Control of intestinal inflammation by regulatory T cells

Control of intestinal inflammation by regulatory T cells

Microbes and Infection, 3, 2001, 929−935 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S128645790101454X/FLA Control o...

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Microbes and Infection, 3, 2001, 929−935 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S128645790101454X/FLA

Control of intestinal inflammation by regulatory T cells Christy Toms, Fiona Powrie* Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

ABSTRACT – Regulatory T(Treg)-cell populations have been identified in a number of disease models. In this review we focus on the role of naturally occurring Treg cells in the control of intestinal inflammation. Specifically, we discuss their mechanism of action with particular emphasis on the role of anti-inflammatory cytokines and cell surface molecules. © 2001 Éditions scientifiques et médicales Elsevier SAS immunity, mucosal / IL-10 / TGF-β

1. Introduction The intestine is an enormously complex microenvironment where cells of the immune system interface with a variety of endogenous and exogenous stimuli. Here, potent host protective cellular and humoral immune responses are activated towards pathogenic bacteria, viruses and parasites, whereas these responses are specifically not activated towards innocuous dietary antigens or members of the normal resident flora. The latter are not completely inert, however, as the number of leukocytes in the intestine is substantially reduced in animals raised under germ-free conditions and these mice manifest impaired oral tolerance induction and delayed-type hypersensitivity (DTH) responses [1, 2]. These results suggest that resident bacteria act as a source of endogenous stimuli to the immune system that is important for both its function and development. However, the constant presence of such a potentially pro-inflammatory stimulus in close proximity to immune cells means that the immune response here must be very tightly controlled to ensure that pathologic chronic inflammatory responses do not develop. The finding that a chronic inflammatory response driven by normal resident bacteria develops in the intestine of mice with genetically induced deficiencies in components of the immune system has provided some clues to the mechanisms which control intestinal homeostasis [3, 4]. There is evidence that cytokines play an important role as mice deficient in interleukin-10 (IL-10) develop chronic entercolitis [5], whereas an ulcerative colitis-like disease develops in mice with targeted disruption of the IL-2 gene [6]. T cells are also essential players as mice which lack a *Correspondence and reprints. E-mail address: [email protected] (F. Powrie). Microbes and Infection 2001, 929-0

functional T-cell receptor α chain and have no αβ TCR+ cells develop inflammatory bowel disease (IBD) [7]. Some of the most direct evidence that distinct subsets of CD4+ T cells can induce and regulate intestinal inflammation has come from studies in which peripheral CD4+ T-cell subsets were transferred to immunodeficient SCID or RAG–/– mice. SCID mice restored with CD45RBhi CD4+ cells developed a chronic colitis within 8–12 weeks [8]. This disease results from the differential expansion of Th1 cells driven by the normal intestinal flora as development of disease was dependent on IL-12, interferon gamma (IFN-γ) and tumour necrosis factor (TNF) and did not develop in T-cell-restored SCID mice raised under germ-free conditions [9, 10]. Reconstitution with CD45RBlow CD4+ cells did not result in colitis, and cotransfer of these cells with the reciprocal CD45RBhi population resulted in the prevention of disease. Taken together these results demonstrate that T cells capable of controlling intestinal inflammation are present in the peripheral T-cell pool of normal mice and that the absence of these cells results in the development of damaging T-cell-mediated immune responses towards intestinal antigens. In this review we summarise recent work to further characterise the immune suppressive properties of regulatory T (Treg) cells which control intestinal inflammation.

