GRAIL is up-regulated in CD4+ CD25+ T regulatory cells and is sufficient for conversion of T cells to a regulatory phenotype

GRAIL (gene related to anergy in lymphocytes) is an ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase necessary for the induction of CD4(+) T cell anergy in vivo. We have extended our previous studies to characterize the expression pattern of GRAIL in other murine CD4(+) T cell types with a described anergic phenotype. These studies revealed that GRAIL expression is increased in naturally occurring (thymically derived) CD4(+) CD25(+) T regulatory cells (mRNA levels 10-fold higher than naive CD25(-) T cells). Further investigation demonstrated that CD25(+) Foxp3(+) antigen-specific T cells were induced after a "tolerizing-administration" of antigen and that GRAIL expression correlated with the CD25(+) Foxp3(+) antigen-specific subset. Lastly, using retroviral transduction, we demonstrated that forced expression of GRAIL in a T cell line was sufficient for conversion of these cells to a regulatory phenotype in the absence of detectable Foxp3. These data demonstrate that GRAIL is differentially expressed in naturally occurring and peripherally induced CD25(+) T regulatory cells and that the expression of GRAIL is linked to their functional regulatory activity.     

Cutting edge: regulatory T cells induce CD4+CD25-Foxp3- T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-beta

Recent studies have shown that TGF-beta together with IL-6 induce the differentiation of IL-17-producing T cells (Th17) T cells. We therefore examined whether CD4(+)CD25(+)Foxp3(+) regulatory T cells, i.e., cells previously shown to produce TGF-beta, serve as Th17 inducers. We found that upon activation purified CD25(+) T cells (or sorted GFP(+) T cells obtained from Foxp3-GFP knockin mice) produce high amounts of soluble TGF-beta and when cultured with CD4(+)CD25(-)Foxp3(-) T cells in the presence of IL-6 induce the latter to differentiate into Th17 cells. Perhaps more importantly, upon activation, CD4(+)CD25(+)Foxp3(+)(GFP(+)) T cells themselves differentiate into Th17 cells in the presence of IL-6 (and in the absence of exogenous TGF-beta). These results indicate that CD4(+)CD25(+)Foxp3(+) regulatory T cells can function as inducers of Th17 cells and can differentiate into Th17 cells. They thus have important implications to our understanding of regulatory T cell function and their possible therapeutic use.     

Th3 cells in peripheral tolerance. I. Induction of Foxp3-positive regulatory T cells by Th3 cells derived from TGF-beta T cell-transgenic mice

TGF-beta has been shown to be critical in the generation of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Because Th3 cells produce large amounts of TGF-beta, we asked whether induction of Th3 cells in the periphery was a mechanism by which CD4(+)CD25(+) Tregs were induced in the peripheral immune compartment. To address this issue, we generated a TGF-beta1-transgenic (Tg) mouse in which TGF-beta is linked to the IL-2 promoter and T cells transiently overexpress TGF-beta upon TCR stimulation but produce little or no IL-2, IL-4, IL-10, IL-13, or IFN-gamma. Naive TGF-beta-Tg mice are phenotypically normal with comparable numbers of lymphocytes and thymic-derived Tregs. We found that repeated antigenic stimulation of pathogenic myelin oligodendrocyte glycoprotein (MOG)-specific CD4(+)CD25(-) T cells from TGF-beta Tg mice crossed to MOG TCR-Tg mice induced Foxp3 expression in both CD25(+) and CD25(-) populations. Both CD25 subsets were anergic and had potent suppressive properties in vitro and in vivo. Furthermore, adoptive transfer of these induced regulatory CD25(+/-) T cells suppressed experimental autoimmune encephalomyelitis when administrated before disease induction or during ongoing experimental autoimmune encephalomyelitis. The suppressive effect of TGF-beta on T cell responses was due to the induction of Tregs and not to the direct inhibition of cell proliferation. The differentiation of Th3 cells in vitro was TGF-beta dependent as anti-TGF-beta abrogated their development. Thus, Ag-specific TGF-beta-producing Th3 cells play a crucial role in inducing and maintaining peripheral tolerance by driving the differentiation of Ag-specific Foxp3(+) regulatory cells in the periphery.     

