RESUMO
Chimeric antigen receptor (CAR)-engineered T and NK cells can cause durable remission of B-cell malignancies; however, limited persistence restrains the full potential of these therapies in many patients. The FAS ligand (FAS-L)/FAS pathway governs naturally-occurring lymphocyte homeostasis, yet knowledge of which cells express FAS-L in patients and whether these sources compromise CAR persistence remains incomplete. Here, we constructed a single-cell atlas of diverse cancer types to identify cellular subsets expressing FASLG, the gene encoding FAS-L. We discovered that FASLG is limited primarily to endogenous T cells, NK cells, and CAR-T cells while tumor and stromal cells express minimal FASLG. To establish whether CAR-T/NK cell survival is regulated through FAS-L, we performed competitive fitness assays using lymphocytes modified with or without a FAS dominant negative receptor (ΔFAS). Following adoptive transfer, ΔFAS-expressing CAR-T and CAR-NK cells became enriched across multiple tissues, a phenomenon that mechanistically was reverted through FASLG knockout. By contrast, FASLG was dispensable for CAR-mediated tumor killing. In multiple models, ΔFAS co-expression by CAR-T and CAR-NK enhanced antitumor efficacy compared with CAR cells alone. Together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS auto-regulatory circuit.
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Inflammation is essential to the disruption of tissue homeostasis and can destabilize the identity of lineage-committed epithelial cells. Here, we employ lineage-traced mouse models, single-cell transcriptomic and chromatin analyses, and CUT&TAG to identify an epigenetic memory of inflammatory injury in the pancreatic acinar cell compartment. Despite resolution of pancreatitis, our data show that acinar cells fail to return to their molecular baseline, with retention of elevated chromatin accessibility and H3K4me1 at metaplasia genes, such that memory represents an incomplete cell fate decision. In vivo, we find this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second insult but increased tumorigenesis with an oncogenic Kras mutation. The lowered threshold for oncogenic transformation, in turn, can be restored by blockade of MAPK signaling. Together, we define the chromatin dynamics, molecular encoding, and recall of a prolonged epigenetic memory of inflammatory injury that impacts future responses but remains reversible.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Camundongos , Animais , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Memória Epigenética , Transformação Celular Neoplásica/patologia , Células Acinares/patologia , Pâncreas/patologia , Cromatina/genética , Metaplasia/patologia , Carcinoma Ductal Pancreático/genéticaRESUMO
IL-15 is under clinical investigation toward the goal of curing HIV infection because of its abilities to reverse HIV latency and enhance immune effector function. However, increased potency through combination with other agents may be needed. 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) enhances IL-15-mediated latency reversal and NK cell function by increasing STAT5 activation. We hypothesized that HODHBt would also synergize with IL-15, via STAT5, to directly enhance HIV-specific cytotoxic T cell responses. We showed that ex vivo IL-15 + HODHBt treatment markedly enhanced HIV-specific granzyme B-releasing T cell responses in PBMCs from antiretroviral therapy-suppressed (ART-suppressed) donors. We also observed upregulation of antigen processing and presentation in CD4+ T cells and increased surface MHC-I. In ex vivo PBMCs, IL-15 + HODHBt was sufficient to reduce intact proviruses in 1 of 3 ART-suppressed donors. Our findings reveal the potential for second-generation IL-15 studies incorporating HODHBt-like therapeutics. Iterative studies layering on additional latency reversal or other agents are needed to achieve consistent ex vivo reservoir reductions.
