Enabling technologies to study how mechanics influence T cell function at the molecular and cellular levels

最近的证据表明，肿瘤微环境(TME)可能形成免疫抑制的避难所。 和逃避。尽管基于检查点抑制剂和TME化学调节的免疫疗法 虽然取得了成功，但仍然存在许多挑战，包括患者反应的异质性和严重的一面 全身性自身免疫引起的影响。这些观察表明，生化刺激并不是 唯一抑制TME内T细胞功能的因素。该领域的一个新兴概念是，物理 TME的特性，如细胞外基质的硬度、组成和结构也与癌症有关 细胞的增殖和存活。然而，TME的机械性能是否特异性地调节T细胞 活动，从而导致免疫逃避，目前尚不清楚。这项提议有两个首要目标。 首先，为了更好地了解机械力在T细胞受体激活和T细胞功能反应中的作用。 第二，了解TME的物理方面如何影响T细胞/肿瘤的相互作用和T细胞功能。 我的博士工作重点是开发使能技术，以便在分子水平上研究机械生物学。 规模，特别关注机械力在T细胞激活中的作用。我的工作表明，I)T 细胞受体在初始识别过程中将PN力传递给其抗原，并在免疫突触和II)T细胞中 在T细胞激活的最早阶段，使用机械能来区分抗原。对于我剩余的F99 在这一阶段，我将重点研究机械力是否对T细胞功能很重要。为了实现这一目标， 我将使用最近开发的邻近标记技术来识别介导T细胞的机械敏感蛋白 细胞信号。这一策略将允许使用蛋白质组分析来确定机械交互作用组 (机械组)在T细胞中。然后，我将确定机械力是否会影响T细胞的长期生物学功能 采用ELISA法和流式细胞术与RNA-SEQ联用。总体而言，这项综合组学研究的结果将 更好地理解T细胞如何利用机械能增强其生物学功能。 对于我的博士后研究(K00阶段)，我的目标是了解TME的物理方面如何影响细胞毒性 T细胞功能(癌症杀伤)。其目标是量化ECM机制如何改变T细胞功能。该数据将支持 一种假说，认为TME通过物理机制增强免疫逃避，从而促进肿瘤转移。 这项工作的意义在于开发促进T细胞抗肿瘤反应的新策略。我 建议在使用3D矩阵模拟TME的实验室工作，并使用这些支架进行联合培养 T细胞和肿瘤移植物。这项工作将更好地定义基质的物理参数(例如水凝胶的范围 僵硬、物理线索组成和结构)，影响T细胞/肿瘤的相互作用和T细胞功能。 总的来说，F99和K00阶段的工作将提供对物理的新的基本理解 T细胞在分子水平和细胞水平上的功能基础。这项研究的结果可能会提供新的设计方案 靶向癌症免疫疗法的原则。

Recent evidence has showed that the tumor microenvironment (TME) may form a sanctuary for immune suppression and evasion. Although Immunotherapies based on checkpoint inhibitors and chemical modulation of the TME have garnered success, many challenges remain, including the heterogeneity of patient response and the serious side effects resulting from systemic autoimmunity. These observations suggested that biochemical stimuli are not the only factor that suppresses T cell functions within the TME. One emerging concept in the field is that the physical properties of the TME such as extracellular matrix stiffness, composition, and architecture also contributes to cancer cell proliferation and survival. However, whether the mechanical properties of the TME specifically modulates T cell activity, and thus contributing to immune evasion, remains unclear. There are two overarching goals for this proposal. First, to better understand the role of mechanical forces in T cell receptor activation, and T cell functional responses. Second, to understand how the physical aspects of TME affect T cell/tumor interactions and T cell function. My PhD work is focused on developing enabling technologies to study mechanobiology at the molecular scale, with a particular focus on the roles of mechanical forces in T cell activation. My work has shown that i) the T cell receptor transmits pN forces to its antigen during initial recognition, and in immunological synapse and ii) T cells use mechanical energy to discriminate antigens during the earliest step of T cell activation. For my remaining F99 phase, I will focus on investigating whether mechanical forces are important for T cell function. To achieve this goal, I will use recently developed proximity labeling technologies to identify mechanosensitive proteins that mediate T cell signaling. This strategy will allow for using proteomic analysis to determine the mechanical interactome (mechanome) in T cells. Then, I will determine whether mechanical forces affect long term T cell biological functions using ELISA and flow cytometry coupled with RNA-SEQ. Overall, the results from this integrative -omics study will offer better understanding of how T cells use mechanical energy to potentiate their biological functions. For my postdoc studies (K00 phase), I aim to understand how the physical aspects of TME affect cytotoxic T cell function (cancer killing). The goal is to quantify how ECM mechanics alter T cell function. This data will support the hypothesis that TME contributes to metastasis by enhancing immune evasion through physical mechanisms. The significance of this work pertains to developing new strategies for promoting T cell anti-tumor response. I propose to work in a lab that employs 3D matrices that mimic the TME and to use these scaffolds for co-culture of T cells and tumor grafts. The work will better define the physical parameters of the matrix (e.g. the extent of hydrogel stiffness, physical cues composition, and architecture) that affect T cell/tumor interaction and T cell function. Collectively, the work from both F99 and K00 phases will provide new fundamental understanding on the physical basis of T cell functions at molecular level and cellular levels. The outcome of this research may offer new design principles for targeted cancer immunotherapies.