Multi-phenotype CRISPR-Cas9 screen identifies p38 kinase as a target for adoptive immunotherapies

T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions. RNA expression profiles of Pmel CD8+ T cells expanded in the presence of Vehicle or p38i (BIRB 796) for 5 or 10 days. Briefly, Pmel T cells received a primary stimulation with its cognate antigen hgp100 in the presence or absence of the p38 inhibitor (BIRB 796). The stimulated cells were expanded for 5 days, and the samples were processed for RNA sequencing. For day 10 RNA sequencing, day 5 cultured cells received a secondary stimulation with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of p38i. The cells were expanded for an additional 5 days in the presence or absence of p38i (BIRB 796) and processed for RNA sequencing.