INTRODUCTION

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The same effector mechanisms that have evolved to protect the host from invading microorganisms can induce immune pathology if not properly regulated. It is now clear that a number of diseases result from the induction of aberrant immune responses toward innocuous antigens derived from dietary proteins, commensal bacteria, and even host tissues. Clearly the immune system has the ability to mount pathological responses, yet despite this, inflammatory diseases attributable to a dysregulated immune response are relatively infrequent, suggesting that mechanisms exist to limit or suppress the immune response. There is accumulating evidence that functionally specialized populations of regulatory T cells (Treg cells) play a dominant role in this process (1).

In this article we describe data obtained in our laboratory regarding Treg cells, which occur naturally in peripheral lymphoid organs and play a key role in the homeostasis of the mucosal immune response.

	REGULATORY T CELLS IN THE MUCOSAL IMMUNE SYSTEM

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Phenotypic and Functional Analysis

A characteristic feature of the immune system at the mucosal surfaces, such as the intestinal and respiratory tracts, is that protective cell-mediated and humoral immune responses against invading pathogens are allowed to proceed while pathogenic responses against innocuous antigens are prevented. The importance of an intact immune system for intestinal homeostasis is revealed by the fact that a number of immune manipulations, including deletion of cytokine genes and alterations in T cell subsets, lead to the development of an inflammatory bowel disease (IBD)-like syndrome in mice (2, 3).

Studies from this laboratory have provided direct evidence that a subset of CD4⁺ T cells controls inflammatory responses in the intestine. Transfer of CD45RB^highCD4⁺ T cells from normal donors into severe combined immunodeficient (SCID) mice led to the development of a severe inflammatory response in the colon (4, 5). Colitis was the result of the development of a helper T cell type 1 (Th1) response as polarized Th1 cells were present in intestinal lesions and disease could be prevented by treatment with an anti-interferon  (IFN-) or anti-tumor necrosis factor  (TNF-) monoclonal antibody (MAb) (6). In contrast, the reciprocal CD45RB^lowCD4⁺ T cell subset transferred into SCID mice did not induce colitis and injected together with pathogenic CD45R^high cells prevented the development of disease. These results showed that T cells capable of inducing intestinal pathology exist normally in healthy animals but that their function is inhibited by a phenotypically distinct population of Treg cells.

Immune suppression mediated by Treg cells was dependent on transforming growth factor  (TGF-) and independent of interleukin 4 (IL-4), suggesting that these cells were functionally distinct from Th2 cells (7). In fact, anti-TGF- MAb was able to abrogate protection from colitis transferred by CD45RB^lowCD4⁺ cells, while IL-4 appeared to play no demonstrable role in either the development or effector function of the regulatory T cell population, as CD45RB^lowCD4⁺ cells from IL-4-deficient mice were equally as potent as wild-type cells in inhibiting colitis (Table 1).

There is evidence that IL-10 plays an important role in mucosal immune regulation, as mice with a targeted disruption of the IL-10 gene developed enterocolitis (8). In addition, administration of murine recombinant IL-10 (rIL-10) prevented colitis in SCID mice restored with CD45RB^highCD4⁺ T cells (6) and in IL-10-deficient (IL-10^/) mice treated from weaning (9). Furthermore, CD45RB^highCD4⁺ cells isolated from transgenic mice that expressed IL-10 under the control of the IL-2 promoter failed to transfer colitis but, rather, were able to inhibit colitis induced by wild-type CD45RB^highCD4⁺ T cells (10). Taken together, these studies provide evidence that IL-10 is able to regulate pathogenic immune responses in the intestine; however, whether this suppression involves the development of regulatory T cells is not known.

Therefore, to investigate whether the CD45RB^lowCD4⁺ population from IL-10^/ mice contained regulatory T cells, this population was compared with CD45RB^lowCD4⁺ cells from wild-type mice for the ability to inhibit colitis. As shown in Figure 1, not only did transfer of CD45RB^lowCD4⁺ cells from IL-10-deficient mice fail to inhibit colitis but this population actually induced disease with characteristics and incidence similar to that induced by wild-type CD45RB^highCD4⁺ cells (11). In support of these results, blockade of IL-10 function by administration of a monoclonal antibody against the IL-10 receptor (IL-10R) completely abrogated protection transferred by CD45RB^lowCD4⁺ cells from wild-type mice, indicating that production of IL-10 by CD45RB^lowCD4⁺ cells was essential for cells within this population to mediate their immune suppressive function.

