RESUMO
Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Proteômica , Análise de Célula Única , Transcriptoma , Humanos , Análise de Célula Única/métodos , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Proteômica/métodos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Hematopoese , Nicho de Células-Tronco , Células da Medula Óssea/metabolismo , Células da Medula Óssea/citologiaRESUMO
T-lineage acute lymphoblastic leukaemia (T-ALL) is a high-risk tumour1 that has eluded comprehensive genomic characterization, which is partly due to the high frequency of noncoding genomic alterations that result in oncogene deregulation2,3. Here we report an integrated analysis of genome and transcriptome sequencing of tumour and remission samples from more than 1,300 uniformly treated children with T-ALL, coupled with epigenomic and single-cell analyses of malignant and normal T cell precursors. This approach identified 15 subtypes with distinct genomic drivers, gene expression patterns, developmental states and outcomes. Analyses of chromatin topology revealed multiple mechanisms of enhancer deregulation that involve enhancers and genes in a subtype-specific manner, thereby demonstrating widespread involvement of the noncoding genome. We show that the immunophenotypically described, high-risk entity of early T cell precursor ALL is superseded by a broader category of 'early T cell precursor-like' leukaemia. This category has a variable immunophenotype and diverse genomic alterations of a core set of genes that encode regulators of hematopoietic stem cell development. Using multivariable outcome models, we show that genetic subtypes, driver and concomitant genetic alterations independently predict treatment failure and survival. These findings provide a roadmap for the classification, risk stratification and mechanistic understanding of this disease.
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The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.
Assuntos
Linfócitos T CD4-Positivos , Imunoterapia Adotiva , Leucemia , Receptores de Antígenos Quiméricos , Antígenos CD19/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Separação Celular , Humanos , Leucemia/imunologia , Leucemia/terapia , Receptores de Antígenos Quiméricos/imunologia , Fatores de TempoRESUMO
Hematopoietic stem cell (HSC) ontogeny is accompanied by dynamic changes in gene regulatory networks. We performed RNA-seq and histone mark ChIP-seq to define the transcriptomes and epigenomes of cells representing key developmental stages of HSC ontogeny in mice. The five populations analyzed were embryonic day 10.5 (E10.5) endothelium and hemogenic endothelium from the major arteries, an enriched population of prehematopoietic stem cells (pre-HSCs), fetal liver HSCs, and adult bone marrow HSCs. Using epigenetic signatures, we identified enhancers for each developmental stage. Only 12% of enhancers are primed, and 78% are active, suggesting the vast majority of enhancers are established de novo without prior priming in earlier stages. We constructed developmental stage-specific transcriptional regulatory networks by linking enhancers and predicted bound transcription factors to their target promoters using a novel computational algorithm, target inference via physical connection (TIPC). TIPC predicted known transcriptional regulators for the endothelial-to-hematopoietic transition, validating our overall approach, and identified putative novel transcription factors, including the broadly expressed transcription factors SP3 and MAZ. Finally, we validated a role for SP3 and MAZ in the formation of hemogenic endothelium. Our data and computational analyses provide a useful resource for uncovering regulators of HSC formation.
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Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Algoritmos , Animais , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Epigênese Genética/genética , Edição de Genes , Camundongos , Fator de Transcrição Sp3/metabolismo , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Differentiation of effector and memory CD8+ T cells is accompanied by extensive changes in the transcriptome and histone modifications at gene promoters; however, the enhancer repertoire and associated gene regulatory networks are poorly defined. Using histone mark chromatin immunoprecipitation coupled with deep sequencing, we mapped the enhancer and super-enhancer landscapes in antigen-specific naive, differentiated effector, and central memory CD8+ T cells during LCMV infection. Epigenomics-based annotation revealed a highly dynamic repertoire of enhancers, which were inherited, de novo activated, decommissioned and re-activated during CD8+ T cell responses. We employed a computational algorithm to pair enhancers with target gene promoters. On average, each enhancer targeted three promoters and each promoter was regulated by two enhancers. By identifying enriched transcription factor motifs in enhancers, we defined transcriptional regulatory circuitry at each CD8+ T cell response stage. These multi-dimensional datasets provide a blueprint for delineating molecular mechanisms underlying functional differentiation of CD8+ T cells.
