DNA-PKcs Regulation of LAT-Mediated Early TCR Signaling in CD4+ and CD8+ T Cells

项目摘要 识别调节T细胞活性的小分子蛋白靶点仍然是一种治疗方法 目的：治疗癌症、自身免疫性疾病和同种异体器官排斥反应患者。为此， 我们实验室已经确定了可药物激酶DNA依赖蛋白激酶催化亚基 (DNA-PKcs)是CD4+和CD8+T细胞功能所必需的。我们观察到DNA-PKcs在以下情况下激活 T细胞刺激，一旦被激活，DNA-PKcs是启动几个关键的免疫原性转录所必需的 程序包括由转录因子nfat、nf𝜅B和egr1驱动的程序。重要的是，这两种化学物质 DNA-PKcs的遗传抑制显著扰乱T细胞的激活、代谢、细胞因子的产生和 细胞毒T细胞杀伤靶细胞的能力。为了深入了解DNA- PKCS调节T细胞活性，我们对用小分子处理的T细胞进行了磷酸化蛋白质组筛选 DNA-PKcs抑制剂。来自这一筛查和我们的后续研究的数据证实了TCR信号蛋白连接物 T细胞活化(LAT)作为DNA-PKcs的重要磷酸化靶点。在这个提案中，我们将利用 一种新型可诱导的转基因小鼠模型，旨在“敲除”DNA-PKcs在成熟小鼠中的特异性表达 评价LAT DNA-PKcs磷酸化与T细胞功能的相关性 以及它对T细胞对抗原刺激反应的影响。在目标1中，我们将描述 Lat和DNA-PKcs通过使用DNA-PKcs缺陷的CD4+或CD8+T细胞和CRISPR- 产生了LAT磷酸化突变体。目标2将集中于确定DNA-PKcs的丢失是在之前还是之后 利用OVA特异性TCR转基因体内T细胞激活影响CD4+和CD8+T细胞对抗原的应答 OTII和OTI小鼠模型。这些目标的实现将为我们提供一个全新的视角 未明确的信号机制，显著影响CD4+和CD8+T细胞介导的免疫 对新的治疗方法有相当大的影响。

Project Summary Identification of small molecule protein targets that modulate T cell activity continues to be a therapeutic goal when treating patients with cancer, autoimmune diseases, and allogeneic organ rejection. To that end, our laboratory has determined that the druggable kinase DNA dependent-protein kinase catalytic subunit (DNA-PKcs) is required for CD4+ and CD8+ T cell function. We observe robust DNA-PKcs activation following T cell stimulation, and once activated, DNA-PKcs is necessary to initiate several key immunogenic transcriptional programs including those driven by the transcription factors NFAT, NF𝜅B, and EGR1. Importantly, both chemical and genetic inhibition of DNA-PKcs significantly disrupts T cell activation, metabolism, cytokine production and the ability of cytotoxic T cells to kill target cells. To gain insight into the molecular mechanisms by which DNA- PKcs regulates T cell activity, we performed a phospho-proteomic screen of T cells treated with a small molecule DNA-PKcs inhibitor. Data from this screen and our follow-up studies confirm the TCR signaling protein Linker for Activation of T cells (LAT) as a significant phosphorylation target of DNA-PKcs. In this proposal, we will utilize novel inducible transgenic mouse models designed to “knockout” DNA-PKcs expression specifically in mature CD4+ or CD8+ lymphocytes to evaluate the relevance of DNA-PKcs phosphorylation of LAT to T cell function and its impact on T cell response to antigen stimulation. In Aim 1, we will characterize the interaction between LAT and DNA-PKcs through in vitro studies using DNA-PKcs-deficient CD4+ or CD8+ T cells and CRISPR- generated LAT phosphomutants. Aim 2 will focus on determining how loss of DNA-PKcs either before or after T cell activation impacts CD4+ and CD8+ T cell response to antigen in vivo using the OVA-specific TCR transgenic OTII and OTI mouse models. Completion of these aims will provide new insights into a completely uncharacterized signaling mechanism that significantly impacts CD4+ and CD8+ T cell-mediated immunity with considerable implications for novel therapy approaches.