IL-18 metabolically reprograms CAR-expressing natural killer T cells and enhances their antitumor activity

Invariant natural killer T cells (NKTs) have intrinsic antitumor properties that make them promising candidates for chimeric antigen receptor (CAR) immunotherapies. Transgenic cytokine expression can enhance cellular therapy potency, and we hypothesized that co-expressing interleukin-18 (IL-18) alone or with IL-15 would boost CAR-NKT therapeutic potential. To test this, we generated retroviral constructs expressing IL-15 and/or IL-18 with an inducible caspase-9 safety switch and co-transduced them with a GD2-specific CAR into human NKTs. Co-expression of IL-18 or IL-15/IL-18 increased CAR-NKT cytotoxicity, proliferation, and cytokine secretion in vitro compared to IL-15 alone. IL-18 also enhanced GPC3.CAR and CD19.CAR NKT activity against hepatocellular carcinoma and B cell leukemia cells, respectively. In a metastatic neuroblastoma model, IL-18-expressing GD2.CAR-NKTs controlled tumors more effectively than IL-15-only cells, but mice in the IL-15/IL-18 group developed severe toxicities not observed in the IL-18-only group. Mechanistically, IL-18 induced a transcriptional program distinct from IL-15, marked by lower exhaustion signatures and enrichment of metabolic pathways. Finally, targeted metabolomics showed that IL-18 drives broad metabolic reprogramming in CAR-NKTs including increased oxidative phosphorylation, glycolysis, glutaminolysis, and purine metabolism. These findings support the use of IL-18 in developing the next generation of cytokine-armed CAR-NKT cancer immunotherapies.