The transcription factor Foxp3 is essential for the development of regulatory T (Treg) cells, yet its expression is insufficient for establishing the Treg cell lineage. Here we showed that Treg cell development was achieved by the combination of two independent processes, i.e., the expression of Foxp3 and the establishment of Treg cell-specific CpG hypomethylation pattern. Both events were induced by T cell receptor stimulation. The Treg cell-type CpG hypomethylation began in the thymus and continued to proceed in the periphery and could be fully established without Foxp3. The hypomethylation was required for Foxp3(+) T cells to acquire Treg cell-type gene expression, lineage stability, and full suppressive activity. Thus, those T cells in which the two events have concurrently occurred are developmentally set into the Treg cell lineage. This model explains how Treg cell fate and plasticity is controlled and can be exploited to generate functionally stable Treg cells.

Foxp3-expressing CD4+CD25+ regulatory T cells (Tregs) constitutively and highly express the immune checkpoint receptor cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), whose Treg-specific deficiency causes severe systemic autoimmunity. As a key mechanism of Treg-mediated suppression, Treg-expressed CTLA-4 down-regulates the expression of CD80/CD86 costimulatory molecules on antigen-presenting cells (APCs). Here, we show that Treg-expressed CTLA-4 facilitated Treg-APC conjugation and immune synapse formation. The immune synapses thus formed provided a stable platform whereby Tregs were able to deplete CD80/CD86 molecules on APCs by extracting them via CTLA-4–dependent trogocytosis. The depletion occurred even with Tregs solely expressing a mutant CTLA-4 form lacking the cytoplasmic portion required for its endocytosis. The CTLA-4–dependent trogocytosis of CD80/CD86 also accelerated in vitro and in vivo passive transfer of other membrane proteins and lipid molecules from APCs to Tregs without their significant reduction on the APC surface. Furthermore, CD80 down-regulation or blockade by Treg-expressed membrane CTLA-4 or soluble CTLA-4-immunoglobulin (CTLA-4-Ig), respectively, disrupted cis-CD80/programmed death ligand-1 (PD-L1) heterodimers and increased free PD-L1 on dendritic cells (DCs), expanding a phenotypically distinct population of CD80lo free PD-L1hi DCs. Thus, Tregs are able to inhibit the T cell stimulatory activity of APCs by reducing their CD80/CD86 expression via CTLA-4–dependent trogocytosis. This CD80/CD86 reduction on APCs is able to exert dual suppressive effects on T cell immune responses by limiting CD80/CD86 costimulation to naïve T cells and by increasing free PD-L1 available for the inhibition of programmed death-1 (PD-1)–expressing effector T cells. Blockade of CTLA-4 and PD-1/PD-L1 in combination may therefore synergistically hinder Treg-mediated immune suppression, thereby effectively enhancing immune responses, including tumor immunity.

Thymus-produced CD4+ regulatory T (Treg) cells, which specifically express the transcription factor forkhead box p3, are potently immunosuppressive and characteristically possess a self-reactive T-cell receptor (TCR) repertoire. To determine the molecular basis of Treg suppressive activity and their self-skewed TCR repertoire formation, we attempted to reconstruct these Treg-specific properties in conventional T (Tconv) cells by genetic manipulation. We show that Tconv cells rendered IL-2 deficient and constitutively expressing transgenic cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) were potently suppressive in vitro when they were preactivated by antigenic stimulation. They also suppressed in vivo inflammatory bowel disease and systemic autoimmunity/ inflammation produced by Treg deficiency. In addition, in the thymus, transgenic CTLA-4 expression in developing Tconv cells skewed their TCR repertoire toward higher self-reactivity, whereas CTLA-4 deficiency specifically in developing thymic Treg cells cancelled their physiological TCR self-skewing. The extracellular portion of CTLA-4 was sufficient for the suppression and repertoire shifting. It interfered with CD28 signaling to responder Tconv cells via outcompeting CD28 for binding to CD80 and CD86, or modulating CD80/CD86 expression on antigen-presenting cells. Thus, a triad of IL-2 repression, CTLA-4 expression, and antigenic stimulation is a minimalistic requirement for conferring Treg-like suppressive activity on Tconv cells, in accordance with the function of forkhead box p3 to strongly repress IL-2 and maintain CTLA-4 expression in natural Treg cells. Moreover, CTLA-4 expression is a key element for the formation of a self-reactive TCR repertoire in natural Treg cells. These findings can be exploited to control immune responses by targeting IL-2 and CTLA-4 in Treg and Tconv cells.immune tolerance | CD25 | thymic selection

