Human CD4+ iNKT cells have highly oxidative metabolism and use glycogen reserves to sustain IFN-γ production in nutrient-limited environments.

Invariant natural killer T (iNKT) cells are promising candidates for allogeneic cellular immunotherapy, but the metabolic pathways that support their effector function in nutrient-limited tumor microenvironments remain poorly defined. Human iNKT cells segregate into CD4+ and CD4⁻CD8⁻ double-negative (DN) subsets with distinct functional profiles, yet whether they use divergent metabolic strategies to sustain IFN-γ production is unknown. Here we show that in vitro-expanded human CD4+ and DN iNKT cells employ distinct metabolic programs that differentially support IFN-γ secretion under nutrient stress. DN iNKT cells exhibit higher Glut1 expression and a more glycolytic phenotype, with IFN-γ production that is sensitive to extracellular glucose withdrawal and glycolytic inhibition. In contrast, CD4+ iNKT cells display high spare respiratory capacity, preferentially engage glutamine-supported mitochondrial respiration, and maintain IFN-γ production despite glucose deprivation or 2-DG inhibitor treatment. CD4+ iNKT cells generated excess ATP through oxidative metabolism, accumulated intracellular glycogen stores during expansion in vitro, and subsequently mobilized this glycogen to support IFN-γ production in tumor-exhausted media. Using a xenograft model of EBV-driven B cell lymphoma, we show that adoptively transferred human CD4+ iNKT cells infiltrate tumors in vivo and are associated with elevated intratumoral IFN-γ. These findings identify a distinctive combination of high mitochondrial oxidative capacity, glutamine utilization, and glycogen storage that endows human CD4+ iNKT cells with exceptional metabolic resilience, suggesting that CD4+ iNKT-based products may provide a particularly valuable platform for adoptive cellular immunotherapy.