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Lineage-determining transcription factors (LD-TFs) drive the differentiation of progenitor cells into a specific lineage. In CD4+ T cells, T-bet dictates differentiation of the TH1 lineage, whereas GATA3 drives differentiation of the alternative TH2 lineage. However, LD-TFs, including T-bet and GATA3, are frequently co-expressed but how this affects LD-TF function is not known. By expressing T-bet and GATA3 separately or together in mouse T cells, we show that T-bet sequesters GATA3 at its target sites, thereby removing GATA3 from TH2 genes. This redistribution of GATA3 is independent of GATA3 DNA binding activity and is instead mediated by the T-bet DNA binding domain, which interacts with the GATA3 DNA binding domain and changes GATA3′s sequence binding preference. This mechanism allows T-bet to drive the TH1 gene expression program in the presence of GATA3. We propose that redistribution of one LD-TF by another may be a common mechanism that could explain how specific cell fate choices can be made even in the presence of other transcription factors driving alternative differentiation pathways.

T-bet is the lineage-specifying transcription factor for CD4+ T helper type 1 (TH 1) cells. T-bet has also been found in other CD4+ T cell subsets, including TH 17 cells and Treg, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells that have naïve cell surface markers and a naïve cell transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4+ T cells. Naïve-like T-bet-experienced cells are polarised to the TH 1 lineage, predisposed to produce IFNγ upon cell activation, and resist repolarisation to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can polarise T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T helper response. This article is protected by copyright. All rights reserved.

T-bet is the lineage-specifying transcription factor for CD4

Resistance to single cytokine blockade, namely anti-tumor necrosis factor (TNF) therapy, is a growing concern for patients with inflammatory bowel disease (IBD). The transcription factor T-bet is a critical regulator of intestinal homeostasis, is genetically linked to mucosal inflammation and controls the expression of multiples genes such as the pro-inflammatory cytokines interferon (IFN)-γ and TNF. Inhibiting T-bet may therefore offer a more attractive prospect for treating IBD but remains challenging to target therapeutically. In this study, we evaluate the effect of targeting the transactivation function of T-bet using inhibitors of P-TEFb (CDK9-cyclin T), a transcriptional elongation factor downstream of T-bet.Using an adaptive immune-mediated colitis model, human colonic lymphocytes from patients with IBD and multiple large clinical datasets, we investigate the effect of cyclin-dependent kinase 9 (CDK9) inhibitors on cytokine production and gene expression in colonic CD4Systemic CDK9 inhibition led to histological improvement of immune-mediated colitis and was associated with targeted suppression of colonic CD4Collectively, our findings reveal CDK9 as a potential target for anti-TNF-resistant IBD, which has the potential for rapid translation to the clinic.

Background and Aims Differential responsiveness to interleukin [IL]-2 between effector CD4+ T cells [Teff] and regulatory T cells [Treg] is a fundamental mechanism of immunoregulation. The single nucleotide polymorphism [SNP] rs61839660, located within IL2RA [CD25], has been associated with the development of Crohn’s disease [CD]. We sought to identify the T cell immune phenotype of IBD patients who carry this SNP. Methods Teff and Treg were isolated from individuals homozygous [TT], heterozygous [CT], or wild-type [CC] for the minor allele at rs61839660, and used for phenotyping [flow cytometry, Cytometry Time Of Flight] functional assays or T cell receptor [TCR] sequencing. Phosphorylation of signal transducer and activator of transcription 5 [STAT5] was assessed in response to IL-2, IL-7, and in the presence of basiliximab, a monoclonal antibody directed against CD25. Teff pro-inflammatory cytokine expression levels were assessed by reverse transcription quantitative polymerase chain reaction after IL-2 and/or TCR stimulation. Results Presence of the minor T allele enhances CD25 expression, leading to increased STAT5 phosphorylation and pro-inflammatory cytokine transcript expression by Teff in response to IL-2 stimulation in vitro. Teff from TT individuals demonstrate a more activated gut homing phenotype. TCR sequencing analysis suggests that TT patients may have a reduced clonal capacity to mount an optimal regulatory T cell response. Conclusions rs61839660 regulates the responsiveness of T cells to IL-2 signalling by modulating CD25 expression. As low-dose IL-2 is being trialled as a selective Treg modulator in CD, these findings highlight the potential for adverse effects in patients with this genotype., Graphical Abstract Graphical Abstract

Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) is an invaluable method to profile of enrichment of histone modifications and transcription factor binding sites across the genome. However, standard ChIP-seq protocols require large numbers of cells (10

