocytes are a potent source of TR cells that adoptively transfer protection from autoimmune diabetes in rodent models (61, 62). CD25 is expressed by 5–10% of CD4+CD8- SP thymocytes in humans (38), rats (31) and mice (30, 37); these cells can inhibit T cell proliferation in vitro and can prevent development of gastritis (37), diabetes (31) and colitis (63) in vivo.

Studies of transplantation tolerance support a key role for thymic epithelium (TE) in the differentiation of TR cells. Grafting of xenogeneic or allogeneic TE induces tolerance to a variety of peripheral tissues of donor origin that is mediated by, and transferable with, CD4+ T cells; this suggests that the selection of T cells on foreign TE generates TR cells that provide dominant tolerance to donor tissues (16, 64). One pathway of intrathymic selection of TR cells has been delineated by elegant studies in which mice expressing a transgenic TCR that recognizes an influenza hemagglutinin (HA) peptide were crossed to a lineage expressing the HA peptide (65, 66). In double-transgenic mice, the HA-specific CD4+T cells are not deleted: instead, approx 50% of them are CD25+ and function as TR cells (65). Radioresistant thymic elements mediate selection of the CD4+CD25+ cells, which arise first in the thymus, before gradually accumulating in secondary lymphoid organs (66). These observations are consistent with the appearance of CD4+CD25+ cells in fetal thymic organ cultures (37) and show that the CD4+CD25+ TR cells found in the thymus are not derived from recirculating activated peripheral T cells. Consistent with published studies (30), CD25 expression is acquired relatively late in thymocyte development, as the cells progress from the CD4+CD8+ double positive stage to the CD4+CD8- SP stage. Strikingly, thymocytes bearing a lower affinity HA-specific transgenic TCR do not develop into CD4+CD25+ thymocytes in double-transgenic mice. This prompted the suggestion that those thymocytes that develop into CD4+CD25+ SP cells may express a TCR with a relatively high affinity for self-peptides present in the thymus (66). Nevertheless, these results are also compatible with an avidity-dependent selection process, similar to the model originally proposed (64).

In this scheme, TR cell development in the thymus would be directed by relatively high avidity interactions between the TCR and self-peptide ligands expressed on thymic APCs (Fig. 1). Lower avidity interactions would predominantly promote the development of conventional CD4+CD25- SP thymocytes, as observed when the affinity of the TCR was reduced in the double transgenic mice. Higher avidity interactions would lead to clonal deletion, as found when the high affinity HA-specific TCR–transgenic mice were crossed with other transgenic lines that expressed higher amounts of the HA peptide (66). A narrow avidity window for the selection of TR cells would ensure that they represented only a small proportion of positively selected thymocytes, perhaps just enough to maintain self-tolerance in the periphery. In addition, those T cells in the peripheral repertoire with the highest intrinsic affinity for self-peptide–major histocompatibility complex (MHC) would be TR cells.

Figure 1. TR cells are a normal product of thymic selection.

TR cells may arise from relatively high-avidity interactions with self-peptide–MHC complexes, just below the threshold for negative selection (green area). This narrow avidity selection window ensures that TR cells will constitute only a small fraction of the mature T cell pool and have a greater sensitivity to self-peptide–MHC than potentially pathogenic autoreactive T cells.

Peripheral maintenance of TR cells

Little is known about how TR cells are incorporated into and maintained within the peripheral T cell pool, although their numbers appear to be relatively stable in the periphery throughout adult life (67). Cytokines and costimulatory molecules are important in the homeostasis of CD25+ TR cells, as this population is absent in IL-2-/- mice (30) and severely diminished in CD28-/- or B7-/- nonobese diabetic (NOD) mice (28). The activation status of APCs may also influence the accumulation of TR cells, as CD40-deficient and CD40 ligand–deficient mice have reduced numbers of CD4+CD25+ cells (63, 68). These findings, together with the fact that TR cells maintain a partially activated phenotype, suggests accumulation of these cells depends on continued peripheral stimulation (69). Evidence that maintenance of TR cells is driven by the presence of the target organs that they regulate comes from the finding that peripheral CD4+ T cells obtained from athyroid rats cannot inhibit the development of thyroiditis in susceptible TxX rats, although their ability to inhibit the development of diabetes is unimpaired (70). Strikingly, thymocytes from athyroid rats can inhibit the development of thyroiditis, which suggests that the failure to maintain this TR cell function in the periphery is due to the absence of the specific autoantigen (70). Similarly, the ability of TR cells to suppress the rejection of allografts in the infectious tolerance transplantation model is highly dependent on the continuous presence of antigen (56).

On the other hand, there is evidence that regulation of harmful T cell responses towards nonself-antigens may be mediated by TR cells that have never been exposed to the target antigens. Thus, although the IBD induced in SCID mice reconstituted with naïve CD4+CD45RBhi T cells is driven by T cell reactivity towards antigens present in the intestinal flora (63), peripheral CD4+CD45RBlo or CD4+CD25+ T cells isolated from immunocompetent germ-free mice can still inhibit the development of colitis (63). It should be noted, however, that CD4+ TR cells are naturally generated that maintain tolerance towards components of the commensal flora and a breakdown in this tolerance is observed in patients with IBD (71, 72). Presently, very little is known about the specificities of naturally occurring TR cells and the existing evidence is supportive of the hypothesis that TR cells are a heterogeneous population that can react with peptides derived from both self-tissues and foreign antigens.

Peripheral de novo development of TR?