Resistance to CD4+CD25+ regulatory T cells and TGF-beta in Cbl-b-/- mice

Cbl-b(-/-) mice have signaling defects that result in CD28-independent T cell activation, increased IL-2 production, hyper-reactive T cells, and increased autoimmunity. Although the increased autoimmunity in these mice is believed to result from the hyper-reactive T cells, the mechanisms leading from T cell hyper-reactivity to autoimmunity remain unclear. Specifically, the function and interaction of CD4(+)CD25(+) regulatory T cells (T(reg)) and CD4(+)CD25(-) effector T cells (T(eff)) in Cbl-b(-/-) mice have not been examined. We now report that Cbl-b(-/-) CD4(+)CD25(+) T(reg) exhibit normal regulatory function in vitro. In contrast, the in vitro response of Cbl-b(-/-) CD4(+)CD25(-) T(eff) is abnormal, in that it is not inhibited by either Cbl-b(-/-) or wild-type T(reg). This resistance of Cbl-b(-/-) T(eff) to in vitro regulation is seen at the levels of both DNA synthesis and cell division. In addition to this resistance to CD4(+)CD25(+) T(reg), Cbl-b(-/-) T(eff) demonstrate in vitro resistance to inhibition by TGF-beta. This second form of resistance in Cbl-b(-/-) T(eff) is seen despite the expression of normal levels of type II TGF-beta receptors and normal levels of phosphorylated Smad3 after TGF-beta stimulation. Coupled with recent reports of resistance to T(reg) in T(eff) exposed to LPS-treated dendritic cells, our present findings suggest that resistance to regulation may be a relevant mechanism in both normal immune function and autoimmunity.     

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.     

Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10

Thymus-derived, natural CD4(+)CD25(+) regulatory T cells can educate peripheral CD4(+)CD25(-) cells to develop suppressive activity by poorly understood mechanisms. TGF-beta has IL-2-dependent costimulatory effects on alloactivated naive, human CD4(+) T cells and induces them ex vivo to become potent contact-dependent, cytokine-independent suppressor cells. In this study, we report that CD4(+)CD25(+) cells are the targets of the costimulatory effects of IL-2 and TGF-beta. These cells do not divide, but, instead, greatly increase the numbers of CD4(+)CD25(-) cells that become CD25(+) cytokine-independent suppressor cells. These CD4(+)CD25(+) regulatory cells, in turn, induce other alloactivated CD4(+)CD25(-) cells to become potent suppressor cells by mechanisms that, surprisingly, require both cell contact and TGF-beta and IL-10. The suppressive effects of these secondary CD4(+)CD25(+) cells depend upon TGF-beta and IL-10. Moreover, both the naive CD4(+) cells induced by IL-2 and TGF-beta to become suppressor cells, and the subsequent CD4(+)CD25(-) cells educated by them to become suppressors express FoxP3. We suggest that the long-term effects of adoptively transferred natural-like CD4(+)CD25(+) regulatory cells induced ex vivo are due to their ability to generate new cytokine-producing CD4(+) regulatory T cells in vivo.     

IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4(+)CD25(+) regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4(+)CD25(+) T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4(+)CD25(-) T cells and counteracts the suppressive activities of CD4(+)CD25(+) T cells on CD4(+)CD25(-) T cells without affecting the percentage of Foxp3(+) cells or survival of Treg. Additionally, CD4(+)CD25(+) T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8(+)CD25(-) T cells but does not revert the CD4(+)CD25(+) T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4(+) T cells resistant to suppression rather than inhibiting CD4(+)CD25(+) T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4(+)CD25(+) T cells. Data indicate that IL-21 renders human CD4(+)CD25(-) T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.     

Cutting edge: TGF-beta signaling is required for the in vivo expansion and immunosuppressive capacity of regulatory CD4+CD25+ T cells

Data regarding the role of TGF-beta for the in vivo function of regulatory CD4(+)CD25(+) T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-beta signaling specifically in T cells was used to assess the role of endogenous TGF-beta for the in vivo function of CD4(+)CD25(+) Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4(+)CD25(+) Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4(+)CD25(+) Treg we could demonstrate that endogenous TGF-beta promotes the expansion of CD4(+)CD25(+) Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4(+)CD25(+) Treg and were more susceptible to the induction of colitis, which could be prevented by the transfer of wild-type Treg. These data indicate that TGF-beta signaling in CD4(+)CD25(+) Treg is required for their in vivo expansion and suppressive capacity.     

