RESUMO
Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.
Assuntos
Diferenciação Celular , Variações do Número de Cópias de DNA , Proteína Proto-Oncogênica N-Myc , Crista Neural , Neuroblastoma , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Crista Neural/metabolismo , Crista Neural/patologia , Feminino , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Aberrações Cromossômicas , Células-Tronco Embrionárias Humanas/metabolismo , Transcriptoma , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice-but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.
Assuntos
Homeostase , Janus Quinases , Macrófagos , Camundongos Knockout , Fatores de Transcrição STAT , Transdução de Sinais , Animais , Camundongos , Macrófagos/imunologia , Macrófagos/metabolismo , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Camundongos Endogâmicos C57BL , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , TYK2 Quinase/metabolismo , TYK2 Quinase/genética , Regulação da Expressão GênicaRESUMO
ABSTRACT: Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which myc-associated zinc finger protein (MAZ) and nuclear transcription factor Y subunit alpha (NFY-A) are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.
Assuntos
Quinase 6 Dependente de Ciclina , Células-Tronco Hematopoéticas , Quinase 6 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Animais , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Humanos , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/citologia , Proliferação de Células , Diferenciação Celular , Camundongos Endogâmicos C57BL , Transplante de Células-Tronco Hematopoéticas , Autorrenovação Celular/efeitos dos fármacosRESUMO
Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.
Assuntos
Neutrófilos , Peixe-Zebra , Animais , Humanos , Camundongos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais Geneticamente Modificados , Medula Óssea/metabolismo , Perfilação da Expressão GênicaRESUMO
INTRODUCTION: Structural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a critical component driving synovial tissue remodelling by interacting with immune cells resulting in distinct synovial pathotypes of RA. METHODS: Automated high-content fluorescence microscopy of co-cultured cytokine-activated FLS and autologous peripheral CD4+ T cells from patients with RA was established to quantify cell-cell interactions. Phenotypic profiling of cytokine-treated FLS and co-cultured T cells was done by flow cytometry and RNA-Seq, which were integrated with publicly available transcriptomic data from patients with different histological synovial pathotypes. Computational prediction and knock-down experiments were performed in FLS to identify adhesion molecules for cell-cell interaction. RESULTS: Cytokine stimulation, especially with TNF-α, led to enhanced FLS-T cell interaction resulting in cell-cell contact-dependent activation, proliferation and differentiation of T cells. Signatures of cytokine-activated FLS were significantly enriched in RA synovial tissues defined as lymphoid-rich or leucocyte-rich pathotypes, with the most prominent effects for TNF-α. FLS cytokine signatures correlated with the number of infiltrating CD4+ T cells in synovial tissue of patients with RA. Ligand-receptor pair interaction analysis identified ICAM1 on FLS as an important mediator in TNF-mediated FLS-T cell interaction. Both, ICAM1 and its receptors were overexpressed in TNF-treated FLS and co-cultured T cells. Knock-down of ICAM1 in FLS resulted in reduced TNF-mediated FLS-T cell interaction. CONCLUSION: Our study highlights the role of cytokine-activated FLS in orchestrating inflammation-associated synovial pathotypes providing novel insights into disease mechanisms of RA.
Assuntos
Artrite Reumatoide , Sinoviócitos , Humanos , Citocinas , Fator de Necrose Tumoral alfa/farmacologia , Membrana Sinovial/patologia , Sinoviócitos/patologia , Fibroblastos/patologia , Células CultivadasRESUMO
Metastasis is the major cause of cancer-related deaths. Neuroblastoma (NB), a childhood tumor has been molecularly defined at the primary cancer site, however, the bone marrow (BM) as the metastatic niche of NB is poorly characterized. Here we perform single-cell transcriptomic and epigenomic profiling of BM aspirates from 11 subjects spanning three major NB subtypes and compare these to five age-matched and metastasis-free BM, followed by in-depth single cell analyses of tissue diversity and cell-cell interactions, as well as functional validation. We show that cellular plasticity of NB tumor cells is conserved upon metastasis and tumor cell type composition is NB subtype-dependent. NB cells signal to the BM microenvironment, rewiring via macrophage mgration inhibitory factor and midkine signaling specifically monocytes, which exhibit M1 and M2 features, are marked by activation of pro- and anti-inflammatory programs, and express tumor-promoting factors, reminiscent of tumor-associated macrophages. The interactions and pathways characterized in our study provide the basis for therapeutic approaches that target tumor-to-microenvironment interactions.
