RESUMO
While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1+ IL-17+ SLAMF6+ population that traffics to the intestine where it is maintained by the microbiota, providing a ready reservoir for the IL-23-driven generation of encephalitogenic GM-CSF+ IFN-γ+ CXCR6+ T cells. Our study defines a direct in vivo relationship between IL-17+ non-pathogenic and GM-CSF+ and IFN-γ+ pathogenic Th17 populations and provides a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease.
Assuntos
Autoimunidade , Intestinos/imunologia , Células-Tronco/metabolismo , Células Th17/imunologia , Animais , Movimento Celular , Células Clonais , Encefalomielite Autoimune Experimental/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Homeostase , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , RNA/metabolismo , RNA-Seq , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CXCR6/metabolismo , Receptores de Interleucina/metabolismo , Reprodutibilidade dos Testes , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Análise de Célula Única , Baço/metabolismoRESUMO
Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA sequencing (RNA-seq) and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses.
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Anticorpos Neutralizantes/imunologia , Linfócitos B/metabolismo , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Anticorpos Antivirais/sangue , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/metabolismo , Reações Antígeno-Anticorpo , Linfócitos B/citologia , Linfócitos B/virologia , COVID-19/patologia , COVID-19/virologia , Microscopia Crioeletrônica , Cristalografia por Raios X , Perfilação da Expressão Gênica , Humanos , Imunoglobulina A/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Domínios Proteicos/imunologia , Multimerização Proteica , Estrutura Quaternária de Proteína , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.
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Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Proteínas Morfogenéticas Ósseas/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Compartimento Celular , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Estudos de Coortes , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade , Inflamação/patologia , Monócitos/patologia , Células Mieloides/patologia , Neutrófilos/patologia , Células Estromais/metabolismo , Linfócitos T/metabolismo , Transcrição GênicaRESUMO
Reciprocal interactions between host T helper cells and gut microbiota enforce local immunological tolerance and modulate extra-intestinal immunity. However, our understanding of antigen-specific tolerance to the microbiome is limited. Here, we developed a systematic approach to predict HLA class-II-specific epitopes using the humanized bacteria-originated T cell antigen (hBOTA) algorithm. We identified a diverse set of microbiome epitopes spanning all major taxa that are compatible with presentation by multiple HLA-II alleles. In particular, we uncovered an immunodominant epitope from the TonB-dependent receptor SusC that was universally recognized and ubiquitous among Bacteroidales. In healthy human subjects, SusC-reactive T cell responses were characterized by IL-10-dominant cytokine profiles, whereas in patients with active Crohn's disease, responses were associated with elevated IL-17A. Our results highlight the potential of targeted antigen discovery within the microbiome to reveal principles of tolerance and functional transitions during inflammation.
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Doença de Crohn , Epitopos Imunodominantes , Linfócitos T CD4-Positivos , Epitopos de Linfócito T , Humanos , Interleucina-10 , Interleucina-17RESUMO
Interleukin-23 receptor plays a critical role in inducing inflammation and autoimmunity. Here, we report that Th1-like cells differentiated in vitro with IL-12 + IL-21 showed similar IL-23R expression to that of pathogenic Th17 cells using eGFP reporter mice. Fate mapping established that these cells did not transition through a Th17 cell state prior to becoming Th1-like cells, and we observed their emergence in vivo in the T cell adoptive transfer colitis model. Using IL-23R-deficient Th1-like cells, we demonstrated that IL-23R was required for the development of a highly colitogenic phenotype. Single-cell RNA sequencing analysis of intestinal T cells identified IL-23R-dependent genes in Th1-like cells that differed from those expressed in Th17 cells. The perturbation of one of these regulators (CD160) in Th1-like cells inhibited the induction of colitis. We thus uncouple IL-23R as a purely Th17 cell-specific factor and implicate IL-23R signaling as a pathogenic driver in Th1-like cells inducing tissue inflammation.
