An intrinsic mechanism predisposes Foxp3-expressing regulatory T cells to Th2 conversion in vivo.
ABSTRACT: Naturally occurring regulatory T (nTreg) cells express Foxp3 and were originally discovered as immune suppressors critical for self-tolerance and immune homeostasis. Through yet-to-be-defined mechanisms, nTreg cells were recently shown to convert into proinflammatory cells. Particularly, attenuation of Foxp3 expression led to Th2 conversion of nTreg cells in vivo. In this paper, we demonstrated an nTreg-specific mechanism controlling their Th2 conversion. We found that wild-type nTreg cells expressing reduced levels of Foxp3 but not those expressing no Foxp3 produced the Th2 cytokine IL-4. Intriguingly, IL-4 production by converted nTreg cells is required for Th2 differentiation of coexisting naive CD4 T cells in vivo, suggesting that Th2 conversion of nTreg cells might be critical for directing Th2 immune responses. Th2 conversion of nTreg cells was not due to their inability to become Th1 cells, because IFN-? was produced by Foxp3-low-expressing cells when IL-4/STAT-6 signaling was abrogated. Surprisingly, however, unlike naive CD4 T cells whose IL-4 production is dependent on STAT-6, Foxp3-low-expressing cells generated IL-4 independent of STAT-6, indicating an intrinsic mechanism that favors nTreg-to-Th2 differentiation. Indeed, compared with naive CD4 T cells, nTreg expressed elevated levels of GATA-3 independent of STAT-6. And GATA-3 was required for nTreg-to-Th2 conversion. Foxp3 may account for this GATA-3 upregulation in nTreg cells, because ectopic expression of Foxp3 preferentially promoted GATA-3 but not T-bet expression. Thus, we have identified an intrinsic mechanism that imposes a Th2/Th1 imbalance and predisposes Foxp3-expressing cells to IL-4 production independent of STAT-6 signaling.
Project description:ROR?t(+) T(H)17 cells are a proinflammatory CD4(+) T-cell population associated with autoimmune tissue injury. In mice, priming of T(H)17 requires TGF-?, which alone directs the priming of FOXP3(+) regulatory T cells (Treg), in association with inflammatory cytokines. Priming of human T(H)17 cells from conventional naive CD4(+) T cells under similar conditions, however, has proved difficult to achieve. Here, we report that differentiation of human T(H)17 cells preferentially occurs from FOXP3(+) naive Treg (NTreg) in the presence of IL-2 and IL-1? and is increased by IL-23 and TGF-?. IL-1?-mediated differentiation correlated with IL-1RI expression in stimulated NTreg and was accompanied by induction of ROR?t along with down-regulation of FOXP3. IL-17-secreting cells in NTreg cultures cosecreted TNF-? and IL-2 and contained distinct subpopulations cosecreting or not cosecreting IFN-? and other T(H)17-associated cytokines. Polarized NTreg contained significant subpopulations of CCR6-expressing cells that were highly enriched in IL-17-secreting cells. Finally, analysis of CCR6 expression with respect to that of IL-1RI identified distinct IL-17-secreting subpopulations that had maintained or lost their suppressive functions. Together our results support the concept that priming of human T(H)17 from naive CD4(+) T cells preferentially takes place from FOXP3(+) Treg precursors in the presence of lineage-specific polarizing factors.
Project description:Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-beta-mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.
Project description:Signaling events affecting thymic selection of un-manipulated polyclonal natural CD25(+)foxp3(+) regulatory T cells (nTreg) have not been established ex vivo. Here, we report a higher frequency of phosphorylated STAT-5 (pSTAT-5) in nTreg cells in the adult murine thymus and to a lesser extent in the periphery, compared to other CD4(+)CD8(-) subsets. In the neonatal thymus, the numbers of pSTAT-5(+) cells in CD25(+)foxp3(-) and nTreg cells increased in parallel, suggesting that pSTAT-5(+)CD25(+)foxp3(-) cells might represent the precursors of foxp3(+) regulatory T cells. This "specific" pSTAT-5 expression detected in nTreg cells ex vivo was likely due to a very recent signal given by IL-2/IL-15 cytokines in vivo since (i) it disappeared rapidly if cells were left unstimulated in vitro and (ii) was also observed if total thymocytes were stimulated in vitro with saturating amounts of IL-2 and/or IL-15 but not IL-7. Interestingly, STAT-5 activation upon IL-2 stimulation correlated better with foxp3 and CD122 than with CD25 expression. Finally, we show that expression of an endogenous superantigen strongly affected the early Treg cell repertoire but not the proportion of pSTAT-5(+) cells within this repertoire. Our results reveal that continuous activation of the CD122/STAT-5 signaling pathway characterize regulatory lineage differentiation in the murine thymus.
