Multimodal stimulation screens reveal unique and shared regulators limiting T-cell fitness [Icam1]

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. Total RNA was isolated in parallel from resting and 24-hour antiCD3-activated Ctrl or Icam1-depleted OT-I/Cas9 T cells. RNA-Seq libraries were prepared after polyA-bead enrichment of mRNAs and sequenced on a NextSeq 550 using High Output Kit v2.0 (Illumina Inc.).