Assuntos
Colite , Receptores de Interleucina , Animais , Inflamação/metabolismo , Interleucina-23/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Células Th1 , Células Th17RESUMO
The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour growth1,2. Here, using high-throughput flow cytometry as well as bulk and single-cell RNA-sequencing and B-cell-receptor-sequencing analysis of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumour-bearing mice. The expanding B cell subset expresses the cell surface molecule T cell immunoglobulin and mucin domain 1 (TIM-1, encoded by Havcr1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. Although conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumour burden, selective deletion of Havcr1 in B cells both substantially inhibited tumour growth and enhanced effector T cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumour-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing B cells enables engagement of the second arm of adaptive immunity to promote anti-tumour immunity and inhibit tumour growth.
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Linfócitos B , Melanoma , Animais , Camundongos , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Ativação Linfocitária , Melanoma/imunologia , Melanoma/patologia , Melanoma/prevenção & controle , Linfócitos T/citologia , Linfócitos T/imunologia , Citometria de Fluxo , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Linfonodos/citologia , Linfonodos/imunologia , Apresentação de Antígeno , Receptores de Antígenos de Linfócitos B/genética , Análise da Expressão Gênica de Célula Única , Carga Tumoral , Interferon Tipo IRESUMO
COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.
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COVID-19/patologia , COVID-19/virologia , Rim/patologia , Fígado/patologia , Pulmão/patologia , Miocárdio/patologia , SARS-CoV-2/patogenicidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Atlas como Assunto , Autopsia , Bancos de Espécimes Biológicos , COVID-19/genética , COVID-19/imunologia , Células Endoteliais , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Fibroblastos , Estudo de Associação Genômica Ampla , Coração/virologia , Humanos , Inflamação/patologia , Inflamação/virologia , Rim/virologia , Fígado/virologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Fagócitos , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/virologia , RNA Viral/análise , Regeneração , SARS-CoV-2/imunologia , Análise de Célula Única , Carga ViralRESUMO
Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.
Assuntos
Células Epiteliais/citologia , Epitélio/metabolismo , Intestino Delgado/citologia , Análise de Célula Única , Animais , Diferenciação Celular , Citocinas/metabolismo , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Homeostase , Antígenos Comuns de Leucócito/metabolismo , Masculino , Camundongos , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/metabolismo , Transcrição Gênica , Linfopoietina do Estroma do TimoRESUMO
Coordinated cell interactions within the esophagus maintain homeostasis, and disruption can lead to eosinophilic esophagitis (EoE), a chronic inflammatory disease with poorly understood pathogenesis. We profile 421,312 individual cells from the esophageal mucosa of 7 healthy and 15 EoE participants, revealing 60 cell subsets and functional alterations in cell states, compositions, and interactions that highlight previously unclear features of EoE. Active disease displays enrichment of ALOX15+ macrophages, PRDM16+ dendritic cells expressing the EoE risk gene ATP10A, and cycling mast cells, with concomitant reduction of TH17 cells. Ligand-receptor expression uncovers eosinophil recruitment programs, increased fibroblast interactions in disease, and IL-9+IL-4+IL-13+ TH2 and endothelial cells as potential mast cell interactors. Resolution of inflammation-associated signatures includes mast and CD4+ TRM cell contraction and cell type-specific downregulation of eosinophil chemoattractant, growth, and survival factors. These cellular alterations in EoE and remission advance our understanding of eosinophilic inflammation and opportunities for therapeutic intervention.
Assuntos
Esofagite Eosinofílica , Humanos , Esofagite Eosinofílica/genética , Esofagite Eosinofílica/patologia , Células Endoteliais/metabolismo , Interleucina-13 , Inflamação/genéticaRESUMO
Whole chromosome losses resulting in near-haploid karyotypes are found in a rare subgroup of treatment-refractory acute lymphoblastic leukemia. To systematically dissect the unique physiology and uncover susceptibilities that can be exploited in near-haploid leukemia, we leveraged single-cell RNA-Seq and computational inference of cell cycle stages to pinpoint key differences between near-haploid and diploid leukemia cells. Combining cell cycle stage-specific differential expression with gene essentiality scores from a genome-wide CRISPR-Cas9-mediated knockout screen, we identified the homologous recombination pathway component RAD51B as an essential gene in near-haploid leukemia. DNA damage analyses revealed significantly increased sensitivity of RAD51-mediated repair to RAD51B loss in the G2/M stage of near-haploid cells, suggesting a unique role of RAD51B in the homologous recombination pathway. Elevated G2/M and G1/S checkpoint signaling was part of a RAD51B signature expression program in response to chemotherapy in a xenograft model of human near-haploid B-ALL, and RAD51B and its associated programs were overexpressed in a large panel of near-haploid B-ALL patients. These data highlight a unique genetic dependency on DNA repair machinery in near-haploid leukemia and demarcate RAD51B as a promising candidate for targeted therapy in this treatment-resistant disease.
