Figure 2.

Glucose is essential for enhancing the proliferation of CD8⁺ T cells by 4-1BB signals. CFSE-labeled CD8⁺ T cells were activated with 0.1 μg/ml anti-CD3 mAb for 16 h. Then the activated CD8⁺ T cells were pre-incubated with the indicated concentration of 2-DG for 1 h and further treated with 5 μg/ml anti-4-1BB mAb for 2 days. (a) The CD8⁺ T cells were stained with anti-CD8-PE-Cy5 and CFSE dilution of CD8⁺ T cells were analyzed by FACSCalibur. (b) Proliferation index was calculated using Modfit LT software (Verity Software House, Topsham, ME, USA) from a. (c) Medium concentrations of lactic acid 48 h after rat IgG or anti-4-1BB treatment. (d–f) Anti-CD3-activated CD8⁺ T cells were cultured in the presence of anti-4-1BB mAb and harvested at the indicated time points for total RNA or protein extraction. First-strand cDNA was synthesized from the total RNA. Transcript levels of GLUT1, HK2 and GAPDH were assessed using specific primer sets (d). Relative expression levels were calculated by dividing the densitometer reading of each band by the value of GAPDH (e). Western blots performed with antibodies specific for GLUT1 and β-actin (f). Naïve indicates freshly isolated CD8⁺ T cells. Data are representative of three independent experiments. Results in b are means±SDs (*P<0.05; **P<0.01). Abbreviation: 2-DG, 2-deoxy-D-glucose.