Gene expression programs controlled by lineage-determining transcription factors are often conserved between species. However, infectious diseases have exerted profound evolutionary pressure, and therefore the genes regulated by immune-specific transcription factors might be expected to exhibit greater divergence. T-bet (Tbx21) is the immune-specific, lineage-specifying transcription factor for T helper type I (Th1) immunity, which is fundamental for the immune response to intracellular pathogens but also underlies inflammatory diseases. We compared T-bet genomic targets between mouse and human CD4+ T cells and correlated T-bet binding patterns with species-specific gene expression. Remarkably, we found that the majority of T-bet target genes are conserved between mouse and human, either via preservation of binding sites or via alternative binding sites associated with transposon-linked insertion. Species-specific T-bet binding was associated with differences in transcription factor–binding motifs and species-specific expression of associated genes. These results provide a genome-wide cross-species comparison of Th1 gene regulation that will enable more accurate translation of genetic targets and therapeutics from pre-clinical models of inflammatory and infectious diseases and cancer into human clinical trials.

Gene expression programmes controlled by lineage-determining transcription factors are often conserved between species. However, infectious diseases have exerted profound evolutionary pressure, and therefore the genes regulated by immune-specific transcription factors might be expected to exhibit greater divergence due to exposure to species-specific pathogens. T-bet (Tbx21) is the immune-specific lineage-defining transcription factor for T helper type I (Th1) immunity, which is fundamental for the immune response to intracellular pathogens but also underlies inflammatory diseases. We therefore compared T-bet genomic targets between mouse and human CD4+ T cells and correlated T-bet binding patterns with species-specific gene expression. Remarkably, we show that the vast majority of T-bet regulated genes are conserved between mouse and human, either via preservation of a binding site or via an alternative binding site associated with transposon-linked insertion. We also identified genes that are specifically targeted by T-bet in humans or mice and which exhibited species-specific expression. These results provide a genome-wide cross-species comparison of T-bet target gene regulation that will enable more accurate translation of genetic targets and therapeutics from pre-clinical models of inflammatory disease into human clinical trials.

Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) is an invaluable method to profile of enrichment of histone modifications and transcription factor binding sites across the genome. However, standard ChIP-seq protocols require large numbers of cells (>107) as starting material, which are often impossible to obtain for rare immune populations. Here we describe a streamlined ChIP protocol optimised for small cell numbers in conjunction with transposon-tagging mediated sequencing library preparation (ChIPmentation) which allows the analysis of samples of as low as 105 cells.

T-bet is the lineage-specifying transcription factor for CD4+ T helper type 1 (TH1) cells. T-bet has also been found in other CD4+ T cell subsets, including TH17 cells and TREG, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells with naïve cell surface markers and that this novel cell population is phenotypically and functionally distinct from conventional naïve CD4+ T cells. These cells are also distinct from previously described populations of memory phenotype and stem cell-like T cells. Naïve-like T-bet-experienced cells are polarised to the TH1 lineage, predisposed to produce IFNγ upon cell activation, and resist repolarisation to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can function to polarise T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T helper response.

Organ transplantation is often lifesaving, but the long-term deleterious effects of combinatorial immunosuppression regimens and allograft failure cause significant morbidity and mortality. Long-term graft survival in the absence of continuing immunosuppression, defined as operational tolerance, has never been described in the context of multiple major histocompatibility complex (MHC) mismatches. Here, we show that miR-142 deficiency leads to indefinite allograft survival in a fully MHC mismatched murine cardiac transplant model in the absence of exogenous immunosuppression. We demonstrate that the cause of indefinite allograft survival in the absence of miR-142 maps specifically to the T cell compartment. Of therapeutic relevance, temporal deletion of miR-142 in adult mice prior to transplantation of a fully MHC mismatched skin allograft resulted in prolonged allograft survival. Mechanistically, miR-142 directly targets Tgfbr1 for repression in regulatory T cells (TREG ). This leads to increased TREG sensitivity to transforming growth factor - beta and promotes transplant tolerance via an augmented peripheral TREG response in the absence of miR-142. These data identify manipulation of miR-142 as a promising approach for the induction of tolerance in human transplantation.

Tregs play a fundamental role in immune tolerance via control of self-reactive effector T cells (Teffs). This function is dependent on maintenance of a high intracellular cAMP concentration. A number of microRNAs are implicated in the maintenance of Tregs. In this study, we demonstrate that peripheral immune tolerance is critically dependent on posttranscriptional repression of the cAMP-hydrolyzing enzyme phosphodiesterase-3b (Pde3b) by microRNA-142-5p (miR-142-5p). In this manner, miR-142-5p acts as an immunometabolic regulator of intracellular cAMP, controlling Treg suppressive function. Mir142 was associated with a super enhancer bound by the Treg lineage–determining transcription factor forkhead box P3 (FOXP3), and Treg-specific deletion of miR-142 in mice (Treg Δ142 ) resulted in spontaneous, lethal, multisystem autoimmunity, despite preserved numbers of phenotypically normal Tregs. Pharmacological inhibition and genetic ablation of PDE3B prevented autoimmune disease and reversed the impaired suppressive function of Tregs in Treg Δ142 animals. These findings reveal a critical molecular switch, specifying Treg function through the modulation of a highly conserved, cell-intrinsic metabolic pathway. Modulation of this pathway has direct relevance to the pathogenesis and treatment of autoimmunity and cancer.

