Cd20 pe

LN tissue was put through a 70 μm (BD Falcon, San Jose, CA) cell strainer to obtain a single cell suspension. Cells were washed with PBS containing 0.01% NaN₃ and 0.5% BSA and stained for 30 min at 4°C with directly labelled antibodies: CD45 PercP-Cy5.5, CD19 Alexa-700, CD27 APC-H7, IgD Pe-Cy7, CD20 PE, IgM FITC, CD69 PE, CD69 PerCP, CD21 APC, CD23 PE, CD25 APC, CD267 PE, BAFF-R FITC, CD16 Percp-Cy5.5, CD56 PE, CD55 PE, CD59 FITC (BD Biosciences, Breda, the Netherlands), HLA-DR Alexa-700 (eBioscience, Vienna, Austria), CD3 FITC (Sanquin, Amsterdam, the Netherlands). After incubation cells were washed and immediately analysed on a FACS CANTO II (BD Biosciences). To enable the measurement of different B cell subsets, a seven-colour FACS panel was set-up using antibodies against CD19, IgD, IgM, CD27, CD21, CD23 and CD45 (for normalization). Data were analysed using FlowJo software (Treestar, Ashland, OR, USA) and presented as frequencies, absolute numbers relative to 100 000 CD45⁺ lymphocytes or geometric mean fluorescence intensity (normalized on negative populations).

Phenotypic analyses from patients’ blood were performed by multicolor flow cytometry of Ficoll-Hypaque-separated cell samples utilizing directly labeled mAb as described previously.^{17 (link)} Briefly, 5 × 10⁵ to 1 × 10⁶ cells were incubated with murine mAb against the appropriate antigens at an optimal dilution for 20 min at 4°C. The following antibodies were used for this study: CD3 PE-Cy7, CD4 PerCP, CD8 PB, CD20 PE (BD Biosciences, Heidelberg, Germany), CD56 APC, CD279 (PD-1) AF488. If not otherwise stated antibodies were purchased from BioLegend (London, UK). Corresponding isotype-matched antibodies were used as controls. Non-specific binding was eliminated by mixing the samples with a 1:5 solution of a commercial human IgG (Octagam; Octapharma, Langenfeld, Germany). After 20 min incubation samples were washed three times in PBS/BSA, and at least 5 × 10⁴ cells per appropriate gate were analyzed using a FACSCanto II flow cytometer with Diva software (Becton Dickinson, Heidelberg, Germany) (gating strategy: see Supplemental material Figure 1). Offline data analysis was performed by using FCS Express software V6 (Denovo Software, Glendale, CA, USA).