Effector CD4+ T cell subsets, whose differentiation is facilitated by distinct cytokine cues, amplify the corresponding type of inflammatory response. Regulatory T (Treg) cells integrate environmental cues to suppress particular types of inflammation. In this regard, STAT3, a transcription factor essential for T helper 17 (Th17) cell differentiation, is necessary for Treg cell-mediated control of Th17 cell responses. Here, we showed that anti-inflammatory interleukin-10 (IL-10), and not proinflammatory IL-6 and IL-23 cytokine signaling, endowed Treg cells with the ability to suppress pathogenic Th17 cell responses. Ablation of the IL-10 receptor in Treg cells resulted in selective dysregulation of Th17 cell responses and colitis similar to that observed in mice harboring STAT3-deficient Treg cells. Thus, Treg cells limit Th17 cell inflammation by serving as principal amplifiers of negative regulatory circuits operating in immune effector cells.

The mammalian gut microbiota provides essential metabolites to the host and promotes the differentiation and accumulation of extrathymically generated regulatory T (pTreg) cells. To explore the impact of these cells on intestinal microbial communities, we assessed the composition of the microbiota in pTreg cell-deficient and -sufficient mice. pTreg cell deficiency led to heightened type 2 immune responses triggered by microbial exposure, which disrupted the niche of border-dwelling bacteria early during colonization. Moreover, impaired pTreg cell generation led to pervasive changes in metabolite profiles, altered features of the intestinal epithelium, and reduced body weight in the presence of commensal microbes. Absence of a single species of bacteria depleted in pTreg cell-deficient animals, Mucispirillum schaedleri, partially accounted for the sequelae of pTreg cell deficiency. These observations suggest that pTreg cells modulate the metabolic function of the intestinal microbiota by restraining immune defense mechanisms that may disrupt a particular bacterial niche.

Innate lymphoid cells (ILCs) are generated early during ontogeny and persist predominantly as tissue-resident cells. Here, we examined how ILCs are maintained and renewed within tissues. We generated a single cell atlas of lung ILC2s and found that Il18r1+ ILCs comprise circulating and tissue-resident ILC progenitors (ILCP) and effector-cells with heterogeneous expression of the transcription factors Tcf7 and Zbtb16, and CD103. Our analyses revealed a continuous differentiation trajectory from Il18r1+ ST2- ILCPs to Il18r- ST2+ ILC2s, which was experimentally validated. Upon helminth infection, recruited and BM-derived cells generated the entire spectrum of ILC2s in parabiotic and shield chimeric mice, consistent with their potential role in the renewal of tissue ILC2s. Our findings identify local ILCPs and reveal ILCP in situ differentiation and tissue adaptation as a mechanism of ILC maintenance and phenotypic diversification. Local niches, rather than progenitor origin, or the developmental window during ontogeny, may dominantly imprint ILC phenotypes in adult tissues.

Regulatory T (Treg) cells reside in lymphoid organs and barrier tissues where they control different types of inflammatory responses. Treg cells are also found in human cancers, and studies in animal models suggest that they contribute to cancer progression. However, properties of human intratumoral Treg cells and those present in corresponding normal tissue remain largely unknown. Here, we analyzed features of Treg cells in untreated human breast carcinomas, normal mammary gland, and peripheral blood. Tumor-resident Treg cells were potently suppressive and their gene-expression pattern resembled that of normal breast tissue, but not of activated peripheral blood Treg cells. Nevertheless, a number of cytokine and chemokine receptor genes, most notably CCR8, were upregulated in tumor-resident Treg cells in comparison to normal tissue-resident ones. Our studies suggest that targeting CCR8 for the depletion of tumor-resident Treg cells might represent a promising immunotherapeutic approach for the treatment of breast cancer.

