RESUMEN
Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity, yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in Treg cell function, coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Transcriptoma , Biomarcadores , Sistemas CRISPR-Cas , Susceptibilidad a Enfermedades , Técnicas de Inactivación de Genes , Marcación de Gen , Enfermedad Injerto contra Huésped/etiología , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor ß (TGF-ß) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.
Asunto(s)
Técnicas de Sustitución del Gen/métodos , Ingeniería Genética/métodos , Inmunoterapia/métodos , Animales , Células Sanguíneas , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , ARN Guía de Kinetoplastida/genética , Análisis de la Célula Individual/métodos , Linfocitos T , Transcriptoma/genéticaRESUMEN
A hallmark of the immune system is the interplay among specialized cell types transitioning between resting and stimulated states. The gene regulatory landscape of this dynamic system has not been fully characterized in human cells. Here we collected assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing data under resting and stimulated conditions for up to 32 immune cell populations. Stimulation caused widespread chromatin remodeling, including response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the importance of these cell states in autoimmunity. Allele-specific read mapping identified variants that alter chromatin accessibility in particular conditions, allowing us to observe evidence of function for a candidate causal variant that is undetected by existing large-scale studies in resting cells. Our results provide a resource of chromatin dynamics and highlight the need to characterize the effects of genetic variation in stimulated cells.
Asunto(s)
Linfocitos B/inmunología , Cromatina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células Asesinas Naturales/inmunología , Elementos de Respuesta/genética , Linfocitos T/inmunología , Desequilibrio Alélico , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Células Cultivadas , Cromatina/efectos de los fármacos , Cromatina/inmunología , Epigénesis Genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-2/farmacología , Interleucina-4/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Polisacáridos/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , TranscriptomaRESUMEN
Regulatory T (Treg) cells maintain immune tolerance through the master transcription factor forkhead box P3 (FOXP3), which is crucial for Treg cell function and homeostasis. We identified an IPEX (immune dysregulation polyendocrinopathy enteropathy X-linked) syndrome patient with a FOXP3 mutation in the domain swap interface of the protein. Recapitulation of this Foxp3 variant in mice led to the development of an autoimmune syndrome consistent with an unrestrained T helper type 2 (Th2) immune response. Genomic analysis of Treg cells by RNA-sequencing, Foxp3 chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-sequencing), and H3K27ac-HiChIP revealed a specific de-repression of the Th2 transcriptional program leading to the generation of Th2-like Treg cells that were unable to suppress extrinsic Th2 cells. Th2-like Treg cells showed increased intra-chromosomal interactions in the Th2 locus, leading to type 2 cytokine production. These findings identify a direct role for Foxp3 in suppressing Th2-like Treg cells and implicate additional pathways that could be targeted to restrain Th2 trans-differentiated Treg cells.
Asunto(s)
Factores de Transcripción Forkhead/inmunología , Mutación , Linfocitos T Reguladores/inmunología , Células Th2/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Niño , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Poliendocrinopatías Autoinmunes/genética , Poliendocrinopatías Autoinmunes/inmunología , Poliendocrinopatías Autoinmunes/metabolismo , Linfocitos T Reguladores/metabolismo , Células Th2/metabolismoRESUMEN
Infection triggers large-scale changes in the phenotype and function of T cells that are critical for immune clearance, yet the gene regulatory mechanisms that control these changes are largely unknown. Using ChIP-seq for specific histone post-translational modifications (PTMs), we mapped the dynamics of â¼25,000 putative CD8+ T cell transcriptional enhancers (TEs) differentially utilized during virus-specific T cell differentiation. Interestingly, we identified a subset of dynamically regulated TEs that exhibited acquisition of a non-canonical (H3K4me3+) chromatin signature upon differentiation. This unique TE subset exhibited characteristics of poised enhancers in the naive CD8+ T cell subset and demonstrated enrichment for transcription factor binding motifs known to be important for virus-specific CD8+ T cell differentiation. These data provide insights into the establishment and maintenance of the gene transcription profiles that define each stage of virus-specific T cell differentiation.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Linaje de la Célula , Elementos de Facilitación Genéticos , Histonas/genética , Gripe Humana/metabolismo , Animales , Linfocitos T CD8-positivos/citología , Diferenciación Celular , Células Cultivadas , Epigénesis Genética , Histonas/metabolismo , Humanos , Gripe Humana/genética , Ratones , Ratones Endogámicos C57BLRESUMEN
Numerous studies have focused on the molecular regulation of perforin (PFP) and granzyme B (GZMB) expression by activated cytotoxic T lymphocytes (CTLs), but little is known about the molecular factors that underpin granzyme A (GZMA) expression. In vitro activation of naïve CD8(+) T cells, in the presence of IL-4, enhanced STAT6-dependent GZMA expression and was associated with GATA3 binding and enrichment of transcriptionally permissive histone posttranslational modifications (PTMs) across the Gzma gene locus. While GZMA expression by effector influenza A virus specific CTLs was also associated with a similar permissive epigenetic signature, memory CTL lacked enrichment of permissive histone PTMs at the Gzma locus, although this was restored within recalled secondary effector CTLs. Importantly, GZMA expression by virus-specific CTLs was associated with GATA3 binding at the Gzma locus, and independent of STAT6-mediated signaling. This suggests regulation of GZMA expression is underpinned by differentiation-dependent regulation of chromatin composition at the Gzma locus and that, given GATA3 is key for CTL differentiation in response to infection, GATA3 expression is regulated by a distinct, IL-4 independent, signaling pathway. Overall, this study provides insights into the molecular mechanisms that control transcription of Gzma during virus-induced CD8(+) T-cell differentiation.
Asunto(s)
Factor de Transcripción GATA3/metabolismo , Granzimas/metabolismo , Histonas/metabolismo , Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos Virales/inmunología , Células Cultivadas , Femenino , Factor de Transcripción GATA3/genética , Granzimas/genética , Memoria Inmunológica , Interleucina-4/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Procesamiento Proteico-Postraduccional , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo , Linfocitos T Citotóxicos/virologíaRESUMEN
The changes in phenotype and function that characterize the differentiation of naïve T cells to effector and memory states are underscored by large-scale, coordinated, and stable changes in gene expression. In turn, these changes are choreographed by the interplay between transcription factors and epigenetic regulators that act to restructure the genome, ultimately ensuring lineage-appropriate gene expression. Here, we focus on the mechanisms that control T cell differentiation, with a particular focus on the role of regulatory elements encoded within the genome, known as transcriptional enhancers (TEs). We discuss the central role of TEs in regulating T cell differentiation, both in health and disease.
RESUMEN
The molecular mechanisms that regulate the rapid transcriptional changes that occur during cytotoxic T lymphocyte (CTL) proliferation and differentiation in response to infection are poorly understood. We have utilized ChIP-seq to assess histone H3 methylation dynamics within naive, effector, and memory virus-specific T cells isolated directly ex vivo after influenza A virus infection. Our results show that within naive T cells, codeposition of the permissive H3K4me3 and repressive H3K27me3 modifications is a signature of gene loci associated with gene transcription, replication, and cellular differentiation. Upon differentiation into effector and/or memory CTLs, the majority of these gene loci lose repressive H3K27me3 while retaining the permissive H3K4me3 modification. In contrast, immune-related effector gene promoters within naive T cells lacked the permissive H3K4me3 modification, with acquisition of this modification occurring upon differentiation into effector/memory CTLs. Thus, coordinate transcriptional regulation of CTL genes with related functions is achieved via distinct epigenetic mechanisms.