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

项目总结/摘要 癌症仍然是美国第二大死亡原因。免疫疗法旨在增强免疫细胞的免疫功能。 靶向恶性细胞的能力，并在该领域带来了巨大的进步。一种重要的抑制剂 T细胞中的蛋白质，程序性细胞死亡蛋白1（PD-1），已经成为癌症的一个无价的目标 免疫疗法虽然抗PD-1抗体治疗在一些患者中非常成功，但在许多其他患者中， 未能帮助或导致并发症，包括癌症过度进展和免疫相关不良事件。 富含鞘糖脂的抑制性跨膜蛋白磷酸化蛋白的研究 微区1（PAG）是PD-1信号传导的下游靶点，将有助于我们更好地了解PD-1通路， 并提供另一个，也许更微妙的，目标，以潜在地提高反应率和/或避免免疫- 相关不良事件。作为富含脂质/富含信号蛋白的膜区域和肌动蛋白之间的联系， 细胞骨架，PAG是一个令人兴奋的和新的目标操纵免疫功能。既往治疗方法 都破坏了配体结合或酶的功能。相反，抗PAG的创新使用 一种抗体只要破坏PAG在突触内的适当定位，就能干扰免疫突触 架构突触组织受到严格的调节，以防止不适当的免疫反应，但 细胞骨架动力学和突触组织之间的精确相互作用尚未完全了解。 调查PAG在这一过程中的作用可以提供更多的清晰度。为了验证PAG 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 在人T细胞系和鼠PAG敲除模型中研究PAG。方法将包括 共聚焦和TIRF显微术、FRET和邻近连接测定、流式细胞术、ELISA、粘附和 细胞毒性测定和鼠肿瘤模型。为了了解肌动蛋白结合结构域的作用， PAG在其定位、蛋白质-蛋白质相互作用以及T细胞活化和效应器功能上的作用， 将通过以下具体目标对这一假设进行审查：目标1。定义PAG-肌动蛋白连接的作用 来驱动T细胞突触的结构和稳定性。目标二。确定PAG-肌动蛋白相互作用的影响 对TCR和PD-1信号传导下游的T细胞功能的影响。目的1将证明PAG在T细胞中的作用 突触解剖学和PAG靶向治疗的物理影响。目标2将说明PD-1 下游靶点依赖于PAG-肌动蛋白连接，并为PAG和PD-1是否 可以作为癌症治疗方案中的良好共同靶点。最终，这项研究将阐明关键的控制， 与T细胞突触组织相关的机制，开辟了更多靶向免疫的途径， 突触此外，Adam莫尔博士实验室和哥伦比亚MSTP的指导和资源将促进 宝贵的技术和专业技能发展的职业生涯在医学和免疫学研究。

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.