Contribution of PAG to Immune Synapse Organization and PD-1 Function

PAG 对免疫突触组织和 PD-1 功能的贡献

• 批准号: 10538164
• 负责人: Emily Kathryn Moore
• 金额: $ 5.18万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2026-07-14
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10538164
• 关键词: Actins; Adhesions; Adoptive Cell Transfers; Adverse effects; Anatomy; Antibodies; Antibody Therapy; Antigen-Presenting Cells; Antitumor Response; Architecture; Autoimmune Diseases; Automobile Driving; Binding; Biological Assay; C-terminal; CD28 gene; Cause of Death; Cell Line; Cell membrane; Cell physiology; Cells; Clinical; Complex; Confocal Microscopy; Cytoskeleton; Enzyme-Linked Immunosorbent Assay; Enzymes; Event; Fellowship; Flow Cytometry; Fluorescence Resonance Energy Transfer; Fostering; Future; Glycosphingolipids; Goals; Human; Imaging Techniques; Immune; Immune Targeting; Immune checkpoint inhibitor; Immune response; Immunology; Immunotherapy; Integral Membrane Protein; Knock-out; Ligand Binding; Ligation; Link; Lipids; MAP Kinase Gene; Malignant Neoplasms; Measures; Mediating; Medicine; Membrane; Membrane Microdomains; Mentorship; Methods; Mind; Modeling; Mus; Mutate; Palmitic Acylation Site; Pathway interactions; Patients; Pattern; Peripheral; Pharmacology; Phosphoproteins; Phosphorylation; Physicians; Process; Production; Proteins; Regimen; Research; Research Personnel; Resources; Role; Shapes; Signal Pathway; Signal Transduction; Signaling Protein; Structure; Synapses; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Technology; Testing; Therapeutic; Therapeutic antibodies; Total Internal Reflection Fluorescent; Tumor-infiltrating immune cells; Tyrosine Phosphorylation Site; Work; anti-PD-1; anti-PD1 antibodies; antibody immunotherapy; cancer cell; cancer immunotherapy; cancer therapy; career; cytokine; cytotoxicity; immune function; immune-related adverse events; immunological synapse; improved; in vivo; innovation; interest; mortality; new therapeutic target; novel; novel therapeutics; polymerization; prevent; programmed cell death protein 1; protein protein interaction; recruit; response; skill acquisition; skills; synaptic function; synaptogenesis; targeted cancer therapy; targeted treatment; therapeutic target; tumor

PROJECT SUMMARY/ABSTRACT Cancer remains the second leading cause of death in the US. Immunotherapy seeks to bolster immune cells’ ability to target malignant cells and has brought immense improvements in the field. One important inhibitory protein in T cells, Programmed Cell Death Protein 1 (PD-1), has become an invaluable target for cancer immunotherapy. While anti-PD-1 antibody therapy is extremely successful in some patients, in many others, it fails to help or causes complications, including cancer hyper-progression and immune-related adverse events. Study of the inhibitory transmembrane protein Phosphoprotein Associated with Glycosphingolipid Rich Microdomains 1 (PAG), a downstream target of PD-1 signaling, will help us better understand the PD-1 pathway, and offer another, perhaps more nuanced, target to potentially improve response rates and/or avoid immune- related adverse events. As a link between lipid-rich/signaling-protein-rich membrane regions and the actin cytoskeleton, PAG is an exciting and novel target for manipulating immune function. Prior therapeutic methods of immune manipulation all disrupt ligand binding or enzyme function. In contrast, innovative use of an anti-PAG antibody to simply disrupt appropriate PAG localization within the synapse could disturb immune synapse architecture. Synapse organization is tightly regulated to prevent inappropriate immune responses, but the precise interaction between cytoskeletal dynamics and synaptic organization is not fully understood. Investigating the role of PAG in this process could provide added clarity. To test the hypothesis that PAG works with actin to control T cell synapse organization and facilitate T cell receptor (TCR) and PD-1 signaling, PAG will be studied in a human T cell line and a murine PAG-knockout model. Methods will include confocal and TIRF microscopy, FRET and proximity ligation assay, flow cytometry, ELISA, adhesion and cytotoxicity assays, and murine tumor models. To understand the contribution of the actin binding domain of PAG on its localization, protein-protein interactions, and T cell activation and effector functions, the primary hypothesis will be examined through the following specific aims: Aim 1. Define the role of the PAG-actin link in driving T cell synapse architecture and stability. Aim 2. Determine the impact of PAG-actin interactions on T cell functions downstream of TCR and PD-1 signaling. Aim 1 will demonstrate the role of PAG in T cells synapse anatomy, and the physical impacts of a PAG-targeting therapy. Aim 2 will illuminate which PD-1 downstream targets are dependent on the PAG-actin link, and provide evidence for whether PAG and PD-1 could serve as good co-targets in cancer therapy regimens. Ultimately, this study will illuminate crucial control mechanisms associated with T cell synapse organization, opening more avenues of targeting the immune synapse. Furthermore, the mentorship and resources in Dr. Adam Mor’s lab and the Columbia MSTP, will foster invaluable technical and professional skill development for a career in medicine and immunology research.

