Low-dose antigen promotes induction of FOXP3 in human CD4+ T cells

Low Ag dose promotes induction and persistence of regulatory T cells (Tregs) in mice, yet few studies have addressed the role of Ag dose in the induction of adaptive CD4(+)FOXP3(+) Tregs in humans. To this end, we examined the level of FOXP3 expression in human CD4(+)CD25(-) T cells upon activation with autologous APCs and varying doses of peptide. Ag-specific T cells expressing FOXP3 were identified by flow cytometry using MHC class II tetramer (Tmr). We found an inverse relationship between Ag dose and the frequency of FOXP3(+) cells for both foreign Ag-specific and self Ag-specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr(+)FOXP3(+) T cells was not due to expansion of natural Tregs, but instead, we found that induction, proliferation, and persistence of FOXP3(+) cells was similar in high- and low-dose cultures, whereas proliferation of FOXP3(-) T cells was favored in high Ag dose cultures. The frequency of FOXP3(+) cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3(+) cells was maintained with IL-2, but not upon restimulation with Ag. Together, these data suggest that low Ag dose favors the transient generation of human Ag-specific adaptive Tregs over the proliferation of Ag-specific FOXP3(-) effector T cells. These adaptive Tregs could function to reduce ongoing inflammatory responses and promote low-dose tolerance in humans, especially when Ag exposure and tolerance is transient.     

CD25+Foxp3+ regulatory T cells facilitate CD4+ T cell clonal anergy induction during the recovery from lymphopenia

T cell clonal anergy induction in lymphopenic nu/nu mice was found to be ineffective. Exposure to a tolerizing peptide Ag regimen instead induced aggressive CD4(+) cell cycle progression and increased Ag responsiveness (priming). Reconstitution of T cell-deficient mice by an adoptive transfer of mature peripheral lymphocytes was accompanied by the development of a CD25(+)Foxp3(+)CTLA-4(+)CD4(+) regulatory T cell population that acted to dampen Ag-driven cell cycle progression and facilitate the induction of clonal anergy in nearby responder CD25(-)CD4(+) T cells. Thus, an early recovery of CD25(+) regulatory T cells following a lymphopenic event can prevent exuberant Ag-stimulated CD4(+) cell cycle progression and promote the development of clonal anergy.     

Regulatory CD8+ T cells control neonatal tolerance to a Th2-mediated autoimmunity

Exposure of newborn animals to a foreign Ag may result in immunological tolerance to that specific Ag, a phenomenon called neonatal tolerance. We have previously reported that neonatal administration to Brown-Norway rats of mercury, a heavy metal toxicant, induces a dominant tolerance, specific for the chemical otherwise responsible for Th2 cell-mediated autoimmune responses in this susceptible strain of rats. Neonatal exposure to Ags can prime immunity, rather than inactivate or delete responses, and sustain regulatory functions effective against autoreactive T cells. Here, we address whether such a tolerant response is due to the generation of regulatory cells. The results suggest that the CD8(+) T cell subset is involved in neonatal tolerance to mercuric salt-induced Th2 autoimmune disease. Thus, we demonstrate that in vivo CD8 depletion breaks tolerance following mercury recall in animals under a neonatal tolerance protocol. Furthermore, adoptive cotransfer of splenocytes from naive and tolerant rats as well as transfer of CD8(+) T cells from tolerant animals prevent naive syngeneic rats from developing pathologic Th2 immune responses. These observations indicate that CD8(+) T cells are endowed with regulatory functions in neonatal tolerance and mediate active suppression. Moreover, neonatal tolerance induced the expansion of CD8(+)CD45RC(high) T cells and the emergence of a high percentage of IFN-gamma-synthesizing CD8(+) T cells, which probably reflects the implication of regulatory Tc1 cells. Thus, in vivo induction of neonatal tolerance suppresses Th2 autoimmune responses via generation of a CD8(+) cell-mediated regulatory response.     

