Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function

Both CTLA-4 and TGF-beta have been implicated in suppression by CD4+CD25+ regulatory T cells (Treg). In this study, the relationship between CTLA-4 and TGF-beta in Treg function was examined. Blocking CTLA-4 on wild-type Treg abrogated their suppressive activity in vitro, whereas neutralizing TGF-beta had no effect, supporting a TGF-beta-independent role for CTLA-4 in Treg-mediated suppression in vitro. In CTLA-4-deficient mice, Treg development and homeostasis was normal. Moreover, Treg from CTLA-4-deficient mice exhibited uncompromised suppressive activity in vitro. These CTLA-4-deficient Treg expressed increased levels of the suppressive cytokines IL-10 and TGF-beta, and in vitro suppression mediated by CTLA-4(-/-) Treg was markedly reduced by neutralizing TGF-beta, suggesting that CTLA-4-deficient Treg develop a compensatory suppressive mechanism through CTLA-4-independent production of TGF-beta. Together, these data suggest that CTLA-4 regulates Treg function by two distinct mechanisms, one during functional development of Treg and the other during the effector phase, when the CTLA-4 signaling pathway is required for suppression. These results help explain contradictions in the literature and support the existence of functionally distinct Treg.     

CD4+ CD25+ [corrected] regulatory T cells render naive CD4+ CD25- T cells anergic and suppressive

CD4(+) CD25(+) Foxp3(+) naturally occurring regulatory T cells (nTreg) are potent inhibitors of almost all immune responses. However, it is unclear how this minor population of cells is capable of exerting its powerful suppressor effects. To determine whether nTreg mediate part of their suppressor function by rendering naive T cells anergic or by converting them to the suppressor phenotype, we cocultured mouse nTreg with naive CD4(+) CD25(-) T cells from T-cell receptor (TCR) transgenic mice on a RAG deficient (RAG(-/-)) background in the presence of anti-CD3 and interleukin-4 (IL-4) to promote cell viability. Two distinct responder cell populations could be recovered from the cocultures. One population remained undivided in the coculture and was non-responsive to restimulation with anti-CD3 or exogenous IL-2, and could not up-regulate IL-2 mRNA or CD25 expression upon TCR restimulation. Those responder cells that had divided in the coculture were anergic to restimulation with anti-CD3 but responded to restimulation with IL-2. The undivided population was capable of suppressing the response of fresh CD4(+) CD25(-) T cells and CD8(+) T cells, while the divided population was only marginally suppressive. Although cell contact between the induced regulatory T cell (iTreg) and the responders was required for suppression to be observed, anti-transforming growth factor-beta partially abrogated their suppressive function. The iTreg did not express Foxp3. Therefore nTreg are not only able to suppress immune responses by inhibiting cytokine production by CD4(+) CD25(-) responder cells, but also appear to modulate the responder cells to render them both anergic and suppressive.     

Recurrent superantigen exposure in vivo leads to highly suppressive CD4+CD25+ and CD4+CD25‐ T cells with anergic and suppressive genetic signatures

Staphylococcal enterotoxin B (SEB) activates T cells via non‐canonical signalling through the T cell receptor and is an established model for T cell unresponsiveness in vivo. In this study, we sought to characterize the suppressive qualities of SEB‐exposed CD4⁺ T cells and correlate this with genetic signatures of anergy and suppression. SEB‐exposed CD25⁺ and CD25^‐Vβ8⁺CD4⁺ T cells expressed forkhead box P3 (FoxP3) at levels comparable to naive CD25⁺ T regulatory cells and were enriched after exposure in vivo. Gene related to anergy in lymphocytes (GRAIL), an anergy‐related E3 ubiquitin ligase, was up‐regulated in the SEB‐exposed CD25⁺ and CD25^‐FoxP3⁺Vβ8⁺CD4⁺ T cells and FoxP3^‐CD25^‐Vβ8⁺CD4⁺ T cells, suggesting that GRAIL may be important for dominant and recessive tolerance. The SEB‐exposed FoxP3⁺GRAIL⁺ T cells were highly suppressive and non‐proliferative independent of CD25 expression level and via a glucocorticoid‐induced tumour necrosis factor R‐related protein‐independent mechanism, whereas naive T regulatory cells were non‐suppressive and partially proliferative with SEB activation in vitro. Lastly, adoptive transfer of conventional T cells revealed that induction of FoxP3⁺ regulatory cells is not operational in this model system. These data provide a novel paradigm for chronic non‐canonical T cell receptor engagement leading to highly suppressive FoxP3⁺GRAIL⁺CD4⁺ T cells.     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Normal human pregnancy is associated with an elevation in the immune suppressive CD25+ CD4+ regulatory T-cell subset

