Live dead red fluorescent reactive dye

The following commercially available antibodies were used for multi-color flow cytometry: BV421-conjugated anti-PD-1, BV521-conjugated anti-CD3, BV605-conjugated anti-CD4, BV786-conjugated anti-chemokine receptor 7 (CCR7), APC/Cy7-conjugated anti-mouse CD4, APC-conjugated anti-mouse PD-1 (Biolegend, San Diego, CA, USA), FITC-conjugated anti-HLA-A2, FITC-conjugated anti-CD45RA, PE-TR-conjugated anti-CD14, CD19, PE-Cy7-conjugated anti-CD127, APC-H7-conjugated anti-CD8, PE-conjugated anti-IFN-γ, PE-Cy7-conjugated anti-tumor necrosis factor α (TNF-α), PE-conjugated anti-major histocompatibility complex (MHC)-pentamer (Proimmune, Oxford, UK), Dead cells were excluded using the LIVE/DEAD red fluorescent reactive dye (Invitrogen, Carlsbad, CA, USA). Multi-color flow cytometry was performed using the LSRII instrument (BD Biosciences), and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). HLA-A^*02 pentamers corresponding to HBV core₁₈₋₂₇ FLPSDFFPSV, HBV core₁₈₋₂₇ FLPSDFFPSI, and HBV polymerase_455−463 were made by Proimmune. For detection of antigen-specific CD8⁺ T cells, PBMCs were incubated with pentamer for 30 min in the dark at 4 degrees Celsius and subsequently phenotyped. HLA class I genotyping was performed by flow cytometry using the anti-HLA-A2 monoclonal antibody.