Ifn γ bv786

For intracellular cytokine staining, PBMCs were stimulated with the corresponding pooled peptide at a final concentration of 8 μg/mL for 1 h at 37 °C. Then, the cells were incubated for an additional 4 h with 1 μg/mL GolgiPlug (Brefeldin A, BD) and 0.7 μg/mL GolgiStop (Monensin, BD) and surface stained with CD107a-PE-CF594 (BD Bioscience). Unstimulated cells were used as negative controls. A combination of 50 ng/mL phorbol-12-myristate-13-acetate and 1 μg/mL ionomycin (both Sigma-Aldrich, Seelze, Germany) was used as a positive control. Dead cells were stained with LIVE/DEADR Fixable Aqua dye (Invitrogen). Surface markers, including CD3-AF700 (BioLegend), CD4-FITC (BD Bioscience), and CD8-APC-H7 (BD Bioscience), were stained. Cells were then washed and permeabilized using Cytofix/Cytoperm (BD Bioscience). Subsequently, the cells were washed with Perm/Wash buffer (BD Bioscience), stained intracellularly with Tumour Necrosis Factor (TNF)-α-APC (BioLegend), IFN-γ-BV786 (BD Bioscience), and IL-2-PE (BioLegend), fixed with 1 × CellFix solution (BD Biosciences) and acquired immediately on a BD LSR Fortessa. The data were analysed using FlowJo (Tree Star Inc., Ashland OR).

Spleen cells were treated with APC (10 µg/mL) for 24 h. 5 h prior to termination of the experiments, cells were stimulated with PMA (20 ng/mL)/Ionomycin (1 mM) in the presence of Monensin (5 µM). After stimulation, cells were collected and stained using a mouse antibody panel including CD3-AF700, CD4-BV711, IFN-γ-BV786, IL-4-PECY7, IL-17-BV421 and IL-22 (PE) (BD Biosciences, Franklin Lakes, NJ, USA) to detect Th cytokines in CD3+ and CD4+ lymphocytes. For specific Th cell phenotype identification, cells were treated with APC for 24 h and then stained with an antibody panel containing CD3-AF700, CD4-BV711, T-bet-BV786 (Th1), GATA3-PECY7 (Th2), Roγ-BV421 (Th17), FOX3-PE and CD25-APC (Treg) (BD Biosciences). Detection was performed using a BD LSR Fortessa flow cytometer (BD Biosciences).