Assuntos
Leucemia Linfoide , Multiômica , Humanos , Haploidia , Aberrações Cromossômicas , Reparo do DNA , ProteínasRESUMO
Plasma cells (PCs) constitute a significant fraction of colonic mucosal cells and contribute to inflammatory infiltrates in ulcerative colitis (UC). While gut PCs secrete bacteria-targeting IgA antibodies, their role in UC pathogenesis is unknown. We performed single-cell V(D)J- and RNA-seq on sorted B cells from the colon of healthy individuals and patients with UC. A large fraction of B cell clones is shared between different colon regions, but inflammation in UC broadly disrupts this landscape, causing transcriptomic changes characterized by an increase in the unfolded protein response (UPR) and antigen presentation genes, clonal expansion, and isotype skewing from IgA1 and IgA2 to IgG1. We also directly expressed and assessed the specificity of 152 mAbs from expanded PC clones. These mAbs show low polyreactivity and autoreactivity and instead target both shared bacterial antigens and specific bacterial strains. Altogether, our results characterize the microbiome-specific colon PC response and how its disruption might contribute to inflammation in UC.
Assuntos
Colite Ulcerativa , Humanos , Colite Ulcerativa/genética , Plasmócitos , Colo , Inflamação/metabolismo , Antígenos de Bactérias , Bactérias , Imunoglobulina A/metabolismo , Mucosa IntestinalRESUMO
E3 ligases regulate key processes, but many of their roles remain unknown. Using Perturb-seq, we interrogated the function of 1,130 E3 ligases, partners and substrates in the inflammatory response in primary dendritic cells (DCs). Dozens impacted the balance of DC1, DC2, migratory DC and macrophage states and a gradient of DC maturation. Family members grouped into co-functional modules that were enriched for physical interactions and impacted specific programs through substrate transcription factors. E3s and their adaptors co-regulated the same processes, but partnered with different substrate recognition adaptors to impact distinct aspects of the DC life cycle. Genetic interactions were more prevalent within than between modules, and a deep learning model, comßVAE, predicts the outcome of new combinations by leveraging modularity. The E3 regulatory network was associated with heritable variation and aberrant gene expression in immune cells in human inflammatory diseases. Our study provides a general approach to dissect gene function.
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In healthy skin, a cutaneous immune system maintains the balance between tolerance towards innocuous environmental antigens and immune responses against pathological agents. In atopic dermatitis (AD), barrier and immune dysfunction result in chronic tissue inflammation. Our understanding of the skin tissue ecosystem in AD remains incomplete with regard to the hallmarks of pathological barrier formation, and cellular state and clonal composition of disease-promoting cells. Here, we generated a multi-modal cell census of 310,691 cells spanning 86 cell subsets from whole skin tissue of 19 adult individuals, including non-lesional and lesional skin from 11 AD patients, and integrated it with 396,321 cells from four studies into a comprehensive human skin cell atlas in health and disease. Reconstruction of human keratinocyte differentiation from basal to cornified layers revealed a disrupted cornification trajectory in AD. This disrupted epithelial differentiation was associated with signals from a unique immune and stromal multicellular community comprised of MMP12 + dendritic cells (DCs), mature migratory DCs, cycling ILCs, NK cells, inflammatory CCL19 + IL4I1 + fibroblasts, and clonally expanded IL13 + IL22 + IL26 + T cells with overlapping type 2 and type 17 characteristics. Cell subsets within this immune and stromal multicellular community were connected by multiple inter-cellular positive feedback loops predicted to impact community assembly and maintenance. AD GWAS gene expression was enriched both in disrupted cornified keratinocytes and in cell subsets from the lesional immune and stromal multicellular community including IL13 + IL22 + IL26 + T cells and ILCs, suggesting that epithelial or immune dysfunction in the context of the observed cellular communication network can initiate and then converge towards AD. Our work highlights specific, disease-associated cell subsets and interactions as potential targets in progression and resolution of chronic inflammation.
