Multi-phenotype CRISPR-Cas9 screen identifies p38 kinase as a target for adoptive immunotherapies. 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. Overall design: 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.

T细胞是当前所有有效癌症免疫疗法的核心，但界定具有治疗功效的抗肿瘤T细胞的特征尚未被全面阐明。本研究阐释了有效抗肿瘤T细胞的四类表型特性：细胞扩增、分化、氧化应激与基因组应激。我们利用基于CRISPR-Cas9的原代T细胞遗传筛选体系，测定了25种由T细胞受体驱动的激酶被敲除后的多表型影响。我们鉴定出p38激酶（p38 kinase）是这四类表型的核心调控因子，并揭示了T细胞内p38所调控的转录通路与抗氧化通路。对p38的药理学抑制可提升小鼠抗肿瘤T细胞的疗效，并增强人类肿瘤反应性及基因工程化T细胞的功能，为临床相关干预策略铺平了道路。实验设计：分别经载体或p38抑制剂（BIRB 796）处理5天、10天的Pmel CD8+ T细胞的RNA表达谱。简言之，Pmel T细胞在存在或不存在p38抑制剂（BIRB 796）的条件下，通过其同源抗原hgp100进行初次刺激。将刺激后的细胞扩增5天后，收集样本进行RNA测序。对于第10天的RNA测序样本，将第5天培养的细胞在存在或不存在p38抑制剂（BIRB 796）的条件下，用固相包被抗CD3抗体与可溶性抗CD28抗体进行二次刺激，随后在相同抑制剂存在与否的条件下继续扩增5天，再收集样本进行RNA测序。