Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses.

Foxp3(+) regulatory T (Treg) cells maintain immune homeostasis by limiting different types of inflammatory responses. Here, we report that miR-146a, one of the miRNAs prevalently expressed in Treg cells, is critical for their suppressor function. The deficiency of miR-146a in Treg cells resulted in a breakdown of immunological tolerance manifested in fatal IFNγ-dependent immune-mediated lesions in a variety of organs. This was likely due to augmented expression and activation of signal transducer and activator transcription 1 (Stat1), a direct target of miR-146a. Likewise, heightened Stat1 activation in Treg cells subjected to a selective ablation of SOCS1, a key negative regulator of Stat1 phosphorylation downstream of the IFNγ receptor, was associated with analogous Th1-mediated pathology. Our results suggest that specific aspects of Treg suppressor function are controlled by a single miRNA and that an optimal range of Stat1 activation is important for Treg-mediated control of Th1 responses and associated autoimmunity.

Transcription factor Foxp3 and its protein partners form a complex regulatory network.

The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (T(reg) cells). To gain insights into the molecular mechanisms of Foxp3-mediated gene expression, we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multiprotein complexes of 400-800 kDa or larger and identified 361 associated proteins, ∼30% of which were transcription related. Foxp3 directly regulated expression of a large proportion of the genes encoding its cofactors. Some transcription factor partners of Foxp3 facilitated its expression. Functional analysis of the cooperation of Foxp3 with one such partner, GATA-3, provided additional evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of T(reg) cell biology.

A Single miRNA-mRNA Interaction Affects the Immune Response in a Context- and Cell-Type-Specific Manner.

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.

Stability and function of regulatory T cells expressing the transcription factor T-bet.

Adaptive immune responses are tailored to different types of pathogens through differentiation of naive CD4 T cells into functionally distinct subsets of effector T cells (T helper 1 (TH1), TH2, and TH17) defined by expression of the key transcription factors T-bet, GATA3, and RORγt, respectively. Regulatory T (Treg) cells comprise a distinct anti-inflammatory lineage specified by the X-linked transcription factor Foxp3 (refs 2, 3). Paradoxically, some activated Treg cells express the aforementioned effector CD4 T cell transcription factors, which have been suggested to provide Treg cells with enhanced suppressive capacity. Whether expression of these factors in Treg cells-as in effector T cells-is indicative of heterogeneity of functionally discrete and stable differentiation states, or conversely may be readily reversible, is unknown. Here we demonstrate that expression of the TH1-associated transcription factor T-bet in mouse Treg cells, induced at steady state and following infection, gradually becomes highly stable even under non-permissive conditions. Loss of function or elimination of T-bet-expressing Treg cells-but not of T-bet expression in Treg cells-resulted in severe TH1 autoimmunity. Conversely, following depletion of T-bet- Treg cells, the remaining T-bet+ cells specifically inhibited TH1 and CD8 T cell activation consistent with their co-localization with T-bet+ effector T cells. These results suggest that T-bet+ Treg cells have an essential immunosuppressive function and indicate that Treg cell functional heterogeneity is a critical feature of immunological tolerance.

Corrigendum: Stability and function of regulatory T cells expressing the transcription factor T-bet.

This corrects the article DOI: 10.1038/nature22360.

Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation.

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.

Th17 cells express interleukin-10 receptor and are controlled by Foxp3⁻ and Foxp3+ regulatory CD4+ T cells in an interleukin-10-dependent manner.

T helper 17 (Th17) cells are important for host defense against extracellular microorganisms. However, they are also implicated in autoimmune and chronic inflammatory diseases, and as such need to be tightly regulated. The mechanisms that directly control committed pathogenic Th17 cells in vivo remain unclear. We showed here that IL-17A-producing CD4+ T cells expressed interleukin-10 receptor α (IL-10Rα) in vivo. Importantly, T cell-specific blockade of IL-10 signaling led to a selective increase of IL-17A+IFN-γ⁻ (Th17) and IL-17A+IFN-γ+ (Th17+Th1) CD4+ T cells during intestinal inflammation in the small intestine. CD4+Foxp3⁻ IL-10-producing (Tr1) cells and CD4+Foxp3+ regulatory (Treg) cells were able to control Th17 and Th17+Th1 cells in an IL-10-dependent manner in vivo. Lastly, IL-10 treatment of mice with established colitis decreased Th17 and Th17+Th1 cell frequencies via direct signaling in T cells. Thus, IL-10 signaling directly suppresses Th17 and Th17+Th1 cells.

BRCT-domain protein BRIT1 influences class switch recombination.

DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs.

Modulation of Naive CD8 T Cell Response Features by Ligand Density, Affinity, and Continued Signaling via Internalized TCRs.

T cell response magnitudes increase with increasing antigenic dosage. However, it is unclear whether ligand density only modulates the proportions of responding ligand-specific T cells or also alters responses at the single cell level. Using brief (3 h) exposure of TCR-transgenic mouse CD8 T cells in vitro to varying densities of cognate peptide-MHC ligand followed by ligand-free culture in IL-2, we found that ligand density determined the frequencies of responding cells but not the expression levels of the early activation marker molecule, CD69. Cells with low glucose uptake capacity and low protein synthesis rates were less ligand-sensitive, implicating metabolic competence in the response heterogeneity of CD8 T cell populations. Although most responding cells proliferated, ligand density was associated with time of entry into proliferation and with the extent of cell surface TCR downmodulation. TCR internalization was associated, regardless of the ligand density, with rapidity of c-myc induction, loss of the cell cycle inhibitor p27kip1, metabolic reprogramming, and cell cycle entry. A low affinity peptide ligand behaved, regardless of ligand density, like a low density, high affinity ligand in all these parameters. Inhibition of signaling after ligand exposure selectively delayed proliferation in cells with internalized TCRs. Finally, internalized TCRs continued to signal and genetic modification of TCR internalization and trafficking altered the duration of signaling in a T cell hybridoma. Together, our findings indicate that heterogeneity among responding CD8 T cell populations in their ability to respond to TCR-mediated stimulation and internalize TCRs mediates detection of ligand density or affinity, contributing to graded response magnitudes.

Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein.

Foxp3(+) regulatory T (Treg) cells limit pathogenic immune responses to self-antigens and foreign antigens. An essential role for microRNA (miRNA) in the maintenance and function of Treg cells, revealed by the Treg cell-specific Dicer ablation, raised a question as to a specific miRNA contribution. We found that Foxp3 controlled the elevated miR155 expression required for maintaining Treg cell proliferative activity and numbers under nonlymphopenic conditions. Moreover, miR155 deficiency in Treg cells resulted in increased suppressor of cytokine signaling 1 (SOCS1) expression accompanied by impaired activation of signal transducer and activator of transcription 5 (STAT5) transcription factor in response to limiting amounts of interleukin-2. Our studies suggest that Foxp3-dependent regulation of miR155 maintains competitive fitness of Treg cell subsets by targeting SOCS1, and they provide experimental support for a proposed role for miRNAs in ensuring the robustness of cellular phenotypes.