Functional properties of Vα24⁺ iNKT cells expanded by distinct subsets of DCs. PBMCs were stimulated with α-GalCer–loaded, syngeneic DCs (MoDCs, mDCs, or tolDCs) for 7 days and enriched for Vα24-positive fractions by magnetic bead separation. Functional assays were performed using the enriched Vα24⁺ iNKT cells, which were designated moDC-NKT, mDC-NKT, and tolDC-NKT cells. (A) Proliferation assay. Proliferation of Vα24⁺ iNKT cells, restimulated by MoDCs with or without α-GalCer for 48 hours, was assessed by [³H]TdR uptake. The results shown represent one of 3 experiments. (B) Cytolysis assay. The enriched Vα24⁺ iNKT cells were tested for cytotoxicity against U937 cells in a standard 6-hour ⁵¹Cr release assay. The data are representative of the results of 2 separate experiments. (C) Cytokine production assay. Culture supernatants from Vα24⁺ iNKT cells stimulated by syngeneic MoDCs loaded with α-GalCer were assayed for IFN-γ, TNF-α, IL-4, and IL-10. The results shown represent 1 of 3 experiments. (D) Intracellular cytokine production assay. IL-10– or IFN-γ–producing Vα24⁺ iNKT cells stimulated by syngeneic MoDCs loaded with α-GalCer were assayed by FACS analysis. Similar results were obtained in 2 additional experiments. (E) Intracellular cytokine production assay. IL-10-producing Vα24⁺ iNKT cells stimulated by syngeneic MoDCs loaded with α-GalCer in the presence of anti-IL-10 mAb were assayed by FACS analysis.