2. The immune suppressive cytokines IL-10 and transforming growth factor beta (TGF-β) play a role in the function of Treg cells IL-10-deficient mice mount hyperactive immune responses to a variety of infectious agents and inflammatory stimuli [11], illustrating the important role that IL-10 929

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plays as a negative regulator of the immune response. Its role in intestinal homeostasis is revealed by the fact that IL-10-deficient mice develop chronic inflammation in the intestine with no obvious inflammatory lesions elsewhere. Direct evidence that under normal circumstances IL-10 is produced in response to intestinal bacteria and that this production is essential to prevent intestinal inflammation comes from studies of Helicobacter hepaticus (Hh) infection in mice. Normal mice infected with Hh produced IL-10 in response to bacterial antigens and did not develop intestinal pathology, whereas IL-10–/– mice mounted a Th1 response to the bacteria and developed intestinal inflammation [10]. Consistent with the properties of endogenous IL-10, administration of exogenous IL-10 inhibited development of colitis in SCID mice reconstituted with CD45RBhi CD4+ cells [9] and in other models of IBD [12]. Whilst these data provide incontrovertible evidence that IL-10 production maintains intestinal homeostasis, they do not identify which cells are the important source of IL-10 in vivo, nor which cells are the major targets. To determine whether IL-10 was involved in the function of Treg cells, CD45RBlow CD4+ cells were isolated from IL-10–/– mice and tested for their ability to inhibit IBD. In contrast to Treg cells isolated from wild-type mice those isolated from IL-10–/– mice failed to inhibit colitis when cotransferred with CD45RBhi CD4+ cells. Indeed, CD45RBlow CD4+ cells from IL-10–/– mice induced colitis when transferred alone to immune deficient mice [13]. From these experiments it could not be determined whether IL-10 was required for the differentiation or effector function of Treg cells, as it was possible that in mice deprived of IL-10 throughout ontogeny there was a developmental abnormality in Treg cells. To address this, CD45RBlow CD4+ cells were isolated from normal mice and transferred together with CD45RBhi CD4+ cells into SCID mice in the presence of a neutralising anti-IL-10 receptor mAb. Under these circumstances CD45RBlow CD4+ cells failed to inhibit colitis demonstrating that in the absence of IL-10, differentiated Treg cells still failed to function. Collectively these results suggest that IL-10 production by the Treg cells themselves is essential for their function. Consistent with these studies CD45RBhi CD4+ cells isolated from transgenic mice which expressed IL-10 under control of the IL-2 promoter failed to induce colitis in SCID mice and were able to inhibit the disease when transferred with CD45RBhi CD4+ cells from normal mice [14]. What precise role IL-10 plays in the immune suppressive properties of Treg cells in vivo is not known. Suppression of colitis by Treg cells is characterised by a significant reduction in the number of Th1 cells that accumulate in the intestine. It is not known whether this is a result of effects on T-cell expansion or migration. IL-10 is known to inhibit T-cell activation and cytokine secretion largely through its action on antigen-presenting cells (APCs), resulting in inhibition of the production of molecules such as IL-12 and B7 which are required for optimal T-cell costimulation. It is also able to inhibit a number of effector functions mediated by activated macrophages [15]. These properties of IL-10 make it likely that IL-10-secreting Treg cells act at several points in the inflammatory cascade 930

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both to impede T-cell activation as well as to prevent the release of inflammatory mediators and chemokines locally in the intestine. Recently mice have been generated in which macrophages and neutrophils were unable to respond to IL-10 as a result of a cell-type specific deletion of STAT-3 [16]. These mice were found to spontaneously develop colitis indicating that IL-10 ’deactivation’ of these cells contributes to the immune suppressive properties of this cytokine. In contrast no intestinal inflammation developed in mice in which only T cells were deficient in STAT-3, suggesting that a STAT-3-dependent response to IL-10 by T cells is not required for normal intestinal homeostasis. Activation of T cells in the presence of IL-10 led to the differentiation of a population of IL-10 and TGF-β-secreting T cells, termed T regulatory-1 (Tr-1) cells. These cells were able to inhibit T-cell activation in vitro and inhibit colitis when transferred into immunodeficient mice [17]. This raised the possibility that one mechanism by which Treg cells act in vivo is to induce the progeny of the CD45RBhi CD4+ population to differentiate into IL-10-secreting cells. However, this does not appear to be the case, as CD45RBlow CD4+ cells could effectively inhibit colitis induced by IL-10–/– CD45RBhi CD4+ cells whose progeny are unable to produce IL-10 [13]. Of course this does not rule out the possibility that IL-10 may induce the differentiation of T cells producing alternative anti-inflammatory cytokines, such as TGF-β.

