Figure 1.

PU.1 is required for optimal IL-9 production in murine T cells. Wild-type and Sfpi1^lck−/− naïve CD4⁺ T cells were cultured under T_H9 conditions. (a) After five days of culture, cells were stimulated with anti-CD3 and supernatants after 1, 2 or 3 days of stimulation were tested for IL-9 using ELISA. (b) RNA was isolated from wild-type and Sfpi1^lck−/− T_H2 and T_H9 cultures after stimulation with anti-CD3. Il9 mRNA was assessed using qPCR. (c) Wild-type and Sfpi1^lck−/– T_H9 cultures were stimulated with PMA-ionomycin for 5 h before intracellular staining for IL-9. Results in (a-c) are the average of 3 mice and representative of more than four experiments. (d) Naïve CD4⁺ T cells cultured under T_H1, T_H2, T_H9 or T_H17 conditions were analyzed for Sfpi1 expression using qPCR. Results are an average of 3-5 experiments. (e) Naïve CD4⁺ T cells were cultured under T_H9 or iTreg conditions for five days and stimulated with anti-CD3 for 6 h before intracellular staining for IL-9 and Foxp3. (f,g) Naïve CD4⁺ T cells were cultured under T_H9 or T_H17 conditions for five days and stimulated with (f) PMA-ionomycin for 6 h before intracellular staining for IL-9 and IL-17 or (g) anti-CD3 for 24 h before supernatants were collected for analysis using ELISA. Results in (e-g) are representative of at least three experiments. (h) T_H2 cultures were separated into IL-4^lo and IL-4^hi populations using magnetic selection. RNA was isolated from separated cells to examine expression of Il4, Il9 and Sfpi1. Results are an average of three experiments. *, P < 0.05 using two-tailed student t test.