Cxcr5 cd185 pe

Total CD4 + T cells were isolated from PBMCs using a Magnisort Human CD4 T-cell negative selection kit (Invitrogen, Ther-moFisher). T-cell purity was always more than 94%. CD4 + T cells were then stained with the following fluorochrome-conjugated antibodies: CXCR5 (CD185) PE (eBioscience), CD4 Pacific Blue, PD-1 APC (Beckman Coulter). Tfh cells were defined as CD4 + CXCR5 + PD-1 + cells. Tfh cells, CD4 + CX-CR5 -PD-1 + and CD4 + CXCR5 -PD-1 -were sorted using a flow cytometer (FACS Aria III; BD Biosciences). B lymphocytes were isolated from PBMCs by a magnetic beads CD19 + positive selection kit (eBioscience). All sorted cell populations exhibited high purity (>90%).

After isolation, PBMCs were stained with the following fluorochrome-conjugated antibodies: CXCR3 (CD183) FITC, CCR6 PE-Vio770 (Miltenyi Biotec, Paris, France); CXCR5 (CD185) PE (eBioscience, ThermoFisher, Villebon, France); CD45RA ECD, PD-1 (CD279) PECY5.5, ICOS (CD278) APC, CD3 AA750 (Beckman Coulter, Villepinte, France); CD45 BV510, CD4 BV650, HLA-DR BV786 (BD Biosciences, Rungis, France). For B-lymphocyte analysis, the following fluorochrome-conjugated antibodies were used: IgD FITC, CD21 BV450, CD45 BV510, IL-21R APC (BD Biosciences); CD27 PE, CD24 ECD, CD38 PECY5.5, CD19 PC7 (Beckman Coulter); CD5 AA700 (Biolegend, Ozyme, Montigny-le-Bretonneux). T-lymphocyte and B-lymphocyte absolute numbers were determined using 50 µL of whole blood from patients and HC using Trucount tubes (BD Biosciences). Samples were stained with the following antibodies: CD19 FITC, CD45 ECD, CD3 AA750 (Beckman Coulter) for 15 min and then incubated for 15 min with 450 µL BD FACS Lysis Solution and subsequently analysed by flow cytometry. Compensation beads were used for compensation setting (VersaComp; Beckman Coulter). Cells were analysed on a Cytoflex flow cytometer (Beckman Coulter). Data were analysed using Kaluza V.5.1 software (Beckman Coulter).