Peptide-Specific CD8+ T Cell Activation Assay

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).