mTORC1 controls glucose uptake and glycolysis via HIF1. (A–D) Immunoblot analysis of HIF1α and HIF1β expression in naive P14-LCMV CD8⁺ T cells ± TCR (gp33-41/anti-CD28) stimulation for 20 h (A and C) and P14-LCMV CTLs (A, B, and D) treated with and without IL-2 (B) or rapamycin (C and D) for 20 h. Phospho-S6K1 and phospho-S6 were used as a measure of mTORC1 activity. (E) Immunoblot analysis of c-myc expression in CTLs treated with or without rapamycin for 20 h. Phospho-S6K1 and phospho-S6 were used as a measure of mTORC1 activity. (F) Flow cytometric analysis of HIF1β^WT/WT CD4Cre (WT) and HIF1β^flox/flox CD4Cre (HIF1^−/−) CD8⁺ T cells after TCR (2c11) stimulation for 20 h. (G) Immunoblot analysis of WT and HIF1^−/− CTLs. (H and I) Analysis of glucose uptake (H) and lactate production (I) in WT and HIF1^−/− naive CD8⁺ T cells after TCR (2c11/anti-CD28) stimulation for 20 h. Glucose uptake in unstimulated WT naive T cells is also shown (uptake in unstimulated HIF1^−/− naive T cells is equivalent to WT; not depicted). (J–L) Analysis of glucose uptake (J), Glut1 expression (K), and lactate production (L) in WT versus HIF1^−/− CTLs. (M) A comparison of the transcriptional profile of HIF1 WT versus HIF1^−/− CTLs was performed by microarray. Shown here are KEGG pathway analysis of genes down-regulated in HIF1^−/− CTLs (top) and a heat map of the relative normalized expression of selected genes that are significantly different in expression in WT versus HIF1^−/− CTLs, as determined by microarray. For all panels, data are mean ± SEM or representative of at least three experiments. All metabolic assays were preformed in triplicate (**, P < 0.01; ***, P < 0.001). Molecular mass is indicated in kilodaltons. Dotted lines indicate that intervening lanes have been spliced out.