HTLV-1 Tax-specific CD8+ T-cell Quantification

HTLV-1 Tax-specific CD8⁺ T-cell frequencies were measured by flow cytometric analysis of gamma interferon (IFN-γ) induction after specific stimulation as described previously (44 (link)). Briefly, PBMCs were cocultured with autologous herpesvirus papio-immortalized B-lymphoblastoid cell lines (B-LCL) pulsed with peptide pools (at a final concentration of 2 μM for each peptide) using panels of overlapping peptides spanning HTLV-1 Tax amino acid sequences in the presence of GolgiStop (monensin; BD) for 6 h. Intracellular IFN-γ staining was performed using a Cytofix/Cytoperm kit (BD) and Live/Dead fixable aqua dead cell stain kit (Invitrogen), anti-CD3 APC-Cy7 (clone SP34-2; BD), anti-CD4 FITC, anti-CD8 PerCP, and anti-IFN-γ PE (clone 4S.B3; BioLegend). Stained cells were analyzed by BD FACSCanto II with FACSDiva v8.0.1 and FlowJo v9.2. Specific CD8⁺ T-cell frequencies were calculated by subtracting nonspecific IFN-γ⁺ CD8⁺ T-cell frequencies from those after peptide-specific stimulation. Specific CD8⁺ T-cell frequencies less than 0.02% of CD8⁺ T cells were considered negative.