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Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1–targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy. Human primary and metastatic tumors harbor CD4+ Treg cells that can suppress antitumor immune responses. Bonnal et al. identify an intratumoral type 1 Treg-like CD4+ T cell subset that expresses the transcription factor EOMES, granzyme K and CHI3L2. This EOMES+ T cell subset correlates with disease progression but is responsive to PD-1 checkpoint blockade immunotherapy.

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.

Ex vivo gene expression and miRNA profiling of Eomes+ Tr1-like cells suggested that they represent a differentiation stage that is intermediate between Th1-cells and cytotoxic CD4+ T-cells. Several microRNAs were downregulated in Eomes+ Tr1-like cells that might inhibit Tr1-cell differentiation. In particular, miR-92a targeted Eomes, while miR-125a inhibited IFN-g and IL-10R expression.

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID‐19, which negatively affects T‐cell activation. The presence of effector T cells in patients with mild disease and dysfunctional T cells in severely ill patients suggests that adequate T‐cell responses limit disease severity. Understanding how cDCs cope with SARS‐CoV‐2 can help elucidate how protective immune responses are generated. Here, we report that cDC2 subtypes exhibit similar infection‐induced gene signatures, with the upregulation of interferon‐stimulated genes and interleukin (IL)‐6 signaling pathways. Furthermore, comparison of cDCs between patients with severe and mild disease showed severely ill patients to exhibit profound downregulation of genes encoding molecules involved in antigen presentation, such as MHCII, TAP, and costimulatory proteins, whereas we observed the opposite for proinflammatory molecules, such as complement and coagulation factors. Thus, as disease severity increases, cDC2s exhibit enhanced inflammatory properties and lose antigen presentation capacity. Moreover, DC3s showed upregulation of anti‐apoptotic genes and accumulated during infection. Direct exposure of cDC2s to the virus in vitro recapitulated the activation profile observed in vivo. Our findings suggest that SARS‐CoV‐2 interacts directly with cDC2s and implements an efficient immune escape mechanism that correlates with disease severity by downregulating crucial molecules required for T‐cell activation. This article is protected by copyright. All rights reserved

Whether human IL-10-producing regulatory Tcells (“Tr1”) represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4 + T-cell subsets, including conventional cytotoxic CD4 + Tcells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4 + Tcells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes + GzmK + Tcells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4 + Eomes + T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes + Tr1-like cells are effector cells of a unique GzmK-expressing CD4 + T-cell subset.

Background Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that is caused by autoreactive T cells and associated with viral infections. However, the phenotype of pathogenic T cells in peripheral blood remains to be defined, and how viruses promote MS is debated. Objective We aimed to identify and characterize potentially pathogenic autoreactive T cells, as well as protective antiviral T cells, in patients with MS. Methods We analyzed CD4 + helper T-cell subsets from peripheral blood or cerebrospinal fluid for cytokine production, gene expression, plasticity, homing potentials, and their reactivity to self-antigens and viral antigens in healthy subjects and patients with MS. Moreover, we monitored their frequencies in untreated and fingolimod- or natalizumab-treated patients with MS. Results T H 1/T H 17 central memory (T H 1/T H 17 CM ) cells were selectively increased in peripheral blood of patients with relapsing-remitting MS with a high disease score. T H 1/T H 17 CM cells were closely related to conventional T H 17 cells but had more pathogenic features. In particular, they could shuttle between lymph nodes and the CNS and produced encephalitogenic cytokines. The cerebrospinal fluid of patients with active MS was enriched for CXCL10 and contained mainly CXCR3-expressing T H 1 and T H 1/T H 17 subsets. However, while T H 1 cells responded consistently to viruses, T H 1/T H 17 CM cells reacted strongly with John Cunningham virus in healthy subjects but responded instead to myelin-derived self-antigens in patients with MS. Fingolimod and natalizumab therapies efficiently targeted autoreactive T H 1/T H 17 CM cells but also blocked virus-specific T H 1 cells. Conclusions We propose that autoreactive T H 1/T H 17 CM cells expand in patients with MS and promote relapses after bystander recruitment to the CNS, whereas T H 1 cells perform immune surveillance. Thus the selective targeting of T H 1/T H 17 cells could inhibit relapses without causing John Cunningham virus–dependent progressive multifocal encephalomyelitis.