CD4+CD25+ and CD4+CD25- T cells act respectively as inducer and effector T suppressor cells in superantigen-induced tolerance

The repeated injection of low doses of bacterial superantigens (SAg) is known to induce specific T cell unresponsiveness. We show in this study that the spleen of BALB/c mice receiving chronically, staphylococcal enterotoxin B (SEB) contains SEB-specific CD4(+) TCRBV8(+) T cells exerting an immune regulatory function on SEB-specific primary T cell responses. Suppression affects IL-2 and IFN-gamma secretion as well as proliferation of T cells. However, the suppressor cells differ from the natural CD4(+) T regulatory cells, described recently in human and mouse, because they do not express cell surface CD25. They are CD152 (CTLA-4)-negative and their regulatory activity is not associated with expression of the NF Foxp3. By contrast, after repeated SEB injection, CD4(+)CD25(+) splenocytes were heterogenous and contained both effector as well as regulatory cells. In vivo, CD4(+)CD25(-) T regulatory cells prevented SEB-induced death independently of CD4(+)CD25(+) T cells. Nevertheless, SEB-induced tolerance could not be achieved in thymectomized CD25(+) cell-depleted mice because repeated injection of SEB did not avert lethal toxic shock in these animals. Collectively, these data demonstrate that, whereas CD4(+)CD25(+) T regulatory cells are required for the induction of SAg-induced tolerance, CD4(+)CD25(-) T cells exert their regulatory activity at the maintenance stage of SAg-specific unresponsiveness.     

Induction of foxP3+ regulatory T cells in the periphery of T cell receptor transgenic mice tolerized to transplants

Transplantation tolerance can be induced in mice by grafting under the cover of nondepleting CD4 plus CD8 or CD154 mAbs. This tolerance is donor Ag specific and depends on a population of CD4(+) regulatory T cells that, as yet, remain poorly defined in terms of their specificity, origin, and phenotype. Blocking of the Ag-specific response in vitro with an anti-CD4 mAb allowed T cells from monospecific female TCR-transgenic mice against the male Ag Dby, presented by H-2E(k), to express high levels of foxP3 mRNA. foxP3 induction was dependent on TGF-beta. The nondepleting anti-CD4 mAb was also able to induce tolerance in vivo in such monospecific TCR-transgenic mice, and this too was dependent on TGF-beta. As in conventional mice, acquired tolerance was dominant, such that naive monospecific T cells were not able to override tolerance. Splenic T cells from tolerant mice proliferated normally in response to Ag, and secreted IFN-gamma and some IL-4, similar to control mice undergoing primary or secondary graft rejection. High levels of foxP3 mRNA, and glucocorticoid-induced TNFR superfamily member 18 (GITR)(+) CD25(+) T cells were found within the tolerated skin grafts of long-term tolerant recipients. These data suggest that regulatory T cells maintaining transplantation tolerance after CD4 Ab blockade can be induced de novo through a TGF-beta-dependent mechanism, and come to accumulate in tolerated grafts.     

Generation of anergic and regulatory T cells following prolonged exposure to a harmless antigen

Regulatory CD4(+) T cells are known to develop during the induction of donor-specific peripheral tolerance to transplanted tissues; it is proposed that such tolerance is a consequence of persistent, danger-free stimulation by Ag. To test this hypothesis, male RAG-1(-/-) mice were recolonized with small numbers of monospecific CD4(+) T cells specific for the male H-2E(k)-restricted Ag Dby. After 6 wk in the male environment, the monospecific CD4(+) T cells, having recolonized the host, had become anergic to stimulation in vitro and had acquired a regulatory capacity. CD4(+) T cells in these mice expressed higher levels of CTLA-4 and glucocorticoid-induced TNF-related receptor than naive CD4(+) T cells, but only 3% of the recolonizing cells were CD25(+) and did not express significant foxP3 mRNA. In vivo, these tolerant T cells could censor accumulation of, and IFN-gamma production by, naive T cells, with only a slight inhibition of proliferation. This suppressive effect was not reversed by the addition of fresh bone marrow-derived male dendritic cells. These results suggest that persistent exposure to Ag in conditions that fail to evoke proinflammatory stimuli leads to the development of T cells that are both anergic and regulatory.     

Cognate CD4+ help elicited by resting dendritic cells does not impair the induction of peripheral tolerance in CD8+ T cells

Peripheral tolerance is required to prevent autoimmune tissue destruction by self-reactive T cells that escape negative selection in the thymus. One mechanism of peripheral tolerance in CD8(+) T cells is their activation by resting dendritic cells (DC). In contrast, DC can be "licensed" by CD4(+) T cells to induce cytotoxic function in CD8(+) T cells. The question that then arises, whether CD4(+) T cell help could impair peripheral tolerance induction in self-reactive CD8(+) T cells, has not been addressed. In this study we show that CD4(+) T cell activation by resting DC results in helper function that transiently promotes the expansion and differentiation of cognate CD8(+) T cells. However, both the CD4(+) and CD8(+) T cell populations ultimately undergo partial deletion and acquire Ag unresponsiveness, disabling their ability to destroy OVA-expressing pancreatic beta cells and cause diabetes. Thus, effective peripheral tolerance can be induced by resting DC in the presence of CD4(+) and CD8(+) T cells with specificity for the same Ag.     

