Exploiting the Mechanobiology of PD-1 for Cancer Immunotherapy

项目摘要 程序性细胞死亡1（PD-1）是在T细胞活化后上调的关键共抑制分子之一， 是T细胞衰竭的标志尽管PD-1封锁已经成为一种革命性的战略， 尽管PD-1用于治疗癌症和感染性疾病，但尚未研究PD-1的机械生物学。这就是差距， 目前的物理科学-肿瘤学项目（PS-OP）旨在填补。PS-OP的独特意义 机械生物学角度在于靶向PD-1的机械调节以治疗广泛的 各种疾病，包括黑色素瘤，这是这个PS-OP的重点。PS-OP的方法 多学科团队联合收割机将四种物理科学（PS）工具与两种黑色素瘤小鼠模型相结合。他们 代表了朱实验室和艾哈迈德实验室的独特优势，并使PD-1的研究成为可能 计算机模拟、体外和体内的机械生物学。第一个PS工具是基于DNA的机械张力探针（MTP） 和张力计系绳（TGT），其分别报告和限制PD-1上的细胞产生的力。第二 PS工具是三种基于生物膜力探针（BFP）的单分子方法， 原位PD-1-PD-配体相互作用与单细胞中细胞内信号的同时成像。第三 PS工具是分子动力学（MD）模拟，揭示PD-1与其配体复合物的结构变化 力和原子水平上的成键动力学。第四个PS工具是基于微流体的细胞 诱捕刺激和分析该项目的初步研究表明：1）细胞主动拉 PD-1; 2）PD-1上的力激发捕获键以调节配体键合; 3）力诱导PD-1的重排。 1-PD-L2结合界面形成新的原子水平相互作用; 4）突变PD-1上的特定氨基酸， 其力、捕获键和功能;以及5）PD-1的抑制信号通过破坏抗原识别来抑制抗原识别。 TCR和CD 8在pMHC结合中的协同作用。这些数据支持了一个假设， 调节PD-1的配体结合和信号传导;因此，靶向PD-1的机械调节可能代表 一种新的免疫疗法这一假设将通过三个具体目标进行检验：1）确定 对PD-1的作用力及其对PD-1配体结合、信号传导和功能的影响; 2）通过调节T细胞功能来调节T细胞功能， 靶向PD-1机械调节;和3）研究操纵PD-1捕获键的治疗潜力 在肿瘤小鼠模型中。这些研究将阐明PD-1信号转导机制，提高人们对PD-1的认识， 了解CD 8 + T细胞对黑色素瘤的反应，并提出新的免疫治疗策略， 癌

PROJECT SUMMARY Programmed cell death 1 (PD-1) is one of the key co-inhibitory molecules upregulated upon T cell activation and is a hallmark of T-cell exhaustion. Despite the fact that PD-1 blockade has become a revolutionary strategy in treating cancer and infectious diseases, the mechanobiology of PD-1 has not been studied. This is the gap that tne present Physical Science – Oncology Project (PS-OP) aims to fill. The significance of the PS-OP’s unique mechanobiology angle lies in the therapeutic potential of targeting the mechanoregulation of PD-1 to treat a wide variety of diseases, including melanoma, which is the focus of this PS-OP. The approaches of the PS-OP’s multidisciplinary team combine four physical science (PS) tools with two mouse models of melanoma. They represent the unique strength of the Zhu lab and the Ahmed lab, and enable investigation of PD-1 mechanobiology in silico, in vitro and in vivo. The first PS tool is DNA-based mechanical tension probes (MTP) and tension gauge tethers (TGT) that report and limit, respectively, cell-generated forces on PD-1. The second PS tool is three biomembrane force probe (BFP)-based single-molecule methods that quantify force regulation of in situ PD-1–PD-ligand interactions with concurrent imaging of intracellular signals in single cells. The third PS tool is molecular dynamics (MD) simulations that reveal structural changes of PD-1 in complex with its ligands under force and the bonding dynamics at atomic level. The fourth PS-tool is microfluidic-based devices for cell trapping, stimulation, and analysis. Preliminary studies of the project demonstrate that: 1) cells actively pull on PD-1; 2) force on PD-1 elicits catch bonds to regulate ligand bonding; 3) force induces rearrangement of the PD- 1–PD-L2 binding interface to form new atomic-level interactions; 4) mutating specific amino acids on PD-1 alters its force, catch bond and function; and 5) PD-1’s inhibitory signal suppresses antigen recognition by disrupting the synergy between TCR and CD8 in pMHC binding. These data support the hypothesis that force critically regulates ligand bonding and signaling of PD-1; as such, targeting the PD-1 mechanoregulation may represent a novel approach to immunotherapy. This hypothesis will be tested by three specific aims: 1) Determine the forces on PD-1 and their impact on PD-1 ligand bonding, signaling and function; 2) Modulate T cell function by targeting PD-1 mechanoregulation; and 3) Investigate the therapeutic potential of manipulating PD-1 catch bonds in tumor mouse models. These studies will elucidate the mechanisim of PD-1 signaling, improve one’s understanding of CD8+ T-cell responses to melanoma, and suggest new immunotheraputic strategies to treating cancer.