Assuntos
Antineoplásicos , Infecções por HIV , Humanos , Fator de Transcrição STAT5/metabolismo , Interleucina-15/farmacologia , Interleucina-15/metabolismo , Latência Viral , Linfócitos T Citotóxicos , Antineoplásicos/uso terapêuticoRESUMO
Tumor-specific CD8+ T cells (TST) in patients with cancer are dysfunctional and unable to halt cancer progression. TST dysfunction, also known as exhaustion, is thought to be driven by chronic T cell antigen receptor (TCR) stimulation over days to weeks. However, we know little about the interplay between CD8+ T cell function, cell division and epigenetic remodeling within hours of activation. Here, we assessed early CD8+ T cell differentiation, cell division, chromatin accessibility and transcription in tumor-bearing mice and acutely infected mice. Surprisingly, despite robust activation and proliferation, TST had near complete effector function impairment even before undergoing cell division and had acquired hallmark chromatin accessibility features previously associated with later dysfunction/exhaustion. Moreover, continued tumor/antigen exposure drove progressive epigenetic remodeling, 'imprinting' the dysfunctional state. Our study reveals the rapid divergence of T cell fate choice before cell division in the context of tumors versus infection.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Camundongos , Animais , Divisão Celular , Antígenos de Neoplasias , Cromatina , Receptores de Antígenos de Linfócitos TRESUMO
The pancreatic islets are composed of discrete hormone-producing cells that orchestrate systemic glucose homeostasis. Here we identify subsets of beta cells using a single-cell transcriptomic approach. One subset of beta cells marked by high CD63 expression is enriched for the expression of mitochondrial metabolism genes and exhibits higher mitochondrial respiration compared with CD63lo beta cells. Human and murine pseudo-islets derived from CD63hi beta cells demonstrate enhanced glucose-stimulated insulin secretion compared with pseudo-islets from CD63lo beta cells. We show that CD63hi beta cells are diminished in mouse models of and in humans with type 2 diabetes. Finally, transplantation of pseudo-islets generated from CD63hi but not CD63lo beta cells into diabetic mice restores glucose homeostasis. These findings suggest that loss of a specific subset of beta cells may lead to diabetes. Strategies to reconstitute or maintain CD63hi beta cells may represent a potential anti-diabetic therapy.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Humanos , Camundongos , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Secreção de Insulina , Insulina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Células Secretoras de Insulina/metabolismo , Glucose/metabolismoRESUMO
The immune system coordinates the response to cardiac injury and is known to control regenerative and fibrotic scar outcomes in the heart and subsequent chronic low-grade inflammation associated with heart failure. Here we profiled the inflammatory response to heart injury using single cell transcriptomics to compare and contrast two experimental models with disparate outcomes. We used adult mice, which like humans lack the ability to fully recover and zebrafish which spontaneously regenerate after heart injury. The extracardiac reaction to cardiomyocyte necrosis was also interrogated to assess the specific peripheral tissue and immune cell reaction to chronic stress. Cardiac macrophages are known to play a critical role in determining tissue homeostasis by healing versus scarring. We identified distinct transcriptional clusters of monocytes/macrophages in each species and found analogous pairs in zebrafish and mice. However, the reaction to myocardial injury was largely disparate between mice and zebrafish. The dichotomous response to heart damage between the mammalian and zebrafish monocytes/macrophages may underlie the impaired regenerative process in mice, representing a future therapeutic target.
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Multiple sclerosis (MS) is a complex disease of the CNS thought to require an environmental trigger. Gut dysbiosis is common in MS, but specific causative species are unknown. To address this knowledge gap, we used sensitive and quantitative PCR detection to show that people with MS were more likely to harbor and show a greater abundance of epsilon toxin-producing (ETX-producing) strains of C. perfringens within their gut microbiomes compared with individuals who are healthy controls (HCs). Isolates derived from patients with MS produced functional ETX and had a genetic architecture typical of highly conjugative plasmids. In the active immunization model of experimental autoimmune encephalomyelitis (EAE), where pertussis toxin (PTX) is used to overcome CNS immune privilege, ETX can substitute for PTX. In contrast to PTX-induced EAE, where inflammatory demyelination is largely restricted to the spinal cord, ETX-induced EAE caused demyelination in the corpus callosum, thalamus, cerebellum, brainstem, and spinal cord, more akin to the neuroanatomical lesion distribution seen in MS. CNS endothelial cell transcriptional profiles revealed ETX-induced genes that are known to play a role in overcoming CNS immune privilege. Together, these findings suggest that ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination in the context of circulating myelin autoreactive lymphocytes.
