RESUMO
Immune-checkpoint blockade (ICB) has shown remarkable clinical success in boosting antitumor immunity. However, the breadth of its cellular targets and specific mode of action remain elusive. We find that tumor-infiltrating follicular regulatory T (TFR) cells are prevalent in tumor tissues of several cancer types. They are primarily located within tertiary lymphoid structures and exhibit superior suppressive capacity and in vivo persistence as compared with regulatory T cells, with which they share a clonal and developmental relationship. In syngeneic tumor models, anti-PD-1 treatment increases the number of tumor-infiltrating TFR cells. Both TFR cell deficiency and the depletion of TFR cells with anti-CTLA-4 before anti-PD-1 treatment improve tumor control in mice. Notably, in a cohort of 271 patients with melanoma, treatment with anti-CTLA-4 followed by anti-PD-1 at progression was associated with better a survival outcome than monotherapy with anti-PD-1 or anti-CTLA-4, anti-PD-1 followed by anti-CTLA-4 at progression or concomitant combination therapy.
Assuntos
Antígeno CTLA-4/antagonistas & inibidores , Inibidores de Checkpoint Imunológico/uso terapêutico , Linfócitos do Interstício Tumoral/imunologia , Melanoma/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T Reguladores/imunologia , Animais , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células T Auxiliares Foliculares/imunologia , Microambiente Tumoral/imunologiaRESUMO
While many genetic variants have been associated with risk for human diseases, how these variants affect gene expression in various cell types remains largely unknown. To address this gap, the DICE (database of immune cell expression, expression quantitative trait loci [eQTLs], and epigenomics) project was established. Considering all human immune cell types and conditions studied, we identified cis-eQTLs for a total of 12,254 unique genes, which represent 61% of all protein-coding genes expressed in these cell types. Strikingly, a large fraction (41%) of these genes showed a strong cis-association with genotype only in a single cell type. We also found that biological sex is associated with major differences in immune cell gene expression in a highly cell-specific manner. These datasets will help reveal the effects of disease risk-associated genetic polymorphisms on specific immune cell types, providing mechanistic insights into how they might influence pathogenesis (https://dice-database.org).
Assuntos
Regulação da Expressão Gênica/imunologia , Genótipo , Polimorfismo de Nucleotídeo Único/imunologia , Locos de Características Quantitativas/imunologia , Caracteres Sexuais , Adolescente , Adulto , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The plant homeodomain zinc-finger protein, PHF6, is a transcriptional regulator, and PHF6 germline mutations cause the X-linked intellectual disability (XLID) Börjeson-Forssman-Lehmann syndrome (BFLS). The mechanisms by which PHF6 regulates transcription and how its mutations cause BFLS remain poorly characterized. Here, we show genome-wide binding of PHF6 in the developing cortex in the vicinity of genes involved in central nervous system development and neurogenesis. Characterization of BFLS mice harbouring PHF6 patient mutations reveals an increase in embryonic neural stem cell (eNSC) self-renewal and a reduction of neural progenitors. We identify a panel of Ephrin receptors (EphRs) as direct transcriptional targets of PHF6. Mechanistically, we show that PHF6 regulation of EphR is impaired in BFLS mice and in conditional Phf6 knock-out mice. Knockdown of EphR-A phenocopies the PHF6 loss-of-function defects in altering eNSCs, and its forced expression rescues defects of BFLS mice-derived eNSCs. Our data indicate that PHF6 directly promotes Ephrin receptor expression to control eNSC behaviour in the developing brain, and that this pathway is impaired in BFLS.
Assuntos
Epilepsia , Face/anormalidades , Dedos/anormalidades , Transtornos do Crescimento , Hipogonadismo , Deficiência Intelectual , Deficiência Intelectual Ligada ao Cromossomo X , Obesidade , Humanos , Camundongos , Animais , Deficiência Intelectual/genética , Proteínas Repressoras , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Fatores de TranscriçãoRESUMO
Single-cell analysis across multiple samples and conditions requires quantitative modeling of the interplay between the continuum of cell states and the technical and biological sources of sample-to-sample variability. We introduce GEDI, a generative model that identifies latent space variations in multi-sample, multi-condition single-cell datasets and attributes them to sample-level covariates. GEDI enables cross-sample cell state mapping on par with state-of-the-art integration methods, cluster-free differential gene expression analysis along the continuum of cell states, and machine learning-based prediction of sample characteristics from single-cell data. GEDI can also incorporate gene-level prior knowledge to infer pathway and regulatory network activities in single cells. Finally, GEDI extends all these concepts to previously unexplored modalities that require joint consideration of dual measurements, such as the joint analysis of exon inclusion/exclusion reads to model alternative cassette exon splicing, or spliced/unspliced reads to model the mRNA stability landscapes of single cells.
