Antigen-driven interactions with dendritic cells and expansion of Foxp3+ regulatory T cells occur in the absence of inflammatory signals

Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.     

Cutting edge: CD47 controls the in vivo proliferation and homeostasis of peripheral CD4+ CD25+ Foxp3+ regulatory T cells that express CD103

Peripheral CD103(+)Foxp3(+) regulatory T cells (Tregs) can develop both from conventional naive T cells upon cognate Ag delivery under tolerogenic conditions and from thymic-derived, expanded/differentiated natural Tregs. We here show that CD47 expression, a marker of self on hematopoietic cells, selectively regulated CD103(+)Foxp3(+) Treg homeostasis at the steady state. First, the proportion of effector/memory-like (CD44(high)CD62L(low)) CD103(+)Foxp3(+) Tregs rapidly augmented with age in CD47-deficient mice (CD47(-/-)) as compared with age-matched control littermates. Yet, the percentage of quiescent (CD44(low)CD62L(high)) CD103(-)Foxp3(+) Tregs remained stable. Second, the increased proliferation rate (BrdU incorporation) observed within the CD47(-/-)Foxp3(+) Treg subpopulation was restricted to those Tregs expressing CD103. Third, CD47(-/-) Tregs maintained a normal suppressive function in vitro and in vivo and their increased proportion in old mice led to a decline of Ag-specific T cell responses. Thus, sustained CD47 expression throughout life is critical to avoid an excessive expansion of CD103(+) Tregs that may overwhelmingly inhibit Ag-specific T cell responses.     

Regulatory CD4+CD25+Foxp3+ T cells selectively inhibit the spontaneous form of lymphopenia-induced proliferation of naive T cells

Regulatory CD4(+)CD25(+)Foxp3(+) T cells play a critical role in controlling autoimmunity and T cell homeostasis. However, their role in regulation of lymphopenia-induced proliferation (LIP), a potential mechanism for generation of autoaggressive T cells, has been poorly defined. Currently, two forms of LIP are recognized: spontaneous and homeostatic. Spontaneous LIP is characterized by fast, burst-like cell-cycle activity, and may allow effector T cell differentiation. Homeostatic LIP is characterized by slow and steady cell cycle activity and is not associated with the acquisition of an effector phenotype. In this study, we demonstrate that CD4(+)CD25(+)Foxp3(+) T cells suppress the spontaneous, but not homeostatic, LIP of naive CD8 and CD4 T cells. However, selective inhibition of spontaneous LIP does not fully explain the tolerogenic role of Tregs in lymphopenia-associated autoimmunity. We show here that suppression of LIP in the lymphoid tissues is independent of Treg-derived IL-10. However, IL-10-deficient Tregs are partially defective in their ability to prevent colitis caused by adoptive transfer of CD4 T cells into RAG(-/-) mice. We propose that Tregs may inhibit emergence of effector T cells during the inductive phase of the immune response in the secondary lymphoid tissues by IL-10-independent mechanisms. In contrast, Treg-mediated inhibition of established effector T cells does require IL-10. Both Treg functions appear to be important in control of lymphopenia-associated autoimmunity.     

G protein-coupled receptor 83 overexpression in naive CD4+CD25- T cells leads to the induction of Foxp3+ regulatory T cells in vivo

Foxp3 functions as a lineage specification factor for the development of naturally occurring thymus-derived CD4+CD25+ regulatory T (Treg) cells. Recent evidence suggests that naive Foxp3-CD4+CD25- T cells can be converted in the periphery into Foxp3+ Treg cells. In this study, we have identified the G protein-coupled receptor (GPR)83 to be selectively up-regulated by CD4+CD25+ Treg cells of both murine and human origin in contrast to naive CD4+CD25- or recently activated T cells. Furthermore, GPR83 was induced upon overexpression of Foxp3 in naive CD4+CD25- T cells. Transduction of naive CD4+CD25- T cells with GPR83-encoding retroviruses did not confer in vitro suppressive activity. Nevertheless, GPR83-transduced T cells were able to inhibit the effector phase of a severe contact hypersensitivity reaction of the skin, indicating that GPR83 itself or GPR83-mediated signals conferred suppressive activity to conventional CD4+ T cells in vivo. Most strikingly, this in vivo acquisition of suppressive activity was associated with the induction of Foxp3 expression in GPR83-transduced CD4+ T cells under inflammatory conditions. Our results suggest that GPR83 might be critically involved in the peripheral generation of Foxp3+ Treg cells in vivo.     