The question of whether TR cells represent a unique lineage of CD4+ T cells imprinted with this function in the thymus or whether TR cell function may be acquired by naïve T cells in the periphery is still open. Evidence for the induction of tissue-specific 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 development of a mature prostate that is not subjected to autoimmune attack. In fact, such mice concommitantly develop TR cells that can inhibit the development of autoimmune prostatitis after transfer to post-d3Tx mice (73). Models of transplantation tolerance provide further support for peripheral differentiation of TR cells. In addition to suppressing allograft rejection by nontolerant T cells, TR cells induced by infectious tolerance protocols, or by engraftment of allogeneic fetal thymic epithelium, can also "educate" naïve CD4+ T cells to differentiate into TR cells in the periphery (56, 58, 74).

Protocols that induce T cell tolerance by mucosal administration of antigen are also supportive of the existence of peripheral pathways of TR cell induction (55). In an ovalbumin (OVA)-specific transgenic CD4+ T cell adoptive transfer model, in vivo tolerization of CD4+ T cells—either by administration of intravenous OVA peptide or by feeding intact OVA—led to the emergence of CD4+CD25+ transgenic T cells with immunoregulatory properties (75). Thus, additional pathways may exist whereby naïve CD4+ T cells can be induced to differentiate into TR cells in the periphery and "education" of naïve CD4+T cells provides a potential mechanism to extend the functional repertoire of TR cells in the periphery.

The differentiation of naïve CD4+ T cells is crucially determined by two interrelated factors: the APC that stimulates them and the cytokine environment in which the activation occurs (76, 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 DCs (78) and stimulation of T cells with immature DCs leads to the development of T cells with regulatory activity (79, 80). The development of CD4+CD25+ TR cells from human naïve T cells has been induced ex vivo by culturing the T cells with TGF-b (81). However, prior depletion of the rare CD25+ cells within the naïve T cell population markedly reduces the efficacy of TR cell development, which suggests that TGF-b may preferentially expand precommitted TR cells, rather than inducing their de novo differentiation (81). Stimulation in vivo with APCs that are overexpressing Serrate-1 (a Notch ligand), also results in the differentiation of CD4+ TR cells (82); however, the cellular and molecular interactions that normally direct TR cell development in vivo remain to be determined.

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 model, protection from colitis does not require IL-4, but is crucially dependent on TGF-b (83) and on IL-10 production by T cells (84). TR1 cells that inhibit colitis via bystander suppression also produce high amounts of IL-10 and TGF-b, but not IL-4 (51). The TH3 cells induced in oral tolerance protocols have also been associated with the same set of suppressive cytokines (55), 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-b and IL-4, but the role of IL-10 remains to be determined (18).

The immune-suppressive properties of IL-10 and TGF-b are most likely explained by the ability of these cytokines to inhibit APC function (85-88) and to mediate direct anti-proliferative effects on T cells (89-91). 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-10 (36). 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 IL-10 develop IBD (92). Conversely, the action of TGF-b on T cells is revealed in mice expressing a T cell–specific dominant-negative form of the TGF-b receptor II, which develop inflammatory infiltrates in both the colon and lungs (90).

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 vitro (25, 26, 38-42). 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 contact–dependent mechanism independent of IL-4, IL-10 and TGF-b (25, 26, 38-42). 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 manner (25, 93). CD4+CD25+ T cells 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 stage (93). 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 response (25, 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 cells (25, 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 cells (26, 41).

There is conflicting data on whether the cell contact–dependent 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 cells (94) and similar observations were made with anergic T cells (47). 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 cell–T cell interaction that is independent of APCs (25, 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 arrest (95), 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-10–independent manner (96, 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-b has not been assessed.

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 activation (98). The fact that CTLA-4 expression is primarily restricted to CD4+CD25+ T cells (23, 28, 29) suggests that it may also be functionally important. Indeed, anti–CTLA-4 treatment abrogates the ability of CD4+CD25+ T cells to inhibit colitis (23) and also induces the development of gastritis in normal mice (29). 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 population (29); 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-4–deficient mice also exhibit some suppressive activity (29). 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 cells (29). Alternatively, cross-linking of CTLA-4 on activated T cells may induce TGF-b secretion (99).

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.

Figure 2. TR cells can mediate their effector function via multiple mechanisms.

The inhibitory functions of TR cells on autoreactive T cells that are potentially pathogenic (TPATH cells) may be mediated through the actions of cytokines and/or via a cell contact–dependent mechanism. Although triggering through the 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 cell–T cell interactions also remains to be established.

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 colon (100). 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 cells (101). 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.

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 APCs (102, 103), the localization and distribution of antigen (14) and the recirculation patterns of T cells (104). 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 tissues (105); here they can efficiently phagocytose proteins and apoptotic debris arising from normal cell turnover in the tissue without becoming activated (106, 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 cells (108-110). 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 cells (79, 80). Consistent with the latter hypothesis, TR cells can respond to much lower concentrations of cognate peptide ligands than conventional naïve CD4+ T cells (25) and immature DCs express relatively low amounts of MHC class II and costimulatory molecules (79).

Figure 3. Model for the control of pathogenic immune responses by TR cells.

(a) In the steady-state, 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.

In contrast, the presence of an infectious agent will induce DC activation and migration (111) 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 vitro–hyperstimulation 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 activity (25, 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 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 tolerance (56). 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 tissue–expressed 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 protection (35). However, such strategies should be approached cautiously, as long-term depletion of TR cells may predispose the host to the development of autoimmunity (112). 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.

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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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