Hes1 is specifically expressed in IL-17–producing γδ T cells. (A) Hes1 is specifically expressed in IL-17–producing hybridoma clones. (A) mRNA expressions of Hes1, Hes5, and RORγt in parental hybridomas (no), IL-17–producing (IL-17⁺), and nonproducing (IL-17⁻) γδ TCR⁺ hybridoma clones were analyzed by qRT-PCR. (B-K) Hes1 is specifically expressed in IL-17–producing γδ T cells. (B,D,F,H) Purified γδ T cells from IELs (B), PECs (D), spleens (F), and thymi (8 weeks old; H) were stimulated with PMA and ionomycin and analyzed for intracellular IL-17. Representative dot plots are shown after gating on lymphocytes identified by FSC/SSC profile (B) or γδ TCR⁺ cells (D,F,H). The numbers in the respective quadrants indicate the percentages of positive cells. (C,E,G,I) Purified γδ T cells from IELs (6 weeks old; C) or sorted CD25^high, CD25^low, CD27^high, and CD27^low γδ T cells from PECs (E), spleens (G), and thymi (I) of CβKO mice (6 weeks old) were analyzed for the relative expression of Hes1. Hes1 expression of s-IELs (C), CD25⁻ γδ T cells (E), or CD27⁺ γδ T cells (G,I) was set to 1. (J) Fetal thymocytes (E18) of IL-17–GFP reporter mice were analyzed after stimulation with PMA and ionomycin for 4 hours. The number in the top right quadrant indicates the percentage of IL-17⁺ cells in γδ TCR⁺ cells. (K) After sorting of GFP⁺ and GFP⁻ cells, Hes1 mRNA expression was analyzed by qRT-PCR. Hes1 expression of GFP- γδ T cells was set to 1. Data are representative of 3 independent experiments.