T cells become dysfunctional when they encounter self antigens or are exposed to chronic infection or to the tumour microenvironment1. The function of T cells is tightly regulated by a combinational co-stimulatory signal, and dominance of negative co-stimulation results in T cell dysfunction2. However, the molecular mechanisms that underlie this dysfunction remain unclear. Here, using an in vitro T cell tolerance induction system in mice, we characterize genome-wide epigenetic and gene expression features in tolerant T cells, and show that they are distinct from effector and regulatory T cells. Notably, the transcription factor NR4A1 is stably expressed at high levels in tolerant T cells. Overexpression of NR4A1 inhibits effector T cell differentiation, whereas deletion of NR4A1 overcomes T cell tolerance and exaggerates effector function, as well as enhancing immunity against tumour and chronic virus. Mechanistically, NR4A1 is preferentially recruited to binding sites of the transcription factor AP-1, where it represses effector-gene expression by inhibiting AP-1 function. NR4A1 binding also promotes acetylation of histone 3 at lysine 27 (H3K27ac), leading to activation of tolerance-related genes. This study thus identifies NR4A1 as a key general regulator in the induction of T cell dysfunction, and a potential target for tumour immunotherapy.

In immune responses, activated T cells migrate to B cell follicles and develop to T follicular helper (Tfh) cells, a new subset of CD4+ T cells specialized in providing help to B lymphocytes in the induction of germinal centers 1,2. Although Bcl6 has been shown to be essential in Tfh cell function, it may not regulate the initial migration of T cells 3 or the induction of Tfh program as exampled by C-X-C chemokine receptor type 5 (CXCR5) upregulation 4. Here, we show that Achaete-Scute homologue 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor 5, is selectively upregulated in its expression in Tfh cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6 and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro and accelerates T cell migration to the follicles and Tfh cell development in vivo. Genome-wide analysis indicates that Ascl2 directly regulates Tfh-related genes while inhibits expression of Th1 and Th17 genes. Acute deletion of Ascl2 as well as blockade of its function with the Id3 protein in CD4+ T cells results in impaired Tfh cell development and the germinal center response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances Tfh cell generation. Thus, Ascl2 directly initiates Tfh cell development.

A novel Bcl6 reporter mouse is used to dissect the developmental requirements, plasticity, and genetic profile of Tfh cells.

Interleukin 17 (IL-17) plays an important role in infection and autoimmunity; how it signals remains poorly understood. In this study, we identified ubiquitin-specific protease 25 (USP25) as a negative regulator of IL-17-mediated signaling and inflammation. Overexpression of USP25 inhibited IL-17-triggered signaling, while USP25 deficiency resulted in increased phosphorylation of IκBα and Jnk, increased expression of chemokines and cytokines as well as prolonged half-life of Cxcl1 mRNA following IL-17 treatment. Consistently, Usp25-/- mice exhibited increased sensitivity to IL-17-dependent inflammation and autoimmunity in vivo. Mechanistically, IL-17 stimulation induced the association of USP25 with TRAF5 and TRAF6 and USP25 induced removal of Act1-mediated K63-linked ubiquitination in TRAF5 and TRAF6. Thus, our results demonstrate that USP25 is a deubiquitinating enzyme (DUB) that negatively regulates IL-17-triggered signaling.

SUMMARY The antitumor effector T helper 1 (Th1) and Th17 cells represent two T cell paradigms: short-lived cytolytic Th1 cells and “stem cell-like” memory Th17 cells. We report that Th9 cells represent a third paradigm—they are less-exhausted, fully cytolytic, and hyperproliferative. Only tumor-specific Th9 cells completely eradicated advanced tumors, maintained a mature effector cell signature with cytolytic activity as strong as Th1 cells, and persisted as long as Th17 cells in vivo. Th9 cells displayed a unique Pu.1-Traf6-NF-κB activation-driven hyperproliferative feature, suggesting a persistence mechanism rather than an antiapoptotic strategy. Th9 antitumor efficacy depended on interleukin-9 and upregulated expression of Eomes and Traf6. Thus, tumor-specific Th9 cells are a more effective CD4+ T cell subset for adoptive cancer therapy.

CD8+ T cells can be polarized into IL-9–secreting (Tc9) cells. Ma et al. show that Tc9 cell differentiation is associated with a low cholesterol reprogramming profile, and manipulating cholesterol content in polarizing Tc9 cells significantly affects Tc9 differentiation and antitumor response.

Nur77, an orphan nuclear receptor, plays a key role in apoptosis in T cells. In cancer cell lines, Nur77 can induce apoptosis through the intrinsic apoptotic pathway, but the mechanism by which Nur77 kills T cells remains controversial. In this study, we provide biochemical, pharmacological, and genetic evidence demonstrating that Nur77 induces apoptosis through the activation of the intrinsic pathway in T cells. We also show that Nur77 is a physiological substrate of the MEK-ERK-RSK cascade. Specifically, we demonstrate that RSK phosphorylates Nur77 at serine 354 and this modulates Nur77 nuclear export and intracellular translocation during T cell death. Our data reveal that Nur77 phosphorylation and mitochondrial targeting, regulated by RSK, defines a role for the MEK1/2-ERK1/2 cascade in T cell apoptosis.

IL-9 is a pro-allergic cytokine produced by a newly proposed T helper cell subset TH9. TH9 cells can be generated by treatment of naïve T cells with TGF-β and IL-4 in vitro. But how TGF-β signaling regulates TH9 differentiation is still not clear. Here we demonstrate that Smad2 and Smad4, two transcriptional factors activated by TGF-β signaling, are required for TH9 differentiation in vitro. Deficiency of Smad2 or Smad4 in T cells resulted in impaired IL-9 expression, which was coincident with enrichment of repressive chromatin modification H3K27Me3 and enhanced EZH2 binding to the Il9 locus. Pharmacologic inhibition of EZH2 partially rescued IL-9 production in Smad deficient TH9 cells. Smad proteins may displace EZH2 directly from Il9 locus since Smad2 and Smad4 can bind EZH2. Our data shed light on the molecular mechanisms underlying TH9 cell differentiation, revealing that TGF-β-Smad2/4 signaling pathway regulates IL-9 production through an epigenetic mechanism.