Regulatory T-cell Identification Protocol

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.