RESUMO
We previously showed that chimeric antigen receptor (CAR) T-cell therapy targeting epidermal growth factor receptor variant III (EGFRvIII) produces upregulation of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Here we conducted a phase 1 trial (NCT03726515) of CAR T-EGFRvIII cells administered concomitantly with the anti-PD1 (aPD1) monoclonal antibody pembrolizumab in patients with newly diagnosed, EGFRvIII+ glioblastoma (GBM) (n = 7). The primary outcome was safety, and no dose-limiting toxicity was observed. Secondary outcomes included median progression-free survival (5.2 months; 90% confidence interval (CI), 2.9-6.0 months) and median overall survival (11.8 months; 90% CI, 9.2-14.2 months). In exploratory analyses, comparison of the TME in tumors harvested before versus after CAR + aPD1 administration demonstrated substantial evolution of the infiltrating myeloid and T cells, with more exhausted, regulatory, and interferon (IFN)-stimulated T cells at relapse. Our study suggests that the combination of CAR T cells and PD-1 inhibition in GBM is safe and biologically active but, given the lack of efficacy, also indicates a need to consider alternative strategies.
Assuntos
Anticorpos Monoclonais Humanizados , Glioblastoma , Humanos , Glioblastoma/terapia , Receptores ErbB , Recidiva Local de Neoplasia/metabolismo , Linfócitos T , Microambiente TumoralRESUMO
Failure of adoptive T-cell therapies in patients with cancer is linked to limited T-cell expansion and persistence, even in memory-prone 41BB-(BBz)-based chimeric antigen receptor (CAR) T cells. We show here that BBz-CAR T-cell stem/memory differentiation and persistence can be enhanced through epigenetic manipulation of the histone 3 lysine 9 trimethylation (H3K9me3) pathway. Inactivation of the H3K9 trimethyltransferase SUV39H1 enhances BBz-CAR T cell long-term persistence, protecting mice against tumor relapses and rechallenges in lung and disseminated solid tumor models up to several months after CAR T-cell infusion. Single-cell transcriptomic (single-cell RNA sequencing) and chromatin opening (single-cell assay for transposase accessible chromatin) analyses of tumor-infiltrating CAR T cells show early reprogramming into self-renewing, stemlike populations with decreased expression of dysfunction genes in all T-cell subpopulations. Therefore, epigenetic manipulation of H3K9 methylation by SUV39H1 optimizes the long-term functional persistence of BBz-CAR T cells, limiting relapses, and providing protection against tumor rechallenges. SIGNIFICANCE: Limited CAR T-cell expansion and persistence hinders therapeutic responses in solid cancer patients. We show that targeting SUV39H1 histone methyltransferase enhances 41BB-based CAR T-cell long-term protection against tumor relapses and rechallenges by increasing stemness/memory differentiation. This opens a safe path to enhancing adoptive cell therapies for solid tumors. See related article by Jain et al., p. 142. This article is featured in Selected Articles from This Issue, p. 5.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Animais , Humanos , Camundongos , Cromatina , Imunoterapia Adotiva , Metiltransferases/genética , Metiltransferases/metabolismo , Neoplasias/genética , Neoplasias/terapia , Recidiva , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Dendritic cells (DCs) play a key role in the induction of the adaptive immune response. They capture antigens in peripheral tissues and prime naïve T lymphocytes, triggering the adaptive immune response. In the course of inflammatory processes DCs face stressful conditions including hypoxia, low pH and high concentrations of reactive oxygen species (ROS), among others. How DCs survive under these adverse conditions remain poorly understood. Clusterin is a protein highly expressed by tumors and usually associated with bad prognosis. It promotes cancer cell survival by different mechanisms such as apoptosis inhibition and promotion of autophagy. Here, we show that, upon maturation, human monocyte-derived DCs (MoDCs) up-regulate clusterin expression. Clusterin protects MoDCs from ROS-mediated toxicity, enhancing DC survival and promoting their ability to induce T cell activation. In line with these results, we found that clusterin is expressed by a population of mature LAMP3+ DCs, called mregDCs, but not by immature DCs in human cancer. The expression of clusterin by intratumoral DCs was shown to be associated with a transcriptomic profile indicative of cellular response to stress. These results uncover an important role for clusterin in DC physiology.