Project description:T helper type 1 (Th1) development is facilitated by interrelated changes in key intracellular factors, particularly signal transducer and activator of transcription (STAT)4, T-bet, and GATA-3. Here we show that CD4+ cells from T-bet-/- mice are skewed toward Th2 differentiation by high endogenous GATA-3 levels but exhibit virtually normal Th1 differentiation provided that GATA-3 levels are regulated at an early stage by anti-interleukin (IL)-4 blockade of IL-4 receptor (R) signaling. In addition, under these conditions, Th1 cells from T-bet-/- mice manifest IFNG promotor accessibility as detected by histone acetylation and deoxyribonuclease I hypersensitivity. In related studies, we show that the negative effect of GATA-3 on Th1 differentiation in T-bet-/- cells arises from its ability to suppress STAT4 levels, because if this is prevented by a STAT4-expressing retrovirus, normal Th1 differentiation is observed. Finally, we show that retroviral T-bet expression in developing and established Th2 cells leads to down-regulation of GATA-3 levels. These findings lead to a model of T cell differentiation that holds that naive T cells tend toward Th2 differentiation through induction of GATA-3 and subsequent down-regulation of STAT4/IL-12Rbeta2 chain unless GATA-3 levels or function is regulated by T-bet. Thus, the principal function of T-bet in developing Th1 cells is to negatively regulate GATA-3 rather than to positively regulate the IFNG gene.
Project description:Genetic and epigenetic programming of T helper (Th) cell subsets during their polarization from naive Th cells establishes long-lived memory Th cells that stably maintain their lineage signatures. However, whether memory Th cells can be redifferentiated into another Th lineage is unclear. In this study, we show that Ag-specific memory Th cells were redifferentiated into Foxp3(+) T cells by TGF-beta when stimulated in the presence of all-trans retinoic acid and rapamycin. The "converted" Foxp3(+) T cells that were derived from Th2 memory cells down-regulated GATA-3 and IRF4 and produced little IL-4, IL-5, and IL-13. Instead, the converted Foxp3(+) T cells suppressed the proliferation and cytokine production of Th2 memory cells. More importantly, the converted Foxp3(+) T cells efficiently accumulated in the airways and significantly suppressed Th2 memory cell-mediated airway hyperreactivity, eosinophilia, and allergen-specific IgE production. Our findings reveal the plasticity of Th2 memory cells and provide a strategy for adoptive immunotherapy for the treatment of allergic diseases.
Project description:Foxp3, a winged-helix family transcription factor, serves as the master switch for CD4(+) regulatory T cells (Treg). We identified a unique and evolutionarily conserved CpG-rich island of the Foxp3 nonintronic upstream enhancer and discovered that a specific site within it was unmethylated in natural Treg (nTreg) but heavily methylated in naive CD4(+) T cells, activated CD4(+) T cells, and peripheral TGFbeta-induced Treg in which it was bound by DNMT1, DNMT3b, MeCP2, and MBD2. Demethylation of this CpG site using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza) induced acetylation of histone 3, interaction with TIEG1 and Sp1, and resulted in strong and stable induction of Foxp3. Conversely, IL-6 resulted in methylation of this site and repression of Foxp3 expression. Aza plus TGFbeta-induced Treg resembled nTreg, expressing similar receptors, cytokines, and stable suppressive activity. Strong Foxp3 expression and suppressor activity could be induced in a variety of T cells, including human CD4(+)CD25(-) T cells. Epigenetic regulation of Foxp3 can be predictably controlled with DNMT inhibitors to generate functional, stable, and specific Treg.