Assuntos
Neoplasias da Medula Óssea , Neuroblastoma , Humanos , Criança , Medula Óssea/patologia , Monócitos/metabolismo , Transcriptoma , Epigenômica , Neoplasias da Medula Óssea/genética , Neoplasias da Medula Óssea/metabolismo , Neoplasias da Medula Óssea/patologia , Neuroblastoma/metabolismo , Microambiente Tumoral/genéticaRESUMO
Promoters of antimicrobial genes function as logic boards, integrating signals of innate immune responses. One such set of genes is stimulated by interferon (IFN) signaling, and the expression of these genes [IFN-stimulated genes (ISGs)] can be further modulated by cell stress-induced pathways. Here, we investigated the global effect of stress-induced p38 mitogen-activated protein kinase (MAPK) signaling on the response of macrophages to IFN. In response to cell stress that coincided with IFN exposure, the p38 MAPK-activated transcription factors CREB and c-Jun, in addition to the IFN-activated STAT family of transcription factors, bound to ISGs. In addition, p38 MAPK signaling induced activating histone modifications at the loci of ISGs and stimulated nuclear translocation of the CREB coactivator CRTC3. These actions synergistically enhanced ISG expression. Disrupting this synergy with p38 MAPK inhibitors improved the viability of macrophages infected with Listeria monocytogenes. Our findings uncover a mechanism of transcriptional synergism and highlight the biological consequences of coincident stress-induced p38 MAPK and IFN-stimulated signal transduction.
Assuntos
Interferon gama , Interferons , Interferons/genética , Interferons/farmacologia , Interferons/metabolismo , Interferon gama/metabolismo , Macrófagos/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transcrição Gênica , Fatores de Transcrição/metabolismo , FosforilaçãoRESUMO
BACKGROUND & AIMS: Previous single-cell RNA-sequencing analyses have shown that Trem2-expressing macrophages are present in the liver during obesity, non-alcoholic steatohepatitis (NASH) and cirrhosis. Herein, we aimed to functionally characterize the role of bone marrow-derived TREM2-expressing macrophage populations in NASH. METHODS: We used bulk RNA sequencing to assess the hepatic molecular response to lipid-dependent dietary intervention in mice. Spatial mapping, bone marrow transplantation in two complementary murine models and single-cell sequencing were applied to functionally characterize the role of TREM2+ macrophage populations in NASH. RESULTS: We found that the hepatic transcriptomic profile during steatohepatitis mirrors the dynamics of recruited bone marrow-derived monocytes that already acquire increased expression of Trem2 in the circulation. Increased Trem2 expression was reflected by elevated levels of systemic soluble TREM2 in mice and humans with NASH. In addition, soluble TREM2 levels were superior to traditionally used laboratory parameters for distinguishing between different fatty liver disease stages in two separate clinical cohorts. Spatial transcriptomics revealed that TREM2+ macrophages localize to sites of hepatocellular damage, inflammation and fibrosis in the steatotic liver. Finally, using multiple murine models and in vitro experiments, we demonstrate that hematopoietic Trem2 deficiency causes defective lipid handling and extracellular matrix remodeling, resulting in exacerbated steatohepatitis, cell death and fibrosis. CONCLUSIONS: Our study highlights the functional properties of bone marrow-derived TREM2+ macrophages and implies the clinical relevance of systemic soluble TREM2 levels in the context of NASH. LAY SUMMARY: Our study defines the origin and function of macrophages (a type of immune cell) that are present in the liver and express a specific protein called TREM2. We find that these cells have an important role in protecting against non-alcoholic steatohepatitis (a progressive form of fatty liver disease). We also show that the levels of soluble TREM2 in the blood could serve as a circulating marker of non-alcoholic fatty liver disease.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Modelos Animais de Doenças , Humanos , Lipídeos , Fígado/patologia , Cirrose Hepática/complicações , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , RNA/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
Characterizing resident immune cells in human skin using single-cell assays provides insight into their role in infections, inflammation, and cancer. We describe an optimized protocol to rapidly isolate viable cells from 6-mm skin punch-biopsies. We provide an example in which we coupled single-cell RNA sequencing (scRNA-seq) with single-cell T-cell receptor sequencing (scTCR-seq) of skin and blood cells to study transcriptional profiles and clonotypes of skin resident and peripheral circulating, memory, and effector T cells. This is an improved protocol based on Saluzzo et al. (2021). For complete details on the use and execution of this protocol, please refer to Saluzzo et al. (2021).