Summary CD4+ CD25+ T regulatory cells (TReg), suppress antigen-specific immune responses and are important for allograft tolerance. During pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus) requiring substantial changes in immune regulation over a programmed period of time. We analysed whether immune-suppressive TReg cells were altered during pregnancy and therefore might play a part in this tolerant state. The presence of TReg cells was assessed in the blood of 25 non-pregnant, 63 pregnant and seven postnatal healthy women by flow cytometry. We observed an increase in circulating TReg cells during early pregnancy, peaking during the second trimester and then a decline postpartum. Isolated CD25+ CD4+ cells expressed FoxP3 messenger RNA, a marker of TReg cells, and suppressed proliferative responses of autologous CD4+ CD25- T cells to allogeneic dendritic cells. These data support the concept that normal pregnancy is associated with an elevation in the number of TReg cells which may be important in maintaining materno-fetal tolerance.     

Deletion is neither sufficient nor necessary for the induction of peripheral tolerance in mature CD8+ T cells

Previous reports have demonstrated clonal deletion of CD8(+) T cells during peripheral tolerance induction to tissue antigens. However, direct evidence demonstrating a causal connection between deletion and tolerance has not been reported because of model limitations in which the tissue antigens were expressed in vital organs. Thus, studies were initiated in a mouse model where expression of a membrane-bound ovalbumin fusion protein (mOVA) was driven by a prostate specific androgen regulated probasin promotor, providing restricted expression in a non-vital organ where antigen levels can be abrogated through androgen deprivation. Adoptive transfer of mOVA specific CD8(+) T cells (OT-I) was used to assess the development of peripheral tolerance. Proliferation of OT-I cells was observed, as was partial deletion of transferred OT-I cells. Although deletion occurred, the long-term persistence of a stable level of OT-I cells was observed. Importantly, the persistent OT-I cells lost antigen responsiveness within 3 weeks of transfer. Castration resulted in loss of high-level prostate mOVA expression, with a resultant abrogation of tolerance induction, but surprisingly did not affect the deletion rate of OT-I cells. In contrast, abrogation of deletion through the adoptive transfer of OT-I cells from third generation CD95-deficient mice had no effect on tolerance induction. These data demonstrate the necessity for continued expression of tissue antigen throughout the establishment of peripheral tolerance. Furthermore, these findings demonstrate that deletion is neither sufficient nor required for CD8(+) T-cell tolerance to tissue antigens, suggesting that regulatory events independent of deletion are necessary for peripheral tolerance induction to prostate antigens.     

Anti‐CD3 treatment up‐regulates programmed cell death protein‐1 expression on activated effector T cells and severely impairs their inflammatory capacity

T cells play a key role in the pathogenesis of type 1 diabetes, and targeting the CD3 component of the T‐cell receptor complex provides one therapeutic approach. Anti‐CD3 treatment can reverse overt disease in spontaneously diabetic non‐obese diabetic mice, an effect proposed to, at least in part, be caused by a selective depletion of pathogenic cells. We have used a transfer model to further investigate the effects of anti‐CD3 treatment on green fluorescent protein (GFP)⁺ islet‐specific effector T cells in vivo. The GFP expression allowed us to isolate the known effectors at different time‐points during treatment to assess cell presence in various organs as well as gene expression and cytokine production. We find, in this model, that anti‐CD3 treatment does not preferentially deplete the transferred effector cells, but instead inhibits their metabolic function and their production of interferon‐γ. Programmed cell death protein 1 (PD‐1) expression was up‐regulated on the effector cells from anti‐CD3‐treated mice, and diabetes induced through anti‐PD‐L1 antibody could only be reversed with anti‐CD3 antibody if the anti‐CD3 treatment lasted beyond the point when the anti‐PD‐L1 antibody was washed out of the system. This suggests that PD‐1/PD‐L1 interaction plays an important role in the anti‐CD3 antibody mediated protection. Our data demonstrate an additional mechanism by which anti‐CD3 therapy can reverse diabetogenesis.     