Summary Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy., Highlights • Transcriptome analysis performed on tumor-resident CD4+ Th1, Th17, and Treg cells • Tumor-infiltrating Treg cells are defined by the expression of signature genes • Treg-specific signature genes correlate with patients’ survival in both CRC and NSCLC, Tumor-infiltrating regulatory T cells can suppress effector T cells specific for tumor antigens. De Simone et al. (2016) demonstrate that tumor-infiltrating Treg cells display specific gene signatures that were also validated at the single-cell level. These data can contribute to dissect the molecular networks underlying the biology of tumor-infiltrating Treg cells. As part of the IHEC consortium, this study integrates genetic, epigenetic, and transcriptomic profiling in three immune cell types from nearly 200 people to characterize the distinct and cooperative contributions of diverse genomic inputs to transcriptional variation. Explore the Cell Press IHEC webportal at www.cell.com/consortium/IHEC.

Background: IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (T(R)1) cells but is also produced by CD25(+) regulatory T (Treg) cells. Objective: We aimed to identify and characterize human intestinal T(R)1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs). Methods: CD4(+) T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4(+) T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1 beta and IL-23 responsiveness was assessed. Results: Intestinal T(R)1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5(+) PD-1(+) T(R)1 cells expressed IFN-gamma and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-gamma(+) T(R)1 cells, but not IL-7 receptor-positive T-H cells or CD25(+) Treg cells, showed lower IL-10 expression in patients with IBDs. T(R)1 cells were responsive to IL-23, and IFN-gamma(+) T(R)1 cells downregulated IL-10 with IL-1 beta and IL-23. Conversely, CD25(+) Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression. Conclusions: We provide the first ex vivo characterization of human intestinal T(R)1 cells. Selective downregulation of IL-10 by IFN-gamma(+) T(R)1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs.

Follicular helper T cells (TFHs) are a key component of adaptive immune responses as they help antibody production by B cells. Differentiation and function of TFH cells are controlled by the master gene BCL6, but it is largely unclear how this transcription repressor specifies the TFH program. Here we asked whether BCL6 controlled helper function through down-regulation of specific microRNAs (miRNAs). We first assessed miRNA expression in TFH cells and defined a TFH-specific miRNA signature. We report that hsa-miR-31-5p (miR-31) is down-regulated in TFH; we showed that BCL6 suppresses miR-31 expression by binding to its promoter; and we demonstrated that miR-31 inhibits the expression of molecules that control T-helper function, such as CD40L and SAP. These findings identify a BCL6-initiated inhibitory circuit that stabilizes the follicular helper T cell program at least in part through the control of miRNA transcription. Although BCL6 controls TFH activity in human and mouse, the role of miR-31 is restricted to human TFH cell differentiation, reflecting a species specificity of the miR-31 action. Our findings highlight miR-31 as a possible target to modulate human T cell dependent antibody responses in the settings of infection, vaccination, or immune dysregulation.

Immunosuppressive regulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Targeting Tregs may be a powerful means to unleash more potent immune responses against cancer, but targeting these cells is challenging because their generalized inactivation may incite severe autoimmune toxicities. To selectively target Tregs in tumors, we investigated the role of the H3K27 methyltransferase EZH2 in Tregs and determined that its enhanced activity at tumor sites in mice and humans leads to more robust and stable Tregs. We demonstrate that blocking EZH2 activity, both pharmacologically and genetically, selectively reprograms the function of tumor-infiltrating Tregs without systemically altering Treg function. Genetic disruption of EZH2 in tumor-resident Tregs led to their acquisition of pro-inflammatory functions that remodeled the tumor microenvironment and enhanced the recruitment and function of effector T cells, leading to the complete elimination of tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a novel strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.

Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.