Mechanisms of human PD-1 gene regulation in T follicular helper cells

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1 Programed Cell death (PD-1), encoded by Pdcd1, is an immune inhibitory receptor expressed on the surface of 2 lymphocytes. Engagement of PD-1 by its ligands results in suppression of immune effector function and 3 subsequent dampening of inflammatory responses. The impact of this signaling pathway to human health has 4 been demonstrated by the clinically successful treatment of numerous cancers with antibody blockade of PD-1 5 or its ligand, resulting in reinvigoration of the immune system and successive anti-tumor responses. Surface 6 PD-1 expression is amongst its highest in a subset of CD4+ T cells called T follicular helper cells (TFH). TFH cells 7 are pivotal for optimal germinal center formation and subsequent generation of pathogen- and vaccine- induced 8 antibodies by B cells. As such, PD-1 resides as a key component of a robust humoral response. Despite the 9 clear connection to human health, the majority of previous work has centered on elucidating the mechanisms 10 surrounding murine Pdcd1 (mPdcd1) regulation. Surprisingly, there is almost nothing known about how human 11 Pdcd1 (hPdcd1) is regulated! Here we seek to close this gap in knowledge by identifying the cis-, trans-, and 12 epigenetic pathways that regulate hPdcd1 in TFH cells. We hypothesize that hPdcd1 expression in TFH cells is 13 controlled by novel regulatory mechanisms aimed at controlling the extraordinary high levels of PD-1 on these 14 cells. These findings could identify novel elements and pathways that might be dysfunctional in other 15 immunological contexts and disease settings. In Aim 1 I will identify and classify the cis-regulatory elements that 16 control hPdcd1 expression in TFH cells. I will acquire primary human TFH cells form blood and discarded tonsil 17 tissue, as well as from a recently described model to generate ex vivo TFH-like cells. Preliminary ATAC-seq data 18 in TFH cells has defined regions of interest that will be interrogated for enhancer functionality by engineering 19 promoter-reporter constructs. Additionally, deactivated Cas9-CRISPR technologies will be used to define the 20 functionality of these regions in primary human TFH cells. To further characterize the hPdcd1 epigenetic 21 landscape in TFH cells, I will conduct ChIP-seq for covalent histone modifications and Hi-ChIP to define the 3D 22 enhancer-promoter architecture. In Aim 2, I will determine the unique transcription factor network that regulates 23 hPdcd1 expression in TFH cells. Integration of previous published work with RNA- and ATAC-seq datasets has 24 identified candidate transcription factors that may play a role in hPdcd1 regulation. I will use ChIP-qPCR to 25 determine transcription factor occupancy at the hPdcd1 locus. Subsequently, I will use CRISPR/Cas9 and 26 lentiviral expression systems in primary human cells to knockout or exogenously express factors respectively, 27 and determine the resulting effect on PD-1 expression. Collectively, unearthing the mechanisms of hPdcd1 28 regulation at the molecular level will aid in the identification of novel therapeutic targets, that could allow for the 29 precise manipulation of PD-1 expression in the treatment of cancer, autoimmunity, and infectious disease.