Flow cytometry

PBMCs were resuspended in PBS supplemented with 0.5% wt/vol BSA, 2 mM EDTA, and 0.01% sodium azide and incubated with saturating concentrations of fluorescently labeled conjugated mAbs for 30 min at 4°C under constant agitation as described before (29). Cells were analyzed using a FACSCanto II flow cytometer and FACSDiva software (BD Biosciences). Using FlowJo software, proliferation of B and T cells was determined by measuring CFSE dilution, whereas activation and differentiation were assessed by the expression of CD25, CD27, CD38, CD138, and SLAMF7. The following mAbs (indicated as panel 1) were used for immunophenotyping: CD3 APC-R700 (557943; BD Biosciences), CD4 PE-Cy7 (348809; BD Biosciences), CD8 PerCP-Cy5.5 (341050; BD Biosciences), CD19 APC-R700 (564977; BD Biosciences), CD20 PerCP-Cy5.5 (332781; BD Biosciences), CD25 APC (340907; BD Biosciences), CD27 APC (337169; BD Biosciences), CD27 APC-eFluor 780 (47-0279-42; eBioscience), CD38 PE (345806; BD Biosciences), CD38 PE-Cy7 (335825; BD Biosciences), CD138 APC (347216; BD Biosciences), BAFFR APC (316916; BioLegend), BCMA PE (357504; BioLegend), CXCR4 PE-Cy7 (306514; BioLegend), IgD PE (555779; BD Biosciences), and SLAMF7 PE-Cy7 (331815; BioLegend).

Cells were harvested, pooled, and pelleted before washing twice with 10 ml of PBS/0.1% BSA. Next, cells were fixed and stained according to previously published protocols for intracellular stainings and TF-flow (44). In short, the (1) intracellular staining protocol uses the Fix/Perm kit from BD Biosciences according to the manufacturer’s instructions (Cytofix 554655; Perm. Buffer 558050) and the (2) TF staining protocol uses the Foxp3 fixation buffer (eBioscience, through Thermo Fisher Scientific) and Foxp3 permeabilization buffer (eBioscience). (1) For the intracellular staining, cells were extracellularly stained for 20 min at 4°C with saturating concentrations of CD19 APC-R700 (564977; BD Biosciences). Labeled cells were fixed and permeabilized using the Fix/Perm kit from BD Biosciences. Cell pellets were divided into two separate stainings (indicated as panels 2 and 3). Next, cells were stained intracellularly in PBA with saturating concentrations of the following fluorescently labeled mAbs: CD3 BV421 (562426; BD Biosciences), pERK 1/2 (T202/Y204) (560115; BD Phosflow), NF-κB p65 PE (653003; BioLegend), NF-κB phospho-p65 (S529) PE-Cy7 (560335; BD Biosciences), IκBα Alexa 647 (8993S; Cell Signaling), or NF-κB acetyl-p65 (k310) (ab19870; Abcam), and in combination with a secondary Ab in APC (709-136-149; Jackson ImmunoResearch) after incubation and washing. (2) For the TF staining (indicated as panel 4), cells were stained with 1:1,000 LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and CD19 BV510 (562947; BD Biosciences), SLAMF7 PE-Cy7 (331815; BioLegend), and CD38 V450 (646851; BD Biosciences) with or without CD27 BUV395 (563815; BD Biosciences) antibodies. Cells were incubated for 15 min in the fridge. Next, cells were washed and fixed using Foxp3 permeabilization buffer (eBioscience) on ice. After a washing step with this buffer, samples were stained with 25 μl of staining mix containing anti-PAX5 PE (649708; BioLegend), IRF4-PerCP-Cy5-5 (646415; BioLegend), anti-BLIMP1 AF647 (IC36081R-025; R&D Systems), pSTAT3 (612569; BD Biosciences), and tSTAT3 (564133; BD Biosciences) diluted in Foxp3 permeabilization buffer and incubated for 30 min in the fridge. The samples were washed and resuspended in a volume and measured on a BD FACSymphony A3 or A5 machine. The flow cytometer was calibrated by compensating for all conjugates using UltraComp eBeads Compensation Beads (Invitrogen). The data were analyzed using FlowJo software, v10.6.2 (Treestar).

In separate experiments, CFSE-labeled PBMCs were resuspended in a culture medium and incubated with saturating concentrations of dye-conjugated mAbs for 30 min at 4°C. Naïve (CD20⁺CD19⁺IgD⁺CD27⁻), non-switched (CD20⁺CD19⁺IgD⁺CD27⁺), and memory (CD20⁺CD19⁺IgD⁻CD27⁺) B-cell populations and non–B-cell populations (CD19⁻) were isolated by FACS with a FACSAria II (BD Biosciences) dependent on the experiment. In addition, from the non–B-cell fraction, total CD3⁺ T cells (CD3⁺CD19⁻) were isolated for specific experiments. The following mAbs were used for isolation: CD3 PE (347247; BD Biosciences), CD19 APC-R700 (564977; BD Biosciences), CD27 APC (337169; BD Biosciences), and IgD PE (555779; BD Biosciences). Different combinations, at a fixed number of cells (25,000 B cells with 125,000 non-B cells) or 100,000 T cells, were then cultured in 96-well U-bottom plates for 6 d. During culture, the cells were stimulated with CpG or αCD40 + IL-21 with or without concentrations of daratumumab as described above. T cells were stimulated with anti-CD3 (αCD3) (clone 1xE; Sanquin) and 10 μg/ml anti-CD28 (αCD28) (clone 15E8; Sanquin) with or without daratumumab. After 6 d, cells were analyzed by flow cytometry as described above. Supernatants were collected for further analysis.