Agonist-driven development of CD4+CD25+Foxp3+ regulatory T cells requires a second signal mediated by Stat6

The factors that induce Foxp3 expression and regulatory T (Treg) cell development remain unknown. In this study, we investigated the role of STAT4 and STAT6 in agonist-driven generation of Ag-specific Foxp3-expressing Treg cells. Our findings indicate that fully efficient induction of Foxp3 expression and development of Ag-specific Treg cells requires the synergistic action of two signals: a TCR-mediated signal and a second signal mediated by STAT6. Indeed, by comparing the development of wild-type and STAT4- and STAT6-deficient hemagglutinin-specific T cells in the presence of hemagglutinin Ag, we found that the absence of STAT6 impaired the generation of Ag-specific CD4+CD25+Foxp3+ cells. Moreover, in transgenic mice expressing a constitutively active form of STAT6, we found that the fraction of CD4+Foxp3+ cells exceeds that of control wild-type littermates. Overall these findings support a role for the STAT6 pathway in Treg cell development and maintenance.     

CTLA-4 and CD4+ CD25+ regulatory T cells inhibit protective immunity to filarial parasites in vivo

The T cell coinhibitory receptor CTLA-4 has been implicated in the down-regulation of T cell function that is a quintessential feature of chronic human filarial infections. In a laboratory model of filariasis, Litomosoides sigmodontis infection of susceptible BALB/c mice, we have previously shown that susceptibility is linked both to a CD4+ CD25+ regulatory T (Treg) cell response, and to the development of hyporesponsive CD4+ T cells at the infection site, the pleural cavity. We now provide evidence that L. sigmodontis infection drives the proliferation and activation of CD4+ Foxp3+ Treg cells in vivo, demonstrated by increased uptake of BrdU and increased expression of CTLA-4, Foxp3, GITR, and CD25 compared with naive controls. The greatest increases in CTLA-4 expression were, however, seen in the CD4+ Foxp3- effector T cell population which contained 78% of all CD4+ CTLA-4+ cells in the pleural cavity. Depletion of CD25+ cells from the pleural CD4+ T cell population did not increase their Ag-specific proliferative response in vitro, suggesting that their hyporesponsive phenotype is not directly mediated by CD4+ CD25+ Treg cells. Once infection had established, killing of adult parasites could be enhanced by neutralization of CTLA-4 in vivo, but only if performed in combination with the depletion of CD25+ Treg cells. This work suggests that during filarial infection CTLA-4 coinhibition and CD4+ CD25+ Treg cells form complementary components of immune regulation that inhibit protective immunity in vivo.     

Cyclophosphamide-induced type-1 diabetes in the NOD mouse is associated with a reduction of CD4+CD25+Foxp3+ regulatory T cells

Regulatory T cells (Tregs) have been implicated as key players in immune tolerance as well as suppression of antitumor responses. The chemotherapeutic alkylating agent cyclophosphamide (CY) is widely used in the treatment of tumors and some autoimmune conditions. Although previous data has demonstrated that Tregs may be preferentially affected by CY, its relevance in promoting autoimmune conditions has not been addressed. The nonobese diabetic mouse spontaneously develops type-1 diabetes (T1D). We demonstrate in this study that CY targets CD4+CD25+Foxp3+ Tregs in vivo. CD4+CD25+ T cells isolated from CY-treated mice display reduced suppressive activity in vitro and increased expression of apoptotic markers. Although Treg numbers rapidly recovered to pretreatment levels in the peripheral lymphoid tissues, Tregs failed to recover proportionally within pancreatic infiltrates. T1D progression was effectively prevented by adoptive transfer of a small number of islet Ag-specific CD4+CD25+ Tregs to CY-treated recipients. Prevention of T1D was associated with reduced T cell activation and higher Treg proportions in the pancreas. We conclude that acceleration of T1D by CY is associated with a reduction in CD4+CD25+Foxp3+ Tregs and can be prevented by transfer of CD4+CD25+ Tregs.     

