Physical Basis for T Cell Receptor Binding and Activity

描述（由申请人提供）：T细胞受体（TCR）对配体的识别是T细胞库的发展和维持以及细胞免疫反应的启动和传播所必需的。tcr的一个决定性特征是其特异性和交叉反应性的双重能力。结构、生物物理和免疫学数据继续为tcr如何实现这种两重性提供见解。然而，关键问题仍然存在，许多假设和概括未经证实或未经测试。这项竞争性更新的工作将研究TCR特异性和交叉反应性的物理基础，重点关注三个密切相关的主题：TCR CDR环的动力学，TCR与pMHC结合的机制，以及TCR- pMHC界面内结合能的分布。动力学将通过使用核磁共振、时间分辨荧光各向异性和计算模拟进行分析，首次获得跨生物学相关时间尺度的TCR环路动力学的清晰测量。动力学测量将与TCR特异性和交叉反应性有关。配体与TCR结合的研究将使用停止流动的稳态荧光各向异性进行，这与表面等离子体共振不同，将允许对结合机制（例如，刚体，诱导拟合或预平衡构象集合）进行严格的评估。同样，机制将与特异性和交叉反应性相关，并在流行的结合模型的背景下考虑。在检查结合能分布时，将使用双突变循环，这是一种在其他领域常用的方法，用于评估区域对结合的贡献，但尚未应用于TCR-pMHC相互作用。双突变周期将解决TCR-pMHC界面内不同区域（即CDR环、肽和MHC螺旋）的相对贡献，并检查不同区域相互独立贡献的程度。将研究多种TCR-pMHC系统，包括交叉反应相互作用，紧密和松散精细特异性的相互作用，以及不同程度构象变化和结合拓扑的相互作用。总体而言，将解决关于pMHC的TCR识别的几个悬而未决的问题，其总体目标是阐明TCR如何实现其卓越的分子识别特性。所获得的结果将进一步了解人体免疫系统的正常和异常功能，并有助于发现和设计基于细胞免疫的新疗法。公共卫生相关性：细胞免疫系统中T细胞受体的一个鲜为人知的特征是它们具有特异性和交叉反应性的双重能力。一项详细的生物物理研究将有助于理解这种二元性，其结果不仅有助于理解人类免疫系统的正常和异常功能，而且有助于发现和设计基于免疫学的诊断和治疗方法。

DESCRIPTION (provided by applicant): Recognition of ligand by the a¿ T cell receptor (TCR) is required for the development and maintenance of the T cell repertoire and the initiation and propagation of a cellular immune response. A defining feature of TCRs is their dual capacity for specificity and cross-reactivity. Structural, biophysical, and immunological data continue to provide insight into how TCRs achieve this duality. Yet key questions remain, and a number of hypotheses and generalizations are unproven or untested. The work in this competitive renewal will study the physical bases for TCR specificity and cross-reactivity, focusing on three closely related topics: the dynamics of TCR CDR loops, the mechanisms by which TCRs engage pMHC, and the distribution of binding energy within TCR- pMHC interfaces. Dynamics will be assayed through the use of NMR, time resolved fluorescence anisotropy, and computational simulations, obtaining for the first time clear measurements of TCR loop dynamics across biologically relevant timescales. Measurements of dynamics will be related to TCR specificity and cross- reactivity. Studies of TCR engagement of ligand will be performed with stopped-flow steady-state fluorescence anisotropy, which unlike surface plasmon resonance will allow a rigorous assessment of binding mechanisms (e.g., rigid body, induced fit, or pre-equilibrium conformational ensembles). Again, mechanisms will be related to specificity and cross-reactivity, and considered in the context of popular binding models. In examining the distribution of binding energy, double mutant cycles, an approach commonly used in other fields to asses regional contributions to binding but yet to be applied to TCR-pMHC interactions, will be used. Double mutant cycles will address the relative contributions of various regions within TCR-pMHC interfaces (i.e., the CDR loops, peptide, and MHC helices), as well as examine the extent to which different regions contribute independently of each other. Multiple TCR-pMHC systems will be studied, including cross-reactive interactions, interactions with tight and loose fine specificity, and interactions that proceed with differing degrees of conformational changes and binding topologies. Overall, several outstanding questions regarding TCR recognition of pMHC will be addressed, with the overall aim of clarifying how TCRs achieve their remarkable molecular recognition properties. The results obtained will further the understanding of the normal and abnormal functioning of the human immune system and help in the discovery and design of novel therapeutics based on cellular immunity. Public Health Relevance: A poorly understood feature of T cell receptors of the cellular immune system is their dual capacity for specificity and cross-reactivity. A detailed biophysical study to help understand this duality will be performed, the results of which will contribute not only to the understanding of the normal and abnormal functioning of the human immune system, but also to efforts to discover and design immunologically-based diagnostics and therapeutics.