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Infecções por Arenaviridae/imunologia , Linfócitos T CD8-Positivos/imunologia , Elementos Facilitadores Genéticos/imunologia , Regulação da Expressão Gênica/imunologia , Ativação Linfocitária/imunologia , Algoritmos , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Modelos Animais de Doenças , Elementos Facilitadores Genéticos/genética , Epigenômica/métodos , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Ativação Linfocitária/genética , Vírus da Coriomeningite Linfocítica , Camundongos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologiaRESUMO
KMT2A-rearranged (KMT2A-r) infant acute lymphoblastic leukemia (ALL) is a devastating malignancy with a dismal outcome, and younger age at diagnosis is associated with increased risk of relapse. To discover age-specific differences and critical drivers that mediate poor outcome in KMT2A-r ALL, we subjected KMT2A-r leukemias and normal hematopoietic cells from patients of different ages to single-cell multiomics analyses. We uncovered the following critical new insights: leukemia cells from patients <6 months have significantly increased lineage plasticity. Steroid response pathways are downregulated in the most immature blasts from younger patients. We identify a hematopoietic stem and progenitor-like (HSPC-like) population in the blood of younger patients that contains leukemic blasts and form an immunosuppressive signaling circuit with cytotoxic lymphocytes. These observations offer a compelling explanation for the ability of leukemias in young patients to evade chemotherapy and immune-mediated control. Our analysis also revealed preexisting lymphomyeloid primed progenitors and myeloid blasts at initial diagnosis of B-ALL. Tracking of leukemic clones in 2 patients whose leukemia underwent a lineage switch documented the evolution of such clones into frank acute myeloid leukemia (AML). These findings provide critical insights into KMT2A-r ALL and have clinical implications for molecularly targeted and immunotherapy approaches. Beyond infant ALL, our study demonstrates the power of single-cell multiomics to detect tumor intrinsic and extrinsic factors affecting rare but critical subpopulations within a malignant population that ultimately determines patient outcome.
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Antineoplásicos , Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Antineoplásicos/uso terapêutico , Rearranjo Gênico , Humanos , Imunoterapia , Lactente , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
Schima superba, commonly known as the Chinese guger tree, is highly adaptable and tolerant of poor soil conditions. It is one of the primary species forming the evergreen broad-leaved forests in southern China. Dirigent proteins (DIRs) play crucial roles in the synthesis of plant lignin and lignans, secondary metabolism, and response to adversity stress. However, research on the DIR gene family in S. superba is currently limited. This study identified 24 SsDIR genes, categorizing them into three subfamilies. These genes are unevenly distributed across 13 chromosomes, with 83% being intronless. Collinearity analysis indicated that tandem duplication played a more significant role in the expansion of the gene family compared to segmental duplication. Additionally, we analyzed the expression patterns of SsDIRs in different tissues of S. superba. The SsDIR genes exhibited distinct expression patterns across various tissues, with most being specifically expressed in the roots. Further screening identified SsDIR genes that may regulate drought stress, with many showing differential expression under drought stress conditions. In the promoter regions of SsDIRs, various cis-regulatory elements involved in developmental regulation, hormone response, and stress response were identified, which may be closely related to their diverse regulatory functions. This study will contribute to the further functional identification of SsDIR genes, providing insights into the biosynthetic pathways of lignin and lignans and the mechanisms of plant stress resistance.
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Evolução Molecular , Regulação da Expressão Gênica de Plantas , Família Multigênica , Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Filogenia , Genoma de Planta , Lignina/biossíntese , Lignina/genética , Lignina/metabolismo , Perfilação da Expressão Gênica , Cromossomos de Plantas/genética , Secas , Duplicação Gênica , Regiões Promotoras GenéticasRESUMO
Hematopoietic stem and progenitor cells (HSPCs) in the bone marrow are derived from a small population of hemogenic endothelial (HE) cells located in the major arteries of the mammalian embryo. HE cells undergo an endothelial to hematopoietic cell transition, giving rise to HSPCs that accumulate in intra-arterial clusters (IAC) before colonizing the fetal liver. To examine the cell and molecular transitions between endothelial (E), HE, and IAC cells, and the heterogeneity of HSPCs within IACs, we profiled â¼40 000 cells from the caudal arteries (dorsal aorta, umbilical, vitelline) of 9.5 days post coitus (dpc) to 11.5 dpc mouse embryos by single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing. We identified a continuous developmental trajectory from E to HE to IAC cells, with identifiable intermediate stages. The intermediate stage most proximal to HE, which we term pre-HE, is characterized by increased accessibility of chromatin enriched for SOX, FOX, GATA, and SMAD motifs. A developmental bottleneck separates pre-HE from HE, with RUNX1 dosage regulating the efficiency of the pre-HE to HE transition. A distal candidate Runx1 enhancer exhibits high chromatin accessibility specifically in pre-HE cells at the bottleneck, but loses accessibility thereafter. Distinct developmental trajectories within IAC cells result in 2 populations of CD45+ HSPCs; an initial wave of lymphomyeloid-biased progenitors, followed by precursors of hematopoietic stem cells (pre-HSCs). This multiomics single-cell atlas significantly expands our understanding of pre-HSC ontogeny.
