4.2. MHC Stabilization Assay

Peptide binding to HLA-A*0201 was analyzed using TAP1- and TAP2-deficient T2 cells which express the HLA-A*0201 allele. T2 cells (2 × 10⁵) were added to increasing amounts of the MUC1 peptides (0–100 μg/mL) and β2-microglobulin (1 μg/mL) in a total volume of 100 μL AIM V medium per well in a round-bottom 96-well plate and incubated for 18 h at 37 °C. Binding of the peptides was measured using flow cytometry for upregulation of HLA-A2 surface expression on the T2 cells. The peptide-pulsed T2 cells were washed and stained with the FITC-labeled HLA-A2 antibody (clone BB7.2, Pharmingen, San Diego, CA, USA) prior to analysis. Mean fluorescence intensity (MFI) was determined and the fluorescence index (FI) was calculated from the formula: FI = (F_S − F_B)/(F_T2 − F_B) × 100 where F_S is the MFI of the test peptides, F_B is the no-peptide isotype antibody-stained control MFI, and F_T2 is the no-peptide HLA-A2 antibody-stained control MFI [52].