Th3 cells in peripheral tolerance. II. TGF-beta-transgenic Th3 cells rescue IL-2-deficient mice from autoimmunity

We developed a transgenic (Tg) mouse that expresses TGF-beta under control of the IL-2 promoter to investigate Th3 cell differentiation both in vitro and in vivo. We previously found that repetitive in vitro Ag stimulation results in constant expression of Foxp3 in TGF-beta-Tg Th3 cells that acquire regulatory function independent of surface expression of CD25. To examine the differentiation and function of Th3 cells in vivo and to compare them with thymic-derived CD4(+)CD25(+) regulatory T cells (Treg), we introduced the TGF-beta transgene into T cells of IL-2-deficient (IL-2(-/-)) mice. We found that the induction, differentiation, and function of TGF-beta-derived Foxp3(+) Th3 cells were independent of IL-2, which differs from thymic Tregs. In an environment that lacks functional CD25(+) thymic-derived Tregs, expression of the TGF-beta transgene in IL-2(-/-) mice led to the induction of distinct CD25(-) regulatory cells in the periphery. These cells expressed Foxp3 and efficiently controlled hyperproliferation of T cells and rescued the IL-2(-/-) mouse from lethal autoimmunity. Unlike IL-2(-/-) animals, TGF-beta/IL-2(-/-) mice had normal numbers of T cells, B cells, macrophages, and dendritic cells and did not have splenomegaly, lymphadenopathy, or inflammation in multiple organs. Accumulation of Foxp3(+) cells over time, however, was dependent on IL-2. Our results suggest that TGF-beta-derived Foxp3(+)CD25(+/-) Th3 regulatory cells represent a different cell lineage from thymic-derived CD25(+) Tregs in the periphery but may play an important role in maintaining thymic Tregs in the peripheral immune compartment by secretion of TGF-beta.     

Thymus exosomes-like particles induce regulatory T cells

Exosomes released from different types of cells have been proposed to contribute to intercellular communication. We report that thymic exosome-like particles (ELPs) released from cells of the thymus can induce the development of Foxp3(+) regulatory T (Treg) cells in the lung and liver. Thymic ELPs also induce the conversion of thymic CD4(+)CD25(-) T cells into Tregs. Tregs induced by thymic ELPs suppress the proliferation of CD4(+)CD25(-) T cells in vitro and in vivo. We further show that neutralization of TGF-beta in ELPs partially reverses thymic ELP-mediated induction of CD4(+)Foxp3(+) T cells in the lung and liver. This study demonstrates that thymic ELPs participate in the induction of Foxp3(+) Tregs. Also, TGF-beta of thymic ELPs might be required for the generation of Tregs in the peripheral tissues.     

CD4+CD25bright T cells in human intestinal lamina propria as regulatory cells

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only active suppression by regulatory T cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of inflammatory bowel disease. In this study, we demonstrate that the CD4(+)CD25(bright) T cells reside in the human intestinal lamina propria (LP) and functionally retain regulatory activities. All human LP CD4(+) T cells regardless of CD25 expression constitutively expressed CTLA-4, glucocorticoid-induced TNFR family-related protein, and Foxp3 and proliferate poorly. Although LP CD4(+)CD25(-) T cells showed an activated and anergic/memory phenotype, they did not retain regulatory activity. In LP CD4(+)CD25(+) T cells, however, cells expressing CD25 at high levels (CD4(+)CD25(bright)) suppressed the proliferation and various cytokine productions of CD4(+)CD25(-) T cells. LP CD4(+)CD25(bright) T cells by themselves produced fewer amounts of IL-2, IFN-gamma, and IL-10. Interestingly, LP CD4(+)CD25(bright) T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease. These results suggest that CD4(+)CD25(bright) T cells found in the normal and inflamed intestinal mucosa selectively inhibit the host immune response and therefore may contribute to the intestinal immune homeostasis.     