3. The role of TGF-β The TGF-β family comprises a highly pleiotropic family of cytokines that have functional affects on a variety of cells in the immune system [18]. The importance of TGF-β1 in immune homeostasis is highlighted by the fact that TGF-β1-deficient mice die within 3–5 weeks after birth from severe multiple organ pathology [19]. TGF-β also appears to be essential for the control of intestinal inflammation as colitis developed in SCID mice restored with a mixture of CD45RBhi CD4+ and CD45RBlow CD4+ cells that were treated with a neutralising anti-TGF-β mAb [20]. TGF-β has also been shown to be involved in the function of a number of regulatory T-cell populations functional in other model systems. These include immune suppressive clones termed Th3 cells, generated after oral exposure to antigen [21] and Tr-1 cells [17] as well as naturally occurring Treg cell populations capable of inhibiting thyroiditis [22] and glomerular nephritis [23]. Whilst some Treg cell populations have been shown to produce TGF-β it remains to be established whether T cells are the important source of this cytokine in vivo. Elucidation of the mechanisms by which TGF-β is involved in the function of Treg cells is complicated by the fact that TGF-β receptors are expressed by a wide variety of cells. TGF-β has been shown to alter APC function favouring differentiation of Th2 cells [24, 25]. Peritoneal exudate cells cultured with TGF-β expressed lower levels of CD40 and IL-12 providing a possible explanation for this effect. TGF-β has also been shown to induce IL-10 secretion by APCs [26]. Consistent with this, intranasal Microbes and Infection 2001, 929-0

Control of intestinal inflammation by regulatory T cells

administration of a TGF-β1-containing plasmid led to the amelioration of TNBS-induced colitis by a mechanism that was dependent on IL-10 production [27]. TGF-β has also been shown to be able to suppress the proliferative capacity of naive T cells following activation, in addition to inducing a downregulation of IL-12Rβ2 expression on T cells [28]. The direct effects of TGF-β on T cells have recently been dissected using transgenic mice which express a T-cell-specific dominant negative form of the TGF-βII receptor [29, 30]. These mice had a significantly increased proportion of T cells capable of differentiating into effector cells and developed multiple organ inflammation and autoantibodies. These data dramatically illustrate the importance of T-cell-mediated TGF-βRII signalling in immune homeostasis. Collectively these studies suggest that TGF-β mediates its inhibitory effects on both T cells and APCs and may favour the development of IL-10secreting regulatory T cells.

4. Regulatory T cells which control intestinal inflammation are enriched within the CD25+ CD45RBlow CD4+ population The CD45RBlow CD4+ population is heterogeneous, containing antigen-experienced cells in addition to Treg cells. The ectoenzyme CD38 was found to be expressed by approximately 50% of the CD45RBlow CD4+ population but not by CD45RBhigh CD4+ cells, making it a useful tool with which to further subdivide the former [31]. This phenotypic heterogeneity was reflected functionally as CD38–CD45RBlow CD4+ cells were able to mount recall responses to antigen and to proliferate and produce cytokines in response to polyclonal stimulation, whereas CD38+ cells were unresponsive and resembled anergic T cells. The CD38+ population was not inert as coculture of these cells with normally reactive CD38– CD45RBlow CD4+ cells suppressed the response of the latter, indicating that CD38+ CD45RBlow CD4+ cells were able to mediate immune suppression in vitro. Functional analysis of the mechanism of immune suppression revealed that CD38+ CD45RBlow CD4+ cells had to be triggered via their TCR and that a cognate interaction between responding and regulatory population was required. In stark contrast to the suppression of colitis, immune suppression in vitro did not require IL-4, IL-10 or TGF-β. Similar functional properties were reported for CD4+ T cells which express the IL-2Rα chain, CD25 [32, 33]. Analysis of CD25 expression amongst CD38+ CD45RBlow CD4+ cells showed that 50% were positive and that the regulatory activity resided within this fraction (Read and Powrie, unpublished). Importantly, earlier studies had shown that regulatory T cells capable of inhibiting gastritis were contained within the CD25+ CD4+ subset, making this a useful marker for identifying cells capable of mediating suppression in vitro and in vivo [34, 35]. To assess the ability of the CD25– and CD25+ fractions of CD45RBlow CD4+ cells to inhibit colitis these subsets were transferred to SCID mice with an inoculum of Microbes and Infection 2001, 929-0