Antigen-specific,CD4+CD25+ regulatory T cell clones induced in Peyer's patches

Since intestine is exposed to numerous exogenous antigens such as food and commensal bacteria, the organ bears efficient mechanisms for establishment of tolerance and induction of regulatory T cells (T(reg)). Intestinal and inducible T(reg) include T(r)1-like and T(h)3 cells whose major effector molecules are IL-10 and transforming growth factor (TGF)-beta. These antigen-specific T(reg) are expected to become clinical targets to modify the inflammatory immune response associated with allergy, autoimmune diseases and transplantation. In the present study, we characterized the antigen-specific T(reg) induced in the intestine by orally administering high-dose beta-lactoglobulin (BLG) to BALB/c mice. Seven days after feeding, only Peyer's patch (PP) cells among different organs exerted significant suppressive effect on antibody production upon in vitro BLG stimulation. This suppressive effect was also prominent in six BLG-specific CD4(+) T cell clones (OPP1-6) established from PP from mice orally administered with high doses of BLG and was partially reversed by antibodies to TGF-beta. Intravenous transfer of OPP2 efficiently suppressed BLG-specific IgG1 production in serum following immunization, indicating the role of such T(reg) in the systemic tolerance after oral administration of antigen (oral tolerance). OPP clones secrete TGF-beta, IFN-gamma and low levels of IL-10, a cytokine pattern similar to that secreted by anergic T cells. OPP clones bear a CD4(+)CD25(+) phenotype and show significantly lower proliferative response compared to T(h)0 clones. This lower response is recovered by the addition of IL-2. Thus, antigen-specific CD4(+)CD25(+) T(reg), which have characteristics of anergic cells and actively suppress antibody production are induced in PP upon oral administration of protein antigen.     

DC-SIGN, but not sDC-SIGN, can modulate IL-2 production from PMA- and anti-CD3-stimulated primary human CD4 T cells

Dendritic cell (DC)-specific intercellular cell adhesion molecule-3 (ICAM-3)-grabbing non-integrin (DC-SIGN) is expressed on the surface of DCs and specialized macrophages and can support T cell proliferation. Antibody-mediated co-ligation of CD3 and ICAM-3, the ligand for both DC-SIGN and leukocyte function-associated antigen-1, leads to T cell activation. Therefore, we tested to see whether DC-SIGN or a splice variant of dendritic cell-specific intercellular cell adhesion molecule-3-grabbing non-integrin (sDC-SIGN) can co-stimulate primary human T cells. The sDC-SIGN lacking the transmembrane domain encoded by exon 3 localizes to the cytoplasm of cells and is not secreted. Both B7 and DC-SIGN co-stimulated phorbol myristate acetate-stimulated CD4+ cells as compared with controls. However, unlike B7, both DC-SIGN and sDC-SIGN failed to co-stimulate CD4+ T cells treated with sub-optimal amounts of anti-CD3 (2 microg ml(-1)) as defined by a lack of CD69 and CD25 up-regulation, cell division and cytokine secretion. Instead, DC-SIGN, and not sDC-SIGN, induced a small but consistent down-regulation of IL-2 production by these CD4+ T cells. In contrast, DC-SIGN in the presence of 30 mug ml(-1) of anti-CD3 modestly up-regulated cytokine production as compared with control. These results suggest that DC-SIGN can differentially modulate T cell stimulation.     

Large-scale expansion of rat CD4+ CD25+ T(reg) cells in the absence of T-cell receptor stimulation

T-cell receptor (TCR) stimulation is both central to homeostatic maintenance of CD4(+) CD25(+) regulatory T cells (T(reg) cells) in vivo and a prerequisite for the initiation of suppression by T(reg) cells, both in vivo and in vitro. However, TCR-independent stimulation of T(reg) cells, e.g. with superagonistic CD28-specific monoclonal antibodies (CD28-SA), not only expands these cells in vivo but, as we show here, also mediates large-scale expansion of rat T(reg) cells in vitro. Interestingly, CD28-SA stimulation plus interleukin (IL)-2 was even superior to conventional costimulation plus IL-2 in promoting T(reg) cell growth in vitro. Despite their highly activated phenotype suppression by T(reg) cells expanded in the absence of TCR stimulation remained fully dependent on TCR-triggering for initiation and cell contact was required to exert suppression. With regard to the regulation of suppression by CD28 stimulation we observed that neither the presence of a conventional anti-CD28 monoclonal antibody nor a CD28-SA generally rendered conventional T cells resistant to suppression by preactivated T(reg) cells. Taken together, we provide a novel protocol for long-term propagation of T(reg) cells in vitro and our data are the first to reveal a difference in the signals required for activation and expansion of T(reg) cells and those, involving the TCR, necessary for the initiation of suppression.     

