Naturally arising CD4+CD25+ regulatory T cells suppress the expansion of colitogenic CD4+CD44highCD62L- effector memory T cells

Naturally arising CD4+CD25+ regulatory T (T(R)) cells have been shown to prevent and cure murine T cell-mediated colitis. However, their exact mechanism of controlling colitogenic memory CD4+ T cells in in vivo systems excluding the initial process of naive T cell activation and differentiation has not been examined to date. Using the colitogenic effector memory (T(EM)) CD4+ cell-mediated colitis model induced by adoptive transfer of colitogenic CD4+CD44(high)CD62L(-) lamina propria (LP) T cells obtained from colitic CD4+CD45RB(high) T cell-transferred mice, we have shown in the present study that CD4+CD25+ T(R) cells are able not only to suppress the development of colitis, Th1 cytokine production, and the expansion of colitogenic LP CD4+ T(EM) cells but also to expand these cells by themselves extensively in vivo. An in vitro coculture assay revealed that CD4+CD25+ T(R) cells proliferated in the presence of IL-2-producing colitogenic LP CD4+ T(EM) cells at the early time point (48 h after culture), followed by the acquisition of suppressive activity at the late time point (96 h after culture). Collectively, these data suggest the distinct timing of the IL-2-dependent expansion of CD4+CD25+ T(R) cells and the their suppressive activity on colitogenic LP CD4+ T(EM) cells.     

MyD88-dependent pathway in T cells directly modulates the expansion of colitogenic CD4+ T cells in chronic colitis

TLRs that mediate the recognition of pathogen-associated molecular patterns are widely expressed on/in cells of the innate immune system. However, recent findings demonstrate that certain TLRs are also expressed in conventional TCRalphabeta(+) T cells that are critically involved in the acquired immune system, suggesting that TLR ligands can directly modulate T cell function in addition to various innate immune cells. In this study, we report that in a murine model of chronic colitis induced in RAG-2(-/-) mice by adoptive transfer of CD4(+)CD45RB(high) T cells, both CD4(+)CD45RB(high) donor cells and the expanding colitogenic lamina propria CD4(+)CD44(high) memory cells expresses a wide variety of TLRs along with MyD88, a key adaptor molecule required for signal transduction through TLRs. Although RAG-2(-/-) mice transferred with MyD88(-/-)CD4(+)CD45RB(high) cells developed colitis, the severity was reduced with the delayed kinetics of clinical course, and the expansion of colitogenic CD4(+) T cells was significantly impaired as compared with control mice transferred with MyD88(+/+)CD4(+)CD45RB(high) cells. When RAG-2(-/-) mice were transferred with the same number of MyD88(+/+) (Ly5.1(+)) and MyD88(-/-) (Ly5.2(+)) CD4(+)CD45RB(high) cells, MyD88(-/-)CD4(+) T cells showed significantly lower proliferative responses assessed by in vivo CFSE division assay, and also lower expression of antiapoptotic Bcl-2/Bcl-x(L) molecules and less production of IFN-gamma and IL-17, compared with the paired MyD88(+/+)CD4(+) T cells. Collectively, the MyD88-dependent pathway that controls TLR signaling in T cells may directly promote the proliferation and survival of colitogenic CD4(+) T cells to sustain chronic colitis.     

IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo

We present evidence that donor-reactive CD4(+) T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4(+) T cells contained within the CD45RB(high) fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RB(low) CD4(+) and CD25(+)CD4(+) populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RB(high) CD4(+) T cells. Analysis of the mechanism of immunoregulation transferred by CD45RB(low) CD4(+) T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RB(low) CD4(+) T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RB(high) CD4(+) T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RB(low) CD4(+) T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.     

