Versacomp

Blood samples were stained with the following fluorochrome-conjugated antibodies: MDC8-FITC, CD303-PEVio770, (Miltenyi Biotec), CD45 Krome Orange, CD14-Pacific Blue, CD3-AA750, CD19-AA750, CD56-AA700, CD123-ECD, CD1c PE/Cy5.5 (from Beckman Coulter), HLA-DR-Super Bright 600, CD16-BV785 (Biolegend), OX40L-PE or isotypic control (Biolegend), CD274 (PDL1) APC (BD Biosciences). For Tfh cell analysis, the following-conjugated antibodies were used: CD3-A750, CD4-Pacific Blue (Beckman Coulter); CD45-FITC, CD45RA-PE/Dazzle, CXCR5-PE/Cy7 (Biolegend); OX40-PE (Miltenyi Biotec). Samples were stained with the antibodies for 15 min and then incubated for 15 min with 1 ml BD FACS lysis solution, washed, and subsequently analyzed by flow cytometry. Compensation beads were used for compensation settings (VersaComp; Beckman Coulter). Cells were analyzed on a Cytoflex flow cytometer (Beckman Coulter). Data were analyzed using Kaluza V5.1 software (Beckman Coulter).

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).