CD1d-restricted invariant Natural Killer T (iNKT) cells play a key role in tumor immunosurveillance by actively remodeling the composition of myelomonocytic cells in the tumor microenvironment, enforcing immunostimulatory conditions. iNKT cell transferred into tumor-bearing mice home to the tumor site and delay tumor progression, but their therapeutic impact is limited by progressive functional exhaustion.
To enhance their anti-tumor potential, we engineered iNKT cells to express different immunostimulatory cytokines: IL-15 (±IL-15Rα); IL-12 and IFNα, each known to support lymphocyte survival and effector function. IL-15 and IFNα supported robust expansion and preserved the TH0 effector phenotype of iNKT cells, while IL-12 promoted a TH1-skewed, pro-inflammatory profile with limited in vitro persistence. RNA sequencing of cytokine-engineered iNKT cells revealed that iNKT-IL12 cells acquired a distinct gene expression program, characterized by high metabolic activity, effector profile and terminal differentiation signatures compared to iNKT-IL-15 or iNKT-IFNα cells. In vivo, iNKT-IL12 cells demonstrated superior tumor control but induced significant systemic toxicity, including body weight loss and elevated plasma cytokines in both tumor- and non-tumor-bearing animals, which critically depended on the induction of a massive endogenous IFNg response in the recipient mice. In contrast, IL-15 and IFNα engineering conferred only moderate anti-tumor efficacy, although with good safety profiles. To mitigate IL-12-associated toxicity, we are developing a membrane-bound form of IL-12 to localize cytokine effects and avoid systemic exposure.
These findings highlight how cytokine engineering differentially shapes iNKT cell behavior and identifies IL-12 as a powerful but potentially toxic enhancer of anti-tumor activity.