Assuntos
Clusterina , Neoplasias , Humanos , Morte Celular , Clusterina/genética , Clusterina/metabolismo , Células Dendríticas , Espécies Reativas de Oxigênio/metabolismo , Linfócitos TRESUMO
In chronic infections and cancer, T cells are exposed to prolonged antigen stimulation, resulting in loss of function (or exhaustion) and impairment of effective immunological protection. Exhausted T cells are heterogeneous and include early progenitors (Tpex) and terminally exhausted cells (Tex). Here, we used bulk and single-cell transcriptomics to analyze expression of transposable elements (TEs) in subpopulations of mouse and human CD8+ tumor-infiltrating T lymphocytes (TILs). We show that in mice, members of the virus-like murine VL30 TE family (mostly intact, evolutionary young ERV1s) are strongly repressed in terminally exhausted CD8+ T cells in both tumor and viral models of exhaustion. Tpex expression of these VL30s, which are mainly intergenic and transcribed independently of their closest gene neighbors, was driven by Fli1, a transcription factor involved in progression from Tpex to Tex. Immune checkpoint blockade (ICB) in both mice and patients with cancer increased TE expression (including VL30 in mice), demonstrating that TEs may be applicable as ICB response biomarkers. We conclude that expression of TEs is tightly regulated in TILs during establishment of exhaustion and reprogramming by ICB. Analyses of TE expression on single cells and bulk populations open opportunities for understanding immune cell identity and heterogeneity, as well as for defining cellular gene expression signatures and disease biomarkers.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Animais , Camundongos , Humanos , Elementos de DNA Transponíveis/genética , Exaustão das Células T , BiomarcadoresRESUMO
Cell polarity is an essential and highly conserved process governing cell function. Cell polarization is generally triggered by an external signal that induces the relocation of the centrosome, thus defining the polarity axis of the cell. Here, we took advantage of B cells as a model to study cell polarity and perform a medium-throughput siRNA-based imaging screen to identify new molecular regulators of polarization. We first identified candidates based on a quantitative proteomic analysis of proteins differentially associated with the centrosome of resting non-polarized and stimulated polarized B cells. We then targeted 233 candidates in a siRNA screen and identified hits regulating the polarization of the centrosome and/or lysosomes in B cells upon stimulation. Our dataset of proteomics, images, and polarity indexes provides a valuable source of information for a broad community of scientists interested in the molecular mechanisms regulating cell polarity.
Assuntos
Linfócitos B , RNA Interferente Pequeno , Centrossomo/metabolismo , Proteômica , Humanos , AnimaisRESUMO
Although most characterized tumor antigens are encoded by canonical transcripts (such as differentiation or tumor-testis antigens) or mutations (both driver and passenger mutations), recent results have shown that noncanonical transcripts including long noncoding RNAs and transposable elements (TEs) can also encode tumor-specific neo-antigens. Here, we investigate the presentation and immunogenicity of tumor antigens derived from noncanonical mRNA splicing events between coding exons and TEs. Comparing human non-small cell lung cancer (NSCLC) and diverse healthy tissues, we identified a subset of splicing junctions that is both tumor specific and shared across patients. We used HLA-I peptidomics to identify peptides encoded by tumor-specific junctions in primary NSCLC samples and lung tumor cell lines. Recurrent junction-encoded peptides were immunogenic in vitro, and CD8+ T cells specific for junction-encoded epitopes were present in tumors and tumor-draining lymph nodes from patients with NSCLC. We conclude that noncanonical splicing junctions between exons and TEs represent a source of recurrent, immunogenic tumor-specific antigens in patients with NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Masculino , Humanos , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Elementos de DNA Transponíveis , Linfócitos T CD8-Positivos/patologia , Recidiva Local de Neoplasia/genética , Éxons/genética , Antígenos de Neoplasias/genéticaRESUMO
Oncogenesis often implicates epigenetic alterations, including derepression of transposable elements (TEs) and defects in alternative splicing. Here, we explore the possibility that noncanonical splice junctions between exons and TEs represent a source of tumor-specific antigens. We show that mouse normal tissues and tumor cell lines express wide but distinct ranges of mRNA junctions between exons and TEs, some of which are tumor specific. Immunopeptidome analyses in tumor cell lines identified peptides derived from exon-TE splicing junctions associated to MHC-I molecules. Exon-TE junction-derived peptides were immunogenic in tumor-bearing mice. Both prophylactic and therapeutic vaccinations with junction-derived peptides delayed tumor growth in vivo. Inactivation of the TE-silencing histone 3-lysine 9 methyltransferase Setdb1 caused overexpression of new immunogenic junctions in tumor cells. Our results identify exon-TE splicing junctions as epigenetically controlled, immunogenic, and protective tumor antigens in mice, opening possibilities for tumor targeting and vaccination in patients with cancer.
