Molecular Pathways of Programmed Cell Death And Viral Cytopathicity

National Institute Of Allergy And Infectious Diseases

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Abstract

Programed cell death (PCD) is an indispensable process that takes place through a variety of different mechanisms in multicellular life. Apoptosis and necroptosis, which are essential for embryonic development and many important biological processes that rely on continuous cellular turn-over, are recognized as the two major forms of PCD that contribute to the contraction phase of immune responses. In the periphery, stimulation of the T cell antigen receptor (TCR) can have several different outcomes, including T cell survival, activation, and proliferation, or PCD. However, T cell proliferation and contraction during an immune response must be balanced to allow for both protection against pathogens and avoidance of autoimmunity and the excessive accumulation of cells that might predispose to lymphoid cancer. Although primary stimulation of TCR in resting T cells and restimulation of TCR in activated cells share many early signaling events, there are significant differences that result in either life or death respectively. We coined the term TCR restimulation-induced cell death (RICD) to conceptually distinguish the TCR process resulting in apoptosis from that leading to activation and proliferation. Clonal specificity is the important feature of RICD, which makes it an appealing method for tolerance induction in autoimmune diseases. Compared with conventional immunosuppressant medicines that debilitate the whole immune system and have many side effects, the RICD-based tolerance strategy targets only undesired T cells while keeping the general immune system intact. Thus, tolerogenic induction of antigen- specific RICD may be an effective therapeutic intervention for progressive autoimmune disorders. It is well known that the T cell undergoes dramatic changes immediately after a resting or naive cell is exposed to an antigen. During the TCR signaling cascade, the related proteins are activated, along with transcriptional activation of several T cell-specific genes, causing a wide scale modification of its metabolome. Currently, few details are known about metabolomics during this process. Liquid chromatography (LC) coupled with mass spectrometry (MS) has become the gold standard in various omics fields. However, the coverage of proteomes or metabolomes in complex biological samples remains challenging due to limited speed, sensitivity, and resolution of current mass spectrometers. Adding trapped ion mobility spectrometry (TIMS) to the equation unlocks the parallel accumulation serial fragmentation acquisition method to provide extremely high MS/MS speed and sensitivity, requiring minimal sample amounts. We have acquired a start-of-the-art TimsTOF LC-MS spectrometer system for lipidomics and metabolomics research. In our first project, we are studying the role lipids play in cell death or survival decisions when T cells are sensitive to RICD. Our preliminary in vitro findings show differences in lipid ceramide levels in T cells undergoing activation versus RICD. Further studies will help to map lipid pathways that are involved with the TCR signaling complex. Additionally, we are carrying out genome-wide CRISPR-cas 9 knockout library screening to identify candidate genes encoding proteins that promote cell survival under selected death-inducing circumstances, such as death-receptor and RICD. Due to availability of resources and reagents, these experiments are being conducted using primary mouse cells, but we will confirm the experiments with human T cells once our genes of interest are identified. Blocking key parts of these pathways highly associated with autoimmune diseases could aid in therapeutic development.