Peptide Binding Assay for HLA-A2 Expression

The candidate peptides were synthesized by GenScript Biotechnology Co., Ltd. with purity >98% and resuspended in DMSO at a concentration of 10 mM. The titration of peptide concentration was performed as described previously^{51 (link)}. The T2 cell line was shared by Anna Gil (University of Massachusetts Medical School). T2 cells are TAP-deficient T cells expressing HLA-A2 protein on the cell surface^{52 (link)}. T2 cells were seeded into 96-well plates and then incubated with peptides at a final concentration of 20 µM at 37 °C for 4 hours. DMSO was set as blank control; the reported HLA-A2-restricted influenza A M1 peptide (M58-66 GILGFVFTL) was set as positive control; and the validated EBV virus peptide (IVTDFSVIK) was set as negative control^{26 (link),27 ,51 (link)}. Cells were stained with PE anti-human HLA-A2 antibody (BioLegend, 343305) at 4 °C in the dark for 30 minutes and acquired on a FACSCanto flow cytometer (BD Biosciences).