View larger version (33K): [in this window] [in a new window]   Figure 1.   The function of regulatory T cells that control inflammatory responses in the colon is dependent on IL-10. CD4+ T cell subsets were isolated from the spleen of normal or IL-10-deficient (IL-10/) mice and injected intraperitoneally into immune-deficient recipients. (Reprinted by permission from Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, and F. Powrie. J. Exp. Med. 1999;190:995-1004 [11].)

Taken together, studies of the SCID model of colitis suggest that IL-10 and TGF- play nonredundant roles in the functioning of regulatory T cells that control inflammatory responses toward intestinal antigens, as the neutralization or absence of one of these cytokines is sufficient to abrogate protection. Furthermore, IL-10 produced by regulatory T cells themselves is crucial for the normal functioning of these cells, as CD45RB^lowCD4⁺ cells incapable of synthesizing IL-10 failed to inhibit colitis despite the fact that both the CD45RB^highCD4⁺ T cells and host cells in the SCID were capable of making IL-10.

Mechanisms of Induction

The factors driving the induction of IL-10- and/or TGF-- expressing cells in the gut are not yet fully understood. It has been suggested that it is the balance between proinflammatory and antiinflammatory signals acting on antigen-presenting cells (APCs) that dictates T cell subset differentiation (12). For example, in the gut-associated lymphoid tissue (GALT), pathogens that induce IL-12 and other proinflammatory cytokines by macrophages and dendritic cells lead to the differentiation of Th1 cells, whereas antigens that do not elicit this inflammatory response induce TGF-- or IL-10-secreting T cells (13). Interestingly, studies of Helicobacter hepaticus infection in mice showed that normal mice mounted an IL-10-dependent response whereas IL-10^/ mice developed a pathogenic Th1 response toward the bacterium (14). These studies support the hypothesis that, in immunocompetent hosts, enteric antigens induce IL-10-secreting T cells that are immunosuppressive and prevent inflammatory responses toward intestinal antigens. Mucosal T cell unresponsiveness to enteric antigens has similarly been shown in humans to be mediated by antigen-specific CD4⁺ T cells and production of IL-10 and TGF- (15). While the precise molecular mechanisms involved have not been defined, studies illustrate the effects of altering costimulatory molecules on APCs on the generation of IL-10-producing cells. Blocking CD40-CD154 and CD80/CD86-CD28 interactions during primary allogeneic stimulation led to the production of T cells that secreted increased levels of IL-10 and had reduced proliferative capacity (16). Similarly, T cells stimulated with suboptimal concentrations of anti-CD3 secreted high levels of IL-10 when LFA-1 was bound to intercellular adhesion molecule 1 (ICAM-1) as opposed to ICAM-2 or ICAM-3 (17).

Effector Functions

Precisely how IL-10 and TGF- induce immune suppression in vivo is not known. Both cytokines have well-characterized immune-suppressive properties active on both the induction and effector phases of T cell-mediated inflammatory responses (18, 19). IL-10 has been shown to mediate a range of antiinflammatory activities both on T cells and APCs, including the downregulation of antigen-induced proliferation, cytokine secretion, and expression of costimulatory molecules (20, 21). It seems likely that IL-10-secreting regulatory T cells act to inhibit Th1 cell activation and that IL-10 produced locally in the intestine acts on macrophages to prevent their activation and elaboration of proinflammatory molecules and chemokines, thus inhibiting T cell recruitment into the intestine. In fact, inhibition of colitis mediated by IL-10-secreting CD45RB^lowCD4⁺ cells was characterized by substantial reductions in total number of Th1 cells recovered from the intestine (11). This suggests that the major activity of IL-10-secreting regulatory T cells is to inhibit the accumulation of pathogenic Th1 cells in the intestine. Whether this is due to reduced expansion, or migration, of these cells is not known. In addition, mice in which macrophages and neutrophils are unable to respond to IL-10 as a result of a cell type-specific deletion of STAT3 (signal transducer and activator of transcription 3) developed enterocolitis, suggesting that IL-10-mediated macrophage and neutrophil deactivation contributes as well to the immune-suppressive properties of IL-10 in the intestine (22). Whether the immune-suppressive functions of IL-10 and TGF- are linked or whether they act entirely separately remains to be elucidated. The finding that IL-10^/ mice have immune pathology restricted to the intestine whereas TGF-1^/ mice develop multiple organ disease (23) suggests that IL-10 is not required for the production of TGF-1. However, TGF- has been shown to induce IL-10 secretion by APCs (24), making it a possibility that TGF- alters antigen presentation in favor of the generation of IL-10-secreting regulatory T cells.

