Fig. 6.

The effect of TNFα on NK cells from mice deficient in either TNFR1 or TNFR2. A NK cells were enriched from the spleens of the naive indicated mice and stained with cell trace violet dye followed by stimulation with the indicated doses of TNFα ex vivo for 3 days, and the percentage of dividing NK cells was analyzed. 100 U/ml rhIL-2 was added to maintain NK cell survival. B–E Enriched splenic NK cells from the indicated mice were stimulated with TNFα (30 ng/ml) ex vivo for 48 h in the presence of 100 U/ml rhIL-2. B The proportion of CD25⁺ cells among the total NK cell population of cytokine-stimulated cells. The graphs indicate the MFI of (C) pNF-κB-p65, (D) glucose uptake as measured by 2-NBDG and (E) representative plots of CD71 and CD98 expression on cytokine-stimulated cells. F Freshly isolated NK cells from the spleens of the naive indicated mice were flow-sorted followed by stimulation with TNFα ex vivo in the presence of 100 U/ml rhIL-2, and a glycolysis stress test was performed in glucose-free medium to analyze the relative extracellular acidification rate. G Cells were prepared as in (B), and the proportion of pS6⁺ cells among the total NK cell population was calculated. Enriched NK cells from the spleens of the indicated naive mice were stimulated ex vivo with IL-18 in the presence or absence of TNFα (30 ng/ml) for 48 h. 100 U/ml rhIL-2 was added to maintain NK cell survival. Graphs demonstrate (H) the proportion of CD25⁺ cells among the total NK cell population and the MFI of (I) CD71 and (J) CD69 expression on cytokine-stimulated NK cells. The MFI expression is presented as a percentage relative to the MFI of control cells as 100. Data are from one experiment representative of at least two independent experiments, with at least three replicates per group. Data represent the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns nonsignificant