RESUMO
Esophageal adenocarcinoma (EAC) is a highly lethal cancer of the upper gastrointestinal tract with rising incidence in western populations. To decipher EAC disease progression and therapeutic response, we performed multiomic analyses of a cohort of primary and metastatic EAC tumors, incorporating single-nuclei transcriptomic and chromatin accessibility sequencing, along with spatial profiling. We identified tumor microenvironmental features previously described to associate with therapy response. We identified five malignant cell programs, including undifferentiated, intermediate, differentiated, epithelial-to-mesenchymal transition, and cycling programs, which were associated with differential epigenetic plasticity and clinical outcomes, and for which we inferred candidate transcription factor regulons. Furthermore, we revealed diverse spatial localizations of malignant cells expressing their associated transcriptional programs and predicted their significant interactions with microenvironmental cell types. We validated our findings in three external single-cell RNA-seq and three bulk RNA-seq studies. Altogether, our findings advance the understanding of EAC heterogeneity, disease progression, and therapeutic response.
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A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole-sporozoite PfSPZ vaccine in African infants. Innate immune activation and myeloid signatures at prevaccination baseline correlated with protection from P. falciparum parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ vaccine dose. Machine learning identified spliceosome, proteosome, and resting DC signatures as prevaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline circumsporozoite protein-specific (CSP-specific) IgG predicted nonprotection. Prevaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8+ T cell responses after vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naive mice while diminishing the CD8+ T cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity by whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8+ T cell responses suggests that PfSPZ vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.
Assuntos
Imunidade Inata , Vacinas Antimaláricas , Malária Falciparum , Plasmodium falciparum , Esporozoítos , Vacinas Atenuadas , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/administração & dosagem , Imunidade Inata/imunologia , Humanos , Animais , Malária Falciparum/prevenção & controle , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Camundongos , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/administração & dosagem , Esporozoítos/imunologia , Esporozoítos/efeitos da radiação , Linfócitos T CD8-Positivos/imunologia , Lactente , Proteínas de Protozoários/imunologia , Anticorpos Antiprotozoários/imunologia , Feminino , Parasitemia/imunologia , Parasitemia/prevenção & controle , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Eficácia de VacinasRESUMO
Clear cell renal cell carcinoma (ccRCC) is molecularly heterogeneous, immune infiltrated, and selectively sensitive to immune checkpoint inhibition (ICI). However, the joint tumor-immune states that mediate ICI response remain elusive. We develop spatially aware deep-learning models of tumor and immune features to learn representations of ccRCC tumors using diagnostic whole-slide images (WSIs) in untreated and treated contexts (n = 1,102 patients). We identify patterns of grade heterogeneity in WSIs not achievable through human pathologist analysis, and these graph-based "microheterogeneity" structures associate with PBRM1 loss of function and with patient outcomes. Joint analysis of tumor phenotypes and immune infiltration identifies a subpopulation of highly infiltrated, microheterogeneous tumors responsive to ICI. In paired multiplex immunofluorescence images of ccRCC, microheterogeneity associates with greater PD1 activation in CD8+ lymphocytes and increased tumor-immune interactions. Our work reveals spatially interacting tumor-immune structures underlying ccRCC biology that may also inform selective response to ICI.
Assuntos
Carcinoma de Células Renais , Carcinoma , Aprendizado Profundo , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , FenótipoRESUMO
Neuroendocrine tumors (NETs) are rare cancers that most often arise in the gastrointestinal tract and pancreas. The fundamental mechanisms driving gastroenteropancreatic (GEP)-NET growth remain incompletely elucidated; however, the heterogeneous clinical behavior of GEP-NETs suggests that both cellular lineage dynamics and tumor microenvironment influence tumor pathophysiology. Here, we investigated the single-cell transcriptomes of tumor and immune cells from patients with gastroenteropancreatic NETs. Malignant GEP-NET cells expressed genes and regulons associated with normal, gastrointestinal endocrine cell differentiation, and fate determination stages. Tumor and lymphoid compartments sparsely expressed immunosuppressive targets commonly investigated in clinical trials, such as the programmed cell death protein-1/programmed death ligand-1 axis. However, infiltrating myeloid cell types within both primary and metastatic GEP-NETs were enriched for genes encoding other immune checkpoints, including VSIR (VISTA), HAVCR2 (TIM3), LGALS9 (Gal-9), and SIGLEC10. Our findings highlight the transcriptomic heterogeneity that distinguishes the cellular landscapes of GEP-NET anatomic subtypes and reveal potential avenues for future precision medicine therapeutics.
