DN and CD4 + NKT cell clones expressing identical TCR are differentially activated after α-GalCer/CD1d activation. (A) Cytokine expression profiles of DN (20.22) and CD4 + (20.49) NKT cell clones. HeLa CD1d cells were pulsed with α-GalCer (1 to 10−5 μg/mL) or DMSO (Veh) and used to stimulate the NKT cell clones 20.22 (open symbols) and 20.49 (closed symbols). After 12 hours, IFN-γ (squares) and IL-4 (triangles) in supernatants was measured by enzyme-linked immunosorbent assay. Data represent the average value of duplicate samples ± SD and are representative of at least 3 independent experiments. (B) Time course of CD3 down-regulation in NKT cell clones 20.22 (open symbols) and 20.49 (closed symbols) stimulated with HeLa CD1d cells pre-pulsed with α-GalCer at 10−3 (squares), 10−4 (triangles) and 10−5 (circles) μg/mL. Data are representative of at least 3 independent experiments. (C) Effect of anti-CD4 mAb on cytokine production. HeLa CD1d cells were pulsed with 10−5 μg/mL of α-GalCer, washed and used to stimulate the iNKT cell clones 20.22 (open squares) and 20.49 (closed squares) in the presence of increasing doses of anti-CD4 mAb RPA-T4 (α-CD4). After 12 hours, IFN-γ in the supernatants was measured by enzyme-linked immunosorbent assay. Data represent the average value of duplicate samples ± SD and are representative of at least 3 independent experiments.