CD4+CD25- T cells that express latency-associated peptide on the surface suppress CD4+CD45RBhigh-induced colitis by a TGF-beta-dependent mechanism

Murine CD4(+)CD25(+) regulatory cells have been reported to express latency-associated peptide (LAP) and TGF-beta on the surface after activation, and exert regulatory function by the membrane-bound TGF-beta in vitro. We have now found that a small population of CD4(+) T cells, both CD25(+) and CD25(-), can be stained with a goat anti-LAP polyclonal Ab without being stimulated. Virtually all these LAP(+) cells are also positive for thrombospondin, which has the ability to convert latent TGF-beta to the active form. In the CD4(+)CD45RB(high)-induced colitis model of SCID mice, regulatory activity was exhibited not only by CD25(+)LAP(+) and CD25(+)LAP(-) cells, but also by CD25(-)LAP(+) cells. CD4(+)CD25(-)LAP(+) T cells were part of the CD45RB(low) cell fraction. CD4(+)CD25(-)LAP(-)CD45RB(low) cells had minimal, if any, regulatory activity in the colitis model. The regulatory function of CD25(-)LAP(+) cells was abrogated in vivo by anti-TGF-beta mAb. These results identify a new TGF-beta-dependent regulatory CD4(+) T cell phenotype that is CD25(-) and LAP(+).     

Defects in the Bcl-2-regulated apoptotic pathway lead to preferential increase of CD25 low Foxp3+ anergic CD4+ T cells

Defects in the Bcl-2-regulated apoptotic pathway inhibit the deletion of self-reactive T cells. What is unresolved, however, is the nature and fate of such self-reactive T cells escaping deletion. In this study, we report that mice with such defects contained increased numbers of CD25(low)Foxp3(+) cells in the thymus and peripheral lymph tissues. The increased CD25(low)Foxp3(+) population contained a large fraction of cells bearing self-reactive TCRs, evident from a prominent increase in self-superantigen-specific Foxp3(+)Vβ5(+)CD4(+) T cells in BALB/c Bim(-/-) mice compared with control animals. The survival rate of the expanded CD25(low)Foxp3(+) cells was similar to that of CD25(high)Foxp3(+) CD4 T cells in vitro and in vivo. IL-2R stimulation, but not TCR ligation, upregulated CD25 on CD25(low)Foxp3(+)CD4(+) T cells in vitro and in vivo. The expanded CD25(low)Foxp3(+)CD4(+) T cells from Bim(-/-) mice were anergic but also had weaker regulatory function than CD25(high)Foxp3(+) CD4(+) T cells from the same mice. Analysis of Bim(-/-) mice that also lacked Fas showed that the peripheral homeostasis of this expanded population was in part regulated by this death receptor. In conclusion, these results show that self-reactive T cell escapes from thymic deletion in mice defective in the Bcl-2-regulated apoptotic pathway upregulate Foxp3 and become unresponsive upon encountering self-Ag without necessarily gaining potent regulatory function. This clonal functional diversion may help to curtail autoaggressiveness of escaped self-reactive CD4(+) T cells and thereby safeguard immunological tolerance.     

Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice

RasGRP1 and Sos are two Ras-guanyl-nucleotide exchange factors that link TCR signal transduction to Ras and MAPK activation. Recent studies demonstrate positive selection of developing thymocytes is crucially dependent on RasGRP1, whereas negative selection of autoreactive thymocytes appears to be RasGRP1 independent. However, the role of RasGRP1 in T regulatory (Treg) cell development and function is unknown. In this study, we characterized the development and function of CD4(+)CD25(+)Foxp3(+) and CD8(+)CD44(high)CD122(+) Treg lineages in RasGRP1(-/-) mice. Despite impaired CD4 Treg cell development in the thymus, the periphery of RasGRP1(-/-) mice contained significantly increased frequencies of CD4(+)Foxp3(+) Treg cells that possessed a more activated cell surface phenotype. Furthermore, on a per cell basis, CD4(+)Foxp3(+) Treg cells from mutant mice are more suppressive than their wild-type counterparts. Our data also suggest that the lymphopenic environment in the mutant mice plays a dominant role of favored peripheral development of CD4 Treg cells. These studies suggest that whereas RasGRP1 is crucial for the intrathymic development of CD4 Treg cells, it is not required for their peripheral expansion and function. By contrast to CD4(+)CD25(+)Foxp3(+) T cells, intrathymic development of CD8(+)CD44(high)CD122(+) Treg cells is unaffected by the RasGRP1(-/-) mutation. Moreover, RasGRP1(-/-) mice contained greater numbers of CD8(+)CD44(high)CD122(+) T cells in the spleen, relative to wild-type mice. Activated CD8 Treg cells from RasGRP1(-/-) mice retained their ability to synthesize IL-10 and suppress the proliferation of wild-type CD8(+)CD122(-) T cells, albeit at a much lower efficiency than wild-type CD8 Treg cells.     

Strong impact of CD4+ Foxp3+ regulatory T cells and limited effect of T cell-derived IL-10 on pathogen clearance during Plasmodium yoelii infection

It is well established that CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) play a crucial role in the course of different infectious diseases. However, contradictory results have been published regarding to malaria infection. In this study, we report that specific ablation of Foxp3(+) Tregs in Plasmodium yoelii-infected DEREG-BALB/c mice leads to an increase in T cell activation accompanied by a significant decrease in parasitemia. To better understand how Foxp3(+) Tregs orchestrate this phenotype, we used microarrays to analyze CD4(+)CD25(+)Foxp3(+) Tregs and CD4(+)CD25(-)Foxp3(-) T cells in the course of P. yoelii infection. Using this approach we identified genes specifically upregulated in CD4(+)CD25(+)Foxp3(+) Tregs in the course of infection, such as G-protein-coupled receptor 83 and Socs2. This analysis also revealed that both CD4(+)CD25(+)Foxp3(+) Tregs and CD4(+)CD25(-)Foxp3(-) T cells upregulate CTLA-4, granzyme B, and, more strikingly, IL-10 during acute blood infection. Therefore, we aimed to define the function of T cell-derived IL-10 in this context by Cre/loxP-mediated selective conditional inactivation of the IL-10 gene in T cells. Unexpectedly, IL-10 ablation in T cells exerts only a minor effect on parasite clearance, even though CD8(+) T cells are more strongly activated, the production of IFN-γ and TNF-α by CD4(+)CD25(-) T cells is increased, and the suppressive activity of CD4(+)CD25(+) Tregs is reduced upon infection. In summary, these results suggest that CD4(+)Foxp3(+) Tregs modulate the course of P. yoelii infection in BALB/c mice. Moreover, CD4(+) T cell-derived IL-10 affects T effector function and Treg activity, but has only a limited direct effect on parasite clearance in this model.     

Cutting edge: deficiency in the E3 ubiquitin ligase Cbl-b results in a multifunctional defect in T cell TGF-beta sensitivity in vitro and in vivo

Mice deficient in the E3 ubiquitin ligase Cbl-b have CD28-independent T cells and develop autoimmunity. We previously reported that Cbl-b-/- CD4+CD25- T effector cells are resistant in vitro to the antiproliferative effects of CD4+CD25+ regulatory T cells and TGF-beta. We have now asked whether the resistance noted in Cbl-b-/- T cells is restricted solely to TGF-beta's antiproliferative effects, whether the TGF-beta resistance has in vivo relevance, and whether a defect can be identified in the TGF-beta signaling pathway. We now demonstrate the following: 1) in vitro, Cbl-b deficiency prevents the TGF-beta-mediated induction of Foxp3+ functional regulatory T cells; 2) in vivo, Cbl-b-/- mice show a significantly enhanced response to a tumor that is strictly TGF-beta regulated; and 3) Cbl-b-/- T effector cells have defective TGF-beta-mediated Smad2 phosphorylation. These studies are the first to document that the E3 ubiquitin ligase Cbl-b plays an integral role in T cell TGF-beta signaling, and that its absence results in multifunctional TGF-beta-related defects that have important disease-related implications.