Tumor-reactive CD4+ T cell responses to the melanoma-associated chondroitin sulphate proteoglycan in melanoma patients and healthy individuals in the absence of autoimmunity

To avoid immune escape by down-regulation or loss of Ag by the tumor cells, target Ags are needed, which are important for the malignant phenotype and survival of the tumor. We could identify a CD4(+) T cell epitope derived from the human melanoma-associated chondroitin sulfate proteoglycan (MCSP) (also known as high m.w.-melanoma-associated Ag), which is strongly expressed on >90% of human melanoma lesions and is important for the motility and invasion of melanoma cells. However, MCSP is not strictly tumor specific, because it is also expressed in a variety of normal tissues. Therefore, self tolerance should prevent the induction of strong T cell responses against these Ags by vaccination strategies. In contrast, breaking self tolerance to this Ag by effectively manipulating the immune system might mediate antitumor responses, although it would bear the risk of autoimmunity. Surprisingly, we could readily isolate CD4(+) Th cells from the blood of a healthy donor-recognizing peptide MCSP(693-709) on HLA-DR11-expressing melanoma cells. Broad T cell reactivity against this Ag could be detected in the peripheral blood of both healthy donors and melanoma patients, without any apparent signs of autoimmune disease. In some patients, a decline of T cell reactivity was observed upon tumor progression. Our data indicate that CD4(+) T cells are capable of recognizing a membrane glycoprotein that is important in melanoma cell function, and it may be possible that the sizable reactivity to this Ag in most normal individuals contributes to immune surveillance against cancer.     

Enhanced engagement of CTLA-4 induces antigen-specific CD4+CD25+Foxp3+ and CD4+CD25- TGF-beta 1+ adaptive regulatory T cells

CTLA-4 is a critical negative regulator of T cell response and is instrumental in maintaining immunological tolerance. In this article, we report that enhanced selective engagement of CTLA-4 on T cells by Ag-presenting dendritic cells resulted in the induction of Ag-specific CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)TGF-beta1(+) adaptive Tregs. These cells were CD62L(low) and hyporesponsive to stimulation with cognate Ag but demonstrated a superior ability to suppress Ag-specific effector T cell response compared with their CD62L(high) counterparts. Importantly, treatment of mice with autoimmune thyroiditis using mouse thyroglobulin (mTg)-pulsed anti-CTLA-4 agonistic Ab-coated DCs, which results in a dominant engagement of CTLA-4 upon self-Ag presentation, not only suppressed thyroiditis but also prevented reemergence of the disease upon rechallenge with mTg. Further, the disease suppression was associated with significantly reduced mTg-specific T cell and Ab responses. Collectively, our results showed an important role for selective CTLA-4 signaling in the induction of adaptive Tregs and suggested that approaches that allow dominant CTLA-4 engagement concomitant with Ag-specific TCR ligation can be used for targeted therapy.     

Targeted CTLA-4 engagement induces CD4+CD25+CTLA-4high T regulatory cells with target (allo)antigen specificity

CTLA-4 (CD152) is actively involved in down-regulating T cell activation and maintaining lymphocyte homeostasis. Our earlier studies showed that targeted engagement of CTLA-4 can down-modulate T cell response and suppress allo- and autoimmune responses. In this study, we report that targeted CTLA-4 engagement can induce immune tolerance to a specific target through selective induction of an Ag-specific CD4(+)CD25(+)CTLA-4(high) regulatory T cell (Treg cell) population. Allogeneic cells coated with anti-CTLA-4 Ab induced immune hyporesponsiveness through suppression of proinflammatory cytokines IFN-gamma and IL-2, and up-regulation of the regulatory cytokines IL-10, TGF-beta1, and IL-4, presumably through the engagement of CTLA-4 on activated T cells. Although rechallenge with alloantigen failed to break the unresponsiveness, a transient recovery from tolerance was observed in the presence of high concentrations of exogenous IL-2, saturating concentrations of neutralizing anti-TGF-beta1 and anti-IL-10 Abs, and blocking anti-CTLA-4 Ab, and upon depletion of CD4(+)CD25(+) Treg cells. The CD4(+)CD25(+)CTLA-4(high) Treg cells from tolerant mice suppressed the effector function of CD25(-) T cells from Ag-primed mice. Adoptive transfer of these Treg cells into Ag-primed mice resulted in a significantly reduced alloantigen-specific response. Further characterization demonstrated that the Treg cells with memory phenotype (CD62L(-)) were more potent in suppressing the alloantigen-specific T cell response. These results strongly support that the targeted engagement of CTLA-4 has therapeutic potential for the prevention of transplant rejection.     

