Multimodal stimulation screens reveal unique and shared regulators limiting T-cell fitness [IntenseAcuteChronic_screen]

It has not been systematically studied whether T-cell fitness regulators uniquely or broadly contribute to specific traits limiting antitumor activity. We performed genome-scale CRISPR/Cas9 knockout screens in primary CD8 T-cells to uncover genes negatively impacting on their fitness upon three modes of stimulation: (1) intense stimulation, causing activation-induced cell death (AICD); (2) acute stimulation, triggering T-cell expansion; (3) chronic stimulation, resulting in dysfunction. Besides established regulators, we uncovered several genes controlling T-cell fitness either specifically or commonly under various stimulation modalities. Ablation of Dap5, 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. Finally, inactivation of Ctbp1 induced T-cell persistence exclusively upon chronic stimulation. Our results functionally annotate T-cell fitness regulators based on their unique or shared contribution to traits limiting T-cell antitumor activity. Naive OT-I/Cas9 T-cells were primed using CD3 and CD28 antibody and transduced with the genome-wide Brie sgRNA library. Day 1 after puromycin selection we harvested a library reference sample (t0). After 6d puro-selection, we harvested a Pre-reactivation reference sample. For different stimulation screens, T-cells were stimulated under three different conditions: (1) Intense: T-cells were stimulated with plate-bound CD3 antibody for two times in two days. (2) Acute: T-cells were stimulated once with CD3 antibody for 24h. Cells were then removed from the plates and cultured for another 3d before harvesting. (3) Chronic: T-cells were co-cultured daily with fresh D4M.OVA tumor cells for 11d (11 times). For the resting condition, cells were refreshed daily without adding tumor cells. Both stimulated and resting cells were harvested for analysis after 11 days (11 times) tumor stimulation. Genomic DNA was isolated, sgRNAs were amplified using a one-step barcoding PCR.