The development of colitogenic CD4(+) T cells is regulated by IL-7 in collaboration with NK cell function in a murine model of colitis

We previously reported that IL-7(-/-)RAG(-/-) mice receiving naive T cells failed to induce colitis. Such abrogation of colitis may be associated with not only incomplete T cell maintenance due to the lack of IL-7, but also with the induction of colitogenic CD4(+) T cell apoptosis at an early stage of colitis development. Moreover, NK cells may be associated with the suppression of pathogenic T cells in vivo, and they may induce apoptosis of CD4(+) T cells. To further investigate these roles of NK cells, RAG(-/-) and IL-7(-/-)RAG(-/-) mice that had received naive T cells were depleted of NK cells using anti-asialo GM1 and anti-NK1.1 Abs. NK cell depletion at an early stage, but not at a later stage during colitogenic effector memory T cell (T(EM)) development, resulted in exacerbated colitis in recipient mice even in the absence of IL-7. Increased CD44(+)CD62L(-) T(EM) and unique CD44(-)CD62L(-) T cell subsets were observed in the T cell-reconstituted RAG(-/-) recipients when NK cells were depleted, although Fas, DR5, and IL-7R expressions in this subset differed from those in the CD44(+)CD62L(-) T(EM) subset. NK cell characteristics were the same in the presence or absence of IL-7 in vitro and in vivo. These results suggest that NK cells suppress colitis severity in T cell-reconstituted RAG(-/-) and IL-7(-/-)RAG(-/-) recipient mice through targeting of colitogenic CD4(+)CD44(+)CD62L(-) T(EM) and, possibly, of the newly observed CD4(+)CD44(-)CD62L(-) subset present at the early stage of T cell development.     

IL-7 Is essential for the development and the persistence of chronic colitis

Although IL-7 has recently emerged as a key cytokine involved in controlling the homeostatic turnover and the survival of peripheral resting memory CD4(+) T cells, its potential to be sustained pathogenic CD4(+) T cells in chronic immune diseases, such as inflammatory bowel diseases, still remains unclear. In this study, we demonstrate that IL-7 is essential for the development and the persistence of chronic colitis induced by adoptive transfer of normal CD4(+)CD45RB(high) T cells or colitogenic lamina propria (LP) CD4(+) memory T cells into immunodeficient IL-7(+/+) x RAG-1(-/-) and IL-7(-/-) x RAG-1(-/-) mice. Although IL-7(+/+) x RAG-1(-/-) recipients transferred with CD4(+)CD45RB(high) splenocytes developed massive inflammation of the large intestinal mucosa concurrent with massive expansion of Th1 cells, IL-7(-/-) x RAG-1(-/-) recipients did not. Furthermore, IL-7(-/-) x RAG-1(-/-), but not IL-7(+/+) x RAG-1(-/-), mice transferred with LP CD4(+)CD44(high)CD62L(-)IL-7Ralpha(high) effector-memory T cells (T(EM)) isolated from colitic CD4(+)CD45RB(high)-transferred mice did not develop colitis. Although rapid proliferation of transferred colitogenic LP CD4(+) T(EM) cells was observed in the in IL-7(-/-) x RAG-1(-/-) mice to a similar extent of those in IL-7(+/+) x RAG-1(-/-) mice, Bcl-2 expression was significantly down-modulated in the transferred CD4(+) T cells in IL-7(-/-) x RAG-1(-/-) mice compared with those in IL-7(+/+) x RAG-1(-/-) mice. Taken together, IL-7 is essential for the development and the persistence of chronic colitis as a critical survival factor for colitogenic CD4(+) T(EM) cells, suggesting that therapeutic approaches targeting IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases.     

Expression of dual TCR on DO11.10 T cells allows for ovalbumin-induced oral tolerance to prevent T cell-mediated colitis directed against unrelated enteric bacterial antigens

The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora. Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4(+) T cells (Treg) cells. We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice. SCID-bg mice given DO11.10 CD4(+)CD45RB(high) T cells developed colitis, and cotransferring DO11.10 CD45RB(low)CD4(+) T cells prevented CD4(+)CD45RB(high) T cell-induced colitis in the absence of OVA. The induction and prevention of disease by DO11.10 CD4(+) T cell subsets were associated with an increase in endogenous TCRalpha chain expression on Tg T cells. Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4(+)CD45RB(high) attenuated the colitis in association with increased TGF-beta and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag. OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RB(high) T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells. The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag. Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells. Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.     

Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells

CD4(+)CD25(+) regulatory T cells have been shown to prevent T cell-mediated immune pathology; however, their ability to ameliorate established inflammation has not been tested. Using the CD4(+)CD45RB(high) T cell transfer model of inflammatory bowel disease, we show that CD4(+)CD25(+) but not CD4(+)CD25(-)CD45RB(low) T cells are able to cure intestinal inflammation. Transfer of CD4(+)CD25(+) T cells into mice with colitis led to resolution of the lamina propria infiltrate in the intestine and reappearance of normal intestinal architecture. CD4(+)CD25(+) T cells were found to proliferate in the mesenteric lymph nodes and inflamed colon. They were located between clusters of CD11c(+) cells and pathogenic T cells and found to be in contact with both cell types. These studies suggest that manipulation of CD4(+)CD25(+) T cells may be beneficial in the treatment of chronic inflammatory diseases.     

CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of IL-10

The mechanisms by which the immune system achieves constant T cell numbers throughout life, thereby controlling autoaggressive cell expansions, are to date not completely understood. Here, we show that the CD25(+) subpopulation of naturally activated (CD45RB(low)) CD4 T cells, but not CD25(-) CD45RB(low) CD4 T cells, inhibits the accumulation of cotransferred CD45RB(high) CD4 T cells in lymphocyte-deficient mice. However, both CD25(+) and CD25(-) CD45RB(low) CD4 T cell subpopulations contain regulatory cells, since they can prevent naive CD4 T cell-induced wasting disease. In the absence of a correlation between disease and the number of recovered CD4(+) cells, we conclude that expansion control and disease prevention are largely independent processes. CD25(+) CD45RB(low) CD4 T cells from IL-10-deficient mice do not protect from disease. They accumulate to a higher cell number and cannot prevent the expansion of CD45RB(high) CD4 T cells upon transfer compared with their wild-type counterparts. Although CD25(+) CD45RB(low) CD4 T cells are capable of expanding when transferred in vivo, they reach a homeostatic equilibrium at lower cell numbers than CD25(-) CD45RB(low) or CD45RB(high) CD4 T cells. We conclude that CD25(+) CD45RB(low) CD4 T cells from nonmanipulated mice control the number of peripheral CD4 T cells through a mechanism involving the production of IL-10 by regulatory T cells.     

Cutting edge: Critical role for A2A adenosine receptors in the T cell-mediated regulation of colitis

A(2A) adenosine receptors (A(2A)AR) inhibit inflammation, although the mechanisms through which adenosine exerts its effects remain unclear. Although the transfer of regulatory Th cells blocks colitis induced by pathogenic CD45RB(high) Th cells, we show that CD45RB(low) or CD25+ Th cells from A(2A)AR-deficient mice do not prevent disease. Moreover, CD45RB(high) Th cells from A(2A)AR-deficient mice were not suppressed by control CD45RB(low) Th cells. A(2A)AR agonists suppressed the production of proinflammatory cytokines by CD45RB(high) and CD45RB(low) T cells in association with a loss of mRNA stability. In contrast, anti-inflammatory cytokines, including IL-10 and TGF-beta, were minimally affected. Oral administration of the A(2A)AR agonist ATL313 attenuated disease in mice receiving CD45RB(high) Th cells. These data suggest that A(2A)AR play a novel role in the control of T cell-mediated colitis by suppressing the expression of proinflammatory cytokines while sparing anti-inflammatory activity mediated by IL-10 and TGF-beta.     

The central memory CD4+ T cell population generated during Leishmania major infection requires IL-12 to produce IFN-gamma

Central memory CD4(+) T cells provide a pool of lymph node-homing, Ag-experienced cells that are capable of responding rapidly after a secondary infection. We have previously described a population of central memory CD4(+) T cells in Leishmania major-infected mice that were capable of mediating immunity to a secondary infection. In this study, we show that the Leishmania-specific central memory CD4(+) T cells require IL-12 to produce IFN-gamma, demonstrating that this population needs additional signals to develop into Th1 cells. In contrast, effector cells isolated from immune mice produced IFN-gamma in vitro or in vivo in the absence of IL-12. In addition, we found that when central memory CD4(+) T cells were adoptively transferred into IL-12-deficient hosts, many of the cells became IL-4 producers. These studies indicate that the central memory CD4(+) T cell population generated during L. major infection is capable of developing into either Th1 or Th2 effectors. Thus, continued IL-12 production may be required to ensure the development of Th1 cells from this central memory T cell pool, a finding that has direct relevance to the design of vaccines dependent upon central memory CD4(+) T cells.     

