293T cells were co-transfected with Vpu and CD1d expression vectors and 24 h post transfection loaded with 100 ng/mL αGalCer (KRN7000; Biomol International) for 2 h. 293T cells were then co-incubated with the human CD4+ iNKT cell clone HDD323 (link) at a 1:2 ratio in the presence of brefeldin A (GolgiPLUG; 2 mg/mL; BD Biosciences) for 6 h. To assess iNKT cell IFN-γ production using flow cytometry, cells were stained with anti-CD3-PerCP (clone SK7; BD Biosciences) followed by saponin permeabilization and intracellular staining with anti-IFN-γ-APC (clone 25723.11; BD Biosciences).
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