Figure 5.
Cytokine production by NKT cell subsets after α-GalCer stimulation. WT mice received 2 μg α-GalCer or vehicle by way of i.p. injection and were killed 2 or 48 h later (A). Liver- and spleen-derived lymphocytes were cultured without further stimulation for 2 h in Brefeldin A. After culture, cells were labeled with surface mAb, fixed, and permeabilized for intracellular cytokine staining. Cells were analyzed by flow cytometry, and CD1d/ α-GalCer+αβTCR+ cells were gated on and CD4+ and CD4− subsets screened for intracellular IFN-γ production. Gates were set based on the isotype control at each time point. (B) Liver lymphocytes and NKT cell–enriched thymocytes (with sorted spleen-derived DCs) were cultured for 12 h with 100 ng/ml α-GalCer. Brefeldin A was added for the final 2 h of culture. After culture, cells were prepared for intracellular cytokine staining (as for A). NK1.1+αβTCR+ cells from the liver and NK1.1+ cells from the thymus were electronically gated and CD4+ and CD4− subsets were analyzed for intracellular IFN-γ production (filled line graph: DCs with α-GalCer; dotted line: nonstimulated control).