Tetramer staining and flow cytometric analyses

Selected custom phycoerethrin (PE) or Allophycocyanin (APC)-conjugated HLA-peptide tetramers (Baylor College of Medicine, USA; MBL, Japan) were successfully generated (online supplemental figure S9A). PBMCs were thawed and resuspended in RPMI-1640 containing 0.5% FBS. One hundred microliters of PBS-1% BSA containing fluorophore-conjugated HLA/peptide tetramer (1:50 dilution) was added to 5×10⁵ PBMCs and incubated at room temperature for 20 min in the dark. Cells were washed with PBS-1% BSA, stained with Fluorescein isothiocyanate (FITC)-conjugated or Phycoerythrin (PE)-conjugated anti-CD8 mAbs (Biolegend, USA, 1:200 dilution), and incubated for 15 min. Cells were then washed and resuspended in 400 µL PBS-1% BSA for flow cytometric analysis (LSRFortessa X-20 Analyzer).