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Diferenciação Celular , Endotélio/embriologia , Hemangioblastos/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Diferenciação Celular/genética , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Embrião de Mamíferos , Endotélio/citologia , Endotélio/metabolismo , Feminino , Dosagem de Genes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hemangioblastos/citologia , Hematopoese/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , RNA-Seq/métodosRESUMO
Dissolved organic matter (DOM) derived from various composts can promote significant changes of soil properties. However, little is known about the DOM compositions and their similarities and differences at the molecular level. In this study, the molecular compositions of DOM derived from kitchen waste compost (KWC), green waste compost (GWC), manure waste compost (MWC), and sewage sludge compost (SSC) were characterized by electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS). The molecular formulas were classified into four subcategories: CHO, CHON, CHOS, and CHONS. The KWC, MWC, and SSC DOM represented the highest fraction (35.8%-47.4%) of CHON subcategory, while the GWC DOM represented the highest fraction (68.4%) of CHO subcategory. The GWC DOM was recognized as the nitrogen- and sulfur-deficient compounds that were less saturated, more aromatic, and more oxidized compared with other samples. Further analysis of the oxygen, nitrogen-containing (N-containing), and sulfur-containing (S-containing) functional groups in the four subcategories revealed higher organic molecular complexity. Comparison of the similarities and differences of the four samples revealed 22.8% ubiquitous formulas and 17.4%, 11.1%, 10.7%, and 6.3% unique formulas of GWC, KWC, SSC, and MWC DOM, respectively, suggesting a large proportion of ubiquitous DOM as well as unique, source-specific molecular signatures. The findings presented herein provide new insight into the molecular characterization of DOM derived from various composts and demonstrated the potential role of these different compounds for agricultural utilization.
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Compostagem , Espectrometria de Massas , Nitrogênio , Esgotos , SoloRESUMO
Ratiometric electrochemiluminescence (ECL) has attracted great attention in the field of electrochemical analysis. In this study, a dual-signal-output ratiometric ECL sensor was developed for the detection of nicotinamide adenine dinucleotide (NADH). Nitrogen-doped graphene quantum dots (NGQDs) exhibit double ECL signal output capability, without the requirement of additional coreactants. NADH can amplify the anodic ECL response of NGQDs, while it can diminish the cathodic ECL response of NGQDs. Based on the principle between relative enhancing ECL intensity ratio and NADH concentrations, the constructed ratiometric ECL sensor was applied to NADH assays, with a wide concentration range of 10-400 µM and a low limit of detection (LOD) of 2.5 µM (S/N = 3). Furthermore, the proposed method was applied for the determination of spiked NADH, which was proved to be feasible in the biological sample matrix. The proposed strategy of modulating multiple-ECL signals of the single NGQD emitter not only provides a new ECL system for the accurate detection of NADH but also broadens the design pathway for ratiometric sensing fabrication.
Assuntos
NAD/sangue , Pontos Quânticos/química , Técnicas Eletroquímicas/métodos , Grafite/química , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Medições Luminescentes/métodos , Nitrogênio/químicaRESUMO
The sensitivity of chromatin immunoprecipitation (ChIP) assays poses a major obstacle for epigenomic studies of low-abundance cells. Here we present a microfluidics-based ChIP-seq protocol using as few as 100 cells via drastically improved collection of high-quality ChIP-enriched DNA. Using this technology, we uncovered many new enhancers and super enhancers in hematopoietic stem and progenitor cells from mouse fetal liver, suggesting that enhancer activity is highly dynamic during early hematopoiesis.