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CD45RBhi CD4+ T cells that would normally induce colitis. Whilst both fractions were able to inhibit development of colitis when transferred at high doses, only the CD25+ subset provided significant protection at lower doses (one regulatory:eight potentially pathogenic cells) [36]. These results suggest that regulatory T cells which control intestinal inflammation are enriched within the CD25+ CD45RBlow CD4+ subset. As the immune suppressive properties of these cells in vitro had been shown to be independent of IL-4, IL-10 and TGF-β we directly tested whether TGF-β was involved in the function of CD25+ cells in vivo. As described for unseparated CD45RBlow CD4+ cells, administration of an anti-TGF-β mAb completely reversed the ability of CD25+ CD45RBlow cells to inhibit colitis [36], indicating that TGF-β was also a crucial component in the function of these cells in vivo. There are several possible reasons for the difference between the in vitro and in vivo findings. Firstly, immune suppression in vitro may reflect only one facet of the effector function of Treg cells, with the immune suppressive cytokines required for either the growth or effector function of these cells in vivo. Alternatively, different functional subsets of cells may be involved in the two assays. It remains to be established whether IL-10 is involved in the function of CD25+ CD45RBlow CD4+ cells in vivo.

5. CTLA-4 is required for the function of Treg cells The fact that suppression of CD4+ proliferative responses by CD25+ CD4+ cells required cell–cell interactions between the responder and regulatory populations in vitro suggested that cell surface molecules may be involved in this interaction. Cytotoxic T-lymphocyteassociated antigen-4 (CTLA-4) is known to be a negative regulator of T-cell activation [37, 38] and recent evidence suggests that it plays a pivotal role in the homeostasis of the immune response. Targeted disruption of the CTLA-4 gene resulted in early lethality of CTLA-4–/– mice as a result of lymphoproliferative disorders and multiple organ inflammatory disease [39, 40]. Recently we have found that CTLA-4 is involved in the function of Treg cells as administration of an anti-CTLA-4 antibody completely abrogated the ability of CD25+ CD45RBlow CD4+ cells to inhibit colitis in SCID mice restored with CD45RBhi CD4+ cells [36]. Antibody treatment did not enhance the pathogenicity of the CD45RBhi population nor did it reveal pathogenic responses amongst the CD45RBlow population supporting the idea that disruption of CTLA-4-B7 interactions impairs the function of Treg cells. Constitutive expression of CTLA-4, comprising both intracellular and cell surface, was restricted primarily to CD25+ CD4+ cells suggesting that expression of CTLA-4 on Treg cells themselves was involved in their function. In accordance with these studies, administration of a combination of antiCD25 and anti-CTLA-4 mAbs led to development of gastritis in normal mice, presumably by inhibiting the function of CD25+ CD4+ Treg cells [41]. The function of CD25+ CD4+ cells in vitro was also found to involve CTLA-4 as blockade of CTLA-4-B7 interactions by addi 931

