Anti cd8 percp cy5.5 clone sk1

Thawed PBMCs were seeded into 24-wells plate at the density of 1.0 × 10⁶ cells/well and the cells were cultured in IMDM containing 10% Human AB serum (Sigma-Aldrich, San Louis, USA). More detailed protocol is written in our previous manuscript [30 (link)]. Cells were stimulated with HPV16 E6 or E7 peptide pools or cultured in medium only and at day 7 they were stained first with surface markers CD25 diluted 1:25, stock concentration 10 μg/mL (Anti-CD25-FITC, clone 2A3, BD Pharmingen, San Jose, CA); CD4, diluted 1:100, no information on stock concentration was provided by manufacturer (Anti-CD4-APC, clone RPA-T4, BD Pharmingen); CD8, diluted 1:30, stock concentration 5 μg/mL (Anti-CD8 PerCP-Cy5.5; clone SK1, BD Pharmingen). Subsequently, the cells were fixed and permeabilized using intra-nuclear staining buffer set (FOXP3 Fix/Perm buffer set, Biolegend, San Diego, CA) according to manufacturer’s instructions. An antigen-induced alteration in the population percentage was defined as a change of at least 2× the corresponding percentage in the medium-only control [7 (link),31 (link)].

Regulatory T-cell identification was performed as previously described [9 (link), 15 (link), 39 (link)]. Briefly, PBMCs were cultured with the same peptide pools as used in the LST at a final concentration of 5 μg/mL per peptide. Cells cultured with MRM were used as a positive control and cells without antigen (medium only) were used as a background control. After 7 days of culturing, the cells were harvested and stained with surface markers CD25 (1:25) (anti-CD25 FITC, clone M-A251, BD Pharmingen, San Diego, CA, USA), CD4 (1:100) (anti-CD4-APC, clone RPA-T4, BD Pharmingen), CD8 (1:30) (anti-CD8 PerCP-Cy5.5; clone SK1, BD Pharmingen), and intracellular marker Foxp3 (PE anti-human FOXP3, clone 206D, BioLegend) or isotype control (PE Mouse IgG1, κ Isotype Ctrl, BioLegend). After staining, the cells were acquired by the flow cytometer BD FACSCalibur (BD Biosciences). Analysis was performed by using Flowing Software, version 2.5.0 (Cell Imaging Core, Turku Centre for Biotechnology, Turku, Finland). The fluorescent intensity of MRM-stimulated and medium-only control cells was used to set the gates for the other samples. An antigen-induced alteration in the population percentage was defined as a change of at least 2× the corresponding percentage in the medium-only controls.