CD1d down-regulation inhibits activation of CD1d-restricted NKTcells. (A) NKT cells were coincubated with αGalCer-loaded MOCK- or 81A-EGFP (green) infected DCs. Cell complexes were fixed, permeabilized, and stained with anti–α-tubulin mAb (red) and DAPI (4,6 diamidino-2-phenylindole; blue). White arrowheads indicate centrosomes; white lines show division of NKT cells for quantification of centrosome polarization. (B) Percentage of centrosomes located proximal, middle, or distal to the site of NKT-DC contact. At least 35 complexes in 2 experiments were analyzed; **P = .001. (C) As in panels A and B, but NKT cells were coincubated with αGalCer-loaded MOCK- or Vpu-transfected 293T-CD1d cells. At least 70 complexes in 2 experiments were analyzed; *P < .05. (D) Staining with anti–IFN-γ mAb (red) was performed after coincubation of NKT cells with αGalCer-loaded 81A-EGFP (green) infected DCs for 2 hours in the presences of brefeldin A. Minuses indicate IFN-γ negative, asterisks IFN-γ positive NKT cells in contact with DCs. (E) Quantification of IFN-γ production in 5 independent experiments with DCs from different donors. **P < .01. Scale bars, 15 μm.