Assuntos
Análise de Célula Única , Análise de Célula Única/métodos , Humanos , Perfilação da Expressão Gênica/métodos , Aprendizado de Máquina , Redes Reguladoras de Genes , Biologia Computacional/métodos , Algoritmos , Processamento AlternativoRESUMO
The impact of genetic variants on cells challenged in biologically relevant contexts has not been fully explored. Here, we activated CD4+ T cells from 89 healthy donors and performed a single-cell RNA sequencing assay with >1 million cells to examine cell type-specific and activation-dependent effects of genetic variants. Single-cell expression quantitative trait loci (sc-eQTL) analysis of 19 distinct CD4+ T cell subsets showed that the expression of over 4000 genes is significantly associated with common genetic polymorphisms and that most of these genes show their most prominent effects in specific cell types. These genes included many that encode for molecules important for activation, differentiation, and effector functions of T cells. We also found new gene associations for disease-risk variants identified from genome-wide association studies and highlighted the cell types in which their effects are most prominent. We found that biological sex has a major influence on activation-dependent gene expression in CD4+ T cell subsets. Sex-biased transcripts were significantly enriched in several pathways that are essential for the initiation and execution of effector functions by CD4+ T cells like TCR signaling, cytokines, cytokine receptors, costimulatory, apoptosis, and cell-cell adhesion pathways. Overall, this DICE (Database of Immune Cell Expression, eQTLs, and Epigenomics) subproject highlights the power of sc-eQTL studies for simultaneously exploring the activation and cell type-dependent effects of common genetic variants on gene expression (https://dice-database.org).
Assuntos
Linfócitos T CD4-Positivos/imunologia , Locos de Características Quantitativas , Análise de Célula Única , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Adulto JovemRESUMO
Brain tumor stem cells (BTSCs) and intratumoral heterogeneity represent major challenges in glioblastoma therapy. Here, we report that the LGALS1 gene, encoding the carbohydrate binding protein, galectin1, is a key regulator of BTSCs and glioblastoma resistance to therapy. Genetic deletion of LGALS1 alters BTSC gene expression profiles and results in downregulation of gene sets associated with the mesenchymal subtype of glioblastoma. Using a combination of pharmacological and genetic approaches, we establish that inhibition of LGALS1 signaling in BTSCs impairs self-renewal, suppresses tumorigenesis, prolongs lifespan, and improves glioblastoma response to ionizing radiation in preclinical animal models. Mechanistically, we show that LGALS1 is a direct transcriptional target of STAT3 with its expression robustly regulated by the ligand OSM. Importantly, we establish that galectin1 forms a complex with the transcription factor HOXA5 to reprogram the BTSC transcriptional landscape. Our data unravel an oncogenic signaling pathway by which the galectin1/HOXA5 complex maintains BTSCs and promotes glioblastoma.
Assuntos
Neoplasias Encefálicas/metabolismo , Galectina 1/metabolismo , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Transcrição Gênica , Idoso , Animais , Antineoplásicos/farmacologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Calixarenos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Autorrenovação Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Galectina 1/antagonistas & inibidores , Galectina 1/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/radioterapia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos SCID , Pessoa de Meia-Idade , Mutação , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Tolerância a Radiação , Radiossensibilizantes/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Common genetic polymorphisms associated with COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Given the importance of immune cells in the pathogenesis of COVID-19 illness, here we assessed the effects of COVID-19-risk variants on gene expression in a wide range of immune cell types. Transcriptome-wide association study and colocalization analysis revealed putative causal genes and the specific immune cell types where gene expression is most influenced by COVID-19-risk variants. Notable examples include OAS1 in non-classical monocytes, DTX1 in B cells, IL10RB in NK cells, CXCR6 in follicular helper T cells, CCR9 in regulatory T cells and ARL17A in TH2 cells. By analysis of transposase accessible chromatin and H3K27ac-based chromatin-interaction maps of immune cell types, we prioritized potentially functional COVID-19-risk variants. Our study highlights the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations.
