Cd20 v450

Multiparameter FACS analysis was performed using a monoclonal antibody (mAb) panel to measure activation in blood [18] . The following mAb combinations, relevant for this manuscript, were used: CD45^BUV496 (Becton & Dickinson, Franklin Lakes, NJ, USA (BD), cat 741185), HLA-DR^BUV805 (BD, cat 748338), CD20^V450 (BD, cat 655872), CD16^BV605 (BD, cat 563916), CD3^BV650 (BD, 563916), CD40^BV750 (BD, cat 746948), CD14^PE-TxRed (Beckman Coulter, cat B92391), CD123^PerCP-Cy5.5 (BD cat 558714), CD86^Alexa647 (Biolegend, San Diego, CA, USA, cat 305416), CD1c^APC (Ebioscience cat 17-0015-42). Acquisition was performed on an Aurora flow cytometer (Cytek, Fremont, Ca, USA) and analyzed using FlowJo software. For analysis, the CD45 positive cells with a low side scatter (SSC) profile were first gated and then singlets were selected using a forward scatter (FSC) height versus area plot. Then, HLA-DR positive/CD3, CD20 and CD14 negative cells were selected and CD123 was plotted against CD1c to identify the plasmacytoid DC (pDC)(CD123 positive/CD1c negative) and classical DC2 (cDC2)(CD123 negative/CD1c positive) populations. Expression of CD40 and CD86 was quantified as the mean geometric mean of the fluorescence intensity (MFI).

Thawed PBMCs were surface stained with a combination of CD3 Q655, CD14 Q655 (Invitrogen), CD56 PECy7, CD16 APC-H7 (BD) to exclude non–B cells, CD19 ECD (Beckman Coulter), CD20 V450, CD27 APC, CD38 PERCPCy5.5, CD86 PE, Ki67 Ax700, IgM PECy5, IgD FITC (all BD) to characterize CD19⁺ B cell subsets.