Physical Basis for T Cell Receptor Binding and Activity

描述（由申请人提供）：T细胞库的发育和维持以及细胞免疫应答的启动和传播需要α T细胞受体（TCR）识别配体。TCR的定义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深入了解TCR如何实现这种双重性。然而，关键问题仍然存在，一些假设和概括未经证实或未经检验。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，重点是三个密切相关的主题：TCR CDR环的动力学，TCR参与pMHC的机制，以及TCR- pMHC界面内结合能的分布。将通过使用NMR、时间分辨荧光各向异性和计算模拟来测定动力学，首次获得跨生物学相关时间尺度的TCR环动力学的清晰测量。动力学的测量将与TCR特异性和交叉反应性相关。TCR与配体结合的研究将用停流稳态荧光各向异性进行，这与表面等离子体共振不同，将允许严格评估结合机制（例如，刚体、诱导配合或预平衡构象集合）。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。在检查结合能的分布时，将使用双突变体循环，这是一种在其他领域中常用的方法，用于评估区域对结合的贡献，但尚未应用于TCR-pMHC相互作用。双突变体循环将解决TCR-pMHC界面内各个区域的相对贡献（即，CDR环、肽和MHC螺旋），以及检查不同区域彼此独立贡献的程度。将研究多个TCR-pMHC系统，包括交叉反应性相互作用，具有紧密和松散精细特异性的相互作用，以及以不同程度的构象变化和结合拓扑结构进行的相互作用。总体而言，几个悬而未决的问题，TCR识别pMHC将得到解决，总的目的是澄清TCR如何实现其显着的分子识别特性。所获得的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型疗法。公共卫生相关性：细胞免疫系统的T细胞受体的一个知之甚少的特征是其特异性和交叉反应性的双重能力。将进行详细的生物物理研究，以帮助理解这种双重性，其结果不仅有助于了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.