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SARS-CoV-2 infection leads to a broad range of outcomes and immune responses, with the development of neutralizing antibodies generally correlated with protection against reinfection. Here, we have characterized both neutralizing activity and T cell responses in a cluster of subjects with mild disease linked to a single spreading event. Surprisingly, we observed sex-specific associations between spike- and particularly nucleoprotein-specific T cell responses and neutralization, with pro-inflammatory cytokines being linked to higher titers only in males. Using single cell immunoprofiling, which provided matched transcriptome and T-cell receptor (TCR) profiles in restimulated CD4 + and CD8 + cells from these subjects, we identified differences in type I IFN signaling that may underlie this difference in antibody generation. Finally, we also identified several TCRs associated with cytokine producing T cells. Altogether, our work maps the breadth of immunological outcomes of SARS-CoV2 infections and highlight the potential role of sex-specific feedback loops during the generation of neutralizing antibodies.
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Establishing causal relationships between genetic alterations of human cancers and specific phenotypes of malignancy remains a challenge. We sequentially introduced mutations into healthy human melanocytes in up to five genes spanning six commonly disrupted melanoma pathways, forming nine genetically distinct cellular models of melanoma. We connected mutant melanocyte genotypes to malignant cell expression programs in vitro and in vivo, replicative immortality, malignancy, rapid tumor growth, pigmentation, metastasis, and histopathology. Mutations in malignant cells also affected tumor microenvironment composition and cell states. Our melanoma models shared genotype-associated expression programs with patient melanomas, and a deep learning model showed that these models partially recapitulated genotype-associated histopathological features as well. Thus, a progressive series of genome-edited human cancer models can causally connect genotypes carrying multiple mutations to phenotype.
Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanócitos/metabolismo , Melanoma/patologia , Mutação , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Microambiente Tumoral/genéticaRESUMO
The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.
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Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment-refractory cancer. Molecular stratification in pancreatic cancer remains rudimentary and does not yet inform clinical management or therapeutic development. Here, we construct a high-resolution molecular landscape of the cellular subtypes and spatial communities that compose PDAC using single-nucleus RNA sequencing and whole-transcriptome digital spatial profiling (DSP) of 43 primary PDAC tumor specimens that either received neoadjuvant therapy or were treatment naive. We uncovered recurrent expression programs across malignant cells and fibroblasts, including a newly identified neural-like progenitor malignant cell program that was enriched after chemotherapy and radiotherapy and associated with poor prognosis in independent cohorts. Integrating spatial and cellular profiles revealed three multicellular communities with distinct contributions from malignant, fibroblast and immune subtypes: classical, squamoid-basaloid and treatment enriched. Our refined molecular and cellular taxonomy can provide a framework for stratification in clinical trials and serve as a roadmap for therapeutic targeting of specific cellular phenotypes and multicellular interactions.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/terapia , Perfilação da Expressão Gênica , Humanos , Terapia Neoadjuvante , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Prognóstico , Transcriptoma/genética , Neoplasias PancreáticasRESUMO
The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.
Assuntos
Evasão da Resposta Imune/imunologia , Imunoterapia/métodos , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Pancreáticas/imunologia , Receptores Virais/imunologia , Animais , Humanos , Camundongos , Neoplasias PancreáticasRESUMO
The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.
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Tumor evolution from a single cell into a malignant, heterogeneous tissue remains poorly understood. Here, we profile single-cell transcriptomes of genetically engineered mouse lung tumors at seven stages, from pre-neoplastic hyperplasia to adenocarcinoma. The diversity of transcriptional states increases over time and is reproducible across tumors and mice. Cancer cells progressively adopt alternate lineage identities, computationally predicted to be mediated through a common transitional, high-plasticity cell state (HPCS). Accordingly, HPCS cells prospectively isolated from mouse tumors and human patient-derived xenografts display high capacity for differentiation and proliferation. The HPCS program is associated with poor survival across human cancers and demonstrates chemoresistance in mice. Our study reveals a central principle underpinning intra-tumoral heterogeneity and motivates therapeutic targeting of the HPCS.