Mechanical basis of T cell receptor triggering

描述（由申请人提供）：哺乳动物有机体包含数百万种不同的T细胞，每种细胞都有自己独特的特异性。T细胞是适应性免疫反应的重要组成部分，对保护我们的身体免受外来病原体和肿瘤的侵害至关重要，这些病原体和肿瘤会产生细胞变化。T细胞受体（TCR）是一种多聚体跨膜复合物，具有高度适应性，能够区分与改变细胞表面的自我主要组织相容性复合物分子（pMHC）结合的外源肽。在T细胞的免疫监视过程中，以这些呈递肽形式显示在抗原呈递细胞上的外部标志被感知并传递到T细胞中。一旦被T细胞识别，就会启动免疫激活来摧毁非自身的“入侵者”。T细胞表面上一个弱相互作用的特异性受体如何识别这种肽引发信号仍然是一个未定义的、长期存在的生物学问题。我们的初步研究通过重点实验确定了物理力在激活T细胞中的重要性，提供了一种机械机制来回答长期存在的生物学问题，即TCR pMHC界面的微小差异如何导致触发。我们的目标是验证TCR作为各向异性机械传感器的假设。机械力在TCR激活中起作用的概念将在生理学相关的模型细胞系统中进行探索，并适用于经典的单分子分析设计。我们结合了单分子方法、结构功能突变分析和重组蛋白表达，专门针对T细胞触发事件。首先，我们确定在细胞上触发TCR的物理和化学要求。其次，我们开发了一种单分子检测方法，用于探测TCR-pMHC键在一系列负载中表现出不同效力的肽的强度。第三，我们确定了在活T细胞中柄结构域作为T细胞触发力传感器的作用。这项工作的结果将为TCR机械传感的时间、化学、空间和物理输入提供更清晰的理解。我们的项目包括培训从本科生到教师的各个层次的人员，在单分子测量和免疫学学科的实验室之间建立了重要的跨学科重叠。

DESCRIPTION (provided by applicant): The mammalian organism contains millions of distinct T cells, each with their own unique specificity. T cells are essential components of the adaptive immune response, being critical to the defense of our bodies against foreign pathogens and tumors that create cellular alterations. The T cell receptor (TCR), a multimeric transmembrane complex, is highly adaptive, capable of distinguishing foreign peptides bound to self- major histocompatibility complex molecules (pMHC) displayed on the surface of altered cells. External flags in the form of these presented peptides displayed on antigen presenting cells are sensed and transmitted into the T cell during its process of immune surveillance. Once recognized by the T cell, immune activation is initiated to destroy the non-self "invader". How recognition of ths peptide by a weakly interacting specific receptor on the T cell surface evokes signaling remains an undefined, longstanding, biological question. Our preliminary studies identified via focused experimentation the importance of physical forces in activating T cells, providing a mechanical mechanism to answer the long standing biological question of how minute differences at the TCR pMHC interface lead to triggering. Our objective is to test the hypothesis that the TCR acts as an anisotropic mechanosensor. The concept that mechanical force plays a role in TCR activation will be explored across physiologically relevant model cell-based systems as well as be adapted to classical single molecule assay design. We combine single molecule methods, structure function mutational analysis, and recombinant protein expression specifically targeted to the T cell triggering event. First, we determine the physical and chemical requirements for TCR triggering on cells. Second, we develop a single molecule assay for probing the strength of the TCR-pMHC bond across a range of loads with peptides exhibiting varying potencies. Third, we determine the role of the stalk domain as a force transducer for T cell triggering in live T cells. Outcome of this work will provide a clearer understanding for the temporal, chemical, spatial and physical inputs to TCR mechanosensing. Our project includes training of personnel at levels ranging from undergraduates to faculty with crucial interdisciplinary overlap between labs founded in single molecule measurement and immunology disciplines.