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tion of anti-CTLA-4 Fab fragment inhibited immune suppression. In these studies CD25+ CD4+ cells from normal mice were able to inhibit the response of CD4+ cells from CTLA-4–/– mice. Immune suppression was dependent on CTLA-4 providing definitive evidence that, in the in vitro assay at least, it is CTLA-4 expression on the Treg cells, as opposed to on the potentially responsive cells, that is important. Consistent with this, addition of normal bone marrow was able to prevent the development of lymphoproliferative disease induced by transfer of CTLA-4–/– bone marrow [42] suggesting that the immunosuppressive functions of CTLA-4 do not require cell autonomous expression. The finding that CTLA-4 is required for the function of Treg cells raises the possibility that some of the described immune suppressive properties of CTLA-4 may be attributable to its role in Treg cell function. Currently, little is known about precisely how CTLA-4 is involved. Recently it has been shown that cross-linking CTLA-4 in the presence of TCR ligation results in the production of TGF-β by murine CD4+ T cells [43]. This raises the possibility that signalling via CTLA-4 leads to TGF-β secretion by CD25+ CD4+ cells. The link between CTLA-4 and TGF-β is further highlighted by the striking similarities between the phenotypes of CTLA-4–/– and TGF-β–/– mice. In both cases mice develop severe lymphoproliferative disorders that lead to fatal multiple organ tissue destruction [19, 39, 40]. In this regard it would be of interest to determine whether CD25+ CD45RBlow CD4+ T cells from CTLA-4–/– mice could inhibit colitis. However, the fact that CTLA-4–/– mice die within 3–4 weeks and have highly abnormal T-cell subsets makes this experiment difficult to do and the results difficult to interpret.

6. Antigen specificity of Treg cells Intestinal inflammation did not develop after transfer of CD45RBhi CD4+ T cells to SCID mice raised under germfree conditions, indicating that the pathogenic T-cell response is driven by resident bacteria [44, 45]. Further support for this comes from studies using newborn mice as recipients of CD45RBhi CD4+ T cells [46]. A diverse bacterial flora in the intestine is known to occur only after weaning [47], which correlates with the finding that significant expansion of CD45RBhi CD4+ T cells did not occur until 3 weeks after transfer. This suggests that exogenous antigens (presumably bacterial) drive the T-cell expansion which occurs after transfer of T cells to immunedeficient mice in the absence of Treg cells. Analysis of the T-cell receptor beta (TCRβ) repertoire of T cells found in the intestine of colitic mice following transfer of CD45RBhi CD4+ cells reveals a T-cell population with restricted TCR diversity [48]. This would suggest that specific peptides presented by MHC class II molecules are driving the activation of these cells as opposed to the intestinal flora having a nonspecific effect on these cells. Exposure to bacterial antigens is not required for the function of Treg cells which inhibit intestinal inflammation as CD45RBlow CD4+ cells isolated from germ-free mice were able to inhibit colitis [46]. CD25+ CD4+ thymocytes 932

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were also able to inhibit disease (Read and Powrie, unpublished) suggesting that Treg cells may recognise self antigens as has been proposed for those that regulate autoimmune disease [49–51]. However, endogenous bacteria may have subtle effects on Treg cells as titration experiments revealed that CD45RBlow CD4+ cells from germfree mice were less potent on a cell per cell basis at inhibiting colitis than those from normal mice (Read and Powrie, unpublished). Precisely how the intestinal flora influences the function of Treg cells is not known. It may be that the presence of a normal flora provides inflammatory signals, which drive the expansion of Treg cells. One group of investigators have consistently reported that CD45RBlow CD4+ cells failed to inhibit colitis in SCID mice restored with CD45RBhi CD4+ cells [52]. Given the fact that the presence of a normal flora enhances the potency of Treg cells it is possible that differences in the ability of CD45RBlow CD4+ cells to inhibit disease may be attributable to differences in the environmental status of the donor mice.