Assuntos
Neoplasias Intestinais , Tumores Neuroendócrinos , Neoplasias Pancreáticas , Neoplasias Gástricas , Humanos , Tumores Neuroendócrinos/genética , Neoplasias Intestinais/genética , Neoplasias Gástricas/genética , Neoplasias Pancreáticas/genética , Microambiente Tumoral/genéticaRESUMO
Clear cell renal cell carcinoma (ccRCC) is molecularly heterogeneous, immune infiltrated, and selectively sensitive to immune checkpoint inhibition (ICI). Established histopathology paradigms like nuclear grade have baseline prognostic relevance for ccRCC, although whether existing or novel histologic features encode additional heterogeneous biological and clinical states in ccRCC is uncertain. Here, we developed spatially aware deep learning models of tumor- and immune-related features to learn representations of ccRCC tumors using diagnostic whole-slide images (WSI) in untreated and treated contexts (n = 1102 patients). We discovered patterns of nuclear grade heterogeneity in WSI not achievable through human pathologist analysis, and these graph-based "microheterogeneity" structures associated with PBRM1 loss of function, adverse clinical factors, and selective patient response to ICI. Joint computer vision analysis of tumor phenotypes with inferred tumor infiltrating lymphocyte density identified a further subpopulation of highly infiltrated, microheterogeneous tumors responsive to ICI. In paired multiplex immunofluorescence images of ccRCC, microheterogeneity associated with greater PD1 activation in CD8+ lymphocytes and increased tumor-immune interactions. Thus, our work reveals novel spatially interacting tumor-immune structures underlying ccRCC biology that can also inform selective response to ICI.
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BACKGROUND: Oncogenes act in a cell-intrinsic way to promote tumorigenesis. Whether oncogenes also have a cell-extrinsic effect on suppressing the immune response to cancer is less well understood. METHODS: We use an in vivo expression screen of known cancer-associated somatic mutations in mouse syngeneic tumor models treated with checkpoint blockade to identify oncogenes that promote immune evasion. We then validated candidates from this screen in vivo and analyzed the tumor immune microenvironment of tumors expressing mutant protein to identify mechanisms of immune evasion. RESULTS: We found that expression of a catalytically active mutation in phospho-inositol 3 kinase (PI3K), PIK3CA c.3140A>G (H1047R) confers a selective growth advantage to tumors treated with immunotherapy that is reversed by pharmacological PI3K inhibition. PIK3CA H1047R-expression in tumors decreased the number of CD8+ T cells but increased the number of inhibitory myeloid cells following immunotherapy. Inhibition of myeloid infiltration by pharmacological or genetic modulation of Ccl2 in PIK3CA H1047R tumors restored sensitivity to programmed cell death protein 1 (PD-1) checkpoint blockade. CONCLUSIONS: PI3K activation enables tumor immune evasion by promoting an inhibitory myeloid microenvironment. Activating mutations in PI3K may be useful as a biomarker of poor response to immunotherapy. Our data suggest that some oncogenes promote tumorigenesis by enabling tumor cells to avoid clearance by the immune system. Identification of those mechanisms can advance rational combination strategies to increase the efficacy of immunotherapy.
Assuntos
Neoplasias , Microambiente Tumoral , Animais , Linfócitos T CD8-Positivos/metabolismo , Carcinogênese , Classe I de Fosfatidilinositol 3-Quinases/genética , Modelos Animais de Doenças , Humanos , Evasão da Resposta Imune , Inositol , Camundongos , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Despite initial responses1-3, most melanoma patients develop resistance4 to immune checkpoint blockade (ICB). To understand the evolution of resistance, we studied 37 tumor samples over 9 years from a patient with metastatic melanoma with complete clinical response to ICB followed by delayed recurrence and death. Phylogenetic analysis revealed co-evolution of seven lineages with multiple convergent, but independent resistance-associated alterations. All recurrent tumors emerged from a lineage characterized by loss of chromosome 15q, with post-treatment clones acquiring additional genomic driver events. Deconvolution of bulk RNA sequencing and highly multiplexed immunofluorescence (t-CyCIF) revealed differences in immune composition among different lineages. Imaging revealed a vasculogenic mimicry phenotype in NGFRhi tumor cells with high PD-L1 expression in close proximity to immune cells. Rapid autopsy demonstrated two distinct NGFR spatial patterns with high polarity and proximity to immune cells in subcutaneous tumors versus a diffuse spatial pattern in lung tumors, suggesting different roles of this neural-crest-like program in different tumor microenvironments. Broadly, this study establishes a high-resolution map of the evolutionary dynamics of resistance to ICB, characterizes a de-differentiated neural-crest tumor population in melanoma immunotherapy resistance and describes site-specific differences in tumor-immune interactions via longitudinal analysis of a patient with melanoma with an unusual clinical course.
Assuntos
Antígeno B7-H1/genética , Inibidores de Checkpoint Imunológico/uso terapêutico , Melanoma/terapia , Proteínas do Tecido Nervoso/genética , Receptores de Fator de Crescimento Neural/genética , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Cromossomos Humanos Par 15/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Imunoterapia/efeitos adversos , Masculino , Melanoma/genética , Melanoma/imunologia , Melanoma/patologia , Metástase Neoplásica , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Proteínas do Tecido Nervoso/imunologia , Filogenia , Receptores de Fator de Crescimento Neural/imunologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Immune checkpoint blockade (ICB) results in durable disease control in a subset of patients with advanced renal cell carcinoma (RCC), but mechanisms driving resistance are poorly understood. We characterize the single-cell transcriptomes of cancer and immune cells from metastatic RCC patients before or after ICB exposure. In responders, subsets of cytotoxic T cells express higher levels of co-inhibitory receptors and effector molecules. Macrophages from treated biopsies shift toward pro-inflammatory states in response to an interferon-rich microenvironment but also upregulate immunosuppressive markers. In cancer cells, we identify bifurcation into two subpopulations differing in angiogenic signaling and upregulation of immunosuppressive programs after ICB. Expression signatures for cancer cell subpopulations and immune evasion are associated with PBRM1 mutation and survival in primary and ICB-treated advanced RCC. Our findings demonstrate that ICB remodels the RCC microenvironment and modifies the interplay between cancer and immune cell populations critical for understanding response and resistance to ICB.
Assuntos
Carcinoma de Células Renais/terapia , Fatores Imunológicos/imunologia , Imunoterapia , Neoplasias Renais/terapia , Microambiente Tumoral/imunologia , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Proteínas de Ligação a DNA/imunologia , Humanos , Imunoterapia/métodos , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Fatores de Transcrição/imunologiaRESUMO
Metastatic castration-resistant prostate cancer is typically lethal, exhibiting intrinsic or acquired resistance to second-generation androgen-targeting therapies and minimal response to immune checkpoint inhibitors1. Cellular programs driving resistance in both cancer and immune cells remain poorly understood. We present single-cell transcriptomes from 14 patients with advanced prostate cancer, spanning all common metastatic sites. Irrespective of treatment exposure, adenocarcinoma cells pervasively coexpressed multiple androgen receptor isoforms, including truncated isoforms hypothesized to mediate resistance to androgen-targeting therapies2,3. Resistance to enzalutamide was associated with cancer cell-intrinsic epithelial-mesenchymal transition and transforming growth factor-ß signaling. Small cell carcinoma cells exhibited divergent expression programs driven by transcriptional regulators promoting lineage plasticity and HOXB5, HOXB6 and NR1D2 (refs. 4-6). Additionally, a subset of patients had high expression of dysfunction markers on cytotoxic CD8+ T cells undergoing clonal expansion following enzalutamide treatment. Collectively, the transcriptional characterization of cancer and immune cells from human metastatic castration-resistant prostate cancer provides a basis for the development of therapeutic approaches complementing androgen signaling inhibition.
