A self-reactive TCR drives the development of Foxp3+ regulatory T cells that prevent autoimmune disease

Although Foxp3(+) regulatory T cells (Tregs) are thought to express autoreactive TCRs, it is not clear how individual TCRs influence Treg development, phenotype, and function in vivo. We have generated TCR transgenic mice (termed SFZ70 mice) using Tcra and Tcrb genes cloned from an autoreactive CD4(+) T cell isolated from a Treg-deficient scurfy mouse. The SFZ70 TCR recognizes a cutaneous autoantigen and drives development of both conventional CD4(+) Foxp3(-) T cells (T(conv)) and Foxp3(+) Tregs. SFZ70 Tregs display an activated phenotype evidenced by robust proliferation and expression of skin-homing molecules such as CD103 and P-selectin ligand. Analysis of Foxp3-deficient SFZ70 mice demonstrates that Tregs inhibit T(conv) cell expression of tissue-homing receptors and their production of proinflammatory cytokines. In addition, Treg suppression of SFZ70 T(conv) cells can be overcome by nonspecific activation of APCs. These results provide new insights into the differentiation and function of tissue-specific Tregs in vivo and provide a tractable system for analyzing the molecular requirements of Treg-mediated tolerance toward a cutaneous autoantigen.     

Mechanisms underlying blockade of allograft acceptance by TLR ligands

Immune activation via TLRs is known to prevent transplantation tolerance in multiple animal models. To investigate the mechanisms underlying this barrier to tolerance induction, we used complementary murine models of skin and cardiac transplantation in which prolonged allograft acceptance is either spontaneous or pharmacologically induced with anti-CD154 mAb and rapamycin. In each model, we found that prolonged allograft survival requires the presence of natural CD4(+)Foxp3(+) T regulatory cells (Tregs), and that the TLR9 ligand CpG prevents graft acceptance both by interfering with natural Treg function and by promoting the differentiation of Th1 effector T cells in vivo. We further demonstrate that although Th17 cells differentiate from naive alloreactive T cells, these cells do not arise from natural Tregs in either CpG-treated or untreated graft recipients. Finally, we show that CpG impairs natural Treg suppressor capability and prevents Treg-dependent allograft acceptance in an IL-6-independent fashion. Our data therefore suggest that TLR signals do not prevent prolonged graft acceptance by directing natural Tregs into the Th17 lineage or by using other IL-6-dependent mechanisms. Instead, graft destruction results from the ability of CpG to drive Th1 differentiation and interfere with immunoregulation established by alloreactive natural CD4(+)Foxp3(+) Tregs.     

The link between the PDL1 costimulatory pathway and Th17 in fetomaternal tolerance

Fetomaternal tolerance has been shown to depend both on regulatory T cells (Tregs) and negative signals from the PD1-PDL1 costimulatory pathway. More recently, IL-17-producing T cells (Th17) have been recognized as a barrier in inducing tolerance in transplantation. In this study, we investigate the mechanisms of PDL1-mediated regulation of fetomaternal tolerance using an alloantigen-specific CD4(+) TCR transgenic mouse model system (ABM-tg mouse). PDL1 blockade led to an increase in embryo resorption and a reduction in litter size. This was associated with a decrease in Tregs, leading to a lower Treg/effector T cell ratio. Moreover, PDL1 blockade inhibited Ag-specific alloreactive T cell apoptosis and induced apoptosis of Tregs and a shift toward higher frequency of Th17 cells, breaking fetomaternal tolerance. These Th17 cells arose predominantly from CD4(+)Foxp3(-) cells, rather than from conversion of Tregs. Locally in the placenta, similar decrease in regulatory and apoptotic markers was observed by real-time PCR. Neutralization of IL-17 abrogated the anti-PDL1 effect on fetal survival rate and restored Treg numbers. Finally, the adoptive transfer of Tregs was also able to improve fetal survival in the setting of PDL1 blockade. This is to our knowledge the first report using an alloantigen-specific model that establishes a link between PDL1, Th17 cells, and fetomaternal tolerance.     

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.     

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.     

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.     

