Analysis of cytokine profiles of Th9 cells developed in response to TGF-β, IL-4, IL-10, and IL-1β. Neonatal naive CD4 T cells from cord blood or adult peripheral blood naive and memory CD4 T cells were stimulated with anti-CD3/28 in the presence of TGF-β, IL-4, IL-10, and IL-1β for 5 days. (A) Cytoplasmic IL-4, IL-5, IFN-γ, and IL-17 were assessed by intracellular flow cytometry. Numbers indicate the percentage of cells in the respective quadrants within viable lymphocytes. Findings from 1 of 4 independent experiments with cells from different donors are shown. (B) Flow cytometric analysis of cytoplasmic IL-9 and nuclear T-bet or GATA-3 expression. Findings from 1 of 3 independent experiments with cells from different donors are shown. (C) Quantitative PCR analysis of RORC gene expression; results were normalized to cyclophilin. The mean ± SEM of 3 independent experiments with cells from different donors is demonstrated.