Project 2: Interrogating immuno-metabolic programs using a novel Flow Cytometry based  assay to reveal novel T cell subsets in SARS-CoV-2 infected patients

项目 2：使用新型流式细胞术检测免疫代谢程序，揭示 SARS-CoV-2 感染患者中的新型 T 细胞亚群

• 批准号: 10221909
• 负责人: JONATHAN D POWELL
• 金额: $ 98.32万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221909
• 关键词: 2019-nCoV; Address; Apoptosis; Automobile Driving; Biological Assay; Biological Markers; COVID-19; Cancer Patient; Carnitine Palmitoyltransferase I; Caspase Inhibitor; Cell physiology; Cell surface; Cells; Cellular Assay; Clinical; Critical Illness; Disease; Disease Outcome; Enzymes; Epitopes; Flow Cytometry; Functional disorder; Generations; Glycolysis; HIV-1; Hexokinase 2; Immune; Immune System Diseases; Immune response; Immunity; Infection; Inflammasome; Inflammation; Link; Lymphopenia; Mediating; Metabolic; Metabolic Marker; Metabolism; Mitochondria; Mitochondrial Membrane Protein; Mitochondrial Proteins; Monitor; Myeloid Cells; Myeloid-derived suppressor cells; Natural Immunity; Nature; Pathogenesis; Pathogenicity; Patients; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Population; Proteins; Regimen; Research Project Grants; Role; Severities; Severity of illness; Stains; Surrogate Markers; T cell response; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; Technology; Testing; Therapeutic Agents; Treatment Efficacy; Treatment Protocols; Variant; Viral Antigens; Virus; Virus Replication; Voltage-Dependent Anion Channel; antigen-specific T cells; antiviral immunity; base; cancer immunotherapy; cell type; effector T cell; fatty acid oxidation; granulocyte; histone methylation; inhibitor/antagonist; innovation; insight; methylation biomarker; monocyte; new therapeutic target; novel; novel marker; novel therapeutics; prevent; programs; response; single cell analysis; therapeutic evaluation; therapy design; tumor

The metabolic activity of immune cells is tightly linked to their function. We have been developing a flow cytometry based platform that enables single cell analysis of traditional cell surface markers combined with intracellular staining for proteins involved in metabolic programming. The intracellular proteins include carnitine palmitoyltransferase Ia (CPT1a), which is the rate-limiting enzyme in fatty acid oxidation in mitochondria, proteins involved in glycolysis such as hexokinase II, and the mitochondrial membrane protein voltage- dependent anion channel (VDAC). Intracellular staining also detects histone methylation markers. While this approach was originally developed to predict and track responses to immunotherapy for cancer, we have recently applied this technology to peripheral blood mononuclear cells (PBMC) from COVID-19 patients. By interrogating immune-metabolic programs we have defined three distinct and novel populations of immune cells in the PBMC of COVID-19 patients: He3K27me3+VDAC+ T cells; Hexokinase II+ Granulocytic Myeloid Derived Suppressor Cells; and CPT1a+VDAC+DR- monocytic MDSC. By interrogating PBMC from SARS- CoV-2 infected patients, we seek to define novel biomarkers in order to predict severity of disease and track the course of disease and to define novel surrogate markers for testing therapeutic regimens and identifying novel therapeutic targets. Furthermore, we will interrogate these three novel cell types as a means of better understanding the nature of protective and pathogenic COVID-19 immune responses. Specifically, RP1 will leverage these unique findings to better understand the role of these myeloid cells in innate immunity in SARS- CoV-2 infection, while this project, RP2, will specifically examine the T cell responses. Indeed, the unique population of H3K27me3+VDAC+ T cells appear to be exquisitely sensitive to apoptosis, which is prevented by the pan-caspase inhibitor ZVAD as well as the VDAC inhibitor VBIT-4. To understand the impact of this population on antiviral immunity, we will investigate whether these T cells are specific for SARS-CoV-2 and suggest destruction or dysfunction of viral antigen-specific T cells disproportionately. To address this, we have developed a unique and powerful assay, the functional expansion of specific T cells (FEST) assay that identifies antigen-specific T cell clonotypes using a T cell receptor sequencing (TCRseq)-based platform. By combining the FEST assay, which was originally developed to track tumor reactive T cells in cancer patients, with flow cytometry and TCRseq, we will determine if SARS-CoV-2-specific T cells are uniquely detected in T cell populations with increased levels of metabolic markers. The completion of these studies will yield a novel assay that will enable us to predict the severity and track the outcome of the disease course, develop a novel biomarker for the testing of therapeutic regimens, provide fundamental insight into the pathogenesis of immune dysfunction, and potentially provide insight and a means for developing novel immune-metabolic based drug regimens.

免疫细胞的代谢活性与其功能紧密相关。我们一直在发展流程 基于细胞仪的平台，可以使传统细胞表面标记的单细胞分析与 涉及代谢编程的蛋白质的细胞内染色。细胞内蛋白包括肉碱 棕榈酰转移酶IA（CPT1A），这是线粒体中脂肪酸氧化的速率限制酶， 参与糖酵解的蛋白质，例如己糖激酶II，线粒体膜蛋白电压 - 依赖性阴离子通道（VDAC）。细胞内染色还检测到组蛋白甲基化标记。同时 最初开发的方法是为了预测和跟踪对癌症免疫疗法的反应，我们有 最近将这项技术应用于COVID-19患者的外周血单核细胞（PBMC）。经过 询问免疫代谢程序我们定义了三个不同的新型免疫种群 COVID-19患者PBMC中的细胞：HE3K27ME3+ VDAC+ T细胞；己糖酶II+粒细胞髓样 衍生的抑制细胞；和CPT1A+VDAC+DR-单核细胞MDSC。通过询问SARS的PBMC- COV-2感染患者，我们试图定义新型的生物标志物，以预测疾病的严重程度并追踪 疾病的进程并定义了用于测试治疗方案并识别的新型替代标记 新型治疗靶标。此外，我们将质疑这三种新型细胞类型作为更好的手段 了解保护性和致病性共证的性质199免疫反应。具体来说，RP1会 利用这些独特的发现，以更好地了解这些髓样细胞在SARS中的先天免疫中的作用 COV-2感染，而该项目RP2将专门检查T细胞反应。确实，独特 H3K27ME3+ VDAC+ T细胞的种群似乎对凋亡非常敏感，这是由 Pan-caspase抑制剂ZVAD以及VDAC抑制剂VBIT-4。了解这一点的影响 抗病毒免疫的种群，我们将研究这些T细胞是否针对SARS-COV-2和 表明病毒抗原特异性T细胞的破坏或功能障碍不成比例。为了解决这个问题，我们有 开发了一种独特而有力的测定法，特定T细胞（Fest）测定的功能扩展 使用基于T细胞受体测序（TCRSEQ）的平台鉴定抗原特异性T细胞clonotypes。经过 结合节日测定最初是为了跟踪癌症患者肿瘤反应性T细胞的开发的， 使用流式细胞术和TCRSEQ，我们将确定在T中是否独特地检测到SARS-COV-2特异性T细胞 具有代谢标记水平增加的细胞群。这些研究的完成将产生小说 测定将使我们能够预测严重程度并跟踪疾病病程的结果，发展新颖的测定 用于测试治疗方案的生物标志物，为免疫发病提供了基本的见解 功能障碍，并有可能提供洞察力和一种发展新型免疫代谢药物的手段 方案。