Cells were stained for 30 min at 4 °C in PBS containing 0.01% NaN3 and 0.5% BSA (Sigma Aldrich) with directly labeled antibodies against: CXCR5 Alexa Fluor 488 (clone RF8B2), CCR7 PE-Cy7 (clone 3D12), CD4 APC-H7 (clone SK3), CD8 V450 (RPA-T8), CD3 V500 (UCHT1) (all from BD Biosciences, San Jose, CA, USA), PD-1 PE (J105), CD45 RA (L307.4) eFluor450 (all from eBioscience Inc., San Diego, CA, USA), CD19 V500 (HIB19), and CD45 APC-H7 (2D1). For intracellular cytokine staining, we used IL-10 Pe-Cy7 (clone JES3-9D7) (Biolegend, San Diego, CA, USA) and IL-21 Alexa Fluor 647 (clone 3A3-N2.1) (BD Biosciences). The gating procedure was carefully set up prior to the study. First, all antibodies used were titrated to an optimal concentration. Fluorescence minus one (FMO) and appropriate compensation controls were determined for each antibody in order to gate positive populations. Additionally, an unstained control (for stimulated and unstimulated cells) for each sample was taken during the acquisition (both intracellular and extracellular) to determine positive events.
Cells were analyzed on a FACS Canto II (BD Biosciences), and data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA). Data were plotted as frequency of positive cells.
Cells were analyzed on a FACS Canto II (BD Biosciences), and data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA). Data were plotted as frequency of positive cells.