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.

项目概要鉴定调节 T 细胞活性的小分子蛋白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的目标我们的实验室已确定可药物激酶 DNA 依赖性蛋白激酶催化亚基 (DNA-PKcs) 是 CD4+ 和 CD8+ T 细胞功能所必需的，我们观察到 DNA-PKcs 强烈激活。 T 细胞刺激，一旦激活，DNA-PKcs 对于启动几个关键的免疫原性转录是必需的重要的是，这些程序都是由转录因子 NFAT、NF𝜅B 和 EGR1 驱动的。 DNA-PKcs 的遗传抑制会显着破坏 T 细胞的活化、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，深入了解 DNA- 的分子机制。 PKcs 调节 T 细胞活性，我们对用小分子处理的 T 细胞进行了磷酸蛋白质组筛选 DNA-PKcs 抑制剂。来自该筛选的数据和我们的后续研究证实了 TCR 信号蛋白连接器的作用。在本提案中，我们将利用 T 细胞 (LAT) 的激活作为 DNA-PKcs 的重要磷酸化靶标。新型诱导型转基因小鼠模型，旨在“敲除”成熟小鼠中的 DNA-PKcs 表达 CD4+ 或 CD8+ 淋巴细胞评估 LAT DNA-PKcs 磷酸化与 T 细胞功能的相关性及其对 T 细胞对抗原刺激反应的影响在目标 1 中，我们将描述两者之间的相互作用。 LAT 和 DNA-PKcs 通过使用 DNA-PKcs 缺陷的 CD4+ 或 CD8+ T 细胞和 CRISPR 进行体外研究目标 2 将重点确定 DNA-PKcs 丢失之前或之后的情况。使用 OVA 特异性 TCR 转基因技术，T 细胞激活会影响体内 CD4+ 和 CD8+ T 细胞对抗原的反应 OTII 和 OTI 小鼠模型的完成将为完整的模型提供新的见解。显着影响 CD4+ 和 CD8+ T 细胞介导的免疫的未表征的信号机制对新的治疗方法具有重大意义。