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Encefalomielite Autoimune Experimental , Microbioma Gastrointestinal , Esclerose Múltipla , Animais , Humanos , Clostridium perfringens/genética , Esclerose Múltipla/genética , Privilégio Imunológico , LinfócitosRESUMO
BACKGROUND: Most Crohn's disease [CD] patients require surgery. Ileitis recurs after most ileocolectomies and is a critical determinant for outcomes. The impacts of ileocolectomy-induced bile acid [BA] perturbations on intestinal microbiota and inflammation are unknown. We characterized the relationships between ileocolectomy, stool BAs, microbiota and intestinal inflammation in inflammatory bowel disease [IBD]. METHODS: Validated IBD clinical and endoscopic assessments were prospectively collected. Stool primary and secondary BA concentrations were compared based on ileocolectomy and ileitis status. Primary BA thresholds for ileitis were evaluated. Metagenomic sequencing was use to profile microbial composition and function. Relationships between ileocolectomy, BAs and microbiota were assessed. RESULTS: In 166 patients, elevated primary and secondary BAs existed with ileocolectomy. With ileitis, only primary BAs [795 vs 398 nmol/g, pâ =â 0.009] were higher compared to without ileitis. The optimal primary BA threshold [≥228 nmol/g] identified ileitis on multivariable analysis [odds ratioâ =â 2.3, pâ =â 0.04]. Microbial diversity, Faecalibacterium prausnitzii and O-acetylhomoserine aminocarboxypropyltransferase [MetY] were decreased with elevated primary BAs. Amongst ileocolectomy patients, only those with elevated primary BAs had diversity, F. prausnitzii and MetY reductions. Those with both ileocolectomy and intermediate [pâ =â 0.002] or high [≥228 nmol/g, pâ =â 9.1e-11]] primary BA concentrations had reduced F. prausnitzii compared to without ileocolectomy. Those with ileocolectomy and low [<29.2 nmol/g] primary BA concentrations had similar F. prausnitzii to those without ileocolectomy [pâ =â 0.13]. MetY was reduced with ileitis [pâ =â 0.02]. CONCLUSIONS: Elevated primary BAs were associated with ileitis, and reduced microbial diversity, F. prausnitzii abundance and enzymatic abundance of MetY [acetate and l-methionine-producing enzyme expressed by F. prausnitzii], and were the only factors associated with these findings after ileocolectomy.
Assuntos
Microbioma Gastrointestinal , Ileíte , Doenças Inflamatórias Intestinais , Humanos , Doenças Inflamatórias Intestinais/cirurgia , Doenças Inflamatórias Intestinais/microbiologia , Inflamação , Ileíte/cirurgia , Ileíte/microbiologia , Colectomia , Ácidos e Sais BiliaresRESUMO
The epicardium, a mesothelial cell tissue that encompasses vertebrate hearts, supports heart regeneration after injury through paracrine effects and as a source of multipotent progenitors. However, the progenitor state in the adult epicardium has yet to be defined. Through single-cell RNA-sequencing of isolated epicardial cells from uninjured and regenerating adult zebrafish hearts, we define the epithelial and mesenchymal subsets of the epicardium. We further identify a transiently activated epicardial progenitor cell (aEPC) subpopulation marked by ptx3a and col12a1b expression. Upon cardiac injury, aEPCs emerge from the epithelial epicardium, migrate to enclose the wound, undergo epithelial-mesenchymal transition (EMT), and differentiate into mural cells and pdgfra+hapln1a+ mesenchymal epicardial cells. These EMT and differentiation processes are regulated by the Tgfß pathway. Conditional ablation of aEPCs blocks heart regeneration through reduced nrg1 expression and mesenchymal cell number. Our findings identify a transient progenitor population of the adult epicardium that is indispensable for heart regeneration and highlight it as a potential target for enhancing cardiac repair.