7. A model for the development of intestinal inflammation and its regulation Taking into account all of the available data, a likely scenario is that bacterial antigens are transported to the mesenteric lymph nodes by migrating dendritic cells (DCs). Presentation to T cells in the presence of IL-12 and in the absence of Treg cells results in the development of Th1 cells which home to the intestine. Here they encounter antigen again and mount an inflammatory response that leads to the recruitment of immature DCs from the blood which are able to amplify the response. Consistent with this hypothesis blockade of CD40–CD40L interactions by administration of anti-CD40L antibody prevented colitis in SCID mice reconstituted with CD45RBhi cells [53]. Whether this interaction is important for T-cell priming and expansion in the mesenteric lymph nodes (MLNs) or for the activation of macrophages and DCs in the intestine or both is not known. Recently we have found that colitis is accompanied by an accumulation of activated DCs in the MLNs. These cells were found to express the costimulatory ligand CD134L (Malmstrom et al., unpublished). This was found to be functionally significant as administration of an anti-CD134L mAb inhibited colitis. Importantly, cotransfer of Treg cells inhibited the accumulation of activated DCs suggesting that modulation of DC function is one mechanism by which Treg cells mediate their immune suppressive function. Whether this is an effect on the migration, activation or life-span of the DC population remains to be established. Whilst it has been clearly established that IL-10 and TGF-β are involved in the function of Treg cells, at what point in the inflammatory cascade they work is not known (figure 1). IL-12 is a pivotal cytokine in the development of Th1 responses and the ability of IL-10 to inhibit IL-12 production by DCs and macrophages may explain its ability to inhibit colitis. This may tip the balance in favour of the generation of TGF-βproducing cells that further inhibit the inflammatory Microbes and Infection 2001, 929-0

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References

Figure 1. At what point in the inflammatory cascade do Treg cells work? Under inflammatory conditions, migrating DCs transport bacteria into the MLNs. Presentation of bacterial peptides to naive T cells in the presence of IL-12 results in the generation of Th1 effector cells that migrate into the intestine to mediate an inflammatory response. A number of mechanisms have been postulated for how Treg cells suppress such a response, none of which are mutually exclusive. 1) Interaction of Treg cells with DCs following priming may impair the costimulatory capacity of DCs via inhibition of costimulatory molecule expression. Alternatively, 2) T reg cells may interfere with the proliferative and homing capacity of activated T cells, preventing the accumulation of pathogenic T cells in the intestine. Or 3) anti-inflammatory cytokines produced by Treg cells in the intestine may prevent the release of chemokines and pro-inflammatory cytokines by macrophages inhibiting the progression of the inflammatory response. response. In support of this, administration of an anti-IL-12 mAb to OVA-TCR transgenic mice led to elevations in TGF-β production in both the spleen and Peyer’s patches and enhanced oral tolerance [54].

8. Heterogeneity amongst Treg cell populations? As documented in this Forum there is now indisputable evidence that CD4+ Treg cells are capable of preventing pathogenic inflammatory responses towards both tissuespecific and bacterial antigens [51, 55]. Comparison of the functional properties of these cells has revealed some similarities and some differences. For example whilst CTLA-4 was involved in the function of Treg cells which could inhibit gastritis and those which inhibited colitis, IL-10 only appeared to be involved in the function of the latter population. This may mean that different effector functions are required depending on the nature of the stimulus. The requirement for IL-10 may be specific to the regulation of bacteria-driven responses that most likely lead to enhanced activation of cells of the innate immune system compared to tissue-specific self antigens. Alternatively, these differences may reflect distinct functional subsets of Treg cells. Further experiments are required to: (i) establish whether TGF-β is involved in the prevention of gastritis transferred by CD25+ CD4+ cells; and (ii) elucidate the relationship between these cells and in-vitroderived Tr1 and Th3 cells. Microbes and Infection 2001, 929-0

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