IL-9 production is inhibited by GTIs. (A) Th9 cells were cultured for five days, harvested and stimulated with media, or PMA/ionomycin (PI) in the presence or absence of brefeldin A (BFA) or monensin (Mon) for 5.5 hours. Representative contour plots are shown for intracellular IL-2, TNF and IL-9 staining. (B) The frequency of intracellular IL-2+, TNF+, and IL-9+ cells at day 5 of culture. (C) Il2 and Il9 mRNA levels of day 5 Th9 cells after stimulation with media, PI, or PI + monensin (PIM). These data represent >3 individual experiments and error bars represent standard deviation of at least 3 animals per group. *, p<0.05, reduced as compared to PIM stimulated controls. #, p<0.05 increased as compared to PIM-treated controls. (D) Intracellular IL-9 and IL-10 in viable, pulmonary CD3+ CD4+ T cells from mice chronically exposed to Aspergillus fumigatus extract for 6 weeks. Purified CD4 T cells were stimulated with PI and monensin added at 0, 4, or 6 hours of stimulation. These studies represent 2 individual experiments with 4-5 mice per group. *, p<0.05 greater as compared to 0 hour monensin-treated cells.