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Traumatismos Cardíacos , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Coração/fisiologia , Pericárdio , Células-Tronco/metabolismo , Traumatismos Cardíacos/genética , Transição Epitelial-Mesenquimal/genética , Proteoglicanas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Efforts to cure HIV have focused on reactivating latent proviruses to enable elimination by CD8+ cytotoxic T-cells. Clinical studies of latency reversing agents (LRA) in antiretroviral therapy (ART)-treated individuals have shown increases in HIV transcription, but without reductions in virologic measures, or evidence that HIV-specific CD8+ T-cells were productively engaged. Here, we show that the SARS-CoV-2 mRNA vaccine BNT162b2 activates the RIG-I/TLR - TNF - NFκb axis, resulting in transcription of HIV proviruses with minimal perturbations of T-cell activation and host transcription. T-cells specific for the early gene-product HIV-Nef uniquely increased in frequency and acquired effector function (granzyme-B) in ART-treated individuals following SARS-CoV-2 mRNA vaccination. These parameters of CD8+ T-cell induction correlated with significant decreases in cell-associated HIV mRNA, suggesting killing or suppression of cells transcribing HIV. Thus, we report the observation of an intervention-induced reduction in a measure of HIV persistence, accompanied by precise immune correlates, in ART-suppressed individuals. However, we did not observe significant depletions of intact proviruses, underscoring challenges to achieving (or measuring) HIV reservoir reductions. Overall, our results support prioritizing the measurement of granzyme-B-producing Nef-specific responses in latency reversal studies and add impetus to developing HIV-targeted mRNA therapeutic vaccines that leverage built-in LRA activity.
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Linfócitos T CD8-Positivos , Vacinas contra COVID-19 , COVID-19 , Infecções por HIV , HIV-1 , Vacina BNT162 , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Granzimas , Infecções por HIV/imunologia , Humanos , RNA Mensageiro/genética , RNA Mensageiro/uso terapêutico , SARS-CoV-2 , Vacinação , Vacinas Sintéticas , Latência Viral , Vacinas de mRNA , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrently mutated driver genes. Here we describe a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant PIK3CA, among the most frequently genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this strategy, we developed a panel of TCRs that recognize an endogenously processed neopeptide encompassing a common PIK3CA hotspot mutation restricted by the prevalent human leukocyte antigen (HLA)-A*03:01 allele. Mechanistically, immunogenicity to this public NeoAg arises from enhanced neopeptide/HLA complex stability caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a comparatively 'featureless' surface dominated by the peptide's backbone. To bind this epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface facilitated by an unusually long CDR3ß loop. In patients with diverse malignancies, we observed NeoAg clonal conservation and spontaneous immunogenicity to the neoepitope. Finally, adoptive transfer of TCR-engineered T cells led to tumor regression in vivo in mice bearing PIK3CA-mutant tumors but not wild-type PIK3CA tumors. Together, these findings establish the immunogenicity and therapeutic potential of a mutant PIK3CA-derived public NeoAg.
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Antígenos de Neoplasias , Neoplasias , Animais , Antígenos de Neoplasias/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Humanos , Camundongos , Mutação/genética , Neoplasias/genética , Receptores de Antígenos de Linfócitos TRESUMO
CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained, and the molecular programmes defining the autoimmune T cell state, are unknown. In type 1 diabetes, ß-cell-specific CD8 T cells destroy insulin-producing ß-cells. Here we followed the fate of ß-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN), which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas, where they differentiate further and destroy ß-cells. Whereas transplantation of as few as 20 autoimmune progenitors induced type 1 diabetes, as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived, and stem-like autoimmune progenitors must continuously seed the pancreas to sustain ß-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes.
