Mechanical basis of T cell receptor triggering

描述(申请人提供)：哺乳动物有机体包含数百万个不同的T细胞，每个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，对我们的身体抵御造成细胞变化的外来病原体和肿瘤至关重要。T细胞受体(TCR)是一种多聚体跨膜复合体，具有高度的适应性，能够识别与细胞表面显示的自身主要组织相容性复合体分子(PMHC)结合的外来肽。在T细胞的免疫监视过程中，这些呈现在抗原提呈细胞上的多肽形式的外部标志被感知并传递到T细胞中。一旦被T细胞识别，免疫激活就会启动，以摧毁非我的“入侵者”。T细胞表面弱相互作用的特定受体如何识别这种多肽，如何引起信号传递，仍然是一个不确定的、长期存在的生物学问题。我们的初步研究通过集中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。我们的目标是检验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相关的基于细胞的模型系统中进行探索，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针对T细胞触发事件的重组蛋白表达。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分析方法，用于探测TCR-pMHC键在不同负载范围内的强度，这些多肽显示出不同的效力。第三，我们确定了茎结构域作为T细胞在活T细胞中触发的力传导器的作用。这项工作的成果将为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职员工的人员，在单分子测量和免疫学学科建立的实验室之间存在关键的跨学科重叠。

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.