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。TCRs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提供对TCRs如何实现这种二元性的洞察。然而，关键问题依然存在，许多假设和概括都未经证实或未经检验。这次竞争性更新的工作将研究TCR特异性和交叉反应的物理基础，重点放在三个密切相关的主题上：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，首次在生物相关的时间尺度上获得TCR环动力学的明确测量。动力学的测量将与TCR的特异性和交叉反应有关。对配体TCR结合的研究将使用停流稳态荧光各向异性，这与表面等离子体共振不同，它将允许对结合机制(例如，刚体、诱导匹配或平衡前构象系综)进行严格评估。同样，机制将与特异性和交叉反应有关，并在流行的结合模型的背景下考虑。在研究结合能的分布时，将使用双突变循环，这是其他领域中常用的一种方法，用于评估区域对结合的贡献，但尚未应用于TCR-pMHC相互作用。双突变循环将解决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的贡献程度。将研究多个TCR-pMHC系统，包括交叉反应相互作用，具有紧密和松散精细特异性的相互作用，以及具有不同程度的构象变化和结合拓扑的相互作用。总体而言，关于pMHC的TCR识别的几个悬而未决的问题将被解决，总体目标是阐明TCR如何实现其显著的分子识别特性。所得结果将进一步了解人类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。与公共卫生的相关性：细胞免疫系统的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的双重能力。将进行一项详细的生物物理研究，以帮助理解这种二元性，其结果不仅将有助于理解人类免疫系统的正常和异常功能，而且将有助于发现和设计基于免疫的诊断和治疗方法。

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.