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Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Células Secretoras de Insulina/imunologia , Células-Tronco/patologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Autorrenovação Celular , Células Clonais/imunologia , Células Clonais/metabolismo , Células Clonais/patologia , Modelos Animais de Doenças , Feminino , Glucose-6-Fosfatase/imunologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Células Secretoras de Insulina/patologia , Linfonodos/imunologia , Masculino , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Célula Única , Transplante de Células-Tronco , Células-Tronco/imunologia , Células-Tronco/metabolismo , TranscriptomaRESUMO
Due to an inability to ethically access developing human brain tissue as well as identify prospective cases, early-arising neurodevelopmental and cell-specific signatures of Schizophrenia (Scz) have remained unknown and thus undefined. To overcome these challenges, we utilized patient-derived induced pluripotent stem cells (iPSCs) to generate 3D cerebral organoids to model neuropathology of Scz during this critical period. We discovered that Scz organoids exhibited ventricular neuropathology resulting in altered progenitor survival and disrupted neurogenesis. This ultimately yielded fewer neurons within developing cortical fields of Scz organoids. Single-cell sequencing revealed that Scz progenitors were specifically depleted of neuronal programming factors leading to a remodeling of cell-lineages, altered differentiation trajectories, and distorted cortical cell-type diversity. While Scz organoids were similar in their macromolecular diversity to organoids generated from healthy controls (Ctrls), four GWAS factors (PTN, COMT, PLCL1, and PODXL) and peptide fragments belonging to the POU-domain transcription factor family (e.g., POU3F2/BRN2) were altered. This revealed that Scz organoids principally differed not in their proteomic diversity, but specifically in their total quantity of disease and neurodevelopmental factors at the molecular level. Single-cell sequencing subsequently identified cell-type specific alterations in neuronal programming factors as well as a developmental switch in neurotrophic growth factor expression, indicating that Scz neuropathology can be encoded on a cell-type-by-cell-type basis. Furthermore, single-cell sequencing also specifically replicated the depletion of BRN2 (POU3F2) and PTN in both Scz progenitors and neurons. Subsequently, in two mechanistic rescue experiments we identified that the transcription factor BRN2 and growth factor PTN operate as mechanistic substrates of neurogenesis and cellular survival, respectively, in Scz organoids. Collectively, our work suggests that multiple mechanisms of Scz exist in patient-derived organoids, and that these disparate mechanisms converge upon primordial brain developmental pathways such as neuronal differentiation, survival, and growth factor support, which may amalgamate to elevate intrinsic risk of Scz.
Assuntos
Células-Tronco Pluripotentes Induzidas , Esquizofrenia , Humanos , Organoides/metabolismo , Proteômica , Esquizofrenia/metabolismo , Fatores de Transcrição/metabolismoRESUMO
T cell receptor (TCR) signal strength is a key determinant of T cell responses. We developed a cancer mouse model in which tumor-specific CD8 T cells (TST cells) encounter tumor antigens with varying TCR signal strength. High-signal-strength interactions caused TST cells to up-regulate inhibitory receptors (IRs), lose effector function, and establish a dysfunction-associated molecular program. TST cells undergoing low-signal-strength interactions also up-regulated IRs, including PD1, but retained a cell-intrinsic functional state. Surprisingly, neither high- nor low-signal-strength interactions led to tumor control in vivo, revealing two distinct mechanisms by which PD1hi TST cells permit tumor escape; high signal strength drives dysfunction, while low signal strength results in functional inertness, where the signal strength is too low to mediate effective cancer cell killing by functional TST cells. CRISPR-Cas9-mediated fine-tuning of signal strength to an intermediate range improved anti-tumor activity in vivo. Our study defines the role of TCR signal strength in TST cell function, with important implications for T cell-based cancer immunotherapies.
Assuntos
Neoplasias/etiologia , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Evasão Tumoral , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia Adotiva/métodos , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
After subarachnoid hemorrhage (SAH), up to 95% of surviving patients suffer from post-SAH syndrome, which includes cognitive deficits with impaired memory, executive functions, and emotional disturbances. Although these long-term cognitive deficits are thought to result from damage to temporomesial-hippocampal areas, the underlying mechanisms remain unknown. To fill this gap in knowledge, we performed a systematic RNA sequencing screen of the hippocampus in a mouse model of SAH. SAH was induced by perforation of the circle of Willis in mice. Four days later, hippocampal RNA was obtained from SAH and control (sham perforation) mice. Next-generation RNA sequencing was used to determine differentially expressed genes in the whole bilateral hippocampi remote from the SAH bleeding site. Functional analyses and clustering tools were used to define molecular pathways. Differential gene expression analysis detected 642 upregulated and 398 downregulated genes (false discovery rate <0.10) in SAH compared to Control group. Functional analyses using IPA suite, Gene Ontology terms, REACTOME pathways, and MsigDB Hallmark gene set collections revealed suppression of oligodendrocytes/myelin related genes, and overexpression of genes related to complement system along with genes associated with innate and adaptive immunity, and extracellular matrix reorganization. Interferon regulatory factors, TGF-ß1, and BMP were identified as major orchestrating elements in the hippocampal tissue response. The MEME-Suite identified binding motifs of Krüppel-like factors, zinc finger transcription factors, and interferon regulatory factors as overrepresented DNA promoter motifs. This study provides the first systematic gene and pathway database of the hippocampal response after SAH. Our findings suggest that damage of the entorhinal cortex by subarachnoid blood may remotely trigger specific hippocampal responses, which include suppression of oligodendrocyte function. Identification of these novel pathways may allow for development of new therapeutic approaches for post-SAH cognitive deficits.
