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

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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.