RESUMEN
In the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, epithelial populations in the distal lung expressing Angiotensin-converting enzyme 2 (ACE2) are infrequent, and therefore, the model of viral expansion and immune cell engagement remains incompletely understood. Using human lungs to investigate early host-viral pathogenesis, we found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations. Human alveolar macrophages (AMs) reliably expressed ACE2 allowing both spike-ACE2-dependent viral entry and infection. In contrast to Influenza A virus, SARS-CoV-2 infection of AMs was productive, amplifying viral titers. While AMs generated new viruses, the interferon responses to SARS-CoV-2 were muted, hiding the viral dissemination from specific antiviral immune responses. The reliable and veiled viral depot in myeloid cells in the very early phases of SARS-CoV-2 infection of human lungs enables viral expansion in the distal lung and potentially licenses subsequent immune pathologies.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Pulmón , Macrófagos Alveolares , Células Mieloides , SARS-CoV-2 , Humanos , SARS-CoV-2/fisiología , COVID-19/virología , COVID-19/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Pulmón/virología , Pulmón/inmunología , Pulmón/patología , Macrófagos Alveolares/virología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Células Mieloides/virología , Células Mieloides/metabolismo , Células Mieloides/inmunología , Internalización del Virus , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Tropismo ViralRESUMEN
As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Inhibidor NF-kappaB alfa , Organoides , SARS-CoV-2 , Replicación Viral , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , COVID-19/virología , COVID-19/metabolismo , COVID-19/genética , FN-kappa B/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Inhibidor NF-kappaB alfa/genética , Organoides/virología , Organoides/metabolismo , SARS-CoV-2/fisiologíaRESUMEN
Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.
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Carcinoma de Pulmón de Células no Pequeñas , Resistencia a Antineoplásicos , Neoplasias Pulmonares , Transducción de Señal , Factores de Transcripción , Proteínas Señalizadoras YAP , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Señalizadoras YAP/metabolismo , Línea Celular Tumoral , Animales , Resistencia a Antineoplásicos/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasia Residual , Ratones , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Quinasa de Linfoma Anaplásico/metabolismo , Quinasa de Linfoma Anaplásico/genética , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ras guanine nucleotide-releasing protein 1 (Rasgrp1) is a Ras guanine nucleotide exchange factor that participates in the activation of the Ras-ERK signaling pathway in developing T cells and is required for efficient thymic T cell positive selection. However, the role of Rasgrp1 in mature peripheral T cells has not been definitively addressed, in part because peripheral T cells from constitutive Rasgrp1-deficient mice show an abnormal activated phenotype. In this study, we generated an inducible Rasgrp1-deficient mouse model to allow acute disruption of Rasgrp1 in peripheral CD4+ T cells in the context of normal T cell development. TCR/CD28-mediated activation of Ras-ERK signaling was blocked in Rasgrp1-deficient peripheral CD4+ T cells. Furthermore, Rasgrp1-deficient CD4+ T cells were unable to synthesize IL-2 and the high-affinity IL-2R and were unable to proliferate in response to TCR/CD28 stimulation. These findings highlight an essential function for Rasgrp1 for TCR/CD28-induced Ras-ERK activation in peripheral CD4+ T cells.
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Antígenos CD28 , Linfocitos T CD4-Positivos , Ratones , Animales , Linfocitos T CD4-Positivos/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
We uncover a tumor-suppressive process in urothelium called transcriptional-translational conflict caused by deregulation of the central chromatin remodeling component ARID1A. Loss of Arid1a triggers an increase in a nexus of pro-proliferation transcripts, but a simultaneous inhibition of the eukaryotic elongation factor 2 (eEF2), which results in tumor suppression. Resolution of this conflict through enhancing translation elongation speed enables the efficient and precise synthesis of a network of poised mRNAs resulting in uncontrolled proliferation, clonogenic growth, and bladder cancer progression. We observe a similar phenomenon in patients with ARID1A-low tumors, which also exhibit increased translation elongation activity through eEF2. These findings have important clinical implications because ARID1A-deficient, but not ARID1A-proficient, tumors are sensitive to pharmacologic inhibition of protein synthesis. These discoveries reveal an oncogenic stress created by transcriptional-translational conflict and provide a unified gene expression model that unveils the importance of the crosstalk between transcription and translation in promoting cancer.