项目摘要/摘要 癌症仍然是美国第二大死亡原因。免疫疗法试图增强免疫细胞 能够靶向恶性细胞并在现场带来巨大改善。一种重要的抑制 T细胞中的蛋白质，程序性细胞死亡蛋白1（PD-1）已成为癌症的宝贵靶标 免疫疗法。虽然抗PD-1抗体疗法在某些患者中非常成功，但在许多其他患者中， 无法帮助或引起并发症，包括癌症过度促进和免疫相关的不良事件。 研究与糖脂富含糖果富的抑制性跨膜蛋白磷酸蛋白 微域1（PAG）是PD-1信号的下游目标，将有助于我们更好地了解PD-1途径， 并提供另一种可能更细微的目标，以提高反应率和/或避免免疫力 - 相关的不良事件。作为富含脂质/信号 - 蛋白质富含膜的膜区域与肌动蛋白之间的联系 细胞骨架，PAG是操纵免疫功能的令人兴奋且新颖的目标。先前的治疗方法 免疫操作都破坏了配体结合或酶功能。相反，抗PAG的创新使用 简单破坏突触中适当的PAG定位的抗体可能会干扰免疫突触 建筑学。突触组织受到严格的监管，以防止不适当的免疫回报，但 细胞骨架动力学与突触组织之间的精确相互作用尚不完全了解。 调查PAG在此过程中的作用可以提供更加清晰。测试PAG的假设 与肌动蛋白一起控制T细胞突触组织并促进T细胞受体（TCR）和PD-1 信号传导，PAG将在人类T细胞系和鼠PAG-KNOCKOUT模型中进行研究。方法将包括 共聚焦和TIRF显微镜，FRET和接近连接测定，流式细胞术，ELISA，粘合剂和粘合剂和 细胞毒性测定和鼠肿瘤模型。了解肌动蛋白结合域的贡献 PAG关于其定位，蛋白质 - 蛋白质相互作用以及T细胞激活和效应函数的PAG，主要 假设将通过以下特定目的进行检查：目标1。定义PAG-ACTIN链接的作用 在驱动T细胞突触体系结构和稳定性中。目标2。确定PAG-ACTIN相互作用的影响 TCR和PD-1信号传导下游的T细胞功能。 AIM 1将证明PAG在T细胞中的作用 突触解剖学以及靶向PAG靶向疗法的物理影响。 AIM 2将阐明哪个PD-1 下游靶标取决于PAG-ACTIN链接，并为PAG和PD-1提供了证据 可以用作癌症治疗方案的良好共同目标。最终，这项研究将阐明关键控制 与T细胞突触组织相关的机制，打开了更多靶向免疫的途径 突触。此外，亚当·莫尔（Adam Mor）博士的实验室和哥伦比亚MSTP的指导和资源将培养 医学和免疫学研究职业的宝贵技术和专业技能发展。