Inducible CD4+LAP+Foxp3- regulatory T cells suppress allergic inflammation

Regulatory T cells (Tregs) play a critical role in the maintenance of airway tolerance. We report that inhaled soluble Ag induces adaptive Foxp3(+) Tregs, as well as a regulatory population of CD4(+) T cells in the lungs and lung-draining lymph nodes that express latency-associated peptide (LAP) on their cell surface but do not express Foxp3. Blocking the cytokine IL-10 or TGF-β prevented the generation of LAP(+) Tregs and Foxp3(+) Tregs in vivo, and the LAP(+) Tregs could also be generated concomitantly with Foxp3(+) Tregs in vitro by culturing naive CD4(+) T cells with Ag and exogenous TGF-β. The LAP(+) Tregs strongly suppressed naive CD4(+) T cell proliferation, and transfer of sorted OVA-specific LAP(+) Tregs in vivo inhibited allergic eosinophilia and Th2 cytokine expression in the lung, either when present at the time of Th2 sensitization or when injected after Th2 cells were formed. Furthermore, inflammatory innate stimuli from house dust mite extract, nucleotide-binding oligomerization domain containing 2 ligand, and LPS, which are sufficient for blocking airway tolerance, strongly decreased the induction of LAP(+) Tregs. Taken together, we concluded that inducible Ag-specific LAP(+) Tregs can suppress asthmatic lung inflammation and constitute a mediator of airway tolerance together with Foxp3(+) Tregs.     

The IL-4 receptor alpha-chain-binding cytokines, IL-4 and IL-13, induce forkhead box P3-expressing CD25+CD4+ regulatory T cells from CD25-CD4+ precursors

The mechanisms underlying the extrathymic generation of CD25+CD4 regulatory T cells (Tregs) are largely unknown. In this study the IL-4R alpha-chain-binding cytokines, IL-4 and IL-13, were identified as inducers of CD25+ Tregs from peripheral CD25-CD4 naive T cells. IL-4-induced CD25+ Tregs phenotypically and functionally resemble naturally occurring Tregs in that they are anergic to mitogenic stimulation, inhibit the proliferation of autologous responder T cells, express high levels of the Forkhead box P3 and the surface receptors glucocorticoid-induced TNFR family-related protein and CTLA-4, and inhibit effector T cells in a contact-dependent, but cytokine-independent, manner. The IL-4-induced generation of peripheral Tregs was independent of the presence of TGF-beta or IL-10, but was dependent on Ag-specific stimulation and B7 costimulation. The significance of the IL-4Ralpha-binding cytokines in the generation of Ag-specific Tregs was emphasized in a mouse model of oral tolerance, in which neutralization of IL-4 and IL-13 in mice transgenic for the TCR specific for OVA completely inhibited the expansion of OVA-specific Tregs that can be induced in untreated mice by feeding the nominal Ag. Together, our results demonstrate that IL-4 and IL-13 play an important role in generating Forkhead box P3-expressing CD25+ Tregs extrathymically in an Ag-dependent manner and therefore provide an intriguing link between the well-established immunoregulatory capacity of Th2 cells and the powerful CD25+ Treg population. Moreover, our findings might provide the basis for the design of novel therapeutic approaches for targeted immunotherapy with Tregs to known Ags in autoimmune diseases or graft-vs-host reactions.     

CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells

Foxp3(+)CD25(+)CD4(+) regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-beta- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8(+) DEC-205/CD205(+) DCs, but not the major fraction of CD8(-) DC inhibitory receptor-2 (DCIR2)(+) DCs, induce functional Foxp3(+) Treg from Foxp3(-) precursors in the presence of low doses of Ag but without added TGF-beta. CD8(+)CD205(+) DCs preferentially express TGF-beta, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-beta. In contrast, CD8(-)DCIR2(+) DCs better induce Foxp3(+) Treg when exogenous TGF-beta is supplied. In vivo, CD8(+)CD205(+) DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG(-/-) Foxp3(-)CD4(+) T cells, whereas the CD8(-)DCIR2(+) DCs better stimulate natural Foxp3(+) Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3(+) Treg, in part through the endogenous formation of TGF-beta. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3(+) Treg for treatment of autoimmune diseases, transplant rejection, and allergy.     

CD4+CD25+ regulatory T cells inhibit the antigen-dependent expansion of self-reactive T cells in vivo

A deficiency of CD4+CD25+ regulatory T cells (CD25+ Tregs) in lymphopenic mice can result in the onset of autoimmune gastritis. The gastric H/K ATPase alpha (H/Kalpha) and beta (H/Kbeta) subunits are the immunodominant autoantigens recognized by effector CD4+ T cells in autoimmune gastritis. The mechanism by which CD25+ Tregs suppress autoimmune gastritis in lymphopenic mice is poorly understood. To investigate the antigenic requirements for the genesis and survival of gastritis-protecting CD25+ Tregs, we analyzed mice deficient in H/Kbeta and H/Kalpha, as well as a transgenic mouse line (H/Kbeta-tsA58 Tg line 224) that lacks differentiated gastric epithelial cells. By adoptive transfer of purified T cell populations to athymic mice, we show that the CD25+ Treg population from mice deficient in either one or both of H/Kalpha and H/Kbeta, or from the H/Kbeta-tsA58 Tg line 224 mice, is equally effective in suppressing the ability of polyclonal populations of effector CD4+ T cells to induce autoimmune gastritis. Furthermore, CD25+ Tregs, from either wild-type or H/Kalpha-deficient mice, dramatically reduced the expansion of pathogenic H/Kalpha-specific TCR transgenic T cells and the induction of autoimmune gastritis in athymic recipient mice. Proliferation of H/Kalpha-specific T cells in lymphopenic hosts occurs predominantly in the paragastric lymph node and was dependent on the presence of the cognate H/Kalpha Ag. Collectively, these studies demonstrate that the gastritis-protecting CD25+ Tregs do not depend on the major gastric Ags for their thymic development or their survival in the periphery, and that CD25+ Tregs inhibit the Ag-specific expansion of pathogenic T cells in vivo.     

Natural regulatory T cells and de novo-induced regulatory T cells contribute independently to tumor-specific tolerance

Thymus-derived, naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (nTregs) and Tregs induced in the periphery (iTregs) have both been implicated in regulating immune responses. However, the relationship between these populations in the same host, and their relative contribution to the overall Treg pool, has not been examined. Using a tumor-induced T cell tolerance model, we find that expansion of nTregs and de novo generation of iTregs both contribute to tumor-specific T cell tolerance. In this system in which the number of tumor-specific nTregs can be controlled, the efficiency of nTreg expansion significantly exceeds that of the induction of Tregs from uncommitted progenitors in the tumor-bearing host. However, pre-existing nTregs are neither required for the induction of Tregs nor measurably impact on the extent of their accumulation. Instead, induction of Ag-specific regulatory cells from naive cells is intrinsically influenced by the tumor microenvironment and the presence of tumor Ag.     