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Cromatina , Neoplasias de la Vejiga Urinaria , Humanos , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy.
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COVID-19 , Subtipo H1N1 del Virus de la Influenza A , Humanos , SARS-CoV-2 , Organoides , Tetraspaninas/genéticaRESUMEN
Normal hematopoiesis requires constant prolific production of different blood cell lineages by multipotent hematopoietic stem cells (HSC). Stem- and progenitor- cells need to balance dormancy with proliferation. How genetic alterations impact frequency, lineage potential, and metabolism of HSC is largely unknown. Here, we compared induced expression of KRAS G12D or RasGRP1 to normal hematopoiesis. At low-resolution, both Ras pathway lesions result in skewing towards myeloid lineages. Single-cell resolution CyTOF proteomics unmasked an expansion of HSC- and progenitor- compartments for RasGRP1, contrasted by a depletion for KRAS G12D . SCENITH™ quantitates protein synthesis with single-cell precision and corroborated that immature cells display low metabolic SCENITH™ rates. Both RasGRP1 and KRAS G12D elevated mean SCENITH™ signals in immature cells. However, RasGRP1-overexpressing stem cells retain a metabolically quiescent cell-fraction, whereas this fraction diminishes for KRAS G12D . Our temporal single cell proteomics and metabolomics datasets provide a resource of mechanistic insights into altered hematopoiesis at single cell resolution.
RESUMEN
In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2, and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with the acute respiratory distress syndrome (ARDS) from COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.
RESUMEN
In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic1, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model2,3 to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2, and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with the acute respiratory distress syndrome (ARDS) from COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.
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Brain metastasis (BrM) is the most common form of brain cancer, characterized by neurologic disability and an abysmal prognosis. Unfortunately, our understanding of the biology underlying human BrMs remains rudimentary. Here, we present an integrative analysis of >100,000 malignant and non-malignant cells from 15 human parenchymal BrMs, generated by single-cell transcriptomics, mass cytometry, and complemented with mouse model- and in silico approaches. We interrogated the composition of BrM niches, molecularly defined the blood-tumor interface, and revealed stromal immunosuppressive states enriched with infiltrated T cells and macrophages. Specific single-cell interrogation of metastatic tumor cells provides a framework of 8 functional cell programs that coexist or anticorrelate. Collectively, these programs delineate two functional BrM archetypes, one proliferative and the other inflammatory, that are evidently shaped through tumor-immune interactions. Our resource provides a foundation to understand the molecular basis of BrM in patients with tumor cell-intrinsic and host environmental traits.
Asunto(s)
Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Adulto , Anciano , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/inmunología , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Variación Genética , Humanos , Evasión Inmune , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Modelos Biológicos , Células Mieloides/patología , Análisis de Componente Principal , RNA-Seq , Análisis de la Célula Individual , Linfocitos T/inmunologíaAsunto(s)
Péptidos y Proteínas de Señalización Intracelular , Leucemia , Proteínas de Unión al GTP Monoméricas , Fosfoproteínas , Células Precursoras de Linfocitos T , Animales , GTP Fosfohidrolasas , Haploinsuficiencia/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia/genética , Proteínas de la Membrana , Ratones , Proteínas de Unión al GTP Monoméricas/genética , Mutación , Proteínas Nucleares , Fosfoproteínas/genéticaRESUMEN
T-cell acute lymphoblastic leukaemia (T-ALL) is a bone marrow (BM) malignancy affecting children and adults. Typically treated with chemotherapy, leukaemia remains a major death cause in people under 20 years old. Understanding molecularly altered pathways in T-ALL may lead to new therapeutic avenues in the future. Ras pathway dysregulation is common in T-ALL. We have shown elevated expression levels of the Ras guanine nucleotide exchange factor RasGRP1 in T-ALL patients, which results in constant production of active Ras (RasGTP). When leukaemia cell lines are exposed to cytokines, RasGTP levels further increase in a RasGRP1-dependent manner. How overexpressed RasGRP1 may impact primary BM cells has remained unknown. We recently published a new RoLoRiG mouse model that allows for pIpC-induced overexpression of RasGRP1 in haematopoietic cells, which can be traced with an ires-EGFP cassette. This novel model revealed that RasGRP1 overexpression bestows a fitness advantage to haematopoietic stem cells (HSCs) over wild-type cells. Intriguingly, this increased fitness only manifests in native Hematopoiesis, and not in BM transplantation (BMT) assays. In this commentary, we summarize key features of our RoLoRiG model, elaborate on BM niche importance, and discuss differences between native Hematopoiesis and BMT in the context of stem cell metabolism.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Ratones , Animales , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Modelos Animales de Enfermedad , CitocinasRESUMEN
SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2- positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19.
