Steady-state CD56^DimNK cells have latent mitochondrial activity and undergo a metabolic switch upon cytokine stimulation. (A-C) Metabolic function analyzed by extracellular flux analysis (EFA) in freshly fluorescence-activated cell-sorted (FACS) NKBr and NKDim cells. Cells were sequentially treated with glucose medium (medium), oligomycin A (Oligo), FCCP, and RO plus AA as indicated. (A) ECARs and maximal glycolysis (average values after glucose injection minus average basal ECAR values). (B) OCR plot. Calculation of the maximal respiration (average maximal OCR after FCCP) and basal respiration (average basal respiration after medium injection). (C) Mitochondrial spare respiratory capacity (SRC; average maximal OCR values after FCCP injection minus baseline OCR; data are representative of 3 independent experiments with 2 or 3 donors each). (D) Average fold increase comparing NKBr and NKDim cells is shown for all genes in OXPHOS complexes. Each dot represents the median relative expression level across donors. (E-G) EFA analysis in FACS NKBr and NKDim cells cultured for 18 hours in presence of IL-15 ± IL-12/18. Cells were sequentially treated as indicated above. ECAR and maximal glycolysis (E), OCR, maximal, and basal respiration (F), and SRC (G) (data are representative of 3 independent experiments; data are mean ± SEM). P > .05 was considered not significant (ns; or not indicated). *P < .05, **P < .01, ***P < .001 using 1-way analysis of variance with Tukey’s correction.