Assuntos
Linfócitos T , HumanosRESUMO
We performed a multi-ethnic Epigenome Wide Association study on 22,774 individuals to describe the DNA methylation signature of chronic low-grade inflammation as measured by C-Reactive protein (CRP). We find 1,511 independent differentially methylated loci associated with CRP. These CpG sites show correlation structures across chromosomes, and are primarily situated in euchromatin, depleted in CpG islands. These genomic loci are predominantly situated in transcription factor binding sites and genomic enhancer regions. Mendelian randomization analysis suggests altered CpG methylation is a consequence of increased blood CRP levels. Mediation analysis reveals obesity and smoking as important underlying driving factors for changed CpG methylation. Finally, we find that an activated CpG signature significantly increases the risk for cardiometabolic diseases and COPD.
Assuntos
Metilação de DNA , Inflamação , Proteína C-Reativa/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Humanos , Inflamação/genética , Motivos de NucleotídeosRESUMO
The growth of skin appendages, such as hair, feathers and scales, depends on terminal differentiation of epidermal keratinocytes. Here, we investigated keratinocyte differentiation in avian scutate scales. Cells were isolated from the skin on the legs of 1-day old chicks and subjected to single-cell transcriptomics. We identified two distinct populations of differentiated keratinocytes. The first population was characterized by mRNAs encoding cysteine-rich keratins and corneous beta-proteins (CBPs), also known as beta-keratins, of the scale type, indicating that these cells form hard scales. The second population of differentiated keratinocytes contained mRNAs encoding cysteine-poor keratins and keratinocyte-type CBPs, suggesting that these cells form the soft interscale epidermis. We raised an antibody against keratin 9-like cysteine-rich 2 (KRT9LC2), which is encoded by an mRNA enriched in the first keratinocyte population. Immunostaining confirmed expression of KRT9LC2 in the suprabasal epidermal layers of scutate scales but not in interscale epidermis. Keratinocyte differentiation in chicken leg skin resembled that in human skin with regard to the transcriptional upregulation of epidermal differentiation complex genes and genes involved in lipid metabolism and transport. In conclusion, this study defines gene expression programs that build scutate scales and interscale epidermis of birds and reveals evolutionarily conserved keratinocyte differentiation genes.