Assuntos
Antígenos de Neoplasias , Elementos de DNA Transponíveis , Animais , Camundongos , Elementos de DNA Transponíveis/genética , Antígenos de Neoplasias/genética , Éxons/genética , RNA Mensageiro , Linhagem Celular TumoralRESUMO
Tumor-infiltrating CD8 + T cells progressively lose functionality and fail to reject tumors. The underlying mechanism and re-programing induced by checkpoint blockers are incompletely understood. We show here that genetic ablation or pharmacological inhibition of histone lysine methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In the absence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival and differentiation of IFNγ and Granzyme B producing effector cells that express negative checkpoint molecules, but do not reach final exhaustion. Their transcriptional program correlates with that of melanoma patients responding to immune-checkpoint blockade and identifies the emergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinical response. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory and pluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effector loci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combined with anti-PD-1, suggesting that Suv39h1 is an "epigenetic checkpoint" for tumor immunity.
Assuntos
Linfócitos T CD8-Positivos , Melanoma , Metiltransferases , Receptor de Morte Celular Programada 1 , Proteínas Repressoras , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Epigênese Genética , Humanos , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/genética , Melanoma/imunologia , Melanoma/terapia , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/imunologia , Metiltransferases/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
We analyze transposable elements (TEs) in glioblastoma (GBM) patients using a proteogenomic pipeline that combines single-cell transcriptomics, bulk RNA sequencing (RNA-seq) samples from tumors and healthy-tissue cohorts, and immunopeptidomic samples. We thus identify 370 human leukocyte antigen (HLA)-I-bound peptides encoded by TEs differentially expressed in GBM. Some of the peptides are encoded by repeat sequences from intact open reading frames (ORFs) present in up to several hundred TEs from recent long interspersed nuclear element (LINE)-1, long terminal repeat (LTR), and SVA subfamilies. Other HLA-I-bound peptides are encoded by single copies of TEs from old subfamilies that are expressed recurrently in GBM tumors and not expressed, or very infrequently and at low levels, in healthy tissues (including brain). These peptide-coding, GBM-specific, highly recurrent TEs represent potential tumor-specific targets for cancer immunotherapies.
Assuntos
Glioblastoma , Antígenos de Histocompatibilidade Classe I , Proteogenômica , Elementos de DNA Transponíveis , Glioblastoma/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Peptídeos/genética , RNA-SeqRESUMO
Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.