Surfactant protein A modulates induction of regulatory T cells via TGF-β

TCR signaling plays a critical role in regulatory T cell (Treg) development. However, the mechanism for tissue-specific induction of Tregs in the periphery remains unclear. We observed that surfactant protein A (SP-A)-deficient mice have impaired expression of Foxp3 and fewer CD25(+)Foxp3(+) Tregs after ex vivo stimulation and after stimulation with LPS in vivo. The addition of exogenous SP-A completely reversed this phenotype. Although SP-A is known to inhibit T cell proliferation under certain activation conditions, both IL-2 levels as well as active TGF-β levels increase on extended culture with exogenous SP-A, providing a key mechanism for the maintenance and induction of Tregs. In addition, kinetic suppression assays demonstrate that SP-A enhances the frequency of functional Foxp3(+) Tregs in responder T cell populations in a TGF-β-dependent manner. In mice treated with LPS in vivo, Tregs increased ～160% in wild-type mice compared with only a 50% increase in LPS-treated SP-A(-/-) mice 8 d after exposure. Taken together, these findings support the hypothesis that SP-A affects T cell immune function by the induction of Tregs during activation.     

Requirement for diverse TCR specificities determines regulatory T cell activity in a mouse model of autoimmune arthritis

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are required to restrain the immune system from mounting an autoaggressive systemic inflammatory response, but why their activity can prevent (or allow) organ-specific autoimmunity remains poorly understood. We have examined how TCR specificity contributes to Treg activity using a mouse model of spontaneous autoimmune arthritis, in which CD4(+) T cells expressing a clonotypic TCR induce disease by an IL-17-dependent mechanism. Administration of polyclonal Tregs suppressed Th17 cell formation and prevented arthritis development; notably, Tregs expressing the clonotypic TCR did not. These clonotypic Tregs exerted Ag-specific suppression of effector CD4(+) T cells using the clonotypic TCR in vivo, but failed to mediate bystander suppression and did not prevent Th17 cells using nonclonotypic TCRs from accumulating in joint-draining lymph nodes of arthritic mice. These studies indicate that the availability of Tregs with diverse TCR specificities can be crucial to their activity in autoimmune arthritis.     

TIM-3: a novel regulatory molecule of alloimmune activation

T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4(+)FoxP3(-) and CD8(+) T cells in acutely rejecting graft recipients. A blocking anti-TIM-3 mAb accelerated allograft rejection only in the presence of host CD4(+) T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ-, IL-6-, and IL-17-producing splenocytes, enhanced CD8(+) cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4(+) T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4(+) T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.     

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.     

Pulmonary CCL18 recruits human regulatory T cells

CCL18 is both a constitutively expressed and an inducible chemokine, whose role in the inflammatory reaction is poorly known. The aim of this study was to evaluate whether CCL18 has the capacity to attract human T cells with a regulatory function (regulatory T cells [Treg]). Results from chemotaxis assays performed on different types of Treg showed that CD4(+)CD25(+)CD127(low) cells, but neither T regulatory type 1 clones nor Treg differentiated in vitro with anti-CD3/CD46 mAbs, were recruited by CCL18 in a dose-dependent manner. CCL18-recruited memory CD4(+) T cells were enriched in CD25(high), CD25(+)CD127(low), latency-associated peptide/TGF-β1, and CCR4-expressing T cells, whereas there was no enrichment in Foxp3(+) cells as compared with controls. Stimulated CCL18-recruited memory T cells produced significantly increased amounts of the regulatory cytokines IL-10 and TGF-β1, as well as IL-4, but not IFN-γ and IL-17. Cell surface CCL18 binding was found predominantly on IL-10(+) (26.3 ± 5.8%) and on a few latency-associated peptide/TGF-β1(+) (18.1 ± 1.9%) and IL-4(+) (14.5 ± 2.9%) memory T cells. In an in vivo model of SCID mice grafted with human skin and reconstituted with autologous PBMCs, the intradermal injection of CCL18 led to the cutaneous recruitment of CD4(+), CD25(+), and IL-10(+) cells, but not Foxp3(+) cells. Furthermore, CCL18-recruited memory T cells inhibited the proliferation of CD4(+)CD25(-) effector T cells through an IL-10-dependent mechanism. These data suggest that CCL18 may contribute to maintaining tolerance and/or suppressing deleterious inflammation by attracting memory Tregs into tissues, particularly in the lung, where it is highly and constitutively expressed.     