Activation of the STAT5 transcription factor downstream of the Interleukin-2 receptor (IL-2R) induces expression of Foxp3, a critical step in the differentiation of regulatory T (Treg) cells. Due to the pleiotropic effects of IL-2R signaling, it is unclear how STAT5 acts directly on the Foxp3 locus to promote its expression. Here, we report that IL-2 - STAT5 signaling converged on an enhancer (CNS0) during Foxp3 induction. CNS0 facilitated the IL-2 dependent CD25+Foxp3- precursor to Treg cell transition in the thymus. Its deficiency resulted in impaired Treg cell generation in neonates, which was partially mitigated with age. While the thymic Treg cell paucity caused by CNS0 deficiency did not result in autoimmunity on its own, it exacerbated autoimmune manifestations caused by disruption of the Aire gene. Thus, CNS0 enhancer activity ensures robust Treg cell differentiation early in postnatal life and cooperatively with other tolerance mechanisms minimizes autoimmunity.

The regulatory T (Treg) cells restrain immune responses through suppressor-function elaboration that is dependent upon expression of the transcription factor Foxp3. Despite a critical role for Treg cells in maintaining lympho-myeloid homeostasis, it remains unclear whether a single mechanism or multiple mechanisms of Treg cell-mediated suppression are operating in vivo and how redundant such mechanisms might be. Here we addressed these questions by examining the role of the immunomodulatory cytokine IL-10 in Treg cell-mediated suppression. Analyses of mice in which the Treg cell-specific ablation of a conditional IL-10 allele was induced by Cre recombinase knocked into the Foxp3 gene locus showed that although IL-10 production by Treg cells was not required for the control of systemic autoimmunity, it was essential for keeping immune responses in check at environmental interfaces such as the colon and lungs. Our study suggests that Treg cells utilize multiple means to limit immune responses. Furthermore, these mechanisms are likely to be nonredundant, in that a distinct suppressor mechanism most likely plays a prominent and identifiable role at a particular tissue and inflammatory setting.

Stable changes in chromatin states and gene expression in cells of the immune system form the basis for memory of infections and other challenges. Here, we used naturally occurring cis-regulatory variation in wild-derived inbred mouse strains to explore the mechanisms underlying long-lasting versus transient gene regulation in CD8 T cells responding to acute viral infection. Stably responsive DNA elements were characterized by dramatic and congruent chromatin remodeling events affecting multiple neighboring sites and required distinct transcription factor (TF) binding motifs for their accessibility. Specifically, we found that cooperative recruitment of T-box and Runx family transcription factors to shared targets mediated stable chromatin remodeling upon T cell activation. Our observations provide insights into the molecular mechanisms driving virus-specific CD8 T cell responses and suggest a general mechanism for the formation of transcriptional and epigenetic memory applicable to other immune and non-immune cells.

MicroRNA (miRNA)-dependent regulation of gene expression confers robustness to cellular phenotypes and controls responses to extracellular stimuli. Although a single miRNA can regulate expression of hundreds of target genes, it is unclear whether any of its distinct biological functions can be due to the regulation of a single target. To explore in vivo the function of a single miRNA-mRNA interaction, we mutated the 3' UTR of a major miR-155 target (SOCS1) to specifically disrupt its regulation by miR-155. We found that under physiologic conditions and during autoimmune inflammation or viral infection, some immunological functions of miR-155 were fully or largely attributable to the regulation of SOCS1, whereas others could be accounted only partially or not at all by this interaction. Our data suggest that the role of a single miRNA-mRNA interaction is dependent on cell type and biological context.

Regulatory T (Treg) cell identity is defined by the lineage-specifying transcription factor (TF) Foxp3. Here we examined mechanisms of Foxp3 function by leveraging naturally occurring genetic variation in wild-derived inbred mice, which enables the identification of DNA sequence motifs driving epigenetic features. Chromatin accessibility, TF binding, and gene expression patterns in resting and activated subsets of Treg cells, conventional CD4 T cells, and cells expressing a Foxp3 reporter null allele revealed that the majority of Foxp3-dependent changes occurred at sites not bound by Foxp3. Chromatin accessibility of these indirect Foxp3 targets depended on the presence of DNA binding motifs for other TFs, including TCF1. Foxp3 expression correlated with decreased TCF1 and reduced accessibility of TCF1-bound chromatin regions. Deleting one copy of the Tcf7 gene recapitulated Foxp3-dependent negative regulation of chromatin accessibility. Thus, Foxp3 defines Treg cell identity in a largely indirect manner by fine-tuning the activity of other major chromatin remodeling TFs such as TCF1.