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Imunoprecipitação da Cromatina/instrumentação , Imunoprecipitação da Cromatina/métodos , Epigenômica/instrumentação , Dispositivos Lab-On-A-Chip , Acetilação , Animais , Elementos Facilitadores Genéticos , Epigenômica/métodos , Desenho de Equipamento , Feminino , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Histonas/metabolismo , Humanos , Fígado/embriologia , Fígado/fisiologia , Masculino , Indústria Manufatureira/métodos , Camundongos Endogâmicos C57BLRESUMO
Enhancer mapping has been greatly facilitated by various genomic marks associated with it. However, little is available in our toolbox to link enhancers with their target promoters, hampering mechanistic understanding of enhancer-promoter (EP) interaction. We develop and characterize multiple genomic features for distinguishing true EP pairs from noninteracting pairs. We integrate these features into a probabilistic predictor for EP interactions. Multiple validation experiments demonstrate a significant improvement over state-of-the-art approaches. Systematic analyses of EP interactions across 12 cell types reveal several global features of EP interactions: (i) a larger fraction of EP interactions are cell type specific than enhancers; (ii) promoters controlled by multiple enhancers have higher tissue specificity, but the regulating enhancers are less conserved; (iii) cohesin plays a role in mediating tissue-specific EP interactions via chromatin looping in a CTCF-independent manner. Our approach presents a systematic and effective strategy to decipher the mechanisms underlying EP communication.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica/fisiologia , Modelos Genéticos , Regiões Promotoras Genéticas/fisiologia , Transdução de Sinais/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Biologia Computacional , Simulação por Computador , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Genômica/métodos , Humanos , Regiões Promotoras Genéticas/genética , CoesinasRESUMO
Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies to treat hematologic diseases. To date, HoxB4 remains the most effective transcription factor (TF) the overexpression of which in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop more efficient protocols for in vitro derivation of HSCs. In the present study, we performed global gene-expression profiling and ChIP coupled with deep sequencing at 4 stages of the HoxB4-mediated ESC differentiation toward HSCs. Joint analyses of ChIP/deep sequencing and gene-expression profiling unveiled several global features of the HoxB4 regulatory network. First, it is highly dynamic and gradually expands during the differentiation process. Second, HoxB4 functions as a master regulator of hematopoiesis by regulating multiple hematopoietic TFs and chromatin-modification enzymes. Third, HoxB4 acts in different combinations with 4 other hematopoietic TFs (Fli1, Meis1, Runx1, and Scl) to regulate distinct sets of pathways. Finally, the results of our study suggest that down-regulation of mitochondria and lysosomal genes by HoxB4 plays a role in the impaired lymphoid lineage development from ESC-derived HSCs.
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Células Sanguíneas/fisiologia , Diferenciação Celular/genética , Células-Tronco Embrionárias/fisiologia , Redes Reguladoras de Genes/genética , Hematopoese/genética , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Animais , Células Sanguíneas/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Linfócitos/metabolismo , Linfócitos/fisiologia , Camundongos , Análise em Microsséries , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The bone marrow is the organ responsible for blood production. Diverse non-hematopoietic cells contribute essentially to hematopoiesis. However, these cells and their spatial organization remain largely uncharacterized as they have been technically challenging to study in humans. Here, we used fresh femoral head samples and performed single-cell RNA sequencing (scRNA-Seq) to profile 29,325 enriched non-hematopoietic bone marrow cells and discover nine transcriptionally distinct subtypes. We next employed CO-detection by inDEXing (CODEX) multiplexed imaging of 18 individuals, including both healthy and acute myeloid leukemia (AML) samples, to spatially profile over one million single cells with a novel 53-antibody panel. We discovered a relatively hyperoxygenated arterio-endosteal niche for early myelopoiesis, and an adipocytic, but not endosteal or perivascular, niche for early hematopoietic stem and progenitor cells. We used our atlas to predict cell type labels in new bone marrow images and used these predictions to uncover mesenchymal stromal cell (MSC) expansion and leukemic blast/MSC-enriched spatial neighborhoods in AML patient samples. Our work represents the first comprehensive, spatially-resolved multiomic atlas of human bone marrow and will serve as a reference for future investigation of cellular interactions that drive hematopoiesis.
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Refractoriness to initial chemotherapy and relapse after remission are the main obstacles to cure in T-cell Acute Lymphoblastic Leukemia (T-ALL). Biomarker guided risk stratification and targeted therapy have the potential to improve outcomes in high-risk T-ALL; however, cellular and genetic factors contributing to treatment resistance remain unknown. Previous bulk genomic studies in T-ALL have implicated tumor heterogeneity as an unexplored mechanism for treatment failure. To link tumor subpopulations with clinical outcome, we created an atlas of healthy pediatric hematopoiesis and applied single-cell multiomic (CITE-seq/snATAC-seq) analysis to a cohort of 40 cases of T-ALL treated on the Children's Oncology Group AALL0434 clinical trial. The cohort was carefully selected to capture the immunophenotypic diversity of T-ALL, with early T-cell precursor (ETP) and Near/Non-ETP subtypes represented, as well as enriched with both relapsed and treatment refractory cases. Integrated analyses of T-ALL blasts and normal T-cell precursors identified a bone-marrow progenitor-like (BMP-like) leukemia sub-population associated with treatment failure and poor overall survival. The single-cell-derived molecular signature of BMP-like blasts predicted poor outcome across multiple subtypes of T-ALL within two independent patient cohorts using bulk RNA-sequencing data from over 1300 patients. We defined the mutational landscape of BMP-like T-ALL, finding that NOTCH1 mutations additively drive T-ALL blasts away from the BMP-like state. We transcriptionally matched BMP-like blasts to early thymic seeding progenitors that have low NR3C1 expression and high stem cell gene expression, corresponding to a corticosteroid and conventional cytotoxic resistant phenotype we observed in ex vivo drug screening. To identify novel targets for BMP-like blasts, we performed in silico and in vitro drug screening against the BMP-like signature and prioritized BMP-like overexpressed cell-surface (CD44, ITGA4, LGALS1) and intracellular proteins (BCL-2, MCL-1, BTK, NF-κB) as candidates for precision targeted therapy. We established patient derived xenograft models of BMP-high and BMP-low leukemias, which revealed vulnerability of BMP-like blasts to apoptosis-inducing agents, TEC-kinase inhibitors, and proteasome inhibitors. Our study establishes the first multi-omic signatures for rapid risk-stratification and targeted treatment of high-risk T-ALL.
