Assessment of the TCR Repertoire of Human Circulating T Follicular Helper Cells.

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.