Brefeldin A Decay Assay (BFA)

A BFA decay assay was performed to evaluate the stability of the class I peptide-HLA-A2 complex on the cell surface. For this, T2 cells were seeded in a 96-well rounded-bottom plate at a concentration of 1×10⁶ cells/100 µL in a serum-free RPMI medium containing 3µg/mL human β2m. Cells were cultured with either the candidate peptides at a final concentration of 50µg (as determined by T2 binding assay) or with DMSO as a negative control and incubated at 26°C overnight. The next day, one batch of cells was washed and incubated with 10µg/mL BFA (BioLegend, 420601) for 1 hour at 37°C (Time 0) and thereafter every two hours until 8 hours. After each incubation, cells were transferred into 12x75 mm polycarbonate tubes, washed and stained with an APC-conjugated human HLA-A2 monoclonal antibody (BioLegend, 343308, Clone: BB7.2) for 30 minutes at 4°C and analysed using the flow cytometer. Dead cells were excluded by staining with LIVE/DEAD™ viability dye (Invitrogen) or PI (Sigma-Aldrich).

The stability of peptide-HLA-A2 complex was assessed by calculating the DC₅₀ value, which is defined as the time required for a 50% reduction in the level of MFI recorded at time 0 (26), the longer the time for this to happen, the more stable the complex. The DC₅₀ was calculated according to the formula: (MFI at a given time point/MFI at time 0) X100%. T2 cells incubated in the absence of peptide, but in the presence of the same concentrations of DMSO were used as negative controls.