CD4+ T cells pass through an effector phase during the process of in vivo tolerance induction

An important process in the generation of tolerance to peripheral self-Ags is the induction of unresponsiveness in mature specific T cells. Although the end stage of this process, termed anergy, is well defined, the pathway by which naive T cells become anergic remains to be elucidated. Using an in vivo self-tolerance model, we demonstrate that CD4(+) T cells pass through a significant effector stage on their way to an anergic state. This stage is characterized by production of effector cytokines, provision of help for CD8(+) T cells, and induction of in vivo pathology within organs that express cognate Ag. These results suggest that the initial activation stage in T cell tolerance is similar to that seen in memory induction. They also suggest that autoimmune pathology can result during the natural process of tolerance induction rather than requiring that tolerance be broken.     

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.     

Evidence for a selective migration of fetus-specific CD4+CD25bright regulatory T cells from the peripheral blood to the decidua in human pregnancy

During pregnancy, the maternal immune system has to tolerate the persistence of fetal alloantigens. Many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. Murine studies suggest that CD4(+)CD25(+) T cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. Previous studies by our group demonstrate that a significantly higher percentage of activated T cells and CD4(+)CD25(bright) T cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy. In this study, we examined the phenotypic and functional properties of CD4(+)CD25(bright) T cells derived from maternal peripheral blood and decidual tissue. Depletion of CD4(+)CD25(bright) T cells from maternal peripheral blood demonstrates regulation to third party umbilical cord blood cells comparable to nonpregnant controls, whereas the suppressive capacity to umbilical cord blood cells of her own child is absent. Furthermore, maternal peripheral blood shows a reduced percentage of CD4(+)CD25(bright)FOXP3(+) and CD4(+)CD25(bright)HLA-DR(+) cells compared with peripheral blood of nonpregnant controls. In contrast, decidual lymphocyte isolates contain high percentages of CD4(+)CD25(bright) T cells with a regulatory phenotype that is able to down-regulate fetus-specific and fetus-nonspecific immune responses. These data suggest a preferential recruitment of fetus-specific regulatory T cells from maternal peripheral blood to the fetal-maternal interface, where they may contribute to the local regulation of fetus-specific responses.     

CD4+ T cells from lupus-prone mice avoid antigen-specific tolerance induction in vivo

Activated T cells in spontaneous lupus presumably bypass normal tolerance mechanisms in the periphery, since thymic tolerance appears intact. To determine whether such T cells indeed avoid in vivo peripheral tolerance mechanisms, we assessed their activation and recall responses after in vivo Ag stimulation in the absence of exogenously supplied costimulatory signals. Naive CD4(+) AND (transgenic mice bearing rearranged TCR specific for pigeon cytochrome c, peptides 88-104) TCR-transgenic T cells, specific for pigeon cytochrome c, from lupus-prone Fas-intact MRL/Mp+(Fas-lpr) and from H-2(k)-matched control CBA/CaJ and B10.BR mice (MRL.AND, CBA.AND, and B10.AND, respectively) were adoptively transferred into (MRL x CBA)F(1) or (MRL x B10)F(1) recipients transgenically expressing membrane-bound pigeon cytochrome c as a self-Ag. MRL.AND and control CBA.AND and B10.AND-transgenic T cells were activated and divided after transfer, indicating encounter with their cognate Ag; however, T cells from CBA.AND and B10.AND mice were impaired in their ability to proliferate and produce IL-2 after challenge with pigeon cytochrome c in ex vivo recall assays, a typical phenotype of anergized cells. By contrast, MRL.AND T cells proliferated more, and a significantly higher percentage of such cells produced IL-2, compared with control T cells. This observation that MRL T cells avoided anergy induction in vivo was confirmed in an in vitro system where the cells were stimulated with an anti-CD3 in the absence of a costimulatory signal. These experiments provide direct evidence that CD4(+) T cells from Fas-intact lupus-prone MRL mice are more resistant than nonautoimmune control cells to anergy induction. Anergy avoidance in the periphery might contribute to the characteristic finding in lupus of inappropriate T cell activation in response to ubiquitous self-Ags.     