Assuntos
COVID-19/genética , COVID-19/imunologia , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Receptores CCR/genética , Receptores CCR/metabolismo , Fatores de Risco , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Expression quantitative trait loci (eQTLs) studies provide associations of genetic variants with gene expression but fall short of pinpointing functionally important eQTLs. Here, using H3K27ac HiChIP assays, we mapped eQTLs overlapping active cis-regulatory elements that interact with their target gene promoters (promoter-interacting eQTLs, pieQTLs) in five common immune cell types (Database of Immune Cell Expression, Expression quantitative trait loci and Epigenomics (DICE) cis-interactome project). This approach allowed us to identify functionally important eQTLs and show mechanisms that explain their cell-type restriction. We also devised an approach to eQTL discovery that relies on HiChIP-based promoter interaction maps as a structural framework for deciding which SNPs to test for association with gene expression, and observe ultra-long-distance pieQTLs (>1 megabase away), including several disease-risk variants. We validated the functional role of pieQTLs using reporter assays, CRISPRi, dCas9-tiling guides and Cas9-mediated base-pair editing. In this article we present a method for functional eQTL discovery and provide insights into relevance of noncoding variants for cell-specific gene regulation and for disease association beyond conventional eQTL mapping.
Assuntos
Regulação da Expressão Gênica , Variação Genética , Regiões Promotoras Genéticas , Locos de Características Quantitativas/genética , Acetilação , Sequência de Bases , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Estudo de Associação Genômica Ampla , Genótipo , Histonas/metabolismo , Humanos , Células Jurkat , Leucócitos/metabolismo , Lisina/metabolismo , Análise de Componente PrincipalRESUMO
Common genetic polymorphisms associated with severity of COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Here, we assessed the effects of 679 COVID-19-risk variants on gene expression in a wide-range of immune cell types. Severe COVID-19-risk variants were significantly associated with the expression of 11 protein-coding genes, and overlapped with either target gene promoter or cis -regulatory regions that interact with target promoters in the cell types where their effects are most prominent. For example, we identified that the association between variants in the 3p21.31 risk locus and the expression of CCR2 in classical monocytes is likely mediated through an active cis-regulatory region that interacted with CCR2 promoter specifically in monocytes. The expression of several other genes showed prominent genotype-dependent effects in non-classical monocytes, NK cells, B cells, or specific T cell subtypes, highlighting the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations.
RESUMO
High numbers of tissue-resident memory T (TRM) cells are associated with better clinical outcomes in cancer patients. However, the molecular characteristics that drive their efficient immune response to tumors are poorly understood. Here, single-cell and bulk transcriptomic analysis of TRM and non-TRM cells present in tumor and normal lung tissue from patients with lung cancer revealed that PD-1-expressing TRM cells in tumors were clonally expanded and enriched for transcripts linked to cell proliferation and cytotoxicity when compared with PD-1-expressing non-TRM cells. This feature was more prominent in the TRM cell subset coexpressing PD-1 and TIM-3, and it was validated by functional assays ex vivo and also reflected in their chromatin accessibility profile. This PD-1+TIM-3+ TRM cell subset was enriched in responders to PD-1 inhibitors and in tumors with a greater magnitude of CTL responses. These data highlight that not all CTLs expressing PD-1 are dysfunctional; on the contrary, TRM cells with PD-1 expression were enriched for features suggestive of superior functionality.
Assuntos
Perfilação da Expressão Gênica , Memória Imunológica/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Análise de Célula Única , Linfócitos T/imunologia , Transcriptoma/genética , Proliferação de Células , Células Clonais , Citotoxicidade Imunológica/genética , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/patologia , Subpopulações de Linfócitos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptor de Morte Celular Programada 1/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
CD4+ cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, and heterogeneity, especially in relation to other well-described CD4+ memory T cell subsets. We performed single-cell RNA sequencing in more than 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile, and clonality in humans. Single-cell differential gene expression analysis revealed a spectrum of known transcripts, including several linked to cytotoxic and costimulatory function that are expressed at higher levels in the TEMRA (effector memory T cells expressing CD45RA) subset, which is highly enriched for CD4-CTLs, compared with CD4+ T cells in the central memory (TCM) and effector memory (TEM) subsets. Simultaneous T cell antigen receptor (TCR) analysis in single cells and bulk subsets revealed that CD4-TEMRA cells show marked clonal expansion compared with TCM and TEM cells and that most of CD4-TEMRA were dengue virus (DENV)-specific in donors with previous DENV infection. The profile of CD4-TEMRA was highly heterogeneous across donors, with four distinct clusters identified by the single-cell analysis. We identified distinct clusters of CD4-CTL effector and precursor cells in the TEMRA subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and, thus, could provide insights into the mechanisms that may be used to generate durable and effective CD4-CTL immunity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T Citotóxicos/imunologia , Transcriptoma/imunologia , Perfilação da Expressão Gênica/métodos , Humanos , Memória Imunológica/imunologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Célula Única/métodosRESUMO
Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however, unipotent neutrophil progenitors are not well defined. Here, we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly, we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models, including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases, including cancer.