Th9 development and expression of the Th9 transcription factors PU.1 and IRF4. Naive cord blood CD4 T cells or adult naive and memory CD4 T cells were stimulated with anti-CD3/28 in the presence of the indicated cytokines. (A-B) Expression of PU.1 and IRF4 mRNA was assessed after the additional stimulation of cells with anti-CD3 for 24 hours; mRNA levels were normalized to cyclophilin and are presented relative to expression in cells differentiated in the absence of any cytokines. (C) IL-9 mRNA was assessed by real-time PCR and normalized for the expression of cyclophilin. (D) Frequencies of IL-9–producing cells were assessed by flow-cytometric analysis after 5 days of culture and restimulation with phorbol myristate acetate and ionomycin. The mean ± SEM of at least 3 independent experiments with cells from different donors is shown. *P < .05; **P < .01; ***P < .001 by ANOVA compared with medium control without any cytokine.