Developing a Bioanalytical Toolkit to Study the Mechanobiology of Juxtacrine Signaling

项目摘要/摘要 这项提议的长期目标是更好地了解T细胞如何防御病原体和 根除我们体内的癌细胞。为了实现这一目标，T细胞不断爬行寻找证据 其他细胞表面的外源多肽碎片。一旦T细胞遇到带有外源基因的目标细胞 或突变的多肽，然后它启动激活机制，释放出强大的免疫反应。 T细胞激活障碍与自身免疫性疾病有关，而增强T细胞激活的药物 被用来治疗癌症。因此，人们对了解T的分子机制很感兴趣。 细胞激活。T细胞活化的第一步涉及T细胞受体(TCR)和 主要组织相容性复合体(PMHC)蛋白提供的短肽(8-11个氨基酸)。 因为T细胞具有高度的迁移性，当T细胞与 对于靶细胞，T细胞是否向其TCR复合体传递特定的力量，这是一个长期存在的问题 以及化学-机械耦合是否影响免疫功能。这些问题不能用 传统的成像方法。这项拟议工作的中心假设是先进的机械-- 成像和机械分析方法将揭示参与T细胞调节的TCR力 发信号。在开发高分辨率成像方法以绘制地图的最新突破的基础上 通过细胞表面受体传递的力，我们将致力于弥合我们理解中的这一差距，并揭开 T细胞活化的力学基础。我们的初步数据清楚地表明，我们已经成功地开发了 第一批成像TCR过程中由TCR传递给其配体的皮牛顿力的分子探测器 激活。我们将首先为TCR开发分子力显微镜，以检验中心假设。 这些探头将测试TCR是否是文献中提出的各向异性机械传感器。下一首 我们将在膜-膜连接处映射TCR力，在那里受体可以自由组装 发信号的微团簇。将使用荧光寿命成像显微镜(FLIM)和比率探头 在空间和时间上绘制这些力的地图。最后，我们将使用机械触发的酶来量化TCR 具有超高灵敏度的作用力，并标记在TCR传输后招募的近端分子 力量。这项工作需要多学科的方法，结合三名调查人员的专业知识 生物物理化学、细胞生物学和分子免疫学领域。重要的是，不仅 为这项提案开发的成像和量化技术对于更好地理解 由于适应性免疫系统的特殊性，我们期待着对优化设计和 过继T细胞转移和嵌合抗原受体在免疫治疗中的应用 了解自身免疫性疾病的原因。

Project Summary/Abstract The long-term goal of this proposal is to better understand how T cells defend against pathogens and eradicate cancerous cells within our bodies. To achieve this goal, T cells continuously crawl seeking evidence of foreign peptide fragments on the surface of other cells. Once the T cell encounters a target cell with foreign or mutant peptides, then it initiates activation mechanisms that unleash a potent immune response. Malfunctions in T cell activation are linked with autoimmune disease, while drugs that enhance T cell activation are used to treat cancer. Therefore, there is much interest in understanding the molecular mechanisms of T cell activation. The very first step in T cell activation involves recognition between the T cell receptor (TCR) and the short peptides (8-11 amino acids) presented by the major histocompatibility complex (pMHC) protein. Because T cells are highly migratory and antigen recognition occurs when the T cell physically contacts a target cell, there are long standing questions of whether T cells transmit defined forces to their TCR complex and if chemo-mechanical coupling influences immune function. These questions cannot be answered using conventional imaging approaches. The central hypothesis of the proposed work is that advanced mechano- imaging and mechano-analytical approaches will reveal the TCR forces involved in regulation of T cell signaling. Building on our recent breakthroughs in developing high-resolution imaging approaches to map the forces transmitted by cell surface receptors, we will aim to close this gap in our understanding and unravel the mechanical basis of T cell activation. Our preliminary data clearly shows that we have successfully developed the first molecular probes to image the piconewton forces transmitted by the TCR to its ligand during TCR activation. We will test the central hypothesis by first developing molecular force microscopy for the TCR. These probes will test whether the TCR is an anisotropic mechanosensor as proposed in the literature. Next we will map TCR forces within membrane-membrane junctions where the receptor is free to assemble into signaling microclusters. Fluorescence lifetime imaging microscopy (FLIM) and ratiometric probes will be used to map these forces in space and time. Finally, we will use mechanically-triggered enzymes to quantify TCR forces with ultrahigh sensitivity and to tag proximal molecules that are recruited following transmission of TCR forces. The work requires multidisciplinary approaches combining expertise from three investigators that cover the areas of biophysical chemistry, cell biology, and molecular immunology. Importantly, not only will the imaging and quantification techniques developed for this proposal be critical for better understanding the specificity of the adaptive immune system, we expect important implications for the optimal design and implementation of adoptive T cell transfer and chimeric antigen receptors (CARs) in immunotherapy as well as understanding the causes of autoimmune disease.