In addition to preventing T cell activation, it is possible that activation of naive T cells in the presence of Treg cells leads to the differentiation of the naive T cells into cells with a similar function, rather than into pathogenic cells. This phenomenon has been termed infectious tolerance (25, 26). Given that production of IL-10 by CD45RB^lowCD4⁺ cells was essential for their regulatory function, it was possible that immune suppression by these cells required IL-10 production by the pathogenic CD45RB^highCD4⁺ population. To test this, CD45RB^high CD4⁺ cells from IL-10^/ mice were transferred alone or in combination with wild-type CD45RB^lowCD4⁺ cells to immune-deficient mice. As shown in Figure 2, mice restored with IL-10^/ CD45RB^highCD4⁺ cells developed a colitis similar in incidence and severity to that transferred by this population isolated from wild-type mice. However, protection from colitis was equally effective when wild-type CD45RB^lowCD4⁺ cells were cotransferred with IL-10^-/-CD45RB^highCD4⁺ cells, as the majority of mice had no colitis. These results indicate that inhibition of inflammatory responses in the intestine mediated by CD45RB^lowCD4⁺ cells is not dependent on the differentiation of the progeny of CD45RB^highCD4⁺ cells into IL-10-secreting cells. However, this result does not rule out the possibility that IL-10 secretion by the CD45RB^low population leads to the differentiation of the progeny of CD45RB^high cells into regulatory T cells secreting other cytokines, for example, TGF-.

View larger version (22K): [in this window] [in a new window]   Figure 2.   Prevention of colitis is independent of IL-10 production by CD45RBhighCD4+ T cells. CD4+ T cell subsets were isolated from the spleen of normal or IL-10/ mice and injected intraperitoneally into immune-deficient recipients. (Reprinted by permission from Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, and F. Powrie. J. Exp. Med. 1999;190:995-1004 [11].)

IL-10 has been shown to induce T cell anergy (27) and it may be tempting to speculate that IL-10 could induce anergic T cells that would mediate suppression in vivo. Interestingly, we found that T cells with the characteristics of anergic cells exist naturally within the IL-10-producing CD45RB^low population and can be identified by expression of the CD38 antigen (28). Antigen-primed T cells responding to antigen were contained within the CD38 subpopulation whereas the CD38⁺ subset was unresponsive to polyclonal stimulation, as assessed by proliferation and cytokine secretion. The CD38⁺ population was, however, not inactive as addition of CD38⁺cells to CD38 CD45RB^low cells led to a dose-dependent inhibition of the response to anti-CD3. Similar results have been observed with CD25⁺CD4⁺ cells (29). Analysis of the mechanism of immune suppression mediated in vitro by CD25⁺ or CD38⁺ CD4⁺ cells showed that it was dependent on cell-to-cell contact between responding and regulatory T cells and did not involve IL-10 or TGF-. The relationship between Treg cells that inhibit T cell activation in vitro and those that inhibit IBD in vivo remains to be established. Cross-linking of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) in the presence of T cell receptor (TCR)-mediated signals has been shown to induce TGF- secretion (32), providing a link between cell contact-dependent negative signals and immune-suppressive cytokines. In Figure 3, we describe a model for the immune suppression mediated by Treg cells.

View larger version (31K): [in this window] [in a new window]   Figure 3.   The regulation of effector CD4+ T cells by regulatory T cells (Treg) may be direct and/or may be mediated by alteration of the antigen-presenting cell (APC) function. IL-10, TGF-, and CTLA-4 are key components of the suppressive response; however, their precise roles in the regulatory pathway remain to be elucidated.