Project description:Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFβ, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts.
Project description:Recent studies have highlighted the importance of peripheral induction of Foxp3-expressing regulatory T cells (Tregs) in the dominant control of immunological tolerance. However, Foxp3(+) Treg differentiation from naïve CD4(+) T cells occurs only under selective conditions, whereas the classical T helper (Th) 1 and 2 effector development often dominate T cell immune responses to antigen stimulation in the periphery. The reason for such disparity remains poorly understood. Here we report that Th1/Th2-polarizing cytokines can potently inhibit Foxp3(+) Treg differentiation from naïve CD4(+) precursors induced by TGF-beta. Furthermore, antigen receptor-primed CD4(+) T cells are resistant to Treg induction because of autocrine production of IFNgamma and/or IL-4, whereas neutralizing IFNgamma and IL-4 not only can potentiate TGF-beta-mediated Foxp3 induction in vitro but can also enhance antigen-specific Foxp3(+) Treg differentiation in vivo. Mechanistically, inhibition of Foxp3(+) Treg development by Th1/Th2-polarizing cytokines involves the activation of Th1/Th2 lineage transcription factors T-bet and GATA-3 through the canonical Stat1-, Stat4-, and Stat6-dependent pathways. Using IFNgamma and IL-4 knockouts and retrovirus-mediated transduction of T-bet and GATA-3, we further demonstrate that enforced expression of the Th1/Th2 lineage-specific transcription factors is sufficient to block Foxp3 induction and Treg differentiation independent of the polarizing/effector cytokines. Thus, our study has unraveled a previously unrecognized mechanism of negative cross-regulation of Foxp3(+) Treg fate choice by Th1/Th2 lineage activities. In addition, these findings also provide an attainable explanation for the general paucity of antigen-triggered de novo generation of Foxp3(+) Tregs in the periphery.
Project description:The peripheral Foxp3(+) Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4(+) T cells can be readily converted to Foxp3(+) iTreg in vitro, and memory CD4(+) T cells are resistant to conversion. In this study, we investigated the induction of Foxp3(+) T cells from various CD4(+) T-cell subsets in human peripheral blood. Though naive CD4(+) T cells were readily converted to Foxp3(+) T cells with TGF-? and IL-2 treatment in vitro, such Foxp3(+) T cells did not express the memory marker CD45RO as do Foxp3(+) T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4(+) T cells, defined as CD62L(+) central memory T cells, could be induced by TGF-? to differentiate into Foxp3(+) T cells. It is well known that Foxp3(+) T cells derived from human CD4(+)CD25(-) T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4(+)CD62L(+) central memory T cell-derived Foxp3(+) T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4(+) T cell-derived Foxp3(+) T cells. But further research showed that mouse CD4(+) central memory T cells also could be induced to differentiate into Foxp3(+) T cells, such Foxp3(+) T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4(+)CD62L(+) central memory T cells as a novel potential source of iTreg.
Project description:FOXP3+ regulatory T cell (Treg) based cellular therapies represent promising therapeutic options in autoimmunity, allergy, transplantation and prevention of Graft Versus Host (GVH) Disease. Among human FOXP3-expressing CD4+T cells, only the CD45RA+ naïve Treg (nTreg) subset is suitable for in vitro expansion. However, FoxP3 expression decays in cells using currently described culture protocols. Rapamycin alone was not able to prevent FOXP3 loss in nTregs cells, as only a half of them maintained FOXP3 expression after 14 days of culture. In contrast we report a novel combined drug regimen that can drastically stabilize FOXP3 expression in cultured Tregs. IL-2, rapamycin, histone deacetylase and DNA methyltransferase inhibitors act in synergy to allow expansion of human regulatory T cells with sustained high expression of FOXP3 and CD15s with potent suppressive capacities in vitro and control of murine xeno-GVH reactions. Of note, an additional subsequent infusion of expanded nTreg cells did not improve survival of mice. Combination of IL-2, rapamycin, histone deacetylase and DNA methyltransferase inhibitors is optimal for the expansion in vitro of pure effective nTreg maintaining high levels of FOXP3 for therapeutic purposes.