Measurement of the peptide/HLA-A*0201 complex stability
Briefly, T2 cells (106/ml) were incubated overnight with the candidate peptides, respectively, at a concentration of 20 μg/ml in serum-free medium supplemented with β2-microglobulin at a concentration of 3 μg/ml at 37 °C.
Thereafter, they were washed four times to remove free peptides, incubated with Brefeldin A (10 lg/ml) for 1 h to block cell surface expression of newly synthesized HLA-A2.1 molecules, washed and incubated at 37 °C for 0, 2, 4, 6, or 8 h. Subsequently, cells were stained with anti-HLA-A2 antibody from BB7.2 cells to evaluate the HLA-A2.1 molecule expression. For each time point, peptide induced HLA-A*0201 expression was evaluated by the formula mentioned above. Dissociation complex50 (DC50) was defined as the time required for the loss of 50 % of the HLA-A*0201/peptide complexes stabilized at time = 0.