Figure 5.

Cdkn2a is required for IL‐10 expression only in CD8⁺ but not CD4⁺ T cells, macrophages, and DCs. (A) Naïve CD8⁺ T cells from B6 WT (black bar) and Cdkn2a^−/− (white bar) mice were sorted and stimulated with immobilized anti‐CD3 (10 μg/ml) and soluble anti‐CD28 (1 μg/ml) in the presence of IL‐2 and IL‐4 for 48 h, expanded 2 days. Cells were then restimulated by PMA and ionomycin for 6 h and then analyzed IL‐10 expression by real‐time PCR. Data are representative of three independent experiments (mean+sem; **P<0.01). (B) Supernatants from the parallel, naïve CD8⁺ T cell, cultured in A at 96 h (“−”, no restimulation) and 6 h after restimulation (“+”), were collected for IL‐10 detection by ELISA. Results from three independent experiments were combined and presented (mean+sem; ***P<0.001). (C) Naïve CD4⁺ T cells were sorted from 10BiT and Cdkn2a^−/−.10BiT mice, cultured under iTreg conditions or Th2 conditions, as described in Materials and Methods. Cells were then analyzed by flow cytometry. Data were representative of three independent experiments. (D) Supernatants from iTreg and Th2 cultures of C were collected and used for IL‐10 detection by ELISA. Data (mean+sem) from three combined experiments were shown. No statistical difference was observed. (E) Splenocytes from 10BiT mice or Cdkn2a^−/−.10BiT mice cultured with LPS (1 μg/ml) for 24 h. Cells were then analyzed by flow cytometry. Data are representative of three independent experiments.