RESUMEN
Ras research has experienced a considerable boost in recent years, not least prompted by the Ras initiative launched by the NCI in 2013 ( https://www.cancer.gov/research/key-initiatives/ras ), accompanied and conditioned by a strongly reinvigorated determination within the Ras community to develop therapeutics attacking directly the Ras oncoproteins. As a member of the small G-protein superfamily, function and transforming activity of Ras all revolve about its GDP/GTP loading status. For one thing, the extent of GTP loading will determine the proportion of active Ras in the cell, with implications for intensity and quality of downstream signaling. But also the rate of nucleotide exchange, i.e., the Ras-GDP/GTP cycling rate, can have a major impact on Ras function, as illustrated perhaps most impressively by newly discovered fast-cycling oncogenic mutants of the Ras-related GTPase Rac1. Thus, while the last years have witnessed memorable new findings and technical developments in the Ras field, leading to an improved insight into many aspects of Ras biology, they have not jolted at the basics, but rather deepened our view of the fundamental regulatory principles of Ras activity control. In this brief review, we revisit the role and mechanisms of Ras nucleotide loading and its implications for cancer in the light of recent findings.
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Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias/patología , Proteínas ras/genética , Humanos , Neoplasias/genéticaRESUMEN
Signaling pathways play critical roles in regulating the activation of T cells. Recognition of foreign peptide presented by MHC to the T cell receptor (TCR) triggers a signaling cascade of proximal kinases and adapter molecules that lead to the activation of Effector kinase pathways. These effector kinase pathways play pivotal roles in T cell activation, differentiation, and proliferation. RNA sequencing-based methods have provided insights into the gene expression programs that support the above-mentioned cell biological responses. The proteome is often overlooked. A recent study by Damasio et al. [Biochem. J. (2021) 478, 79-98. doi:10.1042/BCJ20200661] focuses on characterizing the effect of extracellular signal-regulated kinase (ERK) on the remodeling of the proteome of activated CD8+ T cells using Mass spectrometric analysis. Surprisingly, the Effector kinase ERK pathway is responsible for only a select proportion of the proteome that restructures during T cell activation. The primary targets of ERK signaling are transcription factors, cytokines, and cytokine receptors. In this commentary, we discuss the recent findings by Damasio et al. [Biochem. J. (2021) 478, 79-98. doi:10.1042/BCJ20200661] in the context of different Effector kinase pathways in activated T cells.
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Receptores de Antígenos de Linfocitos T , Linfocitos T , Citocinas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Transducción de Señal , Linfocitos T/metabolismoRESUMEN
3D models of cancer have the potential to improve basic, translational, and clinical studies. Patient-derived xenografts, spheroids, and organoids are broad categories of 3D models of cancer, and to date, these 3D models of cancer have been established for a variety of cancer types. In lung cancer, for example, 3D models offer a promising new avenue to gain novel insights into lung tumor biology and improve outcomes for patients afflicted with the number one cancer killer worldwide. However, the adoption and utility of these 3D models of cancer vary, and demonstrating the fidelity of these models is a critical first step before seeking meaningful applications. Here, we review use cases of current 3D lung cancer models and bioinformatic approaches to assessing model fidelity. Bioinformatics approaches play a key role in both validating 3D lung cancer models and high dimensional functional analyses to support downstream applications.
