PU.1 expression and Th9-cell development in CD31⁺ RTEs. (A) Naive CD4 T cells were purified from cord blood or from peripheral blood of adult subjects and stained for CD31. Adult naive CD4 T cells were sorted into CD31⁺ and CD31⁻ subpopulations. (B) CD31⁺ and CD31⁻ cells were processed for ChIP analysis with anti-H3K4me3, anti-H3ac, or anti-H3K27me3 Abs. Eluted DNA was analyzed by quantitative PCR using primers spanning the promoter region of PU.1, T-bet, GATA-3, IRF4, and RORC. The Log₂ of the ratio of relative amounts of DNA precipitated with anti-H3K4me3 to anti-H3K27me3 (top panel) and with anti-H3ac to anti-H3K27me3 (bottom panel) are shown. (C) Cells were stimulated with anti-CD3/28 in the presence of the indicated cytokines. Expression of PU.1 mRNA was assessed after additional stimulation of cells with anti-CD3 for 24 hours. mRNA levels were normalized to cyclophilin and are presented relative to expression in CD31⁺ cells differentiated in the presence of TGF-β only. Frequencies of IL-9–producing cells were assessed by flow cytometry 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 demonstrated. *P < .05; **P < .01; n.s. indicates not significant by Student t test.