Lipopolysaccharide-activated IL-10-secreting dendritic cells suppress experimental autoimmune uveoretinitis by MHCII-dependent activation of CD62L-expressing regulatory T cells

Dendritic cells (DC) are key regulators of immune responses. Mature DC are traditionally considered to be immunogenic, although there is accumulating evidence that they can also be tolerogenic and induce Ag-specific regulatory T cells (Tregs). However, the mechanism of this Treg induction and the site of Treg action in vivo are yet to be defined. In this study, using the experimental model of interphotoreceptor retinoid-binding protein peptide (1-20)-induced experimental autoimmune uveoretinitis, we show that s.c. inoculation of IRBP-peptide-pulsed IL-10-producing LPS-activated mature DC (IL-10-DC) at one site (the cervical region) suppresses autoimmunity induced at a separate site (the inguinal region). Our data show that s.c. IL-10-DC correlates with an increase in the number of CD4(+)CD25(+)Foxp3(+) Tregs at the DC-draining lymph nodes (DC-dLN). However, although MHCII(-/-) IL-10-DC also induces Treg expansion at this DC-dLN, they failed to suppress experimental autoimmune uveoretinitis. Furthermore, unlike wild-type IL-10-DC, MHCII(-/-) IL-10-DC did not correlate with an increase in the percentage of Tregs expressing CD62L at the DC-dLN, nor did they associate with an increase in Treg number at a distal site. Similar effects were also observed after s.c. hen egg lysozyme-pulsed IL-10-DC, which produced a strong reduction in the number and activation of proliferating Ag-specific CD4(+) 3A9 T effector cells. We therefore propose that IL-10-DC require MHCII-dependent Ag presentation, and hence TCR ligation, to promote CD62L-mediated trafficking of Tregs to the site of T effector cell priming, where they suppress autoimmunity.     

Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells

CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-beta. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110delta PI3K (p110deltaD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110deltaD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110deltaD910A/D910A T cells failed to protect against experimental colitis. The identification of p110delta as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.     

Cutting edge: Foxp3-mediated induction of pim 2 allows human T regulatory cells to preferentially expand in rapamycin

Addition of rapamycin to cultures of expanding natural CD4+CD25+Foxp3+ T regulatory cells (Tregs) helps maintain their suppressive activity, but the underlying mechanism is unclear. Pim 2 is a serine/threonine kinase that can confer rapamycin resistance. Unexpectedly, pim 2 was found to be constitutively expressed in freshly isolated, resting Tregs, but not in CD4+CD25- T effector cells. Introduction of Foxp3, but not Foxp3Delta2, into effector T cells induced pim 2 expression and conferred preferential expansion in the presence of rapamycin, indicating that Foxp3 can regulate pim 2 expression. Finally, we determined there is a positive correlation between Treg expansion and Foxp3 expression in the presence of rapamycin. Together, these results indicate that Tregs are programmed to be resistant to rapamycin, providing further rationale for why this immunosuppressive drug should be used in conjunction with expanded Tregs.     

Foxp3+ regulatory T cells impede the priming of protective CD8+ T cells

T cell activation is controlled by incompletely defined opposing stimulation and suppression signals that together sustain the balance between optimal host defense against infection and peripheral tolerance. In this article, we explore the impacts of Foxp3(+) regulatory T cell (Treg) suppression in priming Ag-specific T cell activation under conditions of noninfection and infection. We find the transient ablation of Foxp3(+) Tregs unleashes the robust expansion and activation of peptide-stimulated CD8(+) T cells that provide protection against Listeria monocytogenes infection in an Ag-specific fashion. By contrast, Treg ablation had nonsignificant impacts on the CD8(+) T cell response primed by infection with recombinant L. monocytogenes. Similarly, nonrecombinant L. monocytogenes administered with peptide stimulated the expansion and activation of CD8(+) T cells that paralleled the response primed by Treg ablation. Interestingly, these adjuvant properties of L. monocytogenes did not require CD8(+) T cell stimulation by IL-12 produced in response to infection, but instead were associated with sharp reductions in Foxp3(+) Treg suppressive potency. Therefore, Foxp3(+) Tregs impose critical barriers that, when overcome naturally during infection or artificially with ablation, allow the priming of protective Ag-specific CD8(+) T cells.     