RESUMEN
Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of T-cell ALL. Although genetic mutations hyperactivating cytokine receptor/Ras signaling are prevalent in ETP-ALL, it remains unknown how activated Ras signaling contributes to ETP-ALL. Here, we find that in addition to the frequent oncogenic RAS mutations, wild-type (WT) KRAS transcript level was significantly downregulated in human ETP-ALL cells. Similarly, loss of WT Kras in NrasQ61R/+ mice promoted hyperactivation of extracellular signal-regulated kinase (ERK) signaling, thymocyte hyperproliferation, and expansion of the ETP compartment. Kras-/-; NrasQ61R/+ mice developed early onset of T-cell malignancy that recapitulates many biological and molecular features of human ETP-ALL. Mechanistically, RNA-sequencing analysis and quantitative proteomics study identified that Rasgrp1, a Ras guanine nucleotide exchange factor, was greatly downregulated in mouse and human ETP-ALL. Unexpectedly, hyperactivated Nras/ERK signaling suppressed Rasgrp1 expression and reduced Rasgrp1 level led to increased ERK signaling, thereby establishing a positive feedback loop to augment Nras/ERK signaling and promote cell proliferation. Corroborating our cell line data, Rasgrp1 haploinsufficiency induced Rasgrp1 downregulation and increased phosphorylated ERK level and ETP expansion in NrasQ61R/+ mice. Our study identifies Rasgrp1 as a negative regulator of Ras/ERK signaling in oncogenic Nras-driven ETP-like leukemia.
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Regulación hacia Abajo , Regulación Leucémica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido , Proteínas de Unión al GTP Monoméricas , Mutación Missense , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Proteínas Proto-Oncogénicas p21(ras)/deficiencia , Sustitución de Aminoácidos , Animales , Proliferación Celular/genética , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
RasGRP1 is a Ras guanine nucleotide exchange factor, and an essential regulator of lymphocyte receptor signaling. In mice, Rasgrp1 deletion results in defective T lymphocyte development. RASGRP1-deficient patients suffer from immune deficiency, and the RASGRP1 gene has been linked to autoimmunity. However, how RasGRP1 levels are regulated, and if RasGRP1 dosage alterations contribute to autoimmunity remains unknown. We demonstrate that diminished Rasgrp1 expression caused defective T lymphocyte selection in C57BL/6 mice, and that the severity of inflammatory disease inversely correlates with Rasgrp1 expression levels. In patients with autoimmunity, active inflammation correlated with decreased RASGRP1 levels in CD4+ T cells. By analyzing H3K27 acetylation profiles in human T cells, we identified a RASGRP1 enhancer that harbors autoimmunity-associated SNPs. CRISPR-Cas9 disruption of this enhancer caused lower RasGRP1 expression, and decreased binding of RUNX1 and CBFB transcription factors. Analyzing patients with autoimmunity, we detected reduced RUNX1 expression in CD4+ T cells. Lastly, we mechanistically link RUNX1 to transcriptional regulation of RASGRP1 to reveal a key circuit regulating RasGRP1 expression, which is vital to prevent inflammatory disease.
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Autoinmunidad/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas de Unión al ADN/genética , Factores de Intercambio de Guanina Nucleótido/genética , Transcripción Genética/genética , Animales , Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/inmunología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/inmunología , Proteínas de Unión al ADN/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Factores de Intercambio de Guanina Nucleótido/inmunología , Histonas/genética , Histonas/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transcripción Genética/inmunologíaRESUMEN
Stimulation of naive T lymphocytes via the T cell receptor (TCR) induces distinct phosphorylation patterns that can be used to explore various signaling pathways within the cell. This protocol can be used to characterize different perturbations to the signaling pathways and the variations in time of stimulation. Here, we provide a method of barcoding and consolidating a maximum of 24 different sample conditions using two florescent dyes. This single sample for phospho-staining and flow cytometry saves time and reagents. For complete details on the use and execution of this protocol, please refer to Krutzik and Nolan (2006), Krutzik et al. (2012), Vercoulen et al. (2017), Ksionda et al. (2018), and Myers et al. (2019).
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Citometría de Flujo/métodos , Colorantes Fluorescentes , Técnicas de Sonda Molecular , Linfocitos T , Animales , Células Cultivadas , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Ratones , Fosforilación , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/química , Linfocitos T/citología , Linfocitos T/metabolismo , Factores de TiempoRESUMEN
Here, we provide a detailed protocol for synthetic lethality screens in a Jurkat T cell leukemia line using cell death as the readout measuring the combinatorial effect of a pan-PI3K inhibitor (GDC0941) with specific gene depletion by shRNA. We describe the use of an ultra-complex shRNA library, coverage considerations, time frames, protocol details, and bottlenecks with images to facilitate similar approaches. We discuss how this protocol resource can be readily adapted by investigators. For complete details on the use and execution of this protocol, please refer to (Mues et al., 2019).