Pd 1 fitc eh12.2h7

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.

Peripheral blood mononuclear cells and BMMCs were stained with anti-CD4 VioGreen (REA623), anti-CD3 Vio770 (REA613) from Miltenyi Biotech, anti-CD45RA PerCp (HI100), anti-CD28 APC (28.2), anti-CTLA-4 PE (BNI3), anti-T cell immunoglobulin and mucin protein-3 (TIM-3) PerCP (F38-2E2), anti-programmed death 1 (PD-1) FITC (EH12.2H7), anti-lymphocyte activation gene-3 (LAG-3) APC (7H2C65) from Biolegend, anti-HLADR PeCy7 (G46-6), anti-CD28 BV421 (28.2), anti-CD25 APC (2A3), and anti-CD69 PE (FN 50) from BD Biosciences. Intracellular detection of FOXP3 with anti-FOXP3 FITC Abs was performed using fixed and permeabilized cells following the manufacturer’s instructions. FMO was used as control. Dead cells were excluded by forward and side scatter characteristics and by using either 7-AAD or fixable viability dye Zombie violet (Biolegend).
For each sample at least 2 × 10⁶ total events were acquired using a FACScanto II and FACSSymphony (BD Biosciences). The data were analyzed using FlowJo software.