Figure 3

HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules can be loaded with synthetic peptides. Purified soluble HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules were loaded with equal amount of p2 peptide. Stability of the peptide-loaded HLA-DR*1101 αβ heterodimers was determined by running complexes in reducing condition on SDS gels without boiling, or by testing their capacity to elicit IFN-γ production in specific CD4⁺ T cell clones. (a) SDS-resistance assay of HLA-DR*1101-Bir molecules. Indicated are the protein bands corresponding to the migration of either the HLA-DR*1101-Bir αβ heterodimer, or the single HLA-DRα-Bir or HLA-DR*1101β chains. Lane nil: unloaded HLA-DR*1101-Bir molecules. Lane p2: p2-loaded HLA-DR*1101-Bir molecules. (b) SDS-resistance assay of HLA-DR*1101-Ig molecules. Indicated are the protein bands corresponding to the migration of either the HLA-DR*1101-Ig αβ heterodimer, or the single HLA-DRα-Ig or HLA-DR*1101β chains. Lane nil and p2 are the same as per HLA-DR*1101-bir. (c) IFN-γ production by the p2-specific CD4⁺ T cell clone (TCC) 162 in response to p2-loaded HLA-DR*1101-Bir or HLA-DR*1101-Ig molecules, attached to plastic. The response of the MAGE-3 p39-specific CD4⁺ TCC 2C3.35 is shown as control. T cells were cultured in the presence of the indicated peptide-pulsed HLA-DR*1101 recombinant molecules, in the presence of a costimulatory dose of PMA. As control, T cells were cultured in the presence only of sub-optimal doses of PMA (PMA), or activated by plastic-bound anti-CD3 mAb (Anti-CD3). The release of IFN-γ in the culture supernatant was determined by ELISA 48 h later.