Single-nucleotide variants (SNVs) in key T cell genes can drive clinical pathologies and could be repurposed to improve cellular cancer immunotherapies. Here, we perform massively parallel base-editing screens to generate thousands of variants at gene loci annotated with known or potential clinical relevance. We discover a broad landscape of putative gain-of-function (GOF) and loss-of-function (LOF) mutations, including in PIK3CD and the gene encoding its regulatory subunit, PIK3R1, LCK, SOS1, AKT1 and RHOA. Base editing of PIK3CD and PIK3R1 variants in T cells with an engineered T cell receptor specific to a melanoma epitope or in different generations of CD19 chimeric antigen receptor (CAR) T cells demonstrates that discovered GOF variants, but not LOF or silent mutation controls, enhanced signaling, cytokine production and lysis of cognate melanoma and leukemia cell models, respectively. Additionally, we show that generations of CD19 CAR T cells engineered with PIK3CD GOF mutations demonstrate enhanced antigen-specific signaling, cytokine production and leukemia cell killing, including when benchmarked against other recent strategies.

关键 T 细胞基因中的单核苷酸变异 (SNV) 可以驱动临床病理，并可重新用于改善细胞癌症免疫疗法。在这里，我们进行大规模并行碱基编辑筛选，在具有已知或潜在临床相关性的基因位点上生成数千个变异。我们发现了广泛的假定功能获得 (GOF) 和功能丧失 (LOF) 突变，包括 PIK3CD 及其编码调节亚基 PIK3R1、LCK、SOS1、AKT1 和 RHOA 的基因。在具有黑色素瘤表位特异性工程 T 细胞受体的 T 细胞中或在不同代的 CD19 嵌合抗原受体 (CAR) T 细胞中对 PIK3CD 和 PIK3R1 变体进行碱基编辑表明，发现的 GOF 变体（而非 LOF 或沉默突变对照）增强了分别是同源黑色素瘤和白血病细胞模型的信号传导、细胞因子产生和裂解。此外，我们还发现，用 PIK3CD GOF 突变改造的几代 CD19 CAR T 细胞表现出增强的抗原特异性信号传导、细胞因子产生和白血病细胞杀伤，包括与其他近期策略进行基准比较时。