Anti igm apc

After PBMