IL-21-Enhanced NK cells: A mitochondrial fitness enhancement for targeting glioblastoma stem cells

Glioblastoma (GBM), an aggressive brain cancer, recurs due to the resistance of glioblastoma stem cells (GSCs) to conventional therapies. We recently showed that cord blood-derived NK cells (CBNK) armed with cytokines like IL-21 exhibit enhanced anti-tumor activity against GSCs, showing superior killing capacity after multiple re-challenges. IL-21 transduction enhances NK cell diversity, polyfunctionality, and mitochondrial fitness, improving tumor control and survival in an in vivo glioblastoma model without toxicity. A deeper dive into the cellular mechanisms revealed that IL-21 transduction triggers significant changes in the metabolic and mitochondrial profiles of NK cells. RNA sequencing revealed that IL-21 transduction upregulates genes involved in metabolic reprogramming, mitophagy, and mitochondrial health, including KLF2 , GZMH , CLIC3 , and CEBPD . Further investigations into mitochondrial function confirmed improved mitochondrial respiration (OCR) and reduced glycolytic activity (ECAR) in IL-21 CBNK cells, indicating enhanced mitochondrial fitness. Confocal microscopy revealed that IL-21 CBNK cells had smaller mitochondria, a hallmark of mitophagy, which was significantly different from other cytokine-transduced or non-transduced CBNK cells. Furthermore, mitochondrial-related proteins, including Mitofusin-2 and DRP1, were upregulated in IL-21 NK cells, confirming improved mitochondrial dynamics. Inhibition of mitophagy reversed the enhanced killing ability of IL-21 transduced CBNK cells. Seahorse Mito Fuel tests identified glucose and fatty acids as the primary fuel sources for IL-21 CBNK mitochondria. Blocking these fuels impaired their anti-tumor activity, further supporting the role of mitochondrial metabolism in IL-21-driven NK cell fitness. Additionally, CEBPD, a key transcription factor involved in mitophagy and mitochondrial metabolism, was upregulated in IL-21 transduced CBNK cells, further enhancing their mitochondrial function and anti-tumor efficacy. These results suggest that IL-21 enhances NK cell anti-tumor potency through metabolic reprogramming, mitochondrial fitness, and CEBPD-driven mitophagy, offering a promising therapeutic strategy for glioblastoma.