Institute of Applied Physics, TU Wien, 1060 Vienna, Austria.
Institute of Agricultural Engineering, University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
Cells. 2021 Jan 25;10(2):235. doi: 10.3390/cells10020235.
T-cell antigen recognition is accompanied by extensive morphological rearrangements of the contact zone between the T-cell and the antigen-presenting cell (APC). This process involves binding of the T-cell receptor (TCR) complex to antigenic peptides presented via MHC on the APC surface, the interaction of costimulatory and adhesion proteins, remodeling of the actin cytoskeleton, and the initiation of downstream signaling processes such as the release of intracellular calcium. However, multiparametric time-resolved analysis of these processes is hampered by the difficulty in recording the different readout modalities at high quality in parallel. In this study, we present a platform for simultaneous quantification of TCR distribution via total internal reflection fluorescence microscopy, of intracellular calcium levels, and of T-cell-exerted forces via atomic force microscopy (AFM). In our method, AFM cantilevers were used to bring single T-cells into contact with the activating surface. We designed the platform specifically to enable the study of T-cell triggering via functionalized fluid-supported lipid bilayers, which represent a widely accepted model system to stimulate T-cells in an antigen-specific manner. In this paper, we showcase the possibilities of this platform using primary transgenic T-cells triggered specifically via their cognate antigen presented by MHCII.
T 细胞抗原识别伴随着 T 细胞与抗原呈递细胞（APC）之间接触区的广泛形态重排。这个过程涉及 T 细胞受体（TCR）复合物与 APC 表面 MHC 上呈现的抗原肽结合、共刺激和黏附蛋白的相互作用、肌动蛋白细胞骨架的重塑以及下游信号转导过程的启动，如细胞内钙离子的释放。然而，由于难以同时以高质量记录不同的读出模式，这些过程的多参数时间分辨分析受到了阻碍。在本研究中，我们提出了一个平台，用于通过全内反射荧光显微镜同时定量 TCR 分布、细胞内钙离子水平以及通过原子力显微镜（AFM）测量 T 细胞施加的力。在我们的方法中，使用 AFM 悬臂将单个 T 细胞与激活表面接触。我们专门设计了这个平台，以能够通过功能化的液支撑脂质双层研究 T 细胞的触发，这是一种广泛接受的刺激 T 细胞的模型系统，可以以抗原特异性的方式刺激 T 细胞。在本文中，我们使用通过 MHCII 呈递的其同源抗原特异性触发的原代转基因 T 细胞展示了这个平台的可能性。