Regulatory T (Treg) cells safeguard against autoimmunity and immune pathology. Because determinants of the Treg cell fate are not completely understood, we have delineated signaling events that control the de novo expression of Foxp3 in naive peripheral CD4 T cells and in thymocytes. We report that premature termination of TCR signaling and inibition of phosphatidyl inositol 3-kinase (PI3K) p110α, p110δ, protein kinase B (Akt), or mammalian target of rapamycin (mTOR) conferred Foxp3 expression and Treg-like gene expression profiles. Conversely, continued TCR signaling and constitutive PI3K/Akt/mTOR activity antagonised Foxp3 induction. At the chromatin level, di- and trimethylation of lysine 4 of histone H3 (H3K4me2 and -3) near the Foxp3 transcription start site (TSS) and within the 5′ untranslated region (UTR) preceded active Foxp3 expression and, like Foxp3 inducibility, was lost upon continued TCR stimulation. These data demonstrate that the PI3K/Akt/mTOR signaling network regulates Foxp3 expression.

Micro RNAs (miRNAs) regulate gene expression at the posttranscriptional level. Here we show that regulatory T (T reg) cells have a miRNA profile distinct from conventional CD4 T cells. A partial T reg cell–like miRNA profile is conferred by the enforced expression of Foxp3 and, surprisingly, by the activation of conventional CD4 T cells. Depleting miRNAs by eliminating Dicer, the RNAse III enzyme that generates functional miRNAs, reduces T reg cell numbers and results in immune pathology. Dicer facilitates, in a cell-autonomous fashion, the development of T reg cells in the thymus and the efficient induction of Foxp3 by transforming growth factor β. These results suggest that T reg cell development involves Dicer-generated RNAs.

The development and homeostasis of multicellular organisms relies on gene regulation within individual constituent cells. Gene regulatory circuits that increase the robustness of gene expression frequently incorporate microRNAs as post-transcriptional regulators. Computational approaches, synthetic gene circuits and observations in model organisms predict that the co-regulation of microRNAs and their target mRNAs can reduce cell-to-cell variability in the expression of target genes. However, whether microRNAs directly regulate variability of endogenous gene expression remains to be tested in mammalian cells. Here we use quantitative flow cytometry to show that microRNAs impact on cell-to-cell variability of protein expression in developing mouse thymocytes. We find two distinct mechanisms that control variation in the activation-induced expression of the microRNA target CD69. First, the expression of miR-17 and miR-20a, two members of the miR-17-92 cluster, is co-regulated with the target mRNA Cd69 to form an activation-induced incoherent feed-forward loop. Another microRNA, miR-181a, acts at least in part upstream of the target mRNA Cd69 to modulate cellular responses to activation. The ability of microRNAs to render gene expression more uniform across mammalian cell populations may be important for normal development and for disease., Author Summary microRNAs are integral to many developmental processes and may 'canalise' development by reducing cell-to-cell variation in gene expression. This idea is supported by computational studies that have modeled the impact of microRNAs on the expression of their targets and the construction of artificial incoherent feedforward loops using synthetic biology tools. Here we show that this interesting principle of microRNA regulation actually occurs in a mammalian developmental system. We examine cell-to-cell variation of protein expression in developing mouse thymocytes by quantitative flow cytometry and find that the absence of microRNAs results in increased cell-to-cell variation in the expression of the microRNA target Cd69. Mechanistically, T cell receptor signaling induces both Cd69 and miR-17 and miR-20a, two microRNAs that target Cd69. Co-regulation of microRNAs and their target mRNA dampens the expression of Cd69 and forms an incoherent feedforward loop that reduces cell-to-cell variation on CD69 expression. In addition, miR-181, which also targets Cd69 and is a known modulator of T cell receptor signaling, also affects cell-to-cell variation of CD69 expression. The ability of microRNAs to control the uniformity of gene expression across mammalian cell populations may be important for normal development and for disease.

Summary The transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates., Graphical Abstract, Highlights • Th1 genes and Th2 genes are associated with RNA pol II in the alternative lineage • T-bet acts to recruit Mediator and the SEC to activate Th1 genes and eRNAs • T-bet and NF-κB-dependent P-TEFb recruitment pathways converge at enhancers • P-TEFb inhibition silences T-bet target genes and abrogates uveitis in vivo, Lineage-specifying transcription factors control T helper cell fate choice, but the mechanisms underlying this are unclear. Hertweck et al. reveal that Th1 genes undergo transcriptional initiation in the alternative Th2 lineage and that the Th1 master regulator T-bet functions to recruit Mediator and the super-elongation complex to activate transcriptional elongation.