FoxP3 is an essential transcription factor (TF) for immunologic homeostasis, but how it utilizes the common forkhead DNA-binding domain (DBD) to perform its unique function remains poorly understood. We here demonstrated that unlike other known forkhead TFs, FoxP3 formed a head-to-head dimer using a unique linker (Runx1-binding region [RBR]) preceding the forkhead domain. Head-to-head dimerization conferred distinct DNA-binding specificity and created a docking site for the cofactor Runx1. RBR was also important for proper folding of the forkhead domain, as truncation of RBR induced domain-swap dimerization of forkhead, which was previously considered the physiological form of FoxP3. Rather, swap-dimerization impaired FoxP3 function, as demonstrated with the disease-causing mutation R337Q, whereas a swap-suppressive mutation largely rescued R337Q-mediated functional impairment. Altogether, our findings suggest that FoxP3 can fold into two distinct dimerization states: head-to-head dimerization representing functional specialization of an ancient DBD and swap dimerization associated with impaired functions.

Regulatory T (Treg) cells maintain immune tolerance through the master transcription factor forkhead box P3 (FOXP3), which is crucial for Treg cell function and homeostasis. We identified an IPEX (immune dysregulation polyendocrinopathy enteropathy X-linked) syndrome patient with a FOXP3 mutation in the domain swap interface of the protein. Recapitulation of this Foxp3 variant in mice led to the development of an autoimmune syndrome consistent with an unrestrained T helper type 2 (Th2) immune response. Genomic analysis of Treg cells by RNA-sequencing, Foxp3 chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-sequencing), and H3K27ac-HiChIP revealed a specific de-repression of the Th2 transcriptional program leading to the generation of Th2-like Treg cells that were unable to suppress extrinsic Th2 cells. Th2-like Treg cells showed increased intra-chromosomal interactions in the Th2 locus, leading to type 2 cytokine production. These findings identify a direct role for Foxp3 in suppressing Th2-like Treg cells and implicate additional pathways that could be targeted to restrain Th2 trans-differentiated Treg cells.

TGF-β1 is a regulatory cytokine that has an important role in controlling T cell differentiation. T cell-produced TGF-β1 acts on T cells to promote Th17 cell differentiation and the development of experimental autoimmune encephalomyelitis (EAE). However, the exact TGF-β1-producing T cell subset required for Th17 cell generation and its cellular mechanism of action remain unknown. Here we showed that deletion of the Tgfb1 gene from activated T cells and Treg cells, but not Treg cells alone, abrogated Th17 cell differentiation, resulting in almost complete protection from EAE. Furthermore, differentiation of T cells both in vitro and in vivo demonstrated that TGF-β1 was highly expressed by Th17 cells and acted in a predominantly autocrine manner to maintain Th17 cells in vivo. These findings reveal an essential role for activated T cell-produced TGF-β1 in promoting the differentiation of Th17 cells and controlling inflammatory diseases.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are an essential suppressive T cell lineage of dual origin: Foxp3 induction in thymocytes and mature CD4+ T cells gives rise to thymic (tTreg) and peripheral (pTreg) Treg cells, respectively. While tTreg cells suppress autoimmunity, pTreg cells enforce tolerance to food and commensal microbiota. However, the role of Foxp3 in pTreg cells and the mechanisms supporting their differentiation remain poorly understood. Here, we used genetic tracing to identify microbiota-induced pTreg cells and found that many of their distinguishing features were Foxp3 independent. Lineage-committed, microbiota-dependent pTreg-like cells persisted in the colon in the absence of Foxp3. While Foxp3 was critical for the suppression of a Th17 cell program, colitis, and mastocytosis, pTreg cells suppressed colonic effector T cell expansion in a Foxp3-independent manner. Thus, Foxp3 and the tolerogenic signals that precede and promote its expression independently confer distinct facets of pTreg functionality.