Fig. 7

Reduced metabolic switch in Nfatc1 ^−/− aCD8⁺ T cells. a Heat map of RNA expression from genes encoding enzymes of glycolysis in Nfatc1 ^−/− (Nfatc1 ^f/f x CD4-cre mice) and Nfatc2 ^−/− aCD8⁺ Ts. b Real-time PCRs of Slc2a1, Slc2a3 and Hk2 RNAs encoding the glucose transporters Glut1 and Glut3, and hexokinase 2, respectively, normalized by Actb and relative to naïve CD8⁺ cells. c Incorporation of 2-NBDG into aCD8⁺ Ts and CTLs upon incubation for 1 h at 37 °C. Data from 4 assays were compiled. d Extracellular flux analysis. Above, 4 × 10⁵ WT and Nfatc1 ^−/− CD8⁺ T cells were stimulated overnight with “low” concentrations of plate-bound αCD3 (0.05 μg/ml) and αCD28 (2 μg/ml), and subjected to extracellular flux analysis. Below, cells activated with “high” (standard) αCD3/CD28 concentrations (5/2 μg/ml) were assayed. e Mito stress test of aCD8⁺Ts treated as in d. f Below, incorporation of 2-NBDG (above) and extracellular flux analysis (below) of aCD8⁺ Ts from c.n.Nfatc1/αA x Nfatc1 ^f/f x dlck-cre mice. Above, immune blots showing the lack of NFATc1 expression in cytoplasmic (CP) and nuclear (N) proteins of aCD8⁺ Ts from Nfatc1 ^f/f x dlck-cre mice (lanes 3 and 4), and the expression of c.n.NFATc1/αA (point) in cells from c.n.Nfatc1/αA x Nfatc1 ^f/f x dlck-cre mice (lanes 5 and 6). In lanes 1 and 2, proteins from WT cells were fractionated. Right, RT-PCRs showing Il2 and Myc RNA levels in aCD8⁺Ts from WT, Nfatc1 ^f/f x dlck-cre and c.n.Nfatc1/αA x Nfatc1 ^f/f x dlck-cre mice. Data of 5 PCR assays are shown, relative to naïve WT CD8⁺ T cells and normalized to Actb. Data are shown as means ± SEM. Unpaired Student’s t-test was used for statistics. g Extracellular flux analysis of CD8⁺ T cells from WT and Nfatc1 ^f/f x CD4-cre mice stimulated by αCD3/CD28 (5/2 μg/ml) without (above) or with 100 U/ml hIL-2 (below) for 3 d. All assays were performed three times or more