Stephens Hannah M, Brazin Kristine N, Mallis Robert J, Feng Yinnian, Banik Debasis, Reinherz Ellis L, Lang Matthew J
Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA.
Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA, USA.
Methods Mol Biol. 2022;2478:727-753. doi: 10.1007/978-1-0716-2229-2_26.
T-cell antigen receptors (TCRs) are mechanosensors, which initiate a signaling cascade upon ligand recognition resulting in T-cell differentiation, homeostasis, effector and regulatory functions. An optical trap combined with fluorescence permits direct monitoring of T-cell triggering in response to force application at various concentrations of peptide-bound major histocompatibility complex molecules (pMHC). The technique mimics physiological shear forces applied as cells crawl across antigen-presenting surfaces during immune surveillance. True single molecule studies performed on single cells profile force-bond lifetime, typically seen as a catch bond, and conformational change at the TCR-pMHC bond on the surface of the cell upon force loading. Together, activation and single molecule single cell studies provide chemical and physical triggering thresholds as well as insight into catch bond formation and quaternary structural changes of single TCRs. The present methods detail assay design, preparation, and execution, as well as data analysis. These methods may be applied to a wide range of pMHC-TCR interactions and have potential for adaptation to other receptor-ligand systems.
T细胞抗原受体（TCRs）是机械传感器，在识别配体后启动信号级联反应，导致T细胞分化、稳态、效应和调节功能。光镊与荧光相结合，可以直接监测在不同浓度的肽结合主要组织相容性复合体分子（pMHC）作用下，T细胞对施加力的触发反应。该技术模拟了免疫监视期间细胞在抗原呈递表面爬行时施加的生理剪切力。对单细胞进行的真正单分子研究描绘了力-键寿命（通常表现为捕获键）以及力加载时细胞表面TCR-pMHC键的构象变化。激活研究和单分子单细胞研究共同提供了化学和物理触发阈值，以及对捕获键形成和单个TCR四级结构变化的深入了解。本文方法详细介绍了实验设计、准备、执行以及数据分析。这些方法可应用于广泛的pMHC-TCR相互作用，并有可能适用于其他受体-配体系统。
