B cell enrichment kit

Prior to stimulation for B10 cells, B cells were negatively isolated from a starting population of 10⁷ PBMCs, using the B cell enrichment kit (Stemcell). Isolated B cells were plated in 96-well flat bottom plates in RPMI + 10% FBS. Cells were stimulated with CpG and rCD40L for 48 h at 37°C in 5% CO₂ incubator. At 43 h, cells were restimulated with PMA and ionomycin. In parallel with the end of the 48-h stimulation, PBMCs from the same patients were enriched for CD4 T cells (Stemcell) and stained with the Violet Proliferation dye (1 µM, BD Bioscience) in PBS and incubated in a 37°C water bath for 15 min. After 48 h, the isolated CD4 cells were combined with B cells at 1:1 and 1:2 ratio of T:B cells along with a CD4 T cell only condition and stimulated with αCD3/αCD28 for 5 days. For experiments using the transwell plates, stimulated B cells were plated in top chamber of 24-well transwell plates while CD4 T cells and αCD3/αCD28 were plated in bottom chamber.
To visualize proliferation of the CD4 T cells, cells were stained with Zombie Violet, CD14 Brilliant Violet 510 (M5E2, Biolegend), CXCR5 AlexaFluor 647 (RF8B2, BDB), CD8 AlexaFluor 700 (SK1, Biolegend), CD3 APC-Cy7 (SK7, Biolegend), CD19 PE (HIB19, Biolegend), and CD4 PE-Cy7 (SK3, Biolegend) conjugate for 25 min at 4°C. Following cell surface staining, cells were fixed with 1% PFA and acquired on a LSRII flow cytometer (BDB).

To assess cytokine production, 2 × 10⁵ PBMCs were stimulated with either 10 μg/ml F(ab′)₂ IgM- and IgG-specific Abs (Southern Biotech and Jackson ImmunoResearch, respectively) in combination with 0.5 μg/ml MEGACD40L (Enzo Life Sciences) or 1 μg/ml R848 (TLR7/8 agonist; Invivogen) diluted in complete RPMI (cRPMI: RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin/streptomycin, 1× nonessential amino acids, 1× essential amino acids, and β-mercaptoethanol; all Life Technologies). Cells were stimulated for 16 to 18 hours in the presence of 10 μg/ml anti–PD-1 mAb or an isotype control (LEAF purified; clone EH12.2H7; BioLegend). All stimulations were performed in the presence of 1 μg/ml Brefaldin A (Sigma-Aldrich) at 37°C. Cytokine responses were analyzed by intracellular cytokine staining for IL-6 and TNF-α along with cell-surface staining to identify MBC populations.
For analysis of apoptosis, 2 × 10⁵ B cells were purified by magnetic separation according to the manufacturer’s instructions (B Cell Enrichment Kit, STEMCELL Technologies) and stimulated for 7 days with 1 μg/ml F(ab′)₂ IgM- and IgG-specific Abs plus 500 ng/ml MEGACD40L. Cells were then stained for B cell phenotype, followed by incubation with annexin V–PerCP/Cy5.5 in the presence of annexin V–binding buffer according to the manufacturer’s instructions (BioLegend).

Human PBMCs were isolated via Ficoll density gradient centrifugation, and B cells were enriched using a B cell enrichment kit from Stemcell following the manufacturer’s protocol. Cells were then incubated with 200 μL APC-conjugated S protein trimer (Invitrogen, catalog A20186) and Alexa Fluor 488–conjugated S1 subunit (Invitrogen, catalog A20181) in combination with PE-Cy7 anti–human CD19 (BioLegend, catalog 302216) and APC-Cy7 anti–human CD20 (BioLegend, catalog 302314). Cells were stained in staining buffer (PBS plus 2% FBS) for 30 to 60 minutes on ice and then washed with 15 mL ice-cold staining buffer. Next, stained samples were sorted into cell lysis buffer in 96-well plates via a FACSAria II Cell Sorter (BD Biosciences). CD19⁺CD20⁺S trimer⁺S1⁺ live cells within the lymphocyte gate determined by forward scatter (FSC) and side scatter (SSC) were collected. The 96-well plates were then immediately frozen at –80°C for future use.