Bone morphogenetic proteins (BMPs) are involved in patterning and cellular fate in various organs including the thymus. However, the redundancy of BMPs and their receptors have made it difficult to analyse their physiological roles. Here, we investigated the role of BMP signalling in peripheral CD4 1 T cells by analysing the effects of an inhibitor of BMP signalling, dorsomorphin. Dorsomorphin suppressed phosphorylation of SMAD1/5/8, suggesting that BMP signalling naturally occurs in T cells. At high doses, dorsomorphin suppressed proliferation of T cells in a dose-dependent manner, inducing G1 arrest. Also, dorsomorphin suppressed Th17 and induced Treg-cell differentiation, while preserving Th2 differentiation. Dorsomorphin efficiently suppressed IL-2 production even at low doses in mouse CD4 1 T cells, suggesting that the BMP-Smad signalling physiologically regulates IL-2 transcription in these cells. In addition, recombinant BMP2 induced a dosedependent multiphasic pattern of IL-2 production, while Noggin suppressed IL-2 production at higher doses in Jurkat cells. Notably, BMP signalling controlled the phosphorylation of RUNX1, revealing the molecular nature of its effect. Collectively, we describe multiple effects of dorsomorphin and Noggin on T-cell activation and differentiation, demonstrating a physiological role for BMP signalling in these processes.

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Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8–. CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

CTLA-4 exists as membrane (mCTLA-4) and soluble (sCTLA-4) forms. Here, we found that effector-type regulatory T cells (Tregs) are main sCTLA-4 producers in basal and inflammatory states. We generated sCTLA-4-intact mCTLA-4-deficient mice, which spontaneously developed systemic inflammation distinct from that in double-deficient mice, with predominant activation/differentiation of Th2, M2-like macrophages, and eosinophils. In contrast, sCTLA-4-deficient mCTLA-4-intact mice exhibited spontaneous activation of Th1, Th17, Tfh, and Tc1 cells, production of autoantibody and IgE, M1-like macrophage polarization, and impaired wound healing. Consistently, recombinant sCTLA-4 inhibited in vitro differentiation of naive T cells towards Th1, while allowing Th2 differentiation, through CD80/CD86 blockade on antigen-presenting cells. Moreover, sCTLA-4-intact mCTLA-4-deficient Tregs effectively suppressed Th1-mediated experimental colitis whereas double-deficient Tregs did not. Thus, sCTLA-4 not only suppresses T-cell activation in the steady state but also selectively inhibits type 1 in favor of type 2 immunity during chronic inflammation. sCTLA-4 is, therefore, a viable therapeutic candidate for inflammatory diseases and cancer.

Regulatory T cells (Tregs) are responsible for the maintenance of immune homeostasis. They express the coinhibitory receptor CTLA-4 in high quantities, whose loss causes severe systemic autoinflammation in both mice and humans. CTLA-4 limits the expression of costimulatory CD80/CD86 proteins by antigen-presenting cells (APCs). However, the details of this mechanism are not fully understood. When we analyzed in vitro cocultures of CD80- or CD86-GFP expressing murine dendritic cells (DCs) with ex vivo sorted Tregs, we found that mutant Tregs, whose CTLA-4 lacked the cytoplasmic tail necessary for its endocytosis function, could still uptake CD80- or CD86-GFP from DCs via CTLA-4-dependent trogocytosis. Moreover, the specific CD80/86 uptake by CTLA-4, also facilitated the indirect transfer of membrane lipid particles and other surface proteins. Both flow cytometry and confocal microscopy revealed that immune synapse formation between Tregs and APCs was enhanced depending on CTLA-4 expression by Tregs. Confocal microscopy also demonstrated that uptaken CD80/CD86-GFP proteins colocalized with LFA-1 capping, an indicator of stable immune synapses. Furthermore, in vivo delivery of CTLA-4 WT and knockout (KO) Tregs into CD45.1+RAG2 KO mice showed that transfer of congenic CD45.1 protein from host cells to donor Tregs was significantly diminished in CTLA-4 KO Tregs compared to WT Tregs. The tail portion of CTLA-4 was also found to be dispensable for CD80/86 downregulation and suppressive functions of Tregs. In conclusion, CTLA-4-promoted immune synapse formation provides a stable platform between Tregs and APCs, which results in both depletion of CD80/CD86 via trogocytosis and indirect uptake of other surface proteins from APCs.