Upregulated IL-7 receptor α expression on colitogenic memory CD4+ T cells may participate in the development and persistence of chronic colitis

We have previously demonstrated that IL-7 is essential for the persistence of colitis as a survival factor of colitogenic IL-7Rα-expressing memory CD4(+) T cells. Because IL-7Rα is broadly expressed on various immune cells, it is possible that the persistence of colitogenic CD4(+) T cells is affected by other IL-7Rα-expressing non-T cells. To test this hypothesis, we conducted two adoptive transfer colitis experiments using IL-7Rα(-/-) CD4(+)CD25(-) donor cells and IL-7Rα(-/-) × RAG-2(-/-) recipient mice, respectively. First, IL-7Rα expression on colitic lamina propria (LP) CD4(+) T cells was significantly higher than on normal LP CD4(+) T cells, whereas expression on other colitic LP immune cells, (e.g., NK cells, macrophages, myeloid dendritic cells) was conversely lower than that of paired LP cells in normal mice, resulting in predominantly higher expression of IL-7Rα on colitogenic LP CD4(+) cells, which allows them to exclusively use IL-7. Furthermore, RAG-2(-/-) mice transferred with IL-7Rα(-/-) CD4(+)CD25(-) T cells did not develop colitis, although LP CD4(+) T cells from mice transferred with IL-7Rα(-/-) CD4(+)CD25(-) T cells were differentiated to CD4(+)CD44(high)CD62L(-) effector-memory T cells. Finally, IL-7Rα(-/-) × RAG-2(-/-) mice transferred with CD4(+)CD25(-) T cells developed colitis similar to RAG-2(-/-) mice transferred with CD4(+)CD25(-) T cells. These results suggest that IL-7Rα expression on colitogenic CD4(+) T cells, but not on other cells, is essential for the development of chronic colitis. Therefore, therapeutic approaches targeting the IL-7/IL-7R signaling pathway in colitogenic CD4(+) T cells may be feasible for the treatment of inflammatory bowel diseases.     

Regulation of murine inflammatory bowel disease by CD25+ and CD25- CD4+ glucocorticoid-induced TNF receptor family-related gene+ regulatory T cells

CD4(+)CD25(+) regulatory T cells in normal animals are engaged in the maintenance of immunological self-tolerance and prevention of autoimmune disease. However, accumulating evidence suggests that a fraction of the peripheral CD4(+)CD25(-) T cell population also possesses regulatory activity in vivo. Recently, it has been shown glucocorticoid-induced TNFR family-related gene (GITR) is predominantly expressed on CD4(+)CD25(+) regulatory T cells. In this study, we show evidence that CD4(+)GITR(+) T cells, regardless of the CD25 expression, regulate the mucosal immune responses and intestinal inflammation. SCID mice restored with the CD4(+)GITR(-) T cell population developed wasting disease and severe chronic colitis. Cotransfer of CD4(+)GITR(+) population prevented the development of CD4(+)CD45RB(high) T cell-transferred colitis. Administration of anti-GITR mAb-induced chronic colitis in mice restored both CD45RB(high) and CD45RB(low) CD4(+) T cells. Interestingly, both CD4(+)CD25(+) and CD4(+)CD25(-) GITR(+) T cells prevented wasting disease and colitis. Furthermore, in vitro studies revealed that CD4(+)CD25(-)GITR(+) T cells as well as CD4(+)CD25(+)GITR(+) T cells expressed CTLA-4 intracellularly, showed anergic, suppressed T cell proliferation, and produced IL-10 and TGF-beta. These data suggest that GITR can be used as a specific marker for regulatory T cells controlling mucosal inflammation and also as a target for treatment of inflammatory bowel disease.     

