Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Cell ; 166(6): 1500-1511.e9, 2016 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-27610572

RESUMEN

Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8(+) tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8(+) TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact.


Asunto(s)
Linfocitos T CD8-positivos/patología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Animales , Linfocitos T CD8-positivos/inmunología , Sistemas CRISPR-Cas , Carcinogénesis/genética , Carcinogénesis/inmunología , Femenino , Factor de Transcripción GATA3/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Melanoma/inmunología , Melanoma/fisiopatología , Metalotioneína/deficiencia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL
3.
Nature ; 576(7786): 293-300, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31802004

RESUMEN

Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1-3, but dysfunction due to T cell exhaustion is an important barrier to progress4-6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7-10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.


Asunto(s)
Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Epigénesis Genética , Regulación de la Expresión Génica , Humanos , Ratones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Receptores de Antígenos de Linfocitos T/genética , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/inmunología , Transcripción Genética
4.
Proc Natl Acad Sci U S A ; 118(30)2021 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-34285077

RESUMEN

Dysfunction in T cells limits the efficacy of cancer immunotherapy. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion. We found widespread, dynamic changes in chromatin accessibility and three-dimensional (3D) chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mice, we find that the regulatory networks of T cell exhaustion differ between species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.


Asunto(s)
Cromatina/metabolismo , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Quiméricos de Antígenos , Linfocitos T/fisiología , Animales , Antígenos CD19 , Línea Celular , Cromatina/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Receptor de Muerte Celular Programada 1/genética
5.
Nat Methods ; 16(6): 489-492, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31133759

RESUMEN

Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS).


Asunto(s)
Cromatina/química , Cromatina/genética , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , ARN Largo no Codificante/genética , ARN/química , Telomerasa/química , Animales , Células Cultivadas , Cromosomas , Células Madre Embrionarias/citología , Genoma , Ratones , Regiones Promotoras Genéticas , ARN/genética , Telomerasa/genética
6.
Nature ; 516(7529): 56-61, 2014 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-25471879

RESUMEN

Pluripotent stem cells (PSCs) are capable of dynamic interconversion between distinct substates; however, the regulatory circuits specifying these states and enabling transitions between them are not well understood. Here we set out to characterize transcriptional heterogeneity in mouse PSCs by single-cell expression profiling under different chemical and genetic perturbations. Signalling factors and developmental regulators show highly variable expression, with expression states for some variable genes heritable through multiple cell divisions. Expression variability and population heterogeneity can be influenced by perturbation of signalling pathways and chromatin regulators. Notably, either removal of mature microRNAs or pharmacological blockage of signalling pathways drives PSCs into a low-noise ground state characterized by a reconfigured pluripotency network, enhanced self-renewal and a distinct chromatin state, an effect mediated by opposing microRNA families acting on the Myc/Lin28/let-7 axis. These data provide insight into the nature of transcriptional heterogeneity in PSCs.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes/fisiología , Animales , Muerte Celular , División Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Perfilación de la Expresión Génica , Ratones , MicroARNs/metabolismo , Células Madre Pluripotentes/citología , Transducción de Señal
7.
Nature ; 496(7446): 461-8, 2013 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-23467089

RESUMEN

Despite their importance, the molecular circuits that control the differentiation of naive T cells remain largely unknown. Recent studies that reconstructed regulatory networks in mammalian cells have focused on short-term responses and relied on perturbation-based approaches that cannot be readily applied to primary T cells. Here we combine transcriptional profiling at high temporal resolution, novel computational algorithms, and innovative nanowire-based perturbation tools to systematically derive and experimentally validate a model of the dynamic regulatory network that controls the differentiation of mouse TH17 cells, a proinflammatory T-cell subset that has been implicated in the pathogenesis of multiple autoimmune diseases. The TH17 transcriptional network consists of two self-reinforcing, but mutually antagonistic, modules, with 12 novel regulators, the coupled action of which may be essential for maintaining the balance between TH17 and other CD4(+) T-cell subsets. Our study identifies and validates 39 regulatory factors, embeds them within a comprehensive temporal network and reveals its organizational principles; it also highlights novel drug targets for controlling TH17 cell differentiation.


Asunto(s)
Diferenciación Celular/genética , Redes Reguladoras de Genes/genética , Células Th17/citología , Células Th17/metabolismo , Animales , Células Cultivadas , ADN/genética , ADN/metabolismo , Factores de Transcripción Forkhead/metabolismo , Técnicas de Silenciamiento del Gen , Genoma/genética , Interferón gamma/biosíntesis , Interleucina-2/genética , Ratones , Ratones Endogámicos C57BL , Nanocables , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Silicio , Células Th17/inmunología , Factores de Tiempo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Receptor fas/metabolismo
8.
Nat Med ; 24(5): 580-590, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29686426

RESUMEN

T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy.


Asunto(s)
Cromatina/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Transposasas/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Transformada , Células Clonales , Epigenómica , Humanos , Inmunidad , Células Jurkat , Leucemia/inmunología , Leucemia/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de la Célula Individual
9.
Nat Biotechnol ; 33(5): 495-502, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25867923

RESUMEN

Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.


Asunto(s)
Hibridación Fluorescente in Situ/métodos , Análisis de la Célula Individual/métodos , Pez Cebra/crecimiento & desarrollo , Animales , Regulación del Desarrollo de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Procesamiento de Imagen Asistido por Computador , Transcriptoma/genética , Pez Cebra/genética
10.
Curr Protoc Mol Biol ; 107: 4.22.1-4.22.17, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24984854

RESUMEN

For the past several decades, due to technical limitations, the field of transcriptomics has focused on population-level measurements that can mask significant differences between individual cells. With the advent of single-cell RNA-Seq, it is now possible to profile the responses of individual cells at unprecedented depth and thereby uncover, transcriptome-wide, the heterogeneity that exists within these populations. This unit describes a method that merges several important technologies to produce, in high-throughput, single-cell RNA-Seq libraries. Complementary DNA (cDNA) is made from full-length mRNA transcripts using a reverse transcriptase that has terminal transferase activity. This, when combined with a second "template-switch" primer, allows for cDNAs to be constructed that have two universal priming sequences. Following preamplification from these common sequences, Nextera XT is used to prepare a pool of 96 uniquely indexed samples ready for Illumina sequencing.


Asunto(s)
Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Mensajero , Análisis de Secuencia de ARN/métodos , Animales , ADN Complementario/química , ADN Complementario/genética , Humanos , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA