Igg1 pb

The following mAbs and reagents were used: IgG1-FITC, IgG1-PerCP-Cy5.5, IgG2a-APC, IgG1-PB, CD14-PE, CD14-PErCP, CD45-HorizonV500, streptavidin-PerCP, anti-IFNγ-FITC, 7AAD and Lineage cocktail-FITC (CD3, 16,19, 20,14, 56) from BD Biosciences, Erembodegem, Belgium; CD80-FITC from Beckman Coulter, Woerden, the Netherlands; anti-BDCA1-PE, anti-BDCA2-FITC, anti-BDCA2-biotin, anti-BDCA4-PE, anti-BDCA3-APC, anti-Clec9a-VioBlue, CD19 microbeads and anti-PE-microbeads from Miltenyi Biotec, Bergisch Gladbach, Germany; CD1a-PB, CD209 (DC-SIGN)-PECy7, CD206-eFluor450, CD40-APC and CD274 (PDL1)-PECy7 from eBioscience, Vienna, Austria; CD86-APC, CD86-PB, CD163-PerCP-Cy5.5, CD40-PerCP-Cy5.5, HLA DR-APC-Cy7 and CD209 (DC-SIGN)-APC from Biolegend, London, United Kingdom. PMA, ionomycin and brefeldin A are from Sigma-Aldrich, St Louis, MO. The fix&perm cell permeabilization kit was obtained from An der Grub, Vienna, Austria.

Heparinized peripheral blood samples were collected from the subjects, and mononuclear cells (PBMCs) were obtained by Ficoll gradient centrifugation following the manufacturer's protocol (GE Healthcare Bio‐Sciences AB, Uppsala, Sweden). PBMCs were incubated for 15 min with human γ‐globulins at 4°C for FcR‐blocking. Then, the cells were washed and immediately labeled with specific antibodies or isotype controls for 30 min at 4°C. Specifically, antibodies against the following molecules were used: Vα24‐PE and Vβ11‐FITC (Beckman Coulter, Brea, CA), CD3‐PerCPCy5.5, CD4‐PE, (BioLegend, San Diego, CA), CD8‐PB (BD Pharmingen, Franklin Lakes, NJ), CRTAM‐APC (R&D, Minneapolis, MN), and CD69‐PETR (Beckman Coulter). The following isotype controls were used: IgG1‐PE, IgG2a‐FITC, IgG1‐PErCPCy5.5 (all from BioLegend), IgG1‐PB, (BD Pharmingen), IgG2b‐APC (R&D), and IgG1‐PETR (Beckman Coulter). After incubation, the cells were washed with PBS/2% FBS and were fixed with 4% paraformaldehyde in PBS.