Assuntos
Antineoplásicos/farmacologia , Neoplasias de Próstata Resistentes à Castração/terapia , Transcrição Gênica/efeitos dos fármacos , Biópsia , Linfócitos T CD8-Positivos/imunologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/imunologia , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismoRESUMO
Sarcomatoid and rhabdoid (S/R) renal cell carcinoma (RCC) are highly aggressive tumors with limited molecular and clinical characterization. Emerging evidence suggests immune checkpoint inhibitors (ICI) are particularly effective for these tumors, although the biological basis for this property is largely unknown. Here, we evaluate multiple clinical trial and real-world cohorts of S/R RCC to characterize their molecular features, clinical outcomes, and immunologic characteristics. We find that S/R RCC tumors harbor distinctive molecular features that may account for their aggressive behavior, including BAP1 mutations, CDKN2A deletions, and increased expression of MYC transcriptional programs. We show that these tumors are highly responsive to ICI and that they exhibit an immune-inflamed phenotype characterized by immune activation, increased cytotoxic immune infiltration, upregulation of antigen presentation machinery genes, and PD-L1 expression. Our findings build on prior work and shed light on the molecular drivers of aggressivity and responsiveness to ICI of S/R RCC.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Carcinoma de Células Renais/imunologia , Regulação Neoplásica da Expressão Gênica , Inibidores de Checkpoint Imunológico/uso terapêutico , Proteínas de Checkpoint Imunológico/imunologia , Neoplasias Renais/imunologia , Tumor Rabdoide/imunologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/mortalidade , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas de Checkpoint Imunológico/genética , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Mutação , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , Estudos Retrospectivos , Tumor Rabdoide/tratamento farmacológico , Tumor Rabdoide/genética , Tumor Rabdoide/mortalidade , Transdução de Sinais , Análise de Sobrevida , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/imunologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Lymph node fibroblastic reticular cells (FRCs) respond to signals from activated T cells by releasing nitric oxide, which inhibits T cell proliferation and restricts the size of the expanding T cell pool. Whether interactions with FRCs also support the function or differentiation of activated CD8+ T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8+ T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8+ T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansion-they can also shape the fate and function of CD8+ T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Fibroblastos/fisiologia , Linfonodos/imunologia , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Reprogramação Celular , Montagem e Desmontagem da Cromatina , Citotoxicidade Imunológica , Epigênese Genética , Regulação da Expressão Gênica , Memória Imunológica , Interleucina-6/genética , Interleucina-6/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismoRESUMO
T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8+ tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8+ TILs include a subpopulation of 'progenitor exhausted' cells that retain polyfunctionality, persist long term and differentiate into 'terminally exhausted' TILs. Consequently, progenitor exhausted CD8+ TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8+ T cells might be an important component of improving the response to checkpoint blockade.
Assuntos
Anticorpos Bloqueadores/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Melanoma Experimental/prevenção & controle , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos Bloqueadores/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Linhagem Celular Tumoral , Feminino , Humanos , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/virologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/virologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/prevenção & controle , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Melanoma Experimental/imunologia , Melanoma Experimental/virologia , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8+ T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance.
Assuntos
Adenosina Desaminase/deficiência , Adenosina Desaminase/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoterapia , Inflamação/genética , Inflamação/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferons/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/radioterapia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Edição de RNA , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Initial molecular details of cellular activation following αßT cell antigen receptor (TCR) ligation by peptide-major histocompatibility complexes (pMHC) remain unexplored. We determined the nuclear magnetic resonance (NMR) structure of the TCRα subunit transmembrane (TM) domain revealing a bipartite helix whose segmentation fosters dynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification caused stepwise reduction in TCR associations with CD3ζζ homodimers and CD3εγ plus CD3εδ heterodimers, respectively, leading to an activated transcriptome. Optical tweezers revealed that Arg251 and Lys256 mutations altered αßTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ CxxC motif juxtamembrane elements selectively attenuated signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate T cell activation via a dissociative mechanism, shifting disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRαß and CD3 associations.
Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Domínios Proteicos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Biomarcadores , Complexo CD3/química , Sequência Conservada , Perfilação da Expressão Gênica , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Transdução de Sinais , TranscriptomaRESUMO
Regulatory T cells (Tregs) are key modulators of immune tolerance, capable of suppressing inflammatory immune responses and promoting nonlymphoid tissue homeostasis. Helios, a transcription factor (TF) that is selectively expressed by Tregs, has been shown to be essential for the maintenance of Treg lineage stability in the face of inflammatory conditions that include autoimmune disease and cancer. Helios-deficient Tregs within tumors acquire effector T cell function and contribute to immune responses against cancer. However, the underlying genetic basis of this Treg reprogramming is not well understood. Here, we report that Helios-deficient Tregs within the chronic inflammatory tumor microenvironment (TME) derepress genetic programs associated with T helper (Th) cell differentiation by up-regulating Th cell-associated TFs and effector cytokines. These genetic changes of Helios-deficient Tregs are most apparent in a Treg subpopulation with high affinity for self-antigens, as detected by both increased GITR/PD-1 expression and increased responsiveness to self-antigens. Their combined effects may promote a phenotype conversion of Tregs into effector T cells within the TME, where TCR engagement and costimulatory receptor expression by Tregs are increased. These data provide a genetic basis for the unstable phenotype of Helios-deficient Tregs within the inflammatory environment of tumors and suggest that immune milieu-dependent alterations in gene expression are a central feature of Treg conversion.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Melanoma/metabolismo , Neoplasias Experimentais/metabolismo , Linfócitos T Reguladores/fisiologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição/genéticaRESUMO
To investigate the role of TCR-pMHC interaction in regulating lung CD8 tissue-resident T cell (TR ) differentiation, polyclonal responses were compared against NP366-374 /Db and PA224-233 /Db , two immunodominant epitopes that arise during influenza A infection in mice. Memory niches distinct from iBALTs develop within the lamina propria, supporting CD103+ and CD103- CD8 TR generation and intraepithelial translocation. Gene set enrichment analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) identify dominant TCR, adherens junction, RIG-I-like and NOD-like pattern recognition receptor as well as TGF-ß signaling pathways and memory signatures among PA224-233 /Db T cells consistent with T resident memory (TRM ) status. In contrast, NP366-374 /Db T cells exhibit enrichment of effector signatures, upregulating pro-inflammatory mediators even among TRM . While NP366-374 /Db T cells manifest transcripts linked to canonical exhaustion pathways, PA224-233 /Db T cells exploit P2rx7 purinoreceptor attenuation. The NP366-374 /Db CD103+ subset expresses the antimicrobial lactotransferrin whereas PA224-233 /Db CD103+ utilizes pore-forming mpeg-1, with <22% of genes correspondingly upregulated in CD103+ (or CD103- ) subsets of both specificities. Thus, TCR-pMHC interactions among TR and antigen presenting cells in a tissue milieu strongly impact CD8 T cell biology.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos CD/biossíntese , Diferenciação Celular/imunologia , Proteína DEAD-box 58/metabolismo , Feminino , Memória Imunológica/imunologia , Cadeias alfa de Integrinas/biossíntese , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas NLR/metabolismo , Infecções por Orthomyxoviridae/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Understanding how decidual CD8+ T cell (CD8+ dT) cytotoxicity is regulated and how these cells integrate the competing needs for maternal-fetal tolerance and immunity to infection is an important research and clinical goal. Gene-expression analysis of effector-memory CD8+ dT demonstrated a mixed transcriptional signature of T cell dysfunction, activation, and effector function. High protein expression of coinhibitory molecules PD1, CTLA4, and LAG3, accompanied by low expression of cytolytic molecules suggests that the decidual microenvironment reduces CD8+ dT effector responses to maintain tolerance to fetal antigens. However, CD8+ dT degranulated, proliferated, and produced IFN-γ, TNF-α, perforin, and granzymes upon in vitro stimulation, demonstrating that CD8+ dT are not permanently suppressed and retain the capacity to respond to proinflammatory events, such as infections. The balance between transient dysfunction of CD8+ dT that are permissive of placental and fetal development, and reversal of this dysfunctional state, is crucial in understanding the etiology of pregnancy complications and prevention of congenital infections.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Decídua/metabolismo , Perfilação da Expressão Gênica/métodos , Tolerância Imunológica/genética , Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Feminino , Granzimas/genética , Granzimas/metabolismo , Humanos , Tolerância Imunológica/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Perforina/genética , Perforina/metabolismo , Fatores de TempoRESUMO
The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described 'naive-like' memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/química , Receptores de Antígenos de Linfócitos T/química , Análise de Sequência de RNA/métodos , Software , Algoritmos , Animais , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Célula Única , Vírus da Febre Amarela/imunologiaRESUMO
Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.