Surface staining and intracellular staining
A total of 1.5x106 and 2x106 fresh PBMCs were used for Tfh and Tfreg analysis, respectively. PBMCs were stained with a fixable viability dye-700-alexa fluorophore at 40C for 15 minutes in the dark (for dead cell discrimination) prior to surface and intracellular staining. PBMCs were subsequently stained with surface markers at 40C 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 13-acetate (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 IFN-γ-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 40C using anti-FoxP3-PE antibody according to the manufacturer’s instructions (eBioscience).