MULTIMODAL STIMULATION SCREENS REVEAL UNIQUE AND SHARED GENES LIMITING T-CELL FITNESS

T-cell dysfunction genes limit antitumor activity and may serve as therapeutic targets. It has not been systematically studied whether there are regulators that either uniquely or broadly contribute to T-cell fitness. We performed genome-scale CRISPR/Cas9 knockout screens in primary CD8 T-cells to uncover genes negatively impacting on fitness upon three modes of stimulation: (1) intense stimulation, triggering activation-induced cell death (AICD); (2) acute stimulation, triggering T-cell expansion; (3) chronic stimulation, causing dysfunction. Besides established regulators, we uncovered genes controlling T-cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increased translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T-cell clustering amplified T-cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induced functional T-cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T-cell antitumor activity.