Assuntos
Escamas de Animais/metabolismo , Proteínas Aviárias/genética , Diferenciação Celular/genética , Galinhas/genética , Perfilação da Expressão Gênica , Queratinócitos/metabolismo , Análise de Célula Única , Transcriptoma , Escamas de Animais/citologia , Animais , Animais Recém-Nascidos , Proteínas Aviárias/metabolismo , Galinhas/metabolismo , Evolução Molecular , Extremidades , RNA-Seq , Especificidade da Espécie , Transcrição GênicaRESUMO
People living with HIV (PLWH) are at increased risk for developing skin and mucosal malignancies despite systemic reconstitution of CD4+ T cells upon antiretroviral therapy (ART). The underlying mechanism of chronic tissue-related immunodeficiency in HIV is unclear. We found that skin CD4+ tissue-resident memory T (Trm) cells were depleted after HIV infection and replenished only upon early ART initiation. TCR clonal analysis following early ART suggested a systemic origin for reconstituting CD4+ Trm cells. Single-cell RNA sequencing in PLWH that received late ART treatment revealed a loss of CXCR3+ Trm cells and a tolerogenic skin immune environment. Human papilloma virus-induced precancerous lesion biopsies showed reduced CXCR3+ Trm cell frequencies in the mucosa in PLWH versus HIV- individuals. These results reveal an irreversible loss of CXCR3+ Trm cells confined to skin and mucosa in PLWH who received late ART treatment, which may be a precipitating factor in the development of HPV-related cancer.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Síndromes de Imunodeficiência/imunologia , Células T de Memória/imunologia , Mucosa/imunologia , Pele/imunologia , Adulto , Terapia Antirretroviral de Alta Atividade , Feminino , Infecções por HIV/tratamento farmacológico , Sobreviventes de Longo Prazo ao HIV , Humanos , Síndromes de Imunodeficiência/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Receptores CXCR3/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Tempo para o Tratamento , Adulto JovemRESUMO
BACKGROUND: In early-stage mycosis fungoides (MF), the most common primary cutaneous T-cell lymphoma, limited skin involvement with patches and plaques is associated with a favorable prognosis. Nevertheless, approximately 20-30% of cases progress to tumors or erythroderma, resulting in poor outcome. At present, factors contributing to this switch from indolent to aggressive disease are only insufficiently understood. METHODS: In patients with advanced-stage MF, we compared patches with longstanding history to newly developed plaques and tumors by using single-cell RNA sequencing, and compared results with early-stage MF as well as nonlesional MF and healthy control skin. RESULTS: Despite considerable inter-individual variability, lesion progression was uniformly associated with downregulation of the tissue residency markers CXCR4 and CD69, the heat shock protein HSPA1A, the tumor suppressors and immunoregulatory mediators ZFP36 and TXNIP, and the interleukin 7 receptor (IL7R) within the malignant clone, but not in benign T cells. This phenomenon was not only found in conventional TCR-αß MF, but also in a case of TCR-γδ MF, suggesting a common mechanism across MF subtypes. Conversely, malignant cells in clinically unaffected skin from MF patients showed upregulation of these markers. CONCLUSIONS: Our data reveal a specific panel of biomarkers that might be used for monitoring MF disease progression. Altered expression of these genes may underlie the switch in clinical phenotype observed in advanced-stage MF.
Assuntos
Biomarcadores , Regulação Neoplásica da Expressão Gênica , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/patologia , RNA-Seq , Análise de Célula Única , Adulto , Idoso , Biópsia , Biologia Computacional/métodos , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Micose Fungoide/genética , Micose Fungoide/patologia , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única/métodosRESUMO
The transcription factors signal transducer and activator of transcription 5A (STAT5A) and STAT5B are critical in hematopoiesis and leukemia. They are widely believed to have redundant functions, but we describe a unique role for STAT5B in driving the self-renewal of hematopoietic and leukemic stem cells (HSCs/LSCs). We find STAT5B to be specifically activated in HSCs and LSCs, where it induces many genes associated with quiescence and self-renewal, including the surface marker CD9. Levels of CD9 represent a prognostic marker for patients with STAT5-driven leukemia, and our findings suggest that anti-CD9 antibodies may be useful in their treatment to target and eliminate LSCs. We show that it is vital to consider STAT5A and STAT5B as distinct entities in normal and malignant hematopoiesis.