OX40/OX40L costimulation affects induction of Foxp3+ regulatory T cells in part by expanding memory T cells in vivo

OX40 is a member of the TNFR superfamily and has potent T cell costimulatory activities. OX40 also inhibits the induction of Foxp3(+) regulatory T cells (Tregs) from T effector cells, but the precise mechanism of such inhibition remains unknown. In the present study, we found that CD4(+) T effector cells from OX40 ligand-transgenic (OX40Ltg) mice are highly resistant to TGF-beta mediated induction of Foxp3(+) Tregs, whereas wild-type B6 and OX40 knockout CD4(+) T effector cells can be readily converted to Foxp3(+) T cells. We also found that CD4(+) T effector cells from OX40Ltg mice are heterogeneous and contain a large population of CD44(high)CD62L(-) memory T cells. Analysis of purified OX40Ltg naive and memory CD4(+) T effector cells showed that memory CD4(+) T cells not only resist the induction of Foxp3(+) T cells but also actively suppress the conversion of naive CD4(+) T effector cells to Foxp3(+) Tregs. This suppression is mediated by the production of IFN-gamma by memory T cells but not by cell-cell contact and also involves the induction of T-bet. Importantly, memory CD4(+) T cells have a broad impact on the induction of Foxp3(+) Tregs regardless of their origins and Ag specificities. Our data suggest that one of the mechanisms by which OX40 inhibits the induction of Foxp3(+) Tregs is by inducing memory T cells in vivo. This finding may have important clinical implications in tolerance induction to transplanted tissues.     

Identification of circulating human antigen-reactive CD4+ FOXP3+ natural regulatory T cells

Circulating human CD4(+)CD25(++)CD127(-)FOXP3(+) T cells with a persistent demethylated regulatory T cell (Treg)-specific demethylated region Foxp3 gene are considered natural Tregs (nTregs). We have shown that it is possible to identify functional Ag-reactive nTregs cells for a range of different common viral and vaccination Ags. The frequency of these Ag-reactive nTregs within the nTreg population is strikingly similar to the frequency of Ag-reactive T effector cells within the CD4(+) T cell population. The Ag-reactive nTregs could be recognized with great specificity by induction of CD154 expression. These CD154(+) Ag-reactive nTregs showed a memory phenotype and shared all phenotypical and functional characteristics of nTregs. The isolated CD154(+) nTregs could be most efficiently expanded by specific antigenic stimulation, while their Ag-reactive suppressive activity was maintained. After an in vivo booster Ag challenge, the ratio of Ag-reactive T cells to Ag-reactive Tregs increased substantially, which could be attributed to the rise in effector T cells but not Tregs. In conclusion, the nTreg population mirrors the effector T cell population in the frequency of Ag-reactive T cells. Isolation and expansion of functional Ag-reactive nTregs is possible and of potential benefit for specific therapeutic goals.     

Autoantigen-specific TGFbeta-induced Foxp3+ regulatory T cells prevent autoimmunity by inhibiting dendritic cells from activating autoreactive T cells

Several strategies are being designed to test the therapeutic potential of Ag-specific regulatory T cells to prevent or treat autoimmune diseases. In this study, we demonstrate that naive CD4+ Foxp3- T cells specific for a naturally expressed autoantigen (H+/K+ ATPase) can be converted to Foxp3+ T regulatory cells (Tregs) when stimulated in presence of TGFbeta. TGFbeta-induced Tregs (iTregs) have all the characteristics of naturally generated regulatory T cells in vitro, and more importantly, are effective at preventing organ-specific autoimmunity in a murine model of autoimmune gastritis. H+/K+ ATPase specific iTregs were able to inhibit the initial priming and proliferation of autoreactive T cells, and appear to do so by acting on H+/K+ ATPase presenting dendritic cells (DC). DC exposed to iTregs in vivo were reduced in their ability to stimulate proliferation and cytokine production by H+/K+ ATPase specific T cells. iTregs specifically reduced CD80 and CD86 expression on the surface of H+/K+ ATPase presenting DC in vitro. These studies reveal the therapeutic potential of Ag specific iTregs to prevent autoimmunity, and provide a mechanism by which this population of regulatory T cells, and perhaps others, mediate their suppressive effects in vivo.