The CD4-CD1d interaction is critical for the antigen-specific activation of CD4 ⁺ iNKT cells clones. The CD4 ⁺ iNKT clones LP17DC2 (A) and LP16DCP6 (B) were cultured with increasing doses of plastic-bound soluble recombinant human CD1d, pulsed with α-GalCer. Cultures received nothing (nil), isotype-matched IgG1 (IgG), anti-CD4 S3.5 blocking mAb (anti-CD4), or recombinant glycosylated monomeric HIV gp120 (HIV gp120). IFN-γ contained in the culture was determined by enzyme-linked immunosorbent assay. One representative experiment of 3 is shown. Similar data were obtained by using the anti-CD4 6D10 blocking mAb. (C) LP16DCP6 clone was cultured in triplicated wells containing increasing doses of plastic-bound antihuman CD3 mAb TR66, in the presence of: isotype-matched control IgG1 (IgG); recombinant glycosylated momomeric HIV gp120; anti-CD4 S3.5 blocking mAb. One representative experiment of 3 is shown. Similar experiments performed with other CD4 ⁺ iNKT cell clones gave comparable results.