RESUMO
The widespread application of genome editing to treat or even cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped Delivery Vehicles (EDVs) are engineered virally-derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication components in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs, and demonstrated increased editing in cell lines and therapeutically-relevant primary human T cells. These results show that virally-derived particles can be streamlined to create efficacious genome editing delivery vehicles that could simplify production and manufacturing.
RESUMO
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.
RESUMO
Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28, CTLA4 and ICOS, encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells, both conventional and regulatory subsets, uncovered gene-, cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.
Assuntos
Antígenos CD28 , Antígeno CTLA-4 , Cromatina , Regulação da Expressão Gênica , Humanos , Antígeno CTLA-4/genética , Antígenos CD28/genética , Cromatina/genética , Cromatina/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Sistemas CRISPR-CasRESUMO
Tumor metastasis requires systemic remodeling of distant organ microenvironments that impacts immune cell phenotypes, population structure, and intercellular communication. However, our understanding of immune phenotypic dynamics in the metastatic niche remains incomplete. Here, we longitudinally assayed lung immune transcriptional profiles in the polyomavirus middle T antigen (PyMT) and 4T1 metastatic breast cancer models from primary tumorigenesis, through pre-metastatic niche formation, to the final stages of metastatic outgrowth at single-cell resolution. Computational analyses of these data revealed a TLR-NFκB inflammatory program enacted by both peripherally derived and tissue-resident myeloid cells that correlated with pre-metastatic niche formation and mirrored CD14+ "activated" myeloid cells in the primary tumor. Moreover, we observed that primary tumor and metastatic niche natural killer (NK) cells are differentially regulated in mice and human patient samples, with the metastatic niche featuring elevated cytotoxic NK cell proportions. Finally, we identified cell-type-specific dynamic regulation of IGF1 and CCL6 signaling during metastatic progression that represents anti-metastatic immunotherapy candidate pathways.
Assuntos
Neoplasias da Mama , Células Matadoras Naturais , Neoplasias Pulmonares , Microambiente Tumoral , Animais , Feminino , Humanos , Camundongos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Células Matadoras Naturais/imunologia , Microambiente Tumoral/imunologia , Progressão da Doença , Linhagem Celular Tumoral , Pulmão/imunologia , Pulmão/patologia , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Fator de Crescimento Insulin-Like I/metabolismo , Regulação Neoplásica da Expressão Gênica , Células Mieloides/imunologia , Células Mieloides/metabolismo , Quimiocinas CC/metabolismo , Quimiocinas CC/genética , Transdução de SinaisRESUMO
Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90-99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000-50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.
Assuntos
Metilação de DNA , Neoplasias da Próstata , Humanos , Animais , Masculino , Neoplasias da Próstata/genética , Camundongos , Análise de Sequência de DNA/métodos , Cromatina/genética , DNA/genética , Elementos de DNA Transponíveis/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ilhas de CpG/genética , Linhagem Celular Tumoral , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , TransposasesRESUMO
Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy.
Acute myeloid leukemia (or AML for short) is a type of blood cancer characterized by abnormally high production of immature white blood cells. Despite advances in AML treatment, many patients relapse after an initially successful first round of treatment. As a result, understanding the factors contributing to relapse is essential for developing effective treatments for the disease. Like most cancers, AML can evolve because of changes to the DNA sequence in cells that cause them to grow uncontrollably or resist treatment. Alongside these genetic mutations, AML cells also undergo 'epigenetic' changes, where regions of the DNA are modified and genes can be switched on or off without altering the DNA sequence. Previous research has demonstrated that epigenetic changes contribute to the development of AML, however, it was not clear if these changes could also make cells resistant to treatment without acquiring new DNA mutations. Nuno, Azizi et al. addressed this question by analyzing the epigenetic states of AML cells from 26 patients at the time of their diagnosis and after treatment when the disease had relapsed. Analysis revealed that almost half of the patients with AML experienced a relapse without acquiring new DNA mutations. Instead, these AML cells developed specific epigenetic changes that helped them to resist cancer treatment. Moreover, studying individual AML cells from different patients showed that the cells became more epigenetically similar at relapse, suggesting that they converge towards a more treatment-resistant disease. Future experiments will determine exactly how these epigenetic changes lead to treatment resistance. Currently, most of the drugs used to treat AML are either chemotherapies or ones that target specific DNA mutations. The findings of Nuno, Azizi et al. suggest that drugs targeting specific epigenetic changes may be more effective for some patients. Further studies will be needed to determine which patients may benefit and which epigenetic drugs could be useful.
Assuntos
Epigênese Genética , Leucemia Mieloide Aguda , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Humanos , Recidiva , Mutação , Evolução Molecular , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Neoplásicas/patologiaRESUMO
Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing (scRNA-seq) identifies many rare populations of interest that express specific marker transcript combinations, traditional flow sorting limits our ability to enrich these populations for further profiling, including requiring cell surface markers with high-fidelity antibodies. Additionally, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. To address these limitations, we describe Programmable Enrichment via RNA Flow-FISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations from complex cellular mixtures defined by the presence or absence of specific RNA transcripts. Across immune populations (n = 141,227 cells) and fresh-frozen and formalin-fixed paraffin-embedded brain tissue (n = 29,522 nuclei), we demonstrate the sorting logic that can be used to enrich for cell populations via RNA-based cytometry followed by high-throughput scRNA-seq. Our approach provides a rational, programmable method for studying rare populations identified by one or more marker transcripts.
RESUMO
A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2-6, suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states.
Assuntos
Proteína Forkhead Box O1 , Memória Imunológica , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Linfócitos T , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/genética , Proteína Forkhead Box O1/metabolismo , Edição de Genes , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/citologiaRESUMO
3-dimensional (3D) genome conformation is central to gene expression regulation, yet our understanding of its contribution to rapid transcriptional responses, signal integration, and memory in immune cells is limited. Here, we study the molecular regulation of the inflammatory response in primary macrophages using integrated transcriptomic, epigenomic, and chromosome conformation data, including base pair-resolution Micro-Capture C. We demonstrate that interleukin-4 (IL-4) primes the inflammatory response in macrophages by stably rewiring 3D genome conformation, juxtaposing endotoxin-, interferon-gamma-, and dexamethasone-responsive enhancers in close proximity to their cognate gene promoters. CRISPR-based perturbations of enhancer-promoter contacts or CCCTC-binding factor (CTCF) boundary elements demonstrated that IL-4-driven conformation changes are indispensable for enhanced and synergistic endotoxin-induced transcriptional responses, as well as transcriptional memory following stimulus removal. Moreover, transcriptional memory mediated by changes in chromosome conformation often occurred in the absence of changes in chromatin accessibility or histone modifications. Collectively, these findings demonstrate that rapid and memory transcriptional responses to immunological stimuli are encoded in the 3D genome.
RESUMO
Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.