Anti hla a2 antibody

The T2 cells expressing HLA-A2 molecules on the cell surface were used as artificial APCs. Thus, the peptide binding affinity on T2 cells was examined. The 10 predicted epitope peptides were dissolved in DMSO at 10 mM stock concentration. Then, the titration of peptide concentration was performed as described previously 24 (link). The T2 cell line was shared by Anna Gil (University of Massachusetts Medical School), which is TAP-deficient T cell expressing HLA-A2 protein on the cell surface 25 (link). T2 cells (0.2×10⁶/well) were seeded into 96-well plates and incubated with 20 µM peptides at 37 °C for 4 hours. DMSO was set as blank control, the HLA-A2 restricted Influenza A (flu) M1 peptide (M58-66 GILGFVFTL) was set as positive control and HLA-A11 restricted EBNA3B (EBV) virus peptide (E416-424 IVTDFSVIK) was set as negative control 26 (link)-28 . Cells were stained with anti-HLA-A2 antibody (BioLegend, Cat#343305, US) and acquired using flow cytometer FACS Canto (BD).

The 171 positional scanning variant peptides were obtained by substituting each residue of the original peptide with the 19 other possible amino acids, as previously described (38 (link), 39 (link)). T2 cells were pulsed with 10 μg/mL β2M and each variant peptide at 10 μM in serum-free RPMI for 4 h. Pulsed cells were cocultured 1:1 with preactivated CD8⁺ T cells from healthy donors and 0.25 nM HA29-scDb. Coculture supernatants were assayed for MIP1β (Human CCL4/MIP-1B Quantikine ELISA, R&D Systems) at 24 h. HLA-A2 levels on pulsed cells were also assessed by surface staining with an anti–HLA-A2 antibody (BioLegend) and viability dye (eFluor 780, ThermoFisher).