Nitric oxide modulates TGF-beta-directive signals to suppress Foxp3+ regulatory T cell differentiation and potentiate Th1 development

TGF-β can induce Foxp3(+) inducible regulatory T cells (Treg) and also synergize with IL-6 and IL-4 to induce Th17 and Th9 cells. We now report that NO modulates TGF-β activity away from Treg but toward the Th1 lineage. NO potentiated Th1 differentiation in the presence of TGF-β in both IL-12-independent and -dependent fashions by augmenting IFN-γ-activated STAT-1 and T-bet. Differentiation into Treg, Th1, and Th17 lineages could be modulated by NO competing with other cofactors, such as IL-6 and retinoic acid. NO antagonized IL-6 to block TGF-β-directed Th17 differentiation, and together with IL-6, NO suppressed Treg development induced by TGF-β and retinoic acid. Furthermore, we show that physiologically produced NO from TNF and inducible NO synthase-producing dendritic cells can contribute to Th1 development predominating over Treg development through a synergistic activity induced when these cells cocluster with conventional dendritic cells presenting Ag to naive Th cells. This illustrates that NO is another cofactor allowing TGF-β to participate in development of multiple Th lineages and suggests a new mechanism by which NO, which is associated with protection against intracellular pathogens, might maintain effective Th1 immunity.     

IL-17+ regulatory T cells in the microenvironments of chronic inflammation and cancer

Foxp3(+)CD4(+) regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17(+)CD4(+) T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17(+)Foxp3(+)CD4(+) T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17(+)Foxp3(+)CD4(+) T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17(+)Foxp3(+)CD4(+) T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17(+)Foxp3(+)CD4(+) T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6(+) T cells or Treg cells. IL-17(+)Foxp3(+)CD4(+) T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17(+)Foxp3(+) cells may be "inflammatory" Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.     

The influence of excessive IL-6 production in vivo on the development and function of Foxp3+ regulatory T cells

IL-6 is a proinflammatory cytokine and its overproduction is implicated in a variety of inflammatory disorders. Recent in vitro analyses suggest that IL-6 is a key cytokine that determines the balance between Foxp3(+) regulatory T cells (Tregs) and Th17 cells. However, it remains unclear whether excessive IL-6 production in vivo alters the development and function of Foxp3(+) Tregs. In this study, we analyzed IL-6 transgenic (Tg) mice in which serum IL-6 levels are constitutively elevated. Interestingly, in IL-6 Tg mice, whereas peripheral lymphoid organs were enlarged, and T cells exhibited activated phenotype, Tregs were not reduced but rather increased compared with wild-type mice. In addition, Tregs from Tg mice normally suppressed proliferation of naive T cells in vitro. Furthermore, Tregs cotransferred with naive CD4 T cells into SCID-IL-6 Tg mice inhibited colitis as successfully as those transferred into control SCID mice. These results indicate that overproduction of IL-6 does not inhibit development or function of Foxp3(+) Tregs in vivo. However, when naive CD4 T cells alone were transferred, Foxp3(+) Tregs retrieved from SCID-IL-6 Tg mice were reduced compared with SCID mice. Moreover, the Helios(-) subpopulation of Foxp3(+) Tregs, recently defined as extrathymic Tregs, was significantly reduced in IL-6 Tg mice compared with wild-type mice. Collectively, these results suggest that IL-6 overproduced in vivo inhibits inducible Treg generation from naive T cells, but does not affect the development and function of natural Tregs.     

Antigen, in the presence of TGF-beta, induces up-regulation of FoxP3gfp+ in CD4+ TCR transgenic T cells that mediate linked suppression of CD8+ T cell responses

CD4(+)CD25(+) regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred K(d)-specific, CD4(+) TCR (TCR75) transgenic mice to Foxp3(gfp) knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3(gfp).TCR75 mice did not express significant numbers of natural FoxP3(+) Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with K(d) plus TGF-beta. The resulting GFP(+) TCR75 cells were anergic, whereas the GFP(-) TCR75 cells proliferated upon restimulation with K(d) peptide. Yet both exhibited severely reduced expression of intracellular IFN-gamma and TNF-alpha upon restimulation. GFP(+), but not GFP(-), TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP(+) TCR75 cells also inhibited polyclonal C57BL/6 anti-K(d) CTL responses if the APC expressed K(d) and both MHC class I and class II, and responses by OT1 T cells to B6.K(d).OVA but not B6.K(d) plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.