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The adoptive transfer of chimeric antigen receptor (CAR) T cells represents a breakthrough in clinical oncology, yet both between- and within-patient differences in autologously derived T cells are a major contributor to therapy failure. To interrogate the molecular determinants of clinical CAR T-cell persistence, we extensively characterized the premanufacture T cells of 71 patients with B-cell malignancies on trial to receive anti-CD19 CAR T-cell therapy. We performed RNA-sequencing analysis on sorted T-cell subsets from all 71 patients, followed by paired Cellular Indexing of Transcriptomes and Epitopes (CITE) sequencing and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on T cells from six of these patients. We found that chronic IFN signaling regulated by IRF7 was associated with poor CAR T-cell persistence across T-cell subsets, and that the TCF7 regulon not only associates with the favorable naïve T-cell state, but is maintained in effector T cells among patients with long-term CAR T-cell persistence. These findings provide key insights into the underlying molecular determinants of clinical CAR T-cell function. SIGNIFICANCE: To improve clinical outcomes for CAR T-cell therapy, there is a need to understand the molecular determinants of CAR T-cell persistence. These data represent the largest clinically annotated molecular atlas in CAR T-cell therapy to date, and significantly advance our understanding of the mechanisms underlying therapeutic efficacy.This article is highlighted in the In This Issue feature, p. 2113.
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Imunoterapia Adotiva , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/transplante , Adolescente , Criança , Intervalo Livre de Doença , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Philadelphia , Linfócitos T/imunologiaRESUMO
Single-cell chromatin accessibility sequencing has become a powerful technology for understanding epigenetic heterogeneity of complex tissues. However, there is a lack of open-source software for comprehensive processing, analysis, and visualization of such data generated using all existing experimental protocols. Here, we present scATAC-pro for quality assessment, analysis, and visualization of single-cell chromatin accessibility sequencing data. scATAC-pro computes a range of quality control metrics for several key steps of experimental protocols, with a flexible choice of methods. It generates summary reports for both quality assessment and downstream analysis. scATAC-pro is available at https://github.com/tanlabcode/scATAC-pro.
Assuntos
Cromatina/metabolismo , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Software , Ontologia Genética , Humanos , Pegadas de Proteínas , Análise de Sequência de DNA/normas , Análise de Célula Única/normas , Fatores de Transcrição/metabolismo , Fluxo de TrabalhoRESUMO
Fast, robust and technology-independent computational methods are needed for supervised cell type annotation of single-cell RNA sequencing data. We present SciBet, a supervised cell type identifier that accurately predicts cell identity for newly sequenced cells with order-of-magnitude speed advantage. We enable web client deployment of SciBet for rapid local computation without uploading local data to the server. Facing the exponential growth in the size of single cell RNA datasets, this user-friendly and cross-platform tool can be widely useful for single cell type identification.
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Algoritmos , Análise de Célula Única , Bases de Dados como Assunto , Regulação da Expressão Gênica , Humanos , Internet , Reprodutibilidade dos TestesRESUMO
Interpreting the function of noncoding mutations in cancer genomes remains a major challenge. Here, we developed a computational framework to identify putative causal noncoding mutations of all classes by joint analysis of mutation and gene expression data. We identified thousands of SNVs/small indels and structural variants as putative causal mutations in five major pediatric cancers. We experimentally validated the oncogenic role of CHD4 overexpression via enhancer hijacking in B-ALL. We observed a general exclusivity of coding and noncoding mutations affecting the same genes and pathways. We showed that integrated mutation profiles can help define novel patient subtypes with different clinical outcomes. Our study introduces a general strategy to systematically identify and characterize the full spectrum of noncoding mutations in cancers.