TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells.

TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4+CD8+ double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).

Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation.

The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.

The transcription factor NFAT promotes exhaustion of activated CD8⁺ T cells.

During persistent antigen stimulation, CD8(+) T cells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of T cell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished T cell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8(+) T cells to protect against Listeria infection and attenuate tumor growth in vivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8(+) T cells and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8(+) T cells in vivo. Our data show that NFAT promotes T cell anergy and exhaustion by binding at sites that do not require cooperation with AP-1.

Autocrine IL-2 is required for secondary population expansion of CD8(+) memory T cells.

Two competing theories have been put forward to explain the role of CD4(+) T cells in priming CD8(+) memory T cells: one proposes paracrine secretion of interleukin 2 (IL-2); the other proposes the activation of antigen-presenting cells (APCs) via the costimulatory molecule CD40 and its ligand CD40L. We investigated the requirement for IL-2 by the relevant three cell types in vivo and found that CD8(+) T cells, rather than CD4(+) T cells or dendritic cells (DCs), produced the IL-2 necessary for CD8(+) T cell memory. Il2(-/-) CD4(+) T cells were able to provide help only if their ability to transmit signals via CD40L was intact. Our findings reconcile contradictory elements implicit in each model noted above by showing that CD4(+) T cells activate APCs through a CD40L-dependent mechanism to enable autocrine production of IL-2 in CD8(+) memory T cells.

Exhaustion-associated regulatory regions in CD8+ tumor-infiltrating T cells.

T-cell exhaustion is a progressive loss of effector function and memory potential due to persistent antigen exposure, which occurs in chronic viral infections and cancer. Here we investigate the relation between gene expression and chromatin accessibility in CD8+ tumor-infiltrating lymphocytes (TILs) that recognize a model tumor antigen and have features of both activation and functional exhaustion. By filtering out accessible regions observed in bystander, nonexhausted TILs and in acutely restimulated CD8+ T cells, we define a pattern of chromatin accessibility specific for T-cell exhaustion, characterized by enrichment for consensus binding motifs for Nr4a and NFAT transcription factors. Anti-PD-L1 treatment of tumor-bearing mice results in cessation of tumor growth and partial rescue of cytokine production by the dysfunctional TILs, with only limited changes in gene expression and chromatin accessibility. Our studies provide a valuable resource for the molecular understanding of T-cell exhaustion in cancer and other inflammatory settings.

Cutting Edge: NFAT Transcription Factors Promote the Generation of Follicular Helper T Cells in Response to Acute Viral Infection.

Follicular CD4(+) Th (Tfh) cells provide B cell help in germinal center reactions that support class switching, somatic hypermutation, and the generation of high-affinity Abs. In this article, we show that deficiency in NFAT1 and NFAT2 in CD4(+) T cells leads to impaired germinal center reactions upon viral infection because of reduced Tfh cell differentiation and defective expression of proteins involved in T/B interactions and B cell help, including ICOS, PD-1, and SLAM family receptors. Genome-wide chromatin immunoprecipitation data suggest that NFAT proteins likely directly participate in regulation of genes important for Tfh cell differentiation and function. NFAT proteins are important TCR and Ca(2+)-dependent regulators of T cell biology, and in this article we demonstrate a major positive role of NFAT family members in Tfh differentiation.

FoxO3 is a negative regulator of primary CD8+ T-cell expansion but not of memory formation.

The generation of CD8(+) T cells by vaccination represents an important goal for protective immunity to infectious pathogens. It is thus of utmost importance to understand the mechanisms involved in the generation of optimal CD8(+) T-cell responses. The forkhead box O (FoxO) family of transcription factors has a crucial role in cellular responses to environmental change. Among them, FoxO3 is critically involved in the regulation of cellular proliferation, apoptosis, metabolism and stress resistance to withdrawal of nutrients or cytokine growth factors. Since the role of FoxO3 has been poorly studied in the immune system, here we have evaluated its involvement in the CD8(+) T-cell response. We observe that CD8(+) T cells deficient for FoxO3 undergo a significantly greater primary expansion than their wild-type (WT) counterparts in response to both infectious (vaccinia virus) or non-infectious (non-replicating cellular vaccine) immunogens, resulting in a larger cohort of cells following contraction. These survivors, however, do not undergo a greater secondary response than WT. Taken together, our data show that FoxO3 is a negative regulator of the CD8(+) T-cell response, specifically during the primary expansion.

Selective inhibition of CD4+ T-cell cytokine production and autoimmunity by BET protein and c-Myc inhibitors.

