Peptide binding affinity assay

To evaluate the binding affinity of each candidate peptides to HLA-A*0201 molecules, the classical T2 peptide-binding assay was performed. T2 cells, a cell line characterized by TAP-deficient and HLA-A*0201-positive, were incubated overnight with peptides (100 μM) in PRMI 1640 medium containing 20% fetal bovine serum and β2-microglobulin (3 μg/ml) at 37 °C for 24 h. Then the cells were harvested, washed, and stained with FITC-conjugated anti-HLA-DR mAb for 30 min at 4 °C, the fluorescence intensity was analyzed by flow cytometry. The mean fluorescence index (FI) were calculated as: FI = [mean fluorescence intensity (MFI)_sample − MFI_background]/MFI_background, where MFI_background represents the value without peptide. FI > 1.5 indicated a high affinity to HLA-A*0201 molecules, 1.0 < FI < 1.5 indicated a moderate affinity to the HLA-A*0201 molecule, and 0.5 < FI < 1.0 indicated that the peptide had low affinity to the HLA-A*0201 molecule. All samples were tested in triplicates.