IL-4 exacerbates disease in a Th1 cell transfer model of colitis

IL-4 is associated with Th2-type immune responses and can either inhibit or, in some cases, promote Th1-type responses. We tested the effect of IL-4 treatment on the development of inflammation in the CD4(+)CD45RB(high) T cell transfer model of colitis, which has been characterized as a Th1-dependent disease. IL-4 treatment significantly accelerated the development of colitis in immunodeficient recipients (recombinase-activating gene-2 (Rag2)(-/-)) of CD4(+)CD45RB(high) T cells. Quantitative analysis of mRNA expression in the colons of IL-4-treated mice showed an up-regulation of both Th1- and Th2-associated molecules, including IFN-gamma, IP-10, MIG, CXCR3, chemokine receptor-8, and IL-4. However, cotreatment with either IL-10 or anti-IL-12 mAb effectively blocked the development of colitis in the presence of exogenous IL-4. These data indicate that IL-4 treatment exacerbates a Th1-mediated disease rather than induces Th2-mediated inflammation. As other cell types besides T cells express the receptor for IL-4, the proinflammatory effects of IL-4 on host cells in Rag2(-/-) recipients were assessed. IL-4 treatment was able to moderately exacerbate colitis in Rag2(-/-) mice that were reconstituted with IL-4Ralpha-deficient (IL-4Ralpha(-/-)) CD4(+)CD45RB(high) T cells, suggesting that the IL-4 has proinflammatory effects on both non-T and T cells in this model. IL-4 did not cause colitis in Rag2(-/-) mice in the absence of T cells, but did induce an increase in MHC class II expression in the lamina propria of the colon, which was blocked by cotreatment with IL-10. Together these results indicate that IL-4 can indirectly promote Th1-type inflammation in the CD4(+)CD45RB(high) T cell transfer model of colitis.     

Cutting edge: Generation of colitogenic Th17 CD4 T cells is enhanced by IL-17+ γδ T cells

Th 17 cells have been implicated in the pathogenesis of colitis; however, a cellular mechanism by which colitogenic Th17 immunity arises in vivo remains unclear. In this study, we report that a subset of IL-17(+) γδ T cells plays a crucial role in enhancing in vivo Th17 differentiation and T cell-mediated colitis. TCRβ(-/-) mice were highly susceptible to T cell-mediated colitis, whereas TCRβδ(-/-) mice were resistant to the disease. Importantly, cotransfer of IL-17(+) but not of IL-17(-) γδ T cells with CD4 T cells was sufficient to enhance Th17 differentiation and induce full-blown colitis in TCRβδ(-/-) recipients. Collectively, our results provide a novel function of IL-17(+) γδ T cell subsets in supporting in vivo Th17 differentiation and possibly in fostering the development of intestinal inflammation.     

CD4+CD25+ regulatory T cells cure murine colitis: the role of IL-10, TGF-beta, and CTLA4

Regulatory T cells are critical in regulating the immune response, and therefore play an important role in the defense against infection and control of autoimmune diseases. However, a therapeutic role of regulatory T cells in an established disease has not been fully established. In this study, we provide direct evidence that CD4(+)CD25(+) regulatory T cells can cure an established, severe, and progressive colitis. SCID mice developed severe colitis when adoptively transferred with naive CD4(+)CD25(-) T cells and infected with the protozoan parasite Leishmania major. The disease development can be completely halted and symptoms reversed, with a healthy outcome, by transferring freshly isolated or activated CD4(+)CD25(+) T cells from syngeneic donors. The therapeutic effect of the regulatory T cells was completely blocked by treatment of the recipients with anti-IL-10R, anti-CTLA4, or anti-TGF-beta Ab. However, the resurgence of colitis under these treatments was not accompanied by the reactivation of Th1 or Th2 response nor was it correlated to the parasite load. These results therefore demonstrate that CD4(+)CD25(+) T cells are therapeutic and that the effect is mediated by both IL-10/TGF-beta-dependent and independent mechanisms. Furthermore, colitis can manifest independent of Th1 and Th2 responses.     

Cutting edge: CD4+CD25+ regulatory T cells impaired for intestinal homing can prevent colitis

Transfer of CD4(+)CD45RB(high) T cells into RAG(-/-) mice causes colitis, which can be prevented by CD4(+)CD25(+) regulatory T cells (Treg). Colitis induction by CD4(+)CD45RB(high) T cells requires beta(7) integrin-dependent intestinal localization, but the importance of beta(7) integrins for Treg function is unknown. In this study, we show that beta(7)(-/-) Treg were effective in preventing colitis. Treg expanded in vivo to the same extent as CD4(+)CD45RB(high) T cells after transfer and they did not inhibit CD4(+)CD45RB(high) T cell expansion in lymphoid tissues, although they prevented the accumulation of Th1 effector cells in the intestine. beta(7)(-/-) Treg were significantly reduced in the large intestine, however, compared with wild-type Treg, and regulatory activity could not be recovered from the intestine of recipients of beta(7)(-/-) Treg. These data demonstrate that Treg can prevent colitis by inhibiting the accumulation of tissue-seeking effector cells and that Treg accumulation in the intestine is dispensable for colitis suppression.