Flow-cytometric measurement of HLA-Ig dimer binding to TCCs mediated by antigen analogs and control peptides.

We incubated 10.0 μg HLA-Ig dimers with 660-fold molar excess peptide for 3 days at 4°C. Aliquots of 1.0 μg peptide-loaded dimers were mixed with 0.2 million of either TCC 3-3F4 (▪) or VB57 () and incubated as described in “Materials and methods.” As controls, dimers were also loaded with the pp65_495-503 native peptide, an irrelevant peptide (pp65 B*0702 epitope), or 2 μL PBS/0.02% NaN₃ in all experiments (data not shown). Cells were washed in PBS/2% FBS/0.02% NaN₃ (WB) and subsequently stained with 0.9 μL per sample of goat antimouse IgG1-PE (phycoerythrin) (Caltag, Burlingame, CA). After 2 additional washings in WB, cells were analyzed on the FACSCalibur. The fluorescence ratio between the mean fluorescence of the peptide-loaded dimers and the mean fluorescence of the PBS/0.02% NaN₃–loaded control dimers was calculated. Peptide names ending in N are amidated, and those ending in C are free acids. Experiments were repeated twice with similar results. Measurements with both cell lines were normalized so they could be displayed on the same axis. HIVpol = HIVpol (18-27), which indicates aa position in HIV polymerase protein.