NK cells show increased lipid accumulation and reduced mitochondrial metabolism in the lymphoma environment. (A) Splenocytes from healthy and Eµ-myc lymphoma-bearing mice were harvested at 14 to 21 days after inoculation, and neutral lipid levels were measured (n = 8). (B) Neutral lipid levels of NK cells derived from PBMC of healthy donors or DLBCL patients were examined (n = 13). (C-D) Mitochondrial mass (MT DeepRed FM) on splenic NK cells derived from healthy and Eµ-myc lymphoma-bearing mice were examined at days 5, 14, and 21 after lymphoma inoculation by (C) flow cytometry or at day 14. Representative flow cytometric histograms were obtained at day 14 (n = 8). (D) Splenic NK cells derived from healthy and Eµ-myc lymphoma bearing mice were examined at day 14 using imaging cytometry (NK1.1⁺ TCRβ⁻) (bright field [B.F.], NK1.1, MT DeepRed FM, merged). The images show 1 representative cell from 5 mice/group and negative controls (MT -ve). The graph shows geometric mean intensity of MT DeepRed FM derived from the imaging cytometry (n = 5). (E) Mitochondrial membrane potential on splenic NK cells were examined as in panel C (n = 8). Phosphorylation of (F) AKT and (G) mTOR of splenic NK cells was assessed by flow cytometry (n = 4). (H) Splenocytes were harvested from healthy and Eµ-myc mice at days 5 and 14 after lymphoma inoculation, then stimulated with IL-15 + IL-12/18 for 4 hours in vitro, before phosphorylation of S6 of splenic NK cells was assessed by flow cytometry (n = 8). (I) IL-12 and IL-18 were administered to healthy and Eµ-myc mice on day 17 after lymphoma inoculation and phosphorylated S6 of splenic NK cells was analyzed 20 hours later by flow cytometry (n = 5). Data show mean ± SEM; statistical significance (*P < .05; **P < .01; ***P < .001; ****P < .0001; NS, not significant) was determined by Mann-Whitney test (A-B,D) or 2-way ANOVA followed by Sidak test (C,E-I).