Il 17 pe

For ex vivo analysis, 1-5×10⁶ PBMC were cultured in 24-well plates for 3 hrs in the presence of PMA, ionomycin and GolgiStop (according to the manufacturer’s instructions; BD, Oxford, UK). Cells were stained for cell surface markers using CD3-PE-Cy7 (1:100) and CD14-APC-Cy7 (1:100) (BioLegend, Cambridge, UK), fixed in 2% paraformaldehyde, permeabilized with 0.5% Saponin and stained for CD4-PacificBlue (1:100) in combination with IL-17-PE (1:20), IFN-γ-PerCP-Cy5.5 (1:200), IL-10-AF488 (1:20), and TNF-α-APC (1:100) (all BioLegend). Co-cultures were re-stimulated at day three with PMA/ionomycin for 6 hrs, with GolgiStop present during the last 3 hrs. Cells were stained for cell surface markers using CD2-Pacific Blue (1:1,000) (BioLegend), CD14-APC-Cy7, fixed in 2% paraformaldehyde, and permeabilized with 0.5% Saponin and stained for IL-17-PE, IFN-γ-PerCP-Cy5.5, IL-10-AF488, and TNF-α-APC. Foxp3 was measured using Foxp3-AF647 (1:20) (BioLegend) according to manufacturer’s instructions. Monocyte phenotype was determined by staining with antibodies to CD14-APC-Cy7 (BioLegend), HLA-DR-PerCP-Cy5.5 (1:50) (BD), CD40-PE (1:50) (AbD Serotec, Kidlington, UK), and CD163-FITC (1:50) (SantaCruz, Santa Cruz, CA, USA). Cells were acquired using a FACSCantoII (BD) and analyzed using FlowJo software (TreeStar, Inc).