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The memory CD8+ T cell pool contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. Here, we examined the epigenetic landscape of CD8+ T cells isolated from seven non-lymphoid organs across four distinct infection models, alongside their circulating T cell counterparts. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we found that tissue-resident memory T (TRM) cells and circulating memory T (TCIRC) cells develop along distinct epigenetic trajectories. We identified organ-specific transcriptional regulators of TRM cell development, including FOSB, FOS, FOSL1, and BACH2, and defined an epigenetic signature common to TRM cells across organs. Finally, we found that although terminal TEX cells share accessible regulatory elements with TRM cells, they are defined by TEX-specific epigenetic features absent from TRM cells. Together, this comprehensive data resource shows that TRM cell development is accompanied by dynamic transcriptome alterations and chromatin accessibility changes that direct tissue-adapted and functionally distinct T cell states.
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Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6)1. Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4+ T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration2 or are in clinical studies3-5, we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials1,6-8 and may influence the design and production of autologous and allogeneic cell therapies.
Asunto(s)
Linfocitos T CD4-Positivos , Herpesvirus Humano 6 , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos , Activación Viral , Latencia del Virus , Humanos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Ensayos Clínicos como Asunto , Regulación Viral de la Expresión Génica , Genómica , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/aislamiento & purificación , Herpesvirus Humano 6/fisiología , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Encefalitis Infecciosa/complicaciones , Encefalitis Infecciosa/virología , Receptores Quiméricos de Antígenos/inmunología , Infecciones por Roseolovirus/complicaciones , Infecciones por Roseolovirus/virología , Análisis de Expresión Génica de una Sola Célula , Carga ViralRESUMEN
Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics.
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ADN Mitocondrial , Enfermedades Mitocondriales , Humanos , ADN Mitocondrial/genética , Multiómica , Enfermedades Mitocondriales/genética , Mitocondrias/genética , MutaciónRESUMEN
Cell cycle (CC) facilitates cell division via robust, cyclical gene expression. Protective immunity requires the expansion of pathogen-responsive cell types, but whether CC confers unique gene expression programs that direct the subsequent immunological response remains unclear. Here, we demonstrate that single macrophages (MFs) adopt different plasticity states in CC, which leads to heterogeneous cytokine-induced polarization, priming, and repolarization programs. Specifically, MF plasticity to interferon gamma (IFNG) is substantially reduced during S-G2/M, whereas interleukin 4 (IL-4) induces S-G2/M-biased gene expression, mediated by CC-biased enhancers. Additionally, IL-4 polarization shifts the CC-phase distribution of MFs toward the G2/M phase, providing a subpopulation-specific mechanism for IL-4-induced, dampened IFNG responsiveness. Finally, we demonstrate CC-dependent MF responses in murine and human disease settings in vivo, including Th2-driven airway inflammation and pulmonary fibrosis, where MFs express an S-G2/M-biased tissue remodeling gene program. Therefore, MF inflammatory and regenerative responses are gated by CC in a cyclical, phase-dependent manner.
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Cromatina , Interleucina-4 , Humanos , Ratones , Animales , Interleucina-4/genética , Interleucina-4/farmacología , Cromatina/genética , Cromatina/metabolismo , Macrófagos/metabolismo , Interferón gamma/genética , Interferón gamma/farmacología , Ciclo Celular/genética , División CelularRESUMEN
Tissue-resident memory T cells (TRM cells) provide rapid and superior control of localized infections. While the transcription factor Runx3 is a critical regulator of CD8+ T cell tissue residency, its expression is repressed in CD4+ T cells. Here, we show that, as a direct consequence of this Runx3-deficiency, CD4+ TRM cells lacked the transforming growth factor (TGF)-ß-responsive transcriptional network that underpins the tissue residency of epithelial CD8+ TRM cells. While CD4+ TRM cell formation required Runx1, this, along with the modest expression of Runx3 in CD4+ TRM cells, was insufficient to engage the TGF-ß-driven residency program. Ectopic expression of Runx3 in CD4+ T cells incited this TGF-ß-transcriptional network to promote prolonged survival, decreased tissue egress, a microanatomical redistribution towards epithelial layers and enhanced effector functionality. Thus, our results reveal distinct programming of tissue residency in CD8+ and CD4+ TRM cell subsets that is attributable to divergent Runx3 activity.
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Memoria Inmunológica , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
CRISPR-Cas9 technologies have transformed the study of genetic pathways governing cellular differentiation and function. Recent advances have adapted these methods to immune cells, which has accelerated the pace of functional genomics in immunology and enabled new avenues for the design of cellular immunotherapies for cancer. In this review, we summarize recent developments in CRISPR-Cas9 technology and discuss how they have been leveraged to discover and manipulate novel genetic regulators of the immune system. We envision that these results will provide a valuable resource to aid in the design, implementation, and interpretation of CRISPR-Cas9-based screens in immunology and immuno-oncology.
