Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes
Figure 9
Anti-CD3 treatment enhances glucose intracellular transportation by the activated lymphocytes. (a) Freshly prepared spleen cells (1 × 107/well) were cultured in a 24-well plate in 1 mL medium containing 10% fetal bovine serum with glucose concentration of 360 mg/dL. Anti-CD3 antibody (3 g/mL) or isotype IgG (3 g/mL) was added to the cultures. The culture wells were quadruplicated and incubated for 24 h. Glucose concentrations were measured before and 24 h after incubation. The results were reproduced in additional two independent experiments. (b) Freshly prepared spleen cells were stimulated with anti-CD3 antibody (3 g/mL) or isotype IgG (3 g/mL) for 3 hours; thereafter, different concentrations of 2-NBDG as indicated were added to the cultures for additional 2 hours. The fluorescent intensity of the cells was examined by BioTec plate reader using a laser filter with 480 nm excitation and 528 nm emission. The values of between anti-CD3 and isotype IgG were compared for each concentration of 2-NBDG (). The similar results were reproduced in additional 3 independently experiments. (c) Spleen cells were stimulated with anti-CD3 antibodies as mentioned above for 3 hours, and then 100 M of 2-NBDG was added to the cultures for additional 2 hours. Thereafter, the cells were harvested and stained with CD5-PerCp, B220-PE, and CD69-PE-Cy7. 2-NBDG+ cells in CD69− and CD69+ T cells and B cells were analyzed through gating CD5+ and B220+ cells, respectively. The percentages of 2-NBDG+CD69− and 2-NBDG+CD69+ T or B cells were compared, respectively (). The data shown were obtained from a representative of three experiments. (d) B6 mice were treated with anti-CD3 (50 g/mouse) or isotype IgG (50 g/mouse). Three mice were included in each group. Six hours later, the spleen cells were prepared and Glut1 expression was examined by real-time PCR. The values were normalized by housekeeping gene actin, and the expression of Glut1 for isotype IgG treated mice was defined as 1. Data shown were obtained from 3 mice in each group.