Assuntos
Células-Tronco Hematopoéticas/patologia , Leucemia/patologia , Células-Tronco Neoplásicas/patologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Tetraspanina 29/metabolismo , Animais , Autorrenovação Celular , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Células Tumorais CultivadasRESUMO
Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. While initially restricted to the skin, malignant cells can appear in blood, bone marrow and secondary lymphoid organs in later disease stages. However, only little is known about phenotypic and functional properties of malignant T cells in relationship to tissue environments over the course of disease progression. We thus profiled the tumor micromilieu in skin, blood and lymph node in a patient with advanced MF using single-cell RNA sequencing combined with V-D-J T-cell receptor sequencing. In skin, we identified clonally expanded T-cells with characteristic features of tissue-resident memory T-cells (TRM, CD69+CD27-NR4A1+RGS1+AHR+ ). In blood and lymph node, the malignant clones displayed a transcriptional program reminiscent of a more central memory-like phenotype (KLF2+TCF7+S1PR1+SELL+CCR7+ ), while retaining tissue-homing receptors (CLA, CCR10). The skin tumor microenvironment contained potentially tumor-permissive myeloid cells producing regulatory (IDO1) and Th2-associated mediators (CCL13, CCL17, CCL22). Given their expression of PVR, TNFRSF14 and CD80/CD86, they might be under direct control by TIGIT+CTLA4+CSF2+TNFSF14+ tumor cells. In sum, this study highlights the adaptive phenotypic and functional plasticity of MF tumor cell clones. Thus, the TRM-like phenotype enables long-term skin residence of MF cells. Their switch to a TCM-like phenotype with persistent skin homing molecule expression in the circulation might explain the multi-focal nature of MF.
Assuntos
Micose Fungoide/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Cutâneas/patologia , Linfócitos T/patologia , Idoso , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Memória Imunológica , Linfonodos/metabolismo , Micose Fungoide/genética , Micose Fungoide/metabolismo , Células-Tronco Neoplásicas/química , Análise de Sequência de RNA , Pele/citologia , Pele/imunologia , Linfócitos T/química , Microambiente TumoralRESUMO
CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.
Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19 , Epitopos de Linfócito T , Antígenos HLA-A/imunologia , Imunidade Celular , Mutação , SARS-CoV-2 , Linfócitos T CD8-Positivos/patologia , COVID-19/genética , COVID-19/imunologia , COVID-19/patologia , Proliferação de Células , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interferon gama/imunologia , Peptídeos/genética , Peptídeos/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologiaRESUMO
Adaptive features of natural killer (NK) cells have been reported in various species with different underlying mechanisms. It is unclear, however, which NK cell populations are capable of mounting antigen-specific recall responses and how such functions are regulated at the molecular level. Here, we identify and characterize a discrete population of CD49a+CD16- NK cells in the human liver that displays increased epigenetic potential to elicit memory responses and has the functional properties to exert antigen-specific immunity in the skin as an effector site. Integrated chromatin-based epigenetic and transcriptomic profiling revealed unique characteristics of hepatic CD49a+CD16- NK cells when compared with conventional CD49a-CD16+ NK cells, thereby defining active genomic regions and molecules underpinning distinct NK cell reactivity. In contrast to conventional NK cells, our results suggest that adaptive CD49a+CD16- NK cells are able to bypass the KIR receptor-ligand system upon antigen-specific stimulation. Furthermore, these cells were highly migratory toward chemokine gradients expressed in epicutaneous patch test lesions as an effector site of adaptive immune responses in the skin. These results define pathways operative in human antigen-specific adaptive NK cells and provide a roadmap for harnessing this NK cell subset for specific therapeutic or prophylactic vaccine strategies.
Assuntos
Imunidade Adaptativa/genética , Dermatite de Contato/imunologia , Epigênese Genética/imunologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Linhagem Celular Tumoral , Separação Celular , Dermatite de Contato/genética , Dermatite de Contato/patologia , Feminino , Citometria de Fluxo , Humanos , Integrina alfa1/metabolismo , Células Matadoras Naturais/metabolismo , Fígado/citologia , Subpopulações de Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Níquel/administração & dosagem , Níquel/imunologia , Testes do Emplastro , Cultura Primária de Células , RNA-Seq , Análise de Célula Única , Pele/citologia , Pele/imunologia , Pele/patologiaRESUMO
The Bruton tyrosine kinase (BTK) inhibitor ibrutinib provides effective treatment for patients with chronic lymphocytic leukemia (CLL), despite extensive heterogeneity in this disease. To define the underlining regulatory dynamics, we analyze high-resolution time courses of ibrutinib treatment in patients with CLL, combining immune-phenotyping, single-cell transcriptome profiling, and chromatin mapping. We identify a consistent regulatory program starting with a sharp decrease of NF-κB binding in CLL cells, which is followed by reduced activity of lineage-defining transcription factors, erosion of CLL cell identity, and acquisition of a quiescence-like gene signature. We observe patient-to-patient variation in the speed of execution of this program, which we exploit to predict patient-specific dynamics in the response to ibrutinib based on the pre-treatment patient samples. In aggregate, our study describes time-dependent cellular, molecular, and regulatory effects for therapeutic inhibition of B cell receptor signaling in CLL, and it establishes a broadly applicable method for epigenome/transcriptome-based treatment monitoring.
