Molecular Pathways of Programmed Cell Death And Viral Cytopathicity

细胞死亡（PCD）是多细胞生命中通过各种不同机制发生的一个不可或缺的过程。细胞凋亡和坏死性凋亡是胚胎发育和许多依赖于连续细胞翻转的重要生物学过程所必需的，被认为是PCD的两种主要形式，有助于免疫反应的收缩期。在外周，T细胞抗原受体（TCR）的刺激可以具有几种不同的结果，包括T细胞存活、活化和增殖或PCD。然而，在免疫应答期间T细胞增殖和收缩必须平衡，以允许保护免受病原体的侵害并避免自身免疫和可能易患淋巴癌的细胞的过度积累。尽管静息T细胞中TCR的初次刺激和活化细胞中TCR的再刺激共享许多早期信号传导事件，但分别导致存活或死亡的显著差异。我们创造了术语TCR再刺激诱导的细胞死亡（RICD），以在概念上区分导致凋亡的TCR过程与导致活化和增殖的TCR过程。克隆特异性是RICD的重要特征，这使其成为诱导自身免疫性疾病耐受的一种有吸引力的方法。与削弱整个免疫系统并具有许多副作用的常规免疫抑制剂药物相比，基于RICD的耐受性策略仅针对不需要的T细胞，同时保持整体免疫系统的完整性。因此，抗原特异性RICD的致耐受性诱导可能是进行性自身免疫性疾病的有效治疗干预。 众所周知，T细胞在静息或幼稚细胞暴露于抗原后立即发生显著变化。在TCR信号级联过程中，相关蛋白被激活，沿着几个T细胞特异性基因的转录激活，导致其代谢组的大规模修饰。目前，在这一过程中，关于代谢组学的细节知之甚少。液相色谱（LC）与质谱（MS）联用已成为各种组学领域的金标准。然而，由于当前质谱仪的速度、灵敏度和分辨率有限，复杂生物样品中蛋白质组或代谢组的覆盖仍然具有挑战性。将捕获离子迁移谱法（TIMS）添加到方程中，可以解锁并行累积串联碎片采集方法，从而提供极高的MS/MS速度和灵敏度，只需最少的样品量。我们已经获得了最先进的TimsTOF LC-MS光谱仪系统，用于脂质组学和代谢组学研究。在我们的第一个项目中，我们正在研究当T细胞对RICD敏感时，脂质在细胞死亡或存活决定中的作用。我们的初步体外研究结果显示，与RICD相比，T细胞活化过程中脂质神经酰胺水平存在差异。进一步的研究将有助于绘制与TCR信号复合物有关的脂质通路。此外，我们正在进行全基因组CRISPR-cas 9敲除文库筛选，以识别编码在选定的死亡诱导环境下促进细胞存活的蛋白质的候选基因，如死亡受体和RICD。由于资源和试剂的可用性，这些实验正在使用原代小鼠细胞进行，但一旦我们的目标基因被鉴定，我们将确认使用人类T细胞的实验。阻断这些与自身免疫性疾病高度相关的通路的关键部分可以有助于治疗的发展。

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.