CRISPR/Cas9 mutagenesis screening identifies PRDM1 as a key regulator of the natural killer T cell central memory program and effector function

Natural killer T cells (NKTs) are a promising platform for cancer immunotherapy, but few genes involved in regulation of NKT therapeutic activity have been identified. To discover regulators of NKT functional fitness, we developed a CRISPR/Cas9-based mutagenesis screen that employs a guide (g)RNA library targeting 1,118 immune-related genes. Unmodified NKTs and NKTs expressing a GD2-specific chimeric antigen receptor (CAR.NKTs) were transduced with the gRNA library and exposed to CD1d+/GD2- leukemia or CD1d-/GD2+ neuroblastoma cells, respectively, over six challenge cycles in vitro. Quantification of gRNA abundance revealed enrichment of PRDM1-specific gRNAs in both NKTs and CAR.NKTs, a result that was validated through targeted PRDM1 knockout. Transcriptional, phenotypic, and functional analyses demonstrated that CAR.NKTs with PRDM1 knockout undergo central memory-like differentiation and resist exhaustion while downregulating granzyme B and mediating reduced cytotoxicity in vitro and moderate antitumor activity in an in vivo xenogeneic neuroblastoma model. In contrast, shRNA-mediated knockdown of PRDM1 expression preserved CAR.NKT effector function while promoting central memory differentiation, resulting in CAR.NKTs with potent in vivo antitumor activity. Thus, we identified PRDM1 as a regulator of NKT memory differentiation and effector function that can be exploited to improve the efficacy of next-generation NKT-based cancer immunotherapy. Overall design: To determine how PRDM1 CRISPR KO impacts GD2.CAR NKT gene expression, we sequenced RNA isolated from PRDM1 KO GD2.CAR NKTs after one and six rounds of tumor challenge. To determine the impact of knocking down PRDM1 on the GD2.CAR NKT transcriptome, we defined the genome-wide expression profile of PRDM1 KD GD2.CAR NKTs and compared with that of scrambled shRNA control cells after one cycle or six cycles of serial tumor challenge.