Assuntos
Tirosina Quinase da Agamaglobulinemia/efeitos dos fármacos , Cromatina/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Pirazóis/antagonistas & inibidores , Pirazóis/metabolismo , Pirazóis/uso terapêutico , Pirimidinas/antagonistas & inibidores , Pirimidinas/metabolismo , Pirimidinas/uso terapêutico , Adenina/análogos & derivados , Epigenoma , Epigenômica , Perfilação da Expressão Gênica , Heterogeneidade Genética/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Aprendizado de Máquina , Piperidinas , Receptores de Antígenos de Linfócitos B/efeitos dos fármacos , Análise de Sequência de RNA , Fatores de Transcrição , TranscriptomaRESUMO
Langerhans cell histiocytosis (LCH) is a rare neoplasm predominantly affecting children. It occupies a hybrid position between cancers and inflammatory diseases, which makes it an attractive model for studying cancer development. To explore the molecular mechanisms underlying the pathophysiology of LCH and its characteristic clinical heterogeneity, we investigated the transcriptomic and epigenomic diversity in primary LCH lesions. Using single-cell RNA sequencing, we identified multiple recurrent types of LCH cells within these biopsies, including putative LCH progenitor cells and several subsets of differentiated LCH cells. We confirmed the presence of proliferative LCH cells in all analyzed biopsies using IHC, and we defined an epigenomic and gene-regulatory basis of the different LCH-cell subsets by chromatin-accessibility profiling. In summary, our single-cell analysis of LCH uncovered an unexpected degree of cellular, transcriptomic, and epigenomic heterogeneity among LCH cells, indicative of complex developmental hierarchies in LCH lesions. SIGNIFICANCE: This study sketches a molecular portrait of LCH lesions by combining single-cell transcriptomics with epigenome profiling. We uncovered extensive cellular heterogeneity, explained in part by an intrinsic developmental hierarchy of LCH cells. Our findings provide new insights and hypotheses for advancing LCH research and a starting point for personalizing therapy.See related commentary by Gruber et al., p. 1343.This article is highlighted in the In This Issue feature, p. 1325.
Assuntos
Epigênese Genética , Epigenômica , Histiocitose de Células de Langerhans/genética , Biomarcadores , Biópsia , Suscetibilidade a Doenças , Epigenômica/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Histiocitose de Células de Langerhans/diagnóstico , Histiocitose de Células de Langerhans/metabolismo , Humanos , Imuno-Histoquímica , Radiografia , Análise de Célula ÚnicaRESUMO
Neural crest cells are embryonic progenitors that generate numerous cell types in vertebrates. With single-cell analysis, we show that mouse trunk neural crest cells become biased toward neuronal lineages when they delaminate from the neural tube, whereas cranial neural crest cells acquire ectomesenchyme potential dependent on activation of the transcription factor Twist1. The choices that neural crest cells make to become sensory, glial, autonomic, or mesenchymal cells can be formalized as a series of sequential binary decisions. Each branch of the decision tree involves initial coactivation of bipotential properties followed by gradual shifts toward commitment. Competing fate programs are coactivated before cells acquire fate-specific phenotypic traits. Determination of a specific fate is achieved by increased synchronization of relevant programs and concurrent repression of competing fate programs.