Differential binding of α-GalCer–loaded CD1d tetramers on CD4 ⁺ and DN human iNKT cell clones expressing identical TCR. (A) iNKT cell clones 20.22 (open symbols) and 20.49 (closed symbols) were incubated with increasing concentrations of α-GalCer–loaded CD1d tetramer. For each point, tetramer fluorescence is normalized to the level of surface expressed TCR (N. Fluorescence). (B) Scatchard transformation of the binding isotherm of α-GalCer–loaded CD1d tetramer to the NKT cell clones 20.22 (open symbols) and 20.49 (closed symbols). The ratio of normalized fluorescence to the tetramer concentration is plotted against the normalized fluorescence. (C) Decay plots of the natural logarithm of binding to the NKT cell clones 20.22 (open symbols) and 20.49 (closed symbols) of α-GalCer-loaded CD1d tetramers. Normalized fluorescence is plotted versus time after addition of anti-CD1d blocking mAb. Cells were incubated with α-GalCer–loaded CD1d tetramer and TCR mAb then brought to either + 4°C or + 22°C. Blocking anti-CD1d (30 μg/mL) was added and, at consecutive time points, an aliquot was analyzed by flow cytometry. Half-lives of tetramer stainings are indicated (t_1/2). (D) α-GalCer–loaded CD1d tetramer binding to iNKT cell clones 20.22 and 20.49 in the absence (filled histograms) or presence of anti-CD4 mAbs (open histograms). One representative experiment (of 3) is shown.