Gamma Delta T cell surveillance of metabolites as signals of cellular stress

 描述（由申请人提供）：人V β 9 V β 2 T细胞是人血液中发现的主要亚群，在健康个体中占T细胞的5%，在感染或疾病期间扩增至20 - 50%。这些细胞在介导针对微生物病原体的免疫中发挥重要作用，包括结核分枝杆菌和麻风分枝杆菌（分别是结核病和麻风病的病原体），并且可以对某些类型的肿瘤细胞产生有效的反应。V β 9 V β 2 T细胞通过识别结构相关的非肽磷酸化抗原（pAg）来应对这些威胁。在肿瘤细胞中，这些是由于甲羟戊酸途径的过度产生而积累的中间代谢物，并且在微生物中，它们在类异戊二烯生物合成期间产生。目前尚不清楚V_（19）V_（12）T细胞如何识别这些pAg;该提案旨在提高我们对V_（19）V_（12）T细胞激活背后机制的整体理解，并提高我们在临床环境中调节这一群体的能力。这些细胞也代表了一个主要的机会，研究替代识别策略的MHC细胞，经典和最非经典的MHC分子似乎不参与识别过程。我们的合作者是第一个确定BTN3A分子在pAg介导的V β 9 V β 2 T细胞刺激中的作用的人，从那时起，我们已经证明了它们中的一个的细胞内结构域，BTN3A1，是pAg积累的分子传感器。我们试图理解pAg结合后发生的事件，我们的目标1："为了确定pAg结合BTN3A1 B30.2细胞内结构域的直接分子后果。"通过使用结构和动力学研究，包括晶体学，NMR和基于荧光的测量（FRET）。在我们的目标2.“为了确定pAg或20.1 mAb结合后BTN3A1分子在质膜中的超分子组织及其在V β 9V β 2 T细胞活化中的作用”，我们将使用基于FRET的方法，显微镜和蛋白质工程来研究介导BTN3A分子细胞表面组装的因素。最后，我们将采用交联和下拉结合基于SILAC的质谱方法，辅以高通量全基因组敲除筛选，以实现我们的目标3。“从BTN3A1到V β 9V β 2 TCR：发现将BTN3A1连接到V β 9V β 2 T细胞活化的相关分子参与者”，其将定义参与pAg识别以诱导直接导致V β 9V β 2 T细胞活化的信号的其他分子参与者。

 DESCRIPTION (provided by applicant): Human V9V2 T cells are the major subset of  T cells that are found in human blood, comprising up to 5% of the T cells in healthy individuals and expanding to 20-50% during infection or disease. These cells play important roles in mediating immunity against microbial pathogens, including Mycobacterium tuberculosis and Mycobacterium leprae (the causative agents of tuberculosis and leprosy, respectively), and can respond potently against certain types of tumor cells. V9V2 T cells respond to these threats through recognition of structurally related non-peptidic phosphorylated antigens (pAgs). In tumor cells these are intermediate metabolites that accumulate due to over-production of the mevalonate pathway and in microbes they are generated during isoprenoid biosynthesis. It is largely unknown how V9V2 T cells recognize these pAgs; this proposal seeks to improve our overall understanding of the mechanisms behind V9V2 T cell activation and improve our ability to modulate this population in clinical settings. These cells also represent a prime opportunity to study alternative recognition strategies by  T cells, as classical and most no-classical MHC molecules do not appear to be involved in the recognition process. Our collaborators were the first to define the role of the BTN3A molecules in pAg-mediated V9V2 T cell stimulation and we have since then demonstrated the intracellular domain of one on them, BTN3A1, is the molecular sensor for pAg accumulation. We seek to understand the events that occur after pAg binding in our Aim1: "To determine the direct molecular consequences of pAg binding to the BTN3A1 B30.2 intracellular domain." by using structural and dynamic studies including crystallography, NMR and fluorescence based measurements (FRET). In our Aim 2. "To determine the supra-molecular organization of BTN3A1 molecules in the plasma membrane upon pAg or 20.1 mAb binding and its role in V9V2 T cell activation", we will use FRET based approaches, microscopy and protein engineering to study the factors that mediate cell surface assembly of BTN3A molecules. Finally, we will employ cross-linking and pull-downs combined with a SILAC-based mass spectrometry approach, complemented with a high-throughput genome-wide knockdown screen to pursue our Aim 3. "From BTN3A1 to the V9V2 TCR: discovery of the relevant molecular players linking BTN3A1 to V9V2 T cell activation", which will define the other molecular players that are involved in pAg recognition in inducing the signals that directly lead to V9V2 T cell activation.