Assuntos
Células Dendríticas/imunologia , Inflamação/imunologia , Mucosa Intestinal/patologia , Linfócitos T/imunologia , Actomiosina/metabolismo , Animais , Apresentação de Antígeno , Antígenos CD/metabolismo , Antígeno CD11b/metabolismo , Diferenciação Celular , Movimento Celular , Células Cultivadas , Tolerância Imunológica , Cadeias alfa de Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-2/imunologia , Tretinoína/metabolismoRESUMO
Tumor-infiltrating lymphocytes (TILs), in general, and especially CD8+ TILs, represent a favorable prognostic factor in non-small cell lung cancer (NSCLC). The tissue origin, regenerative capacities, and differentiation pathways of TIL subpopulations remain poorly understood. Using a combination of single-cell RNA and T cell receptor (TCR) sequencing, we investigate the functional organization of TIL populations in primary NSCLC. We identify two CD8+ TIL subpopulations expressing memory-like gene modules: one is also present in blood (circulating precursors) and the other one in juxtatumor tissue (tissue-resident precursors). In tumors, these two precursor populations converge through a unique transitional state into terminally differentiated cells, often referred to as dysfunctional or exhausted. Differentiation is associated with TCR expansion, and transition from precursor to late-differentiated states correlates with intratumor T cell cycling. These results provide a coherent working model for TIL origin, ontogeny, and functional organization in primary NSCLC.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Pulmonares/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T CD8-Positivos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Diferenciação Celular/imunologia , Feminino , Humanos , Pulmão/imunologia , Pulmão/patologia , Pulmão/cirurgia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pneumonectomia , Microambiente Tumoral/imunologiaRESUMO
Transposable elements (TEs) compose nearly half of mammalian genomes and provide building blocks for cis-regulatory elements. Using high-throughput sequencing, we show that 84 TE subfamilies are overrepresented, and distributed in a lineage-specific fashion in core and boundary domains of CD8+ T cell enhancers. Endogenous retroviruses are most significantly enriched in core domains with accessible chromatin, and bear recognition motifs for immune-related transcription factors. In contrast, short interspersed elements (SINEs) are preferentially overrepresented in nucleosome-containing boundaries. A substantial proportion of these SINEs harbor a high density of the enhancer-specific histone mark H3K4me1 and carry sequences that match enhancer boundary nucleotide composition. Motifs with regulatory features are better preserved within enhancer-enriched TE copies compared to their subfamily equivalents located in gene deserts. TE-rich and TE-poor enhancers associate with both shared and unique gene groups and are enriched in overlapping functions related to lymphocyte and leukocyte biology. The majority of T cell enhancers are shared with other immune lineages and are accessible in common hematopoietic progenitors. A higher proportion of immune tissue-specific enhancers are TE-rich compared to enhancers specific to other tissues, correlating with higher TE occurrence in immune gene-associated genomic regions. Our results suggest that during evolution, TEs abundant in these regions and carrying motifs potentially beneficial for enhancer architecture and immune functions were particularly frequently incorporated by evolving enhancers. Their putative selection and regulatory cooption may have accelerated the evolution of immune regulatory networks.
Assuntos
Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos/genética , Evolução Molecular , Linfócitos T/imunologia , Animais , Cromatina/genética , Cromatina/imunologia , Elementos de DNA Transponíveis/imunologia , Retrovirus Endógenos/genética , Retrovirus Endógenos/imunologia , Elementos Facilitadores Genéticos/imunologia , Redes Reguladoras de Genes/genética , Genoma Humano/genética , Genoma Humano/imunologia , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Elementos Nucleotídeos Curtos e Dispersos/genética , Elementos Nucleotídeos Curtos e Dispersos/imunologiaRESUMO
Naive CD4+ T lymphocytes differentiate into different effector types, including helper and regulatory cells (Th and Treg, respectively). Heritable gene expression programs that define these effector types are established during differentiation, but little is known about the epigenetic mechanisms that install and maintain these programs. Here, we use mice defective for different components of heterochromatin-dependent gene silencing to investigate the epigenetic control of CD4+ T cell plasticity. We show that, upon T cell receptor (TCR) engagement, naive and regulatory T cells defective for TRIM28 (an epigenetic adaptor for histone binding modules) or for heterochromatin protein 1 ß and γ isoforms (HP1ß/γ, 2 histone-binding factors involved in gene silencing) fail to effectively signal through the PI3K-AKT-mTOR axis and switch to glycolysis. While differentiation of naive TRIM28-/- T cells into cytokine-producing effector T cells is impaired, resulting in reduced induction of autoimmune colitis, TRIM28-/- regulatory T cells also fail to expand in vivo and to suppress autoimmunity effectively. Using a combination of transcriptome and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses for H3K9me3, H3K9Ac, and RNA polymerase II, we show that reduced effector differentiation correlates with impaired transcriptional silencing at distal regulatory regions of a defined set of Treg-associated genes, including, for example, NRP1 or Snai3. We conclude that TRIM28 and HP1ß/γ control metabolic reprograming through epigenetic silencing of a defined set of Treg-characteristic genes, thus allowing effective T cell expansion and differentiation into helper and regulatory phenotypes.
