Multicolor Flow Cytometry to Characterize Tfh Cells

Immunophenotyping of lymphocytes was performed by multicolor flow cytometry. Heparinized whole blood samples were immediately stained for 15 minutes with the following antibodies: CD3-APC/Cy7 (UCHT, BioLegend; San Diego, CA, USA), CD4-HorizonV500 (RPA-T4, BD Biosciences; San Jose, CA, USA), CD45RA-BV421 (HI-100, BioLegend), CXCR5-PerCP/Cy5.5 (J252D4, BioLegend), CXCR3-APC (1C6, BD Biosciences), CCR6-PE (11A9, BD Biosciences), CCR7-PE/Cy7 (G043H7, BioLegend), PD-1-FITC (EH12.2H7, BioLegend), CD19-BV421 (HIB19, BioLegend), CD20-APC (2H7, BioLegend), CD27-APC/Cy7 (O323, BioLegend), and CD38-FITC (HIT2, BioLegend). Circulating Tfh cells were defined as CD3⁺CD4⁺CD45RA^-CXCR5⁺ cells, and Tfh1, Tfh2, or Tfh17 cell subsets as CXCR3⁺CCR6^- cells, CXCR3^-CCR6^- cells, or CXCR3^-CCR6⁺ cells among Tfh cells, respectively [19 (link), 20 (link)]. Activated Tfh cells were defined as the CCR7^lowPD-1^high cells among Tfh cells [26 (link)]. CD19⁺CD20^-CD27⁺CD38⁺ cells were defined as plasmablasts. Red blood cells were lysed with FACS Lysing Solution (BD Biosciences). All samples were analyzed with a FACS Aria III (BD Biosciences), and data were analyzed with FlowJo v.7.6.4 Software (Tree Star, Stanford University, CA, USA). Proportions of lymphocyte subsets were determined by the combination of surface marker staining, with exclusion of doublets by forward and side scatter.