Analysis of NKT cells derived from NKT-ES cells. (A) FACS profiles of in vitro–generated NKT cells. Cells developed from NKT-ES cells on day 20 of culture with OP9/Dll-1 (Th2-like NKT) or by the switch culture using OP9/control (Th1-like NKT) were gated on the α-GalCer/CD1d dimer⁺ TCRβ⁺ fraction and further analyzed for the expression of the indicated markers, NK1.1 versus CD3ϵ and CD4 versus CD8. (B) Surface phenotypes of in vitro–generated NKT cells. Gated fractions shown in panel A were further analyzed for the expression of the indicated markers. Shadowed profiles indicate isotype-matched control staining. (C) Proliferation and cytokine production of in vitro–generated NKT cells upon stimulation with α-GalCer. In vitro–generated NKT cells (10⁶/mL) were cocultured with bone marrow–derived dendritic cells (10⁵/mL) in the presence of the indicated dose of α-GalCer (100 ng/mL). ND indicates not detected. Average of triplicate and SD is shown. One representative of 3 experiments is shown.