Lymphocyte recruitment maintains intestinal immune homeostasis but also contributes to inflammation. The orphan chemoattractant receptor GPR15 mediates regulatory T cell homing and immunosuppression in the mouse colon. We show that GPR15 is also expressed by mouse TH17 and TH1 effector cells and is required for colitis in a model that depends on the trafficking of these cells to the colon. In humans GPR15 is expressed by effector cells, including pathogenic TH2 cells in ulcerative colitis, but is expressed poorly or not at all by colon regulatory T (Treg) cells. The TH2 transcriptional activator GATA-3 and the Treg-associated transcriptional repressor FOXP3 robustly bind human, but not mouse, GPR15 enhancer sequences, correlating with receptor expression. Our results highlight species differences in GPR15 regulation and suggest it as a potential therapeutic target for colitis.

T-bet and GATA3 regulate the CD4+ T cell Th1/Th2 cell fate decision but little is known about the interplay between these factors outside of the murine Ifng and Il4/Il5/Il13 loci. Here we show that T-bet and GATA3 bind to multiple distal sites at immune regulatory genes in human effector T cells. These sites display markers of functional elements, act as enhancers in reporter assays and are associated with a requirement for T-bet and GATA3. Furthermore, we demonstrate that both factors bind distal sites at Tbx21 and that T-bet directly activates its own expression. We also show that in Th1 cells, GATA3 is distributed away from Th2 genes, instead occupying T-bet binding sites at Th1 genes, and that T-bet is sufficient to induce GATA3 binding at these sites. We propose these aspects of T-bet and GATA3 function are important for Th1/Th2 differentiation and for understanding transcription factor interactions in other T cell lineage decisions., T-bet and GATA3 regulate differentiation of T cells into Th1 or Th2 cell fates, but little is known about their functional interaction outside of the IFNγ and Il4 /Il5/Il13 loci. Kanhere et al. map these factors across the genome in human T cells, revealing unappreciated breadth of function and interplay between them.

Naive CD4 T cells differentiate into functionally distinct T helper (Th) cells subsets or into regulatory T (Treg) cells in response to the cytokine milieu in which they encounter antigen. A recurring theme in post-thymic CD4 T cell differentiation is the cross-regulation of lineage choice by cytokines and transcription factors that are expressed in alternative lineages. For example, TGFbeta induces the de novo expression of the Treg cell signature transcription factor Foxp3, but iTreg differentiation is blocked by high concentrations of the Th2 cytokine IL4. However, whether IL4 can antagonise Foxp3 induction in more physiological settings remains to be addressed. Here we use a co-culture system to demonstrate that IL4 provided by Th2 cells in vitro is sufficient to block Foxp3 induction in naive CD4 T cells. In addition, we find that Foxp3 induction is efficiently blocked not only by the Th2 transcription factor Gata3, but also by PU.1, which is transiently induced during Th2 differentiation. These data suggest that iTreg differentiation may be affected by the polarity of immune responses.

Coreceptor expression is tightly regulated during thymocyte development. Deletion of specific Cd8 enhancers leads to variegated expression of CD8αβ heterodimers in double-positive (DP) thymocytes. Here we show CD8 variegation was correlated with an epigenetic “off” state, linking Cd8 enhancer function with chromatin remodeling of the adjacent genes Cd8a and Cd8b1 (Cd8). We show the zinc finger protein MAZR, encoded by the Zfp278 gene, bound the Cd8 enhancer and interacted with the corepressor N-CoR complex in double-negative thymocytes. MAZR was down-regulated in DP and single-positive CD8+ thymocytes. Enforced expression of MAZR led to impaired Cd8 activation and variegated CD8 expression. Our results demonstrate epigenetic control of the Cd8 gene loci and identify MAZR as an important regulator of Cd8 gene expression.

Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regulate the initiation and the maintenance of Foxp3 gene expression in CD4 T cells. Full-length Runx promoted the de novo expression of Foxp3 during inducible T reg cell differentiation, whereas the isolated dominant-negative Runt DNA binding domain antagonized de novo Foxp3 expression. Foxp3 expression in natural T reg cells remained dependent on Runx proteins and correlated with the binding of Runx/core-binding factor β to regulatory elements within the Foxp3 locus. Our data show that Runx and Foxp3 are components of a feed-forward loop in which Runx proteins contribute to the expression of Foxp3 and cooperate with Foxp3 proteins to regulate the expression of downstream target genes.