Antigen loading of CD40–B cells.

(A) Specific binding of hepatitis-B core peptide was demonstrated by means of HLA-A*0201⁺ (left) and HLA-A*0201⁻(right) CD40–B cells. As negative control, autofluorescence of CD40–B cells and isotype control are shown. Staining for MHC class I was used as positive control. Identical imaging parameters were used throughout the experiment. (B) Binding affinity of the HLA-A2–binding peptides I540 (filled circles) and F18 (filled diamonds) and the pan-DR–binding peptide PADRE (filled squares) to CD40–B cells was determined by competition with the hepatitis-B core F18-FITC reference peptide. Fluorescence was analyzed by flow cytometry, and molecules of reference peptide were calculated by means of standardization beads (see “Materials and methods”).