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Sistemas CRISPR-Cas , Neoplasias , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Neoplasias/genética , Neoplasias/terapiaRESUMEN
Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X-linked hyper-IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one-size-fits-all editing strategy for effective T-cell correction, selection, and depletion and investigated the therapeutic potential of T-cell and HSPC therapies in the HIGM1 mouse model. Edited patients' derived CD4 T cells restored physiologically regulated CD40L expression and contact-dependent B-cell helper function. Adoptive transfer of wild-type T cells into conditioned HIGM1 mice rescued antigen-specific IgG responses and protected mice from a disease-relevant pathogen. We then obtained ~ 25% CD40LG editing in long-term repopulating human HSPC. Transplanting such proportion of wild-type HSPC in HIGM1 mice rescued immune functions similarly to T-cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T-cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.
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Síndrome de Inmunodeficiencia con Hiper-IgM Tipo 1 , Síndrome de Inmunodeficiencia con Hiper-IgM , Animales , Edición Génica , Células Madre Hematopoyéticas , Humanos , Ratones , Linfocitos TRESUMEN
Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency and stability of the resulting translocations. A thorough translocation frequency analysis using three orthogonal methods (droplet digital PCR, unidirectional sequencing, and metaphase fluorescence in situ hybridization) yielded comparable results and an overall translocation rate of â¼7% between two simultaneous CRISPR-Cas9 induced edits. In addition, we show that chromosomal translocations can be reduced when using different nuclease combinations, or by the presence of a homologous single stranded oligo donor for multiplexed genome editing. Importantly, the two different approaches for translocation reduction are compatible with cell therapy applications.
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Sistemas CRISPR-Cas , Edición Génica/métodos , Linfocitos T , Translocación Genética , Linfocitos T CD4-Positivos , Proteína 9 Asociada a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , Endonucleasas/genética , Genoma Humano , Humanos , Hibridación Fluorescente in Situ , Herencia Multifactorial , ARN Guía de Kinetoplastida , Streptococcus pyogenesRESUMEN
Long non-coding RNAs (lncRNAs) regulate critical cellular processes and their dysregulation contributes to multiple diseases. Although only a few lncRNAs have defined mechanisms, many of these characterized lncRNAs interact with transcription factors to regulate gene expression, suggesting a common mechanism of action. Identifying RNA-bound transcription factors is especially challenging due to inefficient RNA immunoprecipitation and low abundance of many transcription factors. Here we describe a highly sensitive, user-friendly, and inexpensive technique called RATA (RNA-associated transcription factor array), which utilizes a MS2-aptamer pulldown strategy coupled with transcription factor activation arrays for identification of transcription factors associated with a nuclear RNA of interest. RATA requires only ~5 million cells and standard molecular biology reagents for multiplexed identification of up to 96 transcription factors in 2-3 d. Thus, RATA offers significant advantages over other technologies for analysis of RNA-transcription factor interactions.
RESUMEN
The CRISPR-Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR-Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies.
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Proteína BRCA2/genética , Sistemas CRISPR-Cas , ADN/genética , Edición Génica/métodos , Genoma Humano , Recombinasa Rad51/genética , Reparación del ADN por Recombinación , Proteína BRCA2/antagonistas & inhibidores , Proteína BRCA2/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/metabolismo , Roturas del ADN de Doble Cadena , Endonucleasas/genética , Endonucleasas/metabolismo , Células HEK293 , Humanos , Células K562 , Osteoblastos/citología , Osteoblastos/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/metabolismoRESUMEN
Long non-coding RNAs (lncRNAs) have been implicated in numerous physiological processes and diseases, most notably cancers. However, little is known about the mechanism of many functional lncRNAs. We identified an abundantly expressed lncRNA associated with decreased melanoma patient survival. Increased expression of this lncRNA, SLNCR1, mediates melanoma invasion through a highly conserved sequence similar to that of the lncRNA SRA1. Using a sensitive technique we term RATA (RNA-associated transcription factor array), we show that the brain-specific homeobox protein 3a (Brn3a) and the androgen receptor (AR) bind within and adjacent to SLNCR1's conserved region, respectively. SLNCR1, AR, and Brn3a are specifically required for transcriptional activation of matrix metalloproteinase 9 (MMP9) and increased melanoma invasion. Our observations directly link AR to melanoma invasion, possibly explaining why males experience more melanoma metastases and have an overall lower survival in comparison to females.