TGF-{beta} signaling of human T cells is modulated by the ancillary TGF-{beta} receptor endoglin

Transforming growth factor beta (TGF-beta) inhibits T cell activation and alters differentiation of naive T cells into effector cells. Although four main cell-surface proteins can interact with TGF-beta, only the signaling receptors type I (TGF-betaR type I) and type II (TGF-betaR type II) have so far been described on T cells. The aim of the present study was to investigate the expression of the ancillary receptor endoglin (CD105) by T cells and its role in TGF-beta-mediated signal transduction and function. CD105 expression was analyzed on resting and activated human CD4(+) T cells by flow cytometry, western blot, immunoprecipitation, proliferation and SMAD-responsive reporter gene assays. CD4(+) T cells constitutively expressed CD105 in memory T cells and partially also in naive T cells; however, surface expression is regulated and is increased following TCR engagement, which induced serine/threonine phosphorylation of CD105. In contrast to the suppressive signal mediated by the TGF-beta, cross-linking of CD105 substantially enhanced T cell proliferation, indicating that CD105 by itself mediates signal transduction. Furthermore, CD105 cross-linking induced SMAD-independent signaling via ERK kinase phosphorylation. The present study demonstrates that CD105 is expressed on the surface by activated CD4(+) T cells and CD3 regulated by post-translational means. Furthermore, CD105 acts as a regulatory receptor, counteracting TGF-beta-mediated suppression.     

Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells

Naturally arising CD25(+)CD4(+) regulatory T (T(R)) cells can be exploited to establish immunologic tolerance to non-self antigens. In vivo exposure of CD25(+)CD4(+) T cells from normal naive mice to alloantigen in a T cell-deficient environment elicited spontaneous expansion of alloantigen-specific CD25(+)CD4(+) T(R) cells, which suppressed allograft rejection mediated by subsequently transferred naive T cells, leading to long-term graft tolerance. The expanded T(R) cells, which became CD25(low) in the absence of other T cells, stably sustained suppressive activity, maintained expression levels of other T(R) cell-associated molecules, including Foxp3, CTLA-4 and GITR, and could adoptively transfer tolerance to normal mice. Furthermore, specific removal of the T(R) cells derived from originally transferred CD25(+)CD4(+) T(R) cells evoked graft rejection in the long-term tolerant mice, indicating that any T(R) cells deriving from CD25(-)CD4(+) naive T cells minimally contribute to graft tolerance and that natural T(R) cells are unable to infectiously confer significant suppressive activity to other T cells. Similar antigen-specific expansion of T(R) cells can also be achieved in vitro by stimulating naturally present CD25(+)CD4(+) T cells with alloantigen in the presence of IL-2. The expanded CD25(+)CD4(+) T cells potently suppressed even secondary MLR in vitro and, by in vivo transfer, established antigen-specific long-term graft tolerance. Thus, in vivo or in vitro, direct or indirect ways of antigen-specific expansion of naturally arising Foxp3(+)CD25(+)CD4(+) T(R) cells can establish antigen-specific dominant tolerance to non-self antigens, and would also be instrumental in re-establishing self-tolerance in autoimmune disease and antigen-specific negative control of pathological immune responses.     

Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vbeta repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RB(low) subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen-specific stimulation in vivo. This suggests that anergic/suppressive CD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non-professional antigen-presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.     

树突状细胞扩增抗原特异性CD4+ CD25+调节性T细胞研究进展

CD4+CD25+调节性T细胞(Tr)是同时具有免疫低反应性和免疫抑制性功能两大特征的T细胞.研究证实,CD4+ CD25+ Tr在抑制器官特异性自身免疫性疾病及GVHD是抗原特异性的,因此,应用器官特异性而不是多克隆性的Tr将大大促进以Tr为基础的免疫治疗.而具有调节活性的CD4+ CD25+ Tr仅占人类外周血CIM+ T细胞的1%～2%,因此,研究体外大量扩增的方法 对于以Tr基础的治疗至关重要.研究表明,树突状细胞(DC)作为机体强有力的专职抗原递呈细胞可以扩增具有抗原特异性的CD4+ CD25+ Tr且能增加后者的抑制活性,这为治疗自身免疫性疾病及GVHD提供了新的治疗前景.