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

免疫细胞的代谢活动与其功能密切相关。我们一直在发展一种 基于流式细胞术的平台，能够对传统细胞表面标志物进行单细胞分析， 参与代谢程序的蛋白质的细胞内染色。胞内蛋白质包括肉毒碱 棕榈酰转移酶Ia（CPT 1a），其是线粒体中脂肪酸氧化的限速酶， 参与糖酵解的蛋白质如己糖激酶II和线粒体膜蛋白电压- 依赖性阴离子通道（VDAC）。细胞内染色还检测组蛋白甲基化标记物。虽然这 这种方法最初是为了预测和跟踪对癌症免疫疗法的反应而开发的，我们已经 最近将该技术应用于来自COVID-19患者的外周血单核细胞（PBMC）。通过 询问免疫代谢程序，我们已经定义了三个不同的和新的免疫群体， COVID-19患者PBMC中的细胞：He 3 K27 me 3 +VDAC+ T细胞;己糖激酶II+粒细胞髓样 衍生的抑制细胞;和CPT 1a +VDAC+DR-单核细胞MDSC。通过询问SARS患者的PBMC- 对于CoV-2感染的患者，我们寻求定义新的生物标志物，以预测疾病的严重程度并跟踪 并定义新的替代标记物，用于测试治疗方案和鉴定 新的治疗靶点。此外，我们将审问这三种新的细胞类型，作为一种更好的手段， 了解保护性和致病性COVID-19免疫反应的性质。具体而言，RP 1将 利用这些独特的发现来更好地了解这些骨髓细胞在SARS先天免疫中的作用， CoV-2感染，而这个项目，RP 2，将专门检查T细胞反应。的确， H3 K27 me 3 +VDAC+ T细胞群体似乎对细胞凋亡非常敏感， 泛半胱天冬酶抑制剂ZVAD以及VDAC抑制剂VBIT-4。为了了解这一点的影响， 我们将研究这些T细胞是否对SARS-CoV-2特异， 表明病毒抗原特异性T细胞不成比例地破坏或功能障碍。为了解决这个问题，我们必须 开发了一种独特而强大的检测方法，特异性T细胞功能扩增（FEST）检测， 使用基于T细胞受体测序（TCRseq）的平台鉴定抗原特异性T细胞克隆型。通过 结合FEST测定，其最初被开发用于跟踪癌症患者中的肿瘤反应性T细胞， 通过流式细胞术和TCRseq，我们将确定SARS-CoV-2特异性T细胞是否在T细胞中唯一检测到， 代谢标志物水平增加的细胞群。这些研究的完成将产生一部小说 该试验将使我们能够预测疾病的严重程度并跟踪疾病过程的结果，开发一种新的 生物标志物的治疗方案的测试，提供了基本的洞察免疫的发病机制， 功能障碍，并潜在地为开发新的基于免疫代谢的药物提供见解和手段 养生法

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.