Figure 3.

PGC-1α Is Required for Optimal NK Cell Bioenergetics In Vitro

(A and B) IL-2-cultured NK cells from WT and Ppargc1a cKO mice were washed of IL-2 and seeded onto V3 PET Cell Culture Microplates in unbuffered RPMI, and oxygen consumption rate (OCR) was determined using a Seahorse XF-96 Extracellular Flux Analyzer. Basal OCR was determined, and oligomycin injection was used to quantify ATP-linked OCR followed by FCCP injection to quantify maximal OCR. Basal and maximal glycolytic rates were also obtained from the same set of cells by measuring extracellular acidification rate before and after oligomycin injection. A representative OCR plot is shown followed by quantification over multiple experiments (n = 7 individual mice for each genotype from 4 independent experiments).

(C) IL-2-cultured NK cells from WT and Ppargc1a cKO mice were seeded in control, or anti-NKG2D-coated wells for 6 h. Cells were removed from plates and lysed in potassium phosphate (oxidized metabolites) or potassium hydroxide (reduced metabolites) solutions to collect metabolites. High-performance liquid chromatography was used to quantify levels of ADP, ATP, NAD, and NADH (n = 3 individual mice for each genotype).

(D) IL-2-cultured NK cells from WT and Ppargc1a cKO mice were washed of IL-2 and stained with NK cell markers (NK1.1/CD3ϵ) along with the MitoTracker Green FM dye to measure mitochondrial mass. Data representative of individual mice are shown as histograms with mitochondrial mass on the x axis. Compiled data from multiple animals are presented as well (n = 3 individual mice for each genotype).

Bar graphs present the mean ± SD or mean ± SEM where multiple experiments are indicated. Statistical significance was calculated using two-way ANOVA corrected for multiple comparisons using the Holm-Sidak method (A–C), whereas Student's t test corrected for multiple comparisons was used to determine significance of individual metabolic parameters (A,B). ∗p < 0.05; ∗∗∗p < 0.001.