Injection of iNKT cell ligands induces maturation of splenic DCs, increasing their immunostimulatory capacity. (A) Animals were treated i.v. with 1 μg of α-GalCer or its analog OCH, or with 25 μg of the TLR4 ligand MPL, and surface expression of CD86 was assessed on splenic CD11c⁺ cells 24 hours later relative to that of cells from vehicle-treated animals (gray filled histograms). C3H/HeJ mice, which have a mutation in TLR4, or iNKT cell–deficient mice were used along with their respective controls (C3H/HeN and C57BL/6). (B) Surface expression of CD86 on CD11c⁺ cells was assessed on splenocytes from wild-type C57BL/6 or CD1d–/– mice or a mixture of splenocytes from both (Mixed), stimulated in vitro with (thick solid lines) or without α-GalCer (gray filled histograms) for 16 hours. (C) Mean fluorescence intensity of CD86 (± SE) assessed on CD11c⁺ cells from the spleens of IFN-γ receptor–deficient and type I IFN receptor–deficient animals that had received α-GalCer or vehicle 16 hours previously. (D) The immunostimulatory capacity of splenic CD11c⁺ cells from α-GalCer– or vehicle-treated C57BL/6 (B6) animals was assessed by loading with OVA_257–264 peptide ex vivo and transferring antigen-loaded cells into naive C57BL/6 and iNKT cell–deficient recipients (n = 5) (arrows indicate direction of DC transfer). OVA_257–264–specific CD8⁺ T cell responses in recipient animals were measured in the blood by FACS analysis using H-2K^b/OVA_257–264 tetramers 7 days after transfer. Mean proportions of tetramer⁺ cells as a percentage of CD8⁺ cells (± SE) are shown. (E) Inset shows expression of CD86 on splenic CD11c⁺ cells from C57BL/6 mice injected with vehicle (gray filled histogram), α-GalCer (thick dashed line), MPL (thin dashed line), or α-GalCer plus MPL (thick solid line). Graph shows OVA_257–264–specific CD8⁺ T cell responses enumerated in the blood by FACS analysis 7 days after administration of 400 μg OVA with various combinations of vehicle, α-GalCer, or MPL.