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.