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HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.
Assuntos
Benzotiazóis/farmacologia , Briostatinas/farmacologia , Reservatórios de Doenças/virologia , Isoquinolinas/farmacologia , Latência Viral/efeitos dos fármacos , Proteína bcl-X/antagonistas & inibidores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Infecções por HIV/prevenção & controle , HIV-1/crescimento & desenvolvimento , Humanos , Replicação Viral/efeitos dos fármacos , Proteína bcl-X/metabolismoRESUMO
Sensory inputs activate sparse neuronal ensembles in the dentate gyrus of the hippocampus, but how eligibility of individual neurons to recruitment is determined remains elusive. We identify thousands of largely bistable (CpG methylated or unmethylated) regions within neuronal gene bodies, established during mouse dentate gyrus development. Reducing DNA methylation and the proportion of the methylated epialleles at bistable regions compromises novel context-induced neuronal activation. Conversely, increasing methylation and the frequency of the methylated epialleles at bistable regions enhances intrinsic excitability. Single-nucleus profiling reveals enrichment of specific epialleles related to a subset of primarily exonic, bistable regions in activated neurons. Genes displaying both differential methylation and expression in activated neurons define a network of proteins regulating neuronal excitability and structural plasticity. We propose a model in which bistable regions create neuron heterogeneity and constellations of exonic methylation, which may contribute to cell-specific gene expression, excitability, and eligibility to a coding ensemble.
Assuntos
Epigênese Genética , Hipocampo/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Alelos , Animais , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , DNA Metiltransferase 3A , Giro Denteado/metabolismo , Hipocampo/embriologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/genéticaRESUMO
Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1-6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, 'non-exhausted' immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular/imunologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/deficiência , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Homeodomínio/genética , Humanos , Memória Imunológica , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Camundongos , Neoplasias/patologia , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologia , Transcrição GênicaRESUMO
Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)-JUN family genes as key barriers of DE differentiation. The JNK-JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK-JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.
Assuntos
Diferenciação Celular/genética , Endoderma/embriologia , Endoderma/metabolismo , Genoma , Genômica , Sistema de Sinalização das MAP Quinases , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Expressão Gênica , Técnicas de Inativação de Genes , Genes Reporter , Genômica/métodos , Humanos , Células-Tronco Pluripotentes Induzidas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Reprodutibilidade dos Testes , Proteínas SmadRESUMO
Proper gene expression requires coordinated interplay among transcriptional coactivators, transcription factors and the general transcription machinery. We report here that MSL1, a central component of the dosage compensation complex in Drosophila melanogaster and Drosophila virilis, displays evolutionarily conserved sex-independent binding to promoters. Genetic and biochemical analyses reveal a functional interaction of MSL1 with CDK7, a subunit of the Cdk-activating kinase (CAK) complex of the general transcription factor TFIIH. Importantly, MSL1 depletion leads to decreased phosphorylation of Ser5 of RNA polymerase II. In addition, we demonstrate that MSL1 is a phosphoprotein, and transgenic flies expressing MSL1 phosphomutants show mislocalization of the histone acetyltransferase MOF and histone H4 K16 acetylation, thus ultimately causing male lethality due to a failure of dosage compensation. We propose that, by virtue of its interaction with components of the general transcription machinery, MSL1 exists in different phosphorylation states, thereby modulating transcription in flies.