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的化学调节的免疫疗法具有 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， 由系统性自身免疫引起的效应。这些观察结果表明，生化刺激不是 是抑制TME内T细胞功能的唯一因素。该领域的一个新兴概念是， TME的特性，如细胞外基质硬度、组成和结构也有助于癌症的发生。 细胞增殖和存活。然而，TME的机械特性是否特异性地调节T细胞 活性，从而有助于免疫逃避，仍然不清楚。这项提议有两个首要目标。 首先，为了更好地理解机械力在T细胞受体活化和T细胞功能反应中的作用。 其次，了解TME的物理方面如何影响T细胞/肿瘤相互作用和T细胞功能。 我的博士学位工作的重点是开发使能技术，在分子水平上研究机械生物学。 规模，特别关注机械力在T细胞活化中的作用。我的工作表明，i）T 细胞受体在初始识别期间和在免疫突触中将pN力传递给其抗原，以及ii）T细胞 在T细胞活化的最早阶段使用机械能来区分抗原。关于F99 在这一阶段，我将重点研究机械力对T细胞功能是否重要。为了实现这一目标， 我将使用最近开发的邻近标记技术来鉴定介导T 细胞信号这种策略将允许使用蛋白质组学分析来确定机械相互作用组 （机械组）在T细胞中。然后，我将确定机械力是否影响长期T细胞生物学功能 使用ELISA和与RNA-SEQ.总的来说，这项整合组学研究的结果将 更好地理解T细胞如何利用机械能来增强其生物功能。 对于我的博士后研究（K 00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）。目标是量化ECM机制如何改变T细胞功能。这些数据将支持 TME通过物理机制增强免疫逃避而促进转移的假设。 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 我建议在一个采用模拟TME的3D基质的实验室工作，并使用这些支架进行共培养， 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，水凝胶的程度 硬度、物理线索组成和结构），其影响T细胞/肿瘤相互作用和T细胞功能。 总的来说，F99和K 00阶段的工作将为物理学提供新的基本理解。 T细胞功能的分子水平和细胞水平的基础。这项研究的结果可能会提供新的设计 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.