Exploring Mechanisms of Human PD-1 Gene Regulation in T Follicular Helper Cells

Abstract

Abstract Programed Cell death (PD-1), encoded by Pdcd1, is an immune inhibitory receptor expressed on the surface of lymphocytes. Surface PD-1 expression is amongst its highest in a subset of CD4+ T cells called T follicular helper cells (TFH). TFH cells are pivotal for optimal germinal center formation and subsequent generation of pathogen- and vaccine-induced antibodies by B cells. Despite the clear connection to human health, the majority of previous work has centered on elucidating the mechanisms surrounding murine Pdcd1 regulation. Surprisingly, there is almost nothing known about how human Pdcd1 (hPdcd1) is regulated! Here we seek to close this gap in knowledge by identifying the cis-, trans-, and epigenetic pathways that regulate hPdcd1 in TFH cells. To this end, human TFH cells were sourced from both discarded tonsil tissue as well as from a recently described model to generate ex vivo human TFH cells through treatment with the cytokines Activin A, IL-12 and TGFβ. RNA-sequencing demonstrated that ex vivo TFH cells exhibit characteristics of bona-fide tonsillar TFH cells, including increased expression of PD-1. Furthermore, ATAC-seq and H3K27Ac Hi-ChIP experiments revealed the presence of 7 conserved accessible regions within the hPdcd1 locus that exhibit unique looping characteristics and potential enhancer properties. Functionality of each regulatory element was further defined through promoter reporter assays. Additionally, PAGE-RANK analysis elucidated transcription factors that may play a role in the induction of hPdcd1 expression. Collectively, these findings begin to unearth the mechanisms that control hPdcd1, informing the design of future therapeutics aimed at precisely manipulating human PD-1 expression.