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

DNA-PKcs 对 CD4 和 CD8 T 细胞中 LAT 介导的早期 TCR 信号转导的调节

基本信息

批准号：
10741023
负责人：
Marie Schluterman Burdine
金额：
$ 23.1万
依托单位：
ARKANSAS CHILDREN'S HOSPITAL RES INST
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-05 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10741023
关键词：
Adaptor Signaling Protein Affect Alanine Allogenic Antigens Autoimmune Diseases B-Lymphocytes Binding Proteins CD8-Positive T-Lymphocytes CD8B1 gene Cell Communication Cell Separation Cell membrane Cell physiology Cells Cellular Immunity Chemicals Clinic Clinical Clustered Regularly Interspaced Short Palindromic Repeats Co-Immunoprecipitations Communicable Diseases Coupled Cytotoxic T-Lymphocytes DNA Repair DNA-PKcs DNA-dependent protein kinase Data Development Disease Effectiveness Flow Cytometry Follow-Up Studies Gene Expression Genetic Genetic Transcription Goals Immunity Immunosuppression Immunotherapy In Vitro Inflammatory Investigation Knock-out Laboratories Laser Scanning Confocal Microscopy LoxP-flanked allele MC38 Malignant Neoplasms Mass Spectrum Analysis Mature Lymphocyte Mature T-Lymphocyte Mediating Mediator Metabolism Modeling Molecular Mouse Strains Mus Mutation Ovalbumin PTPRC gene Patients Phosphorylation Phosphotransferases Production Proliferating Proteins Proteomics Receptor Signaling Regulation Role Signal Pathway Signal Transduction Signaling Protein Site Synapses T cell infiltration T cell response T-Cell Activation T-Cell Receptor T-Lymphocyte Tamoxifen Therapeutic Time Transgenic Mice Transgenic Organisms Translating Transplantation Tumor Antigens Tumor-Infiltrating Lymphocytes V(D)J Recombination Vertebrates Work adaptive immune response cancer immunotherapy cancer therapy congenic cytokine cytotoxicity experimental study genetic approach immunogenic improved in vivo inducible Cre inhibitor insight loss of function mutation model design mouse model novel novel therapeutic intervention nuclear factors of activated T-cells organ transplant rejection pharmacologic phosphoproteomics programs receptor binding small molecule tool tumor tumor growth

项目摘要

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.

项目摘要鉴定调节T细胞活性的小分子蛋白靶标继续是一种治疗治疗癌症患者，自身免疫性疾病和同种异体器官排斥时的目标。为此，我们的实验室确定可吸毒激酶DNA依赖性蛋白激酶催化亚基（DNA-PKC）是CD4+和CD8+ T细胞功能所必需的。我们观察到强大的DNA-PKC激活后 T细胞刺激，一旦激活，DNA-PKC是必要的包括转录因子NFAT，NF𝜅B和EGR1驱动的程序。重要的是，两者都化学 DNA-PKC的遗传抑制显着破坏了T细胞激活，代谢，细胞因子的产生和细胞毒性T细胞杀死靶细胞的能力。为了深入了解DNA-的分子机制 PKCS调节T细胞活性，我们进行了用小分子处理的T细胞的磷酸蛋白质筛选 DNA-PKC抑制剂。来自此屏幕的数据和我们的后续研究证实了TCR信号蛋白接头 T细胞（LAT）的激活作为DNA-PKC的显着磷酸化靶标。在此提案中，我们将利用新型诱导的转基因小鼠模型，旨在“敲除” DNA-PKCS表达，专门在成熟中 CD4+或CD8+淋巴细胞评估LAT与T细胞功能的DNA-PKCS磷酸化的相关性及其对T细胞对抗原刺激反应的影响。在AIM 1中，我们将表征 LAT和DNA-PKC通过使用DNA-PKCS缺陷CD4+或CD8+ T细胞和CRISPR-的体外研究以及产生的LAT磷化剂。 AIM 2将集中于确定在之前或之后如何丢失DNA-PKC T细胞激活使用OVA特异性TCR转基因影响CD4+和CD8+ T细胞对体内抗原的反应 OTII和OTI鼠标模型。这些目标的完成将为您提供新的见解未表征的信号传导机制显着影响CD4+和CD8+ T细胞介导的免疫力对新型治疗方法的影响很大。