The CD4-CD1d interaction is critical for the antigen-specific activation of CD4 ⁺ invTCR transfectants. (A) 58αβ-mouse T-cell hybridoma was transfected with the invVα24-JαQ/Vβ11 TCR (left panel) and human CD4 (right panel). (B) CD4 ⁺ invTCR transfectants were activated with increasing doses of either plastic-bound soluble recombinant human CD1d, pulsed with α-GalCer (left panel) or plastic-bound antimouse CD3 2C11 (right panel), in the presence of: isotype matched control IgG (IgG1 + IgG2a); recombinant glycosylated momomeric HIV gp120 (gp120); anti-CD4 S3.5 (S3.5) or 6D10 (6D10) blocking mAbs. IL-2 contained in the culture was determined by enzyme-linked immunosorbent assay. (C) Human CD4 ⁺ iNKT cell clone CL1CD4 was cocultured in duplicate wells with RMA/S-hCD1d or C1R-CD1d, with or without either isotype-matched control IgG or anti-CD4 mAb S3.5. After 48 hours, culture supernatants were collected and tested by enzyme-linked immunosorbent assay for secreted IFN-γ. Nontransfected C1R and RMA/S cells were not recognized by the clone (data not shown). One representative of 3 independent experiments is shown.