1124 Discovering transcriptional regulators of T cell exhaustion for epigenetic reversal of T cell dysfunction

<h3>Background</h3> T cells are part of the immune system’s adaptive defence; they specifically target and kill both virally infected and cancerous cells through antigen recognition. Unfortunately, cancer can exploit intrinsic T cell mechanisms to survive attack by the immune system. T cell exhaustion arises from chronic antigen stimulation, which shifts a portion of the T cell population to the exhausted state (T_EX), resulting in diminished T cell proliferation and tumor/viral clearance. Modern genome engineering technologies have the potential to dramatically advance T cell therapy by programming T_EX cells into desirable phenotypic states. The transcription factor (TF) TOX drives and maintains the T_EX state through epigenetic regulation of exhaustion-associated programs. <h3>Methods</h3> To recapitulate T cell exhaustion in vitro, we repeatedly stimulated primary human T cells with anti-CD3/CD28 beads to mimic chronic stimulation and confirmed induction of the exhaustion program via RNA- and ATAC-seq. To understand what other TF regulators might be driving or preventing T cell exhaustion upstream of TOX, we designed screens using an Open Reading Frame (ORF) library encoding all the TFs in the human genome with TOX protein expression as a readout. CD8+ T cells from three distinct donors were used in an acute stimulation setting (one TCR stimulation) and a chronic stimulation setting (two additional stimulations). Comparing screen results in the context of acute versus chronic stimulation facilitated identification of factors specific to the T_EX state. <h3>Results</h3> Over 100 TF ORFs were enriched in TOX-high and TOX-low bins, including both known and novel regulators of TOX. To comprehensively look at the transcriptomic, epigenomic, and functional effects top TFs had on acutely or chronically stimulated T cells, we sub pooled top TFs and screened in chronic versus acute stimulation setting with a final Phorbol myristate acetate and ionomycin (PMA/IO) stimulation using Simultaneous High-throughput ATAC and RNA expression with sequencing (SHARE-seq) as a readout. Computational analysis revealed THAP6 to be improving stimulation response and pushing away from the exhausted chromatin state. Individual validation of THAP6 confirmed downregulation of multiple T cell exhaustion markers via FACS and an improved functional response via a bead-based cytokine expression assay. <h3>Conclusions</h3> Ongoing work is focused on validating the effects of THAP6 and other top TFs on T cell function (T cell killing and cytokine expression assays), transcriptome (bulk RNA-seq), and epigenome (bulk ATAC-seq). Through epigenetic reprogramming, we hope to achieve clinically relevant T cell phenotypes to improve current immunotherapies in both viral infection and cancer contexts.