	REGULATORY CELLS IN SYSTEMIC IMMUNITY

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Regulatory T cells that control inflammatory responses in the gut by IL-10- and TGF--dependent mechanisms are not unique examples. Thus, TGF--dependent regulatory T cells capable of inhibiting autoimmune nephritis (33) or thyroiditis (34) have been shown to exist naturally in rats, suggesting that Treg cells that function via the secretion of immune-suppressive cytokines play a role in the maintenance of peripheral tolerance. Inhibition of diabetes in nonobese diabetic mice by NK1.1 T cells was dependent on IL-4 and IL-10 (35), and although the mechanism of inhibition in vivo is not known, the CD4⁺CD25⁺ population inhibited multiple organ autoimmune disease induced in nude mice by transfer of CD4⁺ CD25 cells (36, 37). It remains to be established whether regulatory T cells that control inflammatory responses in the gut are the same as those shown to regulate organ-specific autoimmune disease.

	ANTIGEN-INDUCED REGULATORY CELLS

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Exposure of the gastrointestinal tract to soluble antigen leads to a state of antigen-specific hyporesponsiveness on antigen challenge. This phenomenon, termed oral tolerance, presumably capitalizes on the natural mechanisms that act to protect the intestine from immune-mediated attack. Indeed, there is considerable evidence that IL-10- and TGF--secreting T cells are generated after oral exposure to antigen. Weiner and coworkers have clearly shown IL-10 and TGF- secretion in the Peyer's patches and lamina propria shortly after oral administration of antigens (38). The ability to produce regulatory cells in response to feeding antigen has been used to therapeutic ends in a number of autoimmune models (39). Thus CD4⁺ T cells cloned from the mesenteric lymph nodes of mice orally tolerized with myelin basic protein (MBP) secreted TGF- along with variable levels of IL-10 and IL-4 (40). These cells have been termed Th3 cells and were able to protect susceptible mice from experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. Importantly, immune suppression was shown to be dependent on TGF-.

Information on the role of IL-10 in the function of Treg cells induced after exposure to antigen via mucosal surfaces is limited. Both IL-4 and IL-10 were required for protection from experimental autoimmune uveitis (EAU) induced by feeding retinal antigens (41). Similarly, it has been shown that inhibition of EAE in MBP-specific TCR transgenic mice as a result of nasal administration of MBP was associated with upregulation of IL-10 (42). Furthermore, administration of anti-IL-10 antibody at the time of disease induction completely abrogated protection induced by intranasal exposure to MBP peptide.

	DIFFERENTIATION OF REGULATORY T CELLS IN VITRO

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While there is compelling evidence that a functionally specialized subset of Treg cells exists or can be induced in vivo, study of these cells has been hampered by the fact that they have proved difficult to grow in vitro. Groux and coworkers reported that repetitive antigenic stimulation of mouse or human CD4⁺ T cells, in the presence of IL-10, resulted in cells that showed reduced proliferative capacity and increased IL-10 secretion (43). One of the most striking features of these cells, termed Tr-1 cells, is their ability to mediate bystander suppression. Tr-1 cells generated from DO11.10 mice that express a transgene for a TCR specific for an ovalbumin peptide were able to protect against CD45RB^highCD4⁺ cell-induced colitis, but only when mice were fed ovalbumin. It is likely that Tr-1 cells represent an in vitro-generated counterpart of the regulatory T cells that exist naturally within the CD45RB^lowCD4⁺ T cell population and are dependent on IL-10 and TGF- for their function. However, it remains to be established whether the function of Tr-1 cells, in vivo, is dependent on IL-10 and TGF-.

	CONCLUDING REMARKS

TOP ABSTRACT INTRODUCTION REGULATORY T CELLS IN... REGULATORY CELLS IN SYSTEMIC... ANTIGEN-INDUCED REGULATORY... DIFFERENTIATION OF REGULATORY T... CONCLUDING REMARKS REFERENCES

There is now compelling evidence that regulatory T cells that secrete immune-suppressive cytokines are one of the host's natural mechanisms for preventing immune pathology. Their ability to inhibit both Th1, as evidenced in our model of colitis, and Th2 responses makes them excellent targets for immune therapy in a number of diseases including allergy, transplantation, and autoimmune disease. Antigen-specific approaches to immune therapy have been hampered by the fact that in many cases the precise antigen triggering disease is unknown. The ability of Treg cells to mediate antigen-driven bystander suppression may circumvent this problem, as Treg cells could be generated in response to antigens known to be present in the target organ. Antigen-specific Treg cells should in theory home to the target organ and inhibit responses against not only their specific antigen, but other antigens present in the local environment. While our understanding of the role of Treg cells in controlling immune pathology has significantly improved, more information on the basic biology of these cells is required before considering the attractive possibility of manipulating these cells as a treatment for inflammatory diseases.