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

项目摘要 鉴定调节T细胞活性的小分子蛋白质靶点仍然是一种治疗方法。 治疗癌症、自身免疫性疾病和同种异体器官排斥患者时的目标。为此， 我们的实验室已经确定，可药物化的激酶DNA依赖性蛋白激酶催化亚基 （DNA-PKcs）是CD 4+和CD 8 + T细胞功能所必需的。我们观察到强有力的DNA-PKcs激活， 一旦激活，DNA-PKcs是启动几个关键的免疫原性转录所必需的。 包括由转录因子NFAT、NFκ B B和EGFR 1驱动的程序。重要的是，化学 并且DNA-PKcs的遗传抑制显著破坏T细胞活化、代谢、细胞因子产生和 细胞毒性T细胞杀死靶细胞的能力。深入了解DNA的分子机制- PKcs调节T细胞活性，我们对用小分子PKcs处理的T细胞进行磷酸化蛋白质组学筛选， DNA-PKcs抑制剂。来自该筛选和我们的后续研究的数据证实了TCR信号传导蛋白连接体用于 活化T细胞（LAT）作为DNA-PKcs的重要磷酸化靶点。在本提案中，我们将利用 新的可诱导转基因小鼠模型设计为“敲除”DNA-PKcs表达，特异性地在成熟的 CD 4+或CD 8+淋巴细胞，以评估LAT的DNA-PKcs磷酸化与T细胞功能的相关性 及其对T细胞对抗原刺激的反应的影响。在目标1中，我们将描述 LAT和DNA-PKcs通过使用DNA-PKcs缺陷型CD 4+或CD 8 + T细胞和CRISPR-1的体外研究 产生LAT磷酸突变体。目标2将重点关注在治疗前或治疗后， 使用OVA特异性TCR转基因的T细胞活化影响体内CD 4+和CD 8 + T细胞对抗原的应答 OTII和OTI小鼠模型。这些目标的完成将为全面实现 显著影响CD 4+和CD 8 + T细胞介导的免疫的未表征的信号传导机制， 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.