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.