CD4+CD25+Foxp3+ regulatory T cells are dispensable for controlling CD8+ T cell-mediated lung inflammation

Every person harbors a population of potentially self-reactive lymphocytes controlled by tightly balanced tolerance mechanisms. Failures in this balance evoke immune activation and autoimmunity. In this study, we investigated the contribution of self-reactive CD8(+) T lymphocytes to chronic pulmonary inflammation and a possible role for naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (nTregs) in counterbalancing this process. Using a transgenic murine model for autoimmune-mediated lung disease, we demonstrated that despite pulmonary inflammation, lung-specific CD8(+) T cells can reside quiescently in close proximity to self-antigen. Whereas self-reactive CD8(+) T cells in the inflamed lung and lung-draining lymph nodes downregulated the expression of effector molecules, those located in the spleen appeared to be partly Ag-experienced and displayed a memory-like phenotype. Because ex vivo-reisolated self-reactive CD8(+) T cells were very well capable of responding to the Ag in vitro, we investigated a possible contribution of nTregs to the immune control over autoaggressive CD8(+) T cells in the lung. Notably, CD8(+) T cell tolerance established in the lung depends only partially on the function of nTregs, because self-reactive CD8(+) T cells underwent only biased activation and did not acquire effector function after nTreg depletion. However, although transient ablation of nTregs did not expand the population of self-reactive CD8(+) T cells or exacerbate the disease, it provoked rapid accumulation of activated CD103(+)CD62L(lo) Tregs in bronchial lymph nodes, a finding suggesting an adaptive phenotypic switch in the nTreg population that acts in concert with other yet-undefined mechanisms to prevent the detrimental activation of self-reactive CD8(+) T cells.     

CD4+ NKT cells,but not conventional CD4+ T cells,are required to generate efferent CD8+ T regulatory cells following antigen inoculation in an immune-privileged site

Following inoculation of Ag into the anterior chamber (a.c.), systemic tolerance develops that is mediated in part by Ag-specific efferent CD8(+) T regulatory (Tr) cells. This model of tolerance is called a.c.-associated immune deviation. The generation of the efferent CD8(+) Tr cell in a.c.-associated immune deviation is dependent on IL-10-producing, CD1d-restricted, invariant Valpha14(+) NKT (iNKT) cells. The iNKT cell subpopulations are either CD4(+) or CD4(-)CD8(-) double negative. This report identifies the subpopulation of iNKT cells that is important for induction of the efferent Tr cell. Because MHC class II(-/-) (class II(-/-)) mice generate efferent Tr cells following a.c. inoculation, we conclude that conventional CD4(+) T cells are not needed for the development of efferent CD8(+) T cells. Furthermore, Ab depletion of CD4(+) cells in both wild-type mice (remove both conventional and CD4(+) NKT cells) and class II(-/-) mice (remove CD4(+) NKT cells) abrogated the generation of Tr cells. We conclude that CD4(+) NKT cells, but not the class II molecule or conventional CD4(+) T cells, are required for generation of efferent CD8(+) Tr cells following Ag introduction into the eye. Understanding the mechanisms that lead to the generation of efferent CD8(+) Tr cells may lead to novel immunotherapy for immune inflammatory diseases.     

Human CD4+CD25+ regulatory T cells share equally complex and comparable repertoires with CD4+CD25- counterparts

CD4(+)CD25(+) T cells are critical mediators of peripheral immune tolerance. However, many developmental and functional characteristics of these cells are unknown, and knowledge of human regulatory T cells is particularly limited. To better understand how human CD4(+)CD25(+) T cells develop and function, we examined the diversity of CD4(+)CD25(+) and CD4(+)CD25(-) T cell repertoires in both thymus and peripheral blood. Levels of T receptor excision circles (TREC) were comparable in purified CD4(+)CD25(+) and CD4(+)CD25(-) thymic populations, but were significantly higher than those in samples derived from peripheral blood, consistent with murine studies demonstrating thymic development of CD4(+)CD25(+) regulatory T cells. Surprisingly, CD4(+)CD25(-) T cells isolated from peripheral blood had greater TREC quantities than their CD4(+)CD25(+) counterparts, supporting the possibility of extrathymic expansion as well. CD4(+)CD25(+) and CD4(+)CD25(-) T cells from a given individual showed overlapping profiles with respect to diversity by Vbeta staining and spectratyping. Interestingly, CD4(+)CD25(+) T cells have lower quantities of CD3 than CD4(+)CD25(-) T cells. Collectively, these data suggest that human CD4(+)CD25(+) T cells recognize a similar array of Ags as CD4(+)CD25(-) T cells. However, reduced levels of TCR on regulatory T cells suggest different requirements for activation and may contribute to how the immune system regulates whether a particular response is suppressed or augmented.