Assuntos
Diferenciação Celular/fisiologia , Reprogramação Celular/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética/fisiologia , Linfócitos T/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Autoimunidade/fisiologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/genética , Plasticidade Celular/fisiologia , Reprogramação Celular/genética , Homólogo 5 da Proteína Cromobox , Colo/patologia , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Histonas/metabolismo , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transcriptoma , Proteína 28 com Motivo Tripartido/genéticaRESUMO
Clusterin is a glycoprotein able to mediate different physiological functions such as control of complement activation, promotion of unfolded protein clearance and modulation of cell survival. Clusterin is overexpressed in many types of cancers and a large body of evidence suggests that it promotes carcinogenesis and tumor progression. We have previously described a novel clusterin glycoform present in human semen, but not in serum, highly enriched in terminal fucose motifs. Here we show that human luminal breast cancer (LBC) clusterin also bears terminal fucosylated glycans, conferring clusterin the ability to interact with DC-SIGN, a C-type lectin receptor expressed by myeloid cells. This clusterin glycosylation pattern was absent or diminished in non-involved juxtatumoral tissue, suggesting that fucosylated clusterin might represent a cancer associated glycoform. We also found that DC-SIGN is expressed by luminal breast cancer intratumoral macrophages. Moreover, experiments performed in vitro using semen fucosylated clusterin and monocyte derived macrophages showed that the interaction of semen clusterin with DC-SIGN promoted a proangiogenic profile, characterized by a high production of VEGF, IL-8 and TNF-α. Our results reveal an unexpected complexity on the structure and function of secretory clusterin produced by tumors and suggest that fucosylated clusterin produced by luminal breast cancer cells might play a role in tumor progression by promoting the release of pro-angiogenic factors by intratumoral macrophages.
RESUMO
CD4+ T follicular helper (Tfh) cells are essential for inducing efficient humoral responses. T helper polarization is classically orientated by dendritic cells (DCs), which are composed of several subpopulations with distinct functions. Whether human DC subsets display functional specialization for Tfh polarization remains unclear. Here we find that tonsil cDC2 and CD14+ macrophages are the best inducers of Tfh polarization. This ability is intrinsic to the cDC2 lineage but tissue dependent for macrophages. We further show that human Tfh cells comprise two effector states producing either IL-21 or CXCL13. Distinct mechanisms drive the production of Tfh effector molecules, involving IL-12p70 for IL-21 and activin A and TGFß for CXCL13. Finally, using imaging mass cytometry, we find that tonsil CD14+ macrophages localize in situ in the B cell follicles, where they can interact with Tfh cells. Our results indicate that human lymphoid organ cDC2 and macrophages play complementary roles in the induction of Tfh responses.
