Fig. 1
Tpath2 cells show limited IL-33-induced production of IL-5. To compare Tpath2 cells and ILC2s, both cell subsets were prepared from the spleen of memory Th2 mice (see Supplementary Fig. 1a and Methods section). Cells were cultured with IL-7 in complete medium for 1 day after cell sorting, then stimulated with indicated stimuli for 6 h. a Intracellular-staining profiles of IL-5 and IL-13 in Tpath2 cells and ILC2s are shown. The cells were stimulated with indicated stimuli for 6 h. The percentages of IL-5 positive ILC2s and Tpath2 cells are shown (mean ± SD; n = 4 for each group). b Quantitative RT-PCR analysis of the indicated cytokines in Tpath2 cells and ILC2s. The cells were stimulated with indicated stimuli for 4 h. Relative expression (normalized to Hprt) is shown with standard deviation. c ELISA analysis of the indicated cytokines in the culture supernatant from 5 × 104 cells of Tpath2 cells and ILC2s, respectively. The cells were stimulated with indicated stimuli for 12 h. d Intracellular-staining profiles of IL-5 and IL-13 in ST2hi MPCD4 T cells and ILC2s purified from normal BALB/c mice are shown and stimulated with the indicated stimuli for 6 h. The percentages of IL-5 positive ILC2s and ST2hi MPCD4 T cells are shown (mean ± SD; n = 4 for each group). e Expression profiles of ST2 on Tpath2 cells and ILC2s before (immediately after sorting) and after cultivation with IL-7, IL-25 and IL-33 are shown. The number in the histogram represents mean fluorescence intensity (MFI). Gray filled histogram shows isotype control staining. f ChIP assays were performed with anti-trimethyl histone H3-K4 and anti-trimethyl histone H3K27 at the Th2 cytokine gene loci from Tpath2 cells, ILC2s, and effector Th1 cells. The relative intensities (relative to input DNA) of these modifications were determined by quantitative RT-PCR analysis. More than five for a to e, and three for f independent experiments were performed and showed similar results (**p < 0.01; n.s. not significant, n.d. not detected; Mann–Whitney U test; a–c). Three technical replicates were performed with quantitative RT-PCR (b, f) and ELISA (c)