Bromodomain-containing proteins bind acetylated lysine residues on histone tails and are involved in the recruitment of additional factors that mediate histone modifications and enable transcription. A compound, I-BET-762, that inhibits binding of an acetylated histone peptide to proteins of the bromodomain and extra-terminal domain (BET) family, was previously shown to suppress the production of proinflammatory proteins by macrophages and block acute inflammation in mice. Here, we investigated the effect of short-term treatment with I-BET-762 on T-cell function. Treatment of naïve CD4(+) T cells with I-BET-762 during the first 2 d of differentiation had long-lasting effects on subsequent gene expression and cytokine production. Gene expression analysis revealed up-regulated expression of several antiinflammatory gene products, including IL-10, Lag3, and Egr2, and down-regulated expression of several proinflammatory cytokines including GM-CSF and IL-17. The short 2-d treatment with I-BET-762 inhibited the ability of antigen-specific T cells, differentiated under Th1 but not Th17 conditions in vitro, to induce pathogenesis in an adoptive transfer model of experimental autoimmune encephalomyelitis. The suppressive effects of I-BET-762 on T-cell mediated inflammation in vivo were accompanied by decreased recruitment of macrophages, consistent with decreased GM-CSF production by CNS-infiltrating T cells. These effects were mimicked by an inhibitor of c-myc function, implicating reduced expression of c-myc and GM-CSF as one avenue by which I-BET-762 suppresses the inflammatory functions of T cells. Our study demonstrates that inhibiting the functions of BET-family proteins during early T-cell differentiation causes long-lasting suppression of the proinflammatory functions of Th1 cells.

TET2 Regulates Mast Cell Differentiation and Proliferation through Catalytic and Non-catalytic Activities.

Dioxygenases of the TET family impact genome functions by converting 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC). Here, we identified TET2 as a crucial regulator of mast cell differentiation and proliferation. In the absence of TET2, mast cells showed disrupted gene expression and altered genome-wide 5hmC deposition, especially at enhancers and in the proximity of downregulated genes. Impaired differentiation of Tet2-ablated cells could be relieved or further exacerbated by modulating the activity of other TET family members, and mechanistically it could be linked to the dysregulated expression of C/EBP family transcription factors. Conversely, the marked increase in proliferation induced by the loss of TET2 could be rescued exclusively by re-expression of wild-type or catalytically inactive TET2. Our data indicate that, in the absence of TET2, mast cell differentiation is under the control of compensatory mechanisms mediated by other TET family members, while proliferation is strictly dependent on TET2 expression.

Cancer-associated ASXL1 mutations may act as gain-of-function mutations of the ASXL1-BAP1 complex.

ASXL1 is the obligate regulatory subunit of a deubiquitinase complex whose catalytic subunit is BAP1. Heterozygous mutations of ASXL1 that result in premature truncations are frequent in myeloid leukemias and Bohring-Opitz syndrome. Here we demonstrate that ASXL1 truncations confer enhanced activity on the ASXL1-BAP1 complex. Stable expression of truncated, hyperactive ASXL1-BAP1 complexes in a haematopoietic precursor cell line results in global erasure of H2AK119Ub, striking depletion of H3K27me3, selective upregulation of a subset of genes whose promoters are marked by both H2AK119Ub and H3K4me3, and spontaneous differentiation to the mast cell lineage. These outcomes require the catalytic activity of BAP1, indicating that they are downstream consequences of H2AK119Ub erasure. In bone marrow precursors, expression of truncated ASXL1-BAP1 complex cooperates with TET2 loss-of-function to increase differentiation to the myeloid lineage in vivo. Our data raise the possibility that ASXL1 truncation mutations confer gain-of-function on the ASXL-BAP1 complex.

Immune adjuvant efficacy of CpG oligonucleotide in cancer treatment is founded specifically upon TLR9 function in plasmacytoid dendritic cells.

The differences in function, location, and migratory pattern of conventional dendritic cells (cDC) and plasmacytoid DCs (pDC) not only point to specialized roles in immune responses but also signify additive and interdependent relationships required to clear pathogens. We studied the in vivo requirement of cross-talk between cDCs and pDCs for eliciting antitumor immunity against in situ released tumor antigens in the absence or presence of the Toll-like receptor (TLR) 9 agonist CpG. Previous data indicated that CpG boosted tumor-specific T-cell responses after in vivo tumor destruction and increased survival after tumor rechallenges. The present study shows that cDCs are indispensable for cross-presentation of ablation-released tumor antigens and for the induction of long-term antitumor immunity. Depletion of pDCs or applying this model in type I IFN receptor-deficient mice abrogated CpG-mediated responses. CD8α(+) cDCs and the recently identified merocytic cDCs were dependent on pDCs for CpG-induced upregulation of CD80. Moreover, DC transfer studies revealed that merocytic cDCs and CD8α(+) cDCs were most susceptible to pDC help and subsequently promoted tumor-free survival in a therapeutic setting. By transferring wild-type pDCs into TLR9-deficient mice, we finally showed that TLR9 expression in pDCs is sufficient to benefit from CpG as an adjuvant. These studies indicate that the efficacy of CpG in cancer immunotherapy is dependent on cross-talk between pDCs and specific subsets of cDCs.