Critical role of Notch-Hes1 pathway for the development of IL-17–producing γδ T cells. (A) Notch1 expression of γδ T cells in the thymus. Sorted CD27^high and CD27^low γδ T cells from E18 fetal (left) or adult (right) thymi of CβKO mice (6 weeks old) were analyzed for the relative expression of Notch1. Notch1 expression of CD27⁺ γδ T cells was set to 1. (B-D) Dll4-mediated Notch signaling induces the development of IL-17–producing γδ T cells. Three thousand fetal thymocytes (E15) were cocultured with TSt-4/no or TSt-4/Dll4 stromal cells. (B) Hes1 expression was analyzed by qRT-PCR 14 days after the culture. Data shown are the means ± SD of 4 individual wells. *P < .05. (C-D) After coculture with TSt-4/no or TSt-4/Dll4 cells, IL-17 production by γδ T cells was analyzed after stimulation with PMA and ionomycin (C). Representative dot plots are shown after gating on CD45.2⁺ cells. The number in top right or bottom right quadrant indicates γδTCR⁺IL-17⁺ or γδTCR⁺IL-17⁻ cells in CD45.2⁺ cells, respectively. The number in the parentheses shows the percentage of IL-17⁺ cells in γδ TCR⁺ cells. (D) Kinetics of percentages (left) or absolute numbers (right) of IL-17–producing γδ T cells are shown at the indicated days. Data shown are the means ± SD of 4 individual wells at each time point. *P < .05; **P < .01. Data are representative of 3 independent experiments.