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

Abstract

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. TFH 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 (cTFH) are considered a helper T cell linage in the blood that to some extent relates with bona fide TFH cells in the germinal centers of secondary lymphoid organs. Due to the limited access to the secondary lymphoid organs, cTFHhave become a more accessible immunological readout.The assessment of the TCR repertoires of TFH and ofcTFH cells is of both fundamental and clinical importance being instrumental to define the linage relationship of cTFH 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 cTFH cells in vitro and subsequent protocols for the high-throughput TCR sequencing, followed by repertoire data analysis.