Figure 4.
Pten deficiency promotes IL-2 production in Th17 cells. (A) Il2 mRNA levels at days 1, 2, and 3 of culture were measured by qRT-PCR (left). Il2 mRNA levels at day 2 are presented separately (right). Naive CD4+ T cells from WT (Ptenfl/fl; black line) and cKO (Ptenfl/flIl17acre; gray line) mice were cultured under Th17-polarizing conditions. Data were normalized to Gapdh expression. (B and C) IL-2 was measured by flow cytometry (B). Cells from WT (red line) and cKO (blue line) mice were cultured under Th17-polarizing conditions for 3 d. (C) IL-2 cytokine levels in the supernatant of cultured cells were measured by ELISA. Cells were cultured under the same conditions as in (B). (D) Il2 promoter activity was measured in a dual luciferase assay. EL4 cells were transfected with varying concentrations of the pGL3-il2 promoter vector and PTEN siRNA. Relative luciferase activity was calculated by dividing Firefly luciferase activity by Renilla luciferase activity. Cont, control siRNA. (E and F) IL-17A and FOXP3 expression was measured by flow cytometry (E), and Il17a, Rorc, Il23r, and Foxp3 mRNA levels were measured by qRT-PCR (F). Data were pooled from three individual experiments (E, right). Naive CD4+ T cells from WT and cKO mice were cultured under Th17-polarizing conditions in the presence of an anti–IL-2 antibody (S4B6). Dot plot data represent three independent experiments, and data in A–D and F were pooled from three independent experiments. Error bars represent the SD. Statistical differences between groups were determined by the Student t test. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. n.s., not significant.