Institute for Research in Biomedicine, Bellinzona, Università della Svizzera Italiana, Lugano, Switzerland.
Institute of Microbiology, ETH Zürich, Zürich, Switzerland.
Methods Mol Biol. 2022;2380:149-163. doi: 10.1007/978-1-0716-1736-6_13.
Every T cell clone has its unique T cell receptor that results from somatic recombination of V(D)J genes in developing T cells. This process leads to a highly diverse TCR repertoire of naïve T cells, which is selected, upon antigenic recognition, to form the repertoires of effector and memory T cells. The advent of next-generation sequencing (NGS) technology allows for the high-throughput analysis of the TCR repertoires in the different T cell populations. T cells, since their initial discovery in human tonsils and in mouse lymphoid organs, have become the subject of intense investigations due to their essential role in regulating B cell responses and the process of antibody affinity maturation. Circulating follicular helper T cells (cT) are considered a helper T cell linage in the blood that to some extent relates with bona fide T cells in the germinal centers of secondary lymphoid organs. Due to the limited access to the secondary lymphoid organs, cT have become a more accessible immunological readout. The assessment of the TCR repertoires of T and of cT cells is of both fundamental and clinical importance being instrumental to define the linage relationship of cT with other T cell subsets and to monitor response to infections or vaccination or disease states. In this chapter, we will provide detailed methods for isolation of antigen-specific cT cells in vitro and subsequent protocols for the high-throughput TCR sequencing, followed by repertoire data analysis.
每个 T 细胞克隆都有其独特的 T 细胞受体，这是在发育中的 T 细胞中 V(D)J 基因的体细胞重组产生的。这个过程导致了初始 T 细胞的 TCR 库具有高度的多样性，这些 TCR 库在抗原识别后被选择，形成效应和记忆 T 细胞的库。下一代测序（NGS）技术的出现使得不同 T 细胞群体中的 TCR 库能够进行高通量分析。T 细胞自最初在人类扁桃体和小鼠淋巴器官中被发现以来，由于其在调节 B 细胞反应和抗体亲和力成熟过程中的重要作用，已成为研究的热点。循环滤泡辅助 T 细胞（cT）被认为是血液中的一种辅助性 T 细胞谱系，在某种程度上与次级淋巴器官生发中心中的真正 T 细胞有关。由于难以进入次级淋巴器官，cT 已成为更易获取的免疫学研究指标。评估 T 细胞和 cT 细胞的 TCR 库具有基础和临床双重重要性，有助于确定 cT 与其他 T 细胞亚群的谱系关系，并监测对感染、疫苗接种或疾病状态的反应。在本章中，我们将提供详细的方法来体外分离抗原特异性 cT 细胞，并随后进行高通量 TCR 测序的方案，最后是对库数据进行分析。
