mTORC1 regulates glucose uptake and glycolysis in TCR-stimulated CD8⁺ T cells. (A) Immunoblot analysis of Glut1 expression in naive OTI CD8⁺ T cells ± TCR (SIINFEKL) stimulation for 20 h. (B and C) Naive P14-LCMV CD8⁺ T cells ± TCR (gp33-41/anti-CD28) stimulation were assayed for glucose uptake (B) and lactate production (C). (D) Immunoblot analysis of naive OTI CD8⁺ T cells ± TCR (SIINFEKL) stimulation for 20 h. mTORC1 activity was determined by analyzing the phosphorylation of target sequences on S6K1 (T389 and S241/242) and phosphorylation of the S6K1 substrate S6 ribosomal protein. PTEN was used as a loading control. (E–G) Immunoblot analysis (E) and analysis of glucose uptake (F) and lactate production (G) for naive P14-LCMV CD8⁺ T cells ± TCR (gp33-41/anti-CD28) stimulation with or without rapamycin for 20 h. 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.