PRDM1 Is a Key Regulator of the NKT-cell Central Memory Program and Effector Function

Cancer Immunol Res. 2025 Apr 2;13(4):577-590. doi: 10.1158/2326-6066.CIR-24-0259.

Abstract

Natural killer T cells (NKTs) are a promising platform for cancer immunotherapy, but few genes involved in the regulation of NKT therapeutic activity have been identified. To find regulators of NKT functional fitness, we developed a CRISPR/Cas9-based mutagenesis screen that uses a guide RNA (gRNA) library targeting 1,118 immune-related genes. Unmodified NKTs and NKTs expressing a GD2-specific chimeric antigen receptor (GD2.CAR) were transduced with the gRNA library and exposed to CD1d+ 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 GD2.CAR NKTs, a result that was validated through targeted PRDM1 knockout. Transcriptional, phenotypic, and functional analyses demonstrated that CAR NKTs with PRDM1 knockout underwent central memory-like differentiation and resisted exhaustion. However, these cells downregulated the cytotoxic mediator granzyme B and showed reduced in vitro cytotoxicity and only moderate in vivo antitumor activity in a xenogeneic neuroblastoma model. In contrast, short hairpin RNA-mediated PRDM1 knockdown preserved effector function while promoting central memory differentiation, resulting in GD2.CAR NKTs with potent in vivo antitumor activity. Thus, we have identified PRDM1 as a regulator of NKT memory differentiation and effector function that can be exploited to improve the efficacy of NKT-based cancer immunotherapies.

MeSH terms

  • Animals
  • CRISPR-Cas Systems
  • Cell Line, Tumor
  • Humans
  • Immunologic Memory*
  • Immunotherapy, Adoptive / methods
  • Mice
  • Natural Killer T-Cells* / immunology
  • Natural Killer T-Cells* / metabolism
  • Neuroblastoma* / immunology
  • Neuroblastoma* / therapy
  • Positive Regulatory Domain I-Binding Factor 1* / genetics
  • Positive Regulatory Domain I-Binding Factor 1* / metabolism
  • Receptors, Chimeric Antigen / genetics
  • Receptors, Chimeric Antigen / immunology
  • Receptors, Chimeric Antigen / metabolism
  • Xenograft Model Antitumor Assays

Substances

  • Positive Regulatory Domain I-Binding Factor 1
  • PRDM1 protein, human
  • Receptors, Chimeric Antigen