2.4. T2 Binding and Stabilization Assays

The binding affinity of these peptides to HLA-A^∗0201 was evaluated according to our previous study [17, 20, 22]. Briefly, T2 cells (1 × 10⁶ cells/ml) were incubated with each peptide (50 μg/ml) and 3 μg/ml human β2-microglobulin (β2-M, Merck, Germany) in serum-free RPMI 1640 medium for 18 h at 37°C in a humidified atmosphere of 5% CO₂. Then cells were washed twice and incubated with the anti-human HLA-A2 PE-Cyanine7 mAb, BB7.2 (eBioscience, USA) for 0.5 h at 4°C. Finally, cells were collected, washed, and analyzed using flow cytometry (FACSCalibur, Becton Dickinson, USA). The fluorescence index (FI) calculation was performed as follows: FI = [(mean fluorescence intensity (MFI) of the given peptide − background) − (MFI of the PBS control group − background)]/[(MFI of the PBS control group − background)]. The MFI value of cells untreated with peptides and antibodies was employed as background. Peptides with an FI factor of more than 1.5 were classed as high-affinity candidates [22].

For the stability assay [23, 24], each of the selected peptides (50 μg/ml) was incubated with T2 cells (1 × 10⁶ cells/ml) for 18 h in serum-free RPMI 1640 medium supplemented with 3 μg/ml β2-M at 37°C. Thereafter, cells were washed twice and incubated with brefeldin A (10 μg/ml, eBiosciences, USA) for 1 h. Subsequently, the cells were washed and incubated for 0, 2, 4, and 6 h at 37°C. Cells were washed twice, stained with BB7.2 for 0.5 h at 4°C. Finally, cells were collected, washed, and analyzed by flow cytometry. Dissociation complex 50 (DC₅₀) was defined as time (t) required for the loss of 50% of the HLA-A^∗0201/peptide complexes stabilized at 0 h [25].