Il 21 bv421

A total of 1.5x10 6 and 2x10 6 fresh PBMCs were used for Tfh and Tfreg analysis, respectively. PBMCs were stained with a fixable viability dye-700-alexa fluorophore at 4 0 C for 15 minutes in the dark (for dead cell discrimination) prior to surface and intracellular staining. PBMCs were subsequently stained with surface markers at 4 0 C for 30 minutes in the dark with the following monoclonal antibodies: anti-CD3-PE-cy7, anti-CD4-Buv496, anti-CD8-FITC, anti-CXCR5-Percp-cy5.5, anti-PD-1-BV786, anti-CXCR3-PE, anti-CCR6-BV711, and anti-CD25-APC-cy-7. All monoclonal antibodies were purchased from BD Bioscience. For intracellular cytokine staining (ICS), PBMCs were stimulated for 5 hours with phorbol 12-myristate 13acetate (PMA) (50 ng/mL), ionomycin (500 ng/mL) (Sigma-Aldrich), and golgistop (monensin; 0.6 μL/mL) (BD Bioscience). Following stimulation, PBMCs were fixed and permeabilized with cytofix/cytoperm cell permeabilization buffer (BD Bioscience). ICS was then performed using monoclonal antibodies against IFNγ-APC, IL-17-BV650, IL21-BV421, IL-10-PE-CF594 (BD Bioscience) and IL-4-BV605 (Bio-legend) following incubation under the same conditions described above for surface staining. Simultaneously, FoxP3 staining was performed for 30 minutes in dark at 4 0 C using anti-FoxP3-PE antibody according to the manufacturer's instructions (eBioscience).