Detecting SIV-Specific CD8+ T Cells

We measured antigen-specific CD8⁺ T-cell responses by flow cytometric analysis detecting gamma interferon (IFN-γ) induction after specific stimulation as described previously [41 (link)]. Autologous herpesvirus papio-immortalized B-lymphoblastoid cell lines (B-LCLs) were pulsed with individual SIVmac239 epitope-coding peptides (at a final concentration of 1–5 μM) or peptide pools (at a final concentration of 1–2 μM for each peptide) using panels of overlapping peptides spanning the entire SIVmac239 Gag, Pol, Vif, Vpx, Vpr, Tat, Rev, Env, and Nef amino acid sequences (Sigma Aldrich Japan). PBMCs were cocultured with these pulsed B-LCLs under GolgiStop (monensin, BD) presence for 6 hours. Intracellular IFN-γ staining was performed with a CytofixCytoperm kit (BD) and fluorescein isothiocyanate (FITC)-conjugated anti-human CD4 (M-T477, BD), peridinin chlorophyll protein (PerCP)-conjugated anti-human CD8 (SK1, BD), allophycocyanin (APC)-conjugated anti-human CD3 (SP34-2, BD), and phycoerythrin (PE)-conjugated anti-human IFN-γ monoclonal antibodies (4S.B3, Biolegend). Specific T-cell frequencies were calculated by subtracting non-specific IFN-γ⁺ T-cell frequencies from those after antigen-specific stimulation. Specific CD8⁺ T-cell frequencies lower than 0.02% of CD8⁺ T cells were considered negative.