Assuntos
Tecido Linfoide/imunologia , Macrófagos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Polaridade Celular , Quimiocina CXCL13/metabolismo , Células Dendríticas , Humanos , Interleucinas/metabolismo , Receptores de Lipopolissacarídeos/imunologia , Tecido Linfoide/citologia , Subpopulações de Linfócitos TRESUMO
Cytosolic DNA activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS), an innate immune sensor pivotal in anti-microbial defense, senescence, auto-immunity, and cancer. cGAS is considered to be a sequence-independent DNA sensor with limited access to nuclear DNA because of compartmentalization. However, the nuclear envelope is a dynamic barrier, and cGAS is present in the nucleus. Here, we identify determinants of nuclear cGAS localization and activation. We show that nuclear-localized cGAS synthesizes cGAMP and induces innate immune activation of dendritic cells, although cGAMP levels are 200-fold lower than following transfection with exogenous DNA. Using cGAS ChIP-seq and a GFP-cGAS knockin mouse, we find nuclear cGAS enrichment on centromeric satellite DNA, confirmed by imaging, and to a lesser extent on LINE elements. The non-enzymatic N-terminal domain of cGAS determines nucleo-cytoplasmic localization, enrichment on centromeres, and activation of nuclear-localized cGAS. These results reveal a preferential functional association of nuclear cGAS with centromeres.
Assuntos
Centrômero/enzimologia , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Imunidade Inata/genética , Nucleotidiltransferases/metabolismo , Adulto , Animais , Linhagem Celular , Núcleo Celular/enzimologia , DNA , DNA Satélite , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nucleotidiltransferases/química , Domínios ProteicosRESUMO
Presentation of exogenous antigens on MHC-I molecules, termed cross-presentation, is essential for cytotoxic CD8+ T cell responses. In mice, dendritic cells (DCs) that arise from monocytes (mo-DCs) during inflammation have a key function in these responses by cross-presenting antigens locally in peripheral tissues. Whether human naturally-occurring mo-DCs can cross-present is unknown. Here, we use human mo-DCs and macrophages directly purified from ascites to address this question. Single-cell RNA-seq data show that ascites CD1c+ DCs contain exclusively monocyte-derived cells. Both ascites mo-DCs and monocyte-derived macrophages cross-present efficiently, but are inefficient for transferring exogenous proteins into their cytosol. Inhibition of cysteine proteases, but not of proteasome, abolishes cross-presentation in these cells. We conclude that human monocyte-derived cells cross-present exclusively using a vacuolar pathway. Finally, only ascites mo-DCs provide co-stimulatory signals to induce effector cytotoxic CD8+ T cells. Our findings thus provide important insights on how to harness cross-presentation for therapeutic purposes.
Assuntos
Apresentação de Antígeno , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Vacúolos/metabolismo , Antígenos CD1/metabolismo , Ascite/etiologia , Ascite/imunologia , Buffy Coat/citologia , Técnicas de Cultura de Células , Células Cultivadas , Citosol/metabolismo , Células Dendríticas/metabolismo , Feminino , Perfilação da Expressão Gênica , Glicoproteínas/metabolismo , Voluntários Saudáveis , Humanos , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Neoplasias Ovarianas/complicações , Neoplasias Ovarianas/imunologia , Tonsila Palatina/citologia , Análise de Sequência de RNA , Análise de Célula Única , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Vacúolos/imunologiaRESUMO
The functions and transcriptional profiles of dendritic cells (DCs) result from the interplay between ontogeny and tissue imprinting. How tumors shape human DCs is unknown. Here we used RNA-based next-generation sequencing to systematically analyze the transcriptomes of plasmacytoid pre-DCs (pDCs), cell populations enriched for type 1 conventional DCs (cDC1s), type 2 conventional DCs (cDC2s), CD14+ DCs and monocytes-macrophages from human primary luminal breast cancer (LBC) and triple-negative breast cancer (TNBC). By comparing tumor tissue with non-invaded tissue from the same patient, we found that 85% of the genes upregulated in DCs in LBC were specific to each DC subset. However, all DC subsets in TNBC commonly showed enrichment for the interferon pathway, but those in LBC did not. Finally, we defined transcriptional signatures specific for tumor DC subsets with a prognostic effect on their respective breast-cancer subtype. We conclude that the adjustment of DCs to the tumor microenvironment is subset specific and can be used to predict disease outcome. Our work also provides a resource for the identification of potential targets and biomarkers that might improve antitumor therapies.