Metabolic Tracing Reveals IL-2 Driven Glutaminolysis and De Novo Pyrimidine Synthesis in Human Natural Killer Cells

Natural Killer (NK) cells are innate lymphocytes that are key to intrinsic cancer immunosurveillance and an important target for cancer immunotherapy. Understanding fundamental human NK cell metabolism provides opportunities for optimizing NK cell therapies. Little is known about how glutamine, an important cell nutrient and carbon source, is utilized by human NK cells. To address this, we performed U13C-glutamine tracing experiments by Liquid Chromatography Mass Spectrometry and Gas Chromatography Mass Spectrometry analysis of human NK cells stimulated with IL-2 for 18 h to provide a global overview of glutamine usage by these cells. Our results show that glutamine is taken up by resting NK cells and that this increases further upon IL-2 stimulation. Metabolite labelling analysis identified that IL-2 stimulation results in greater conversion of glutamine to glutamate, allowing for anaplerotic flux into the TCA cycle. The fate of the glutamine-derived carbons diverged at oxaloacetate allowing both bioenergetic and biosynthetic outcomes - some carbons continued around the TCA cycle while others were exported, converted to aspartate and subsequently used for pyrimidine synthesis. Nucleotide synthesis by IL-2 activated NK cells was found to be essential for expression of the activation marker CD69. The data indicate that glutamine is a key nutrient taken up by human NK cells, and that IL-2 drives glutaminolysis. Subsequent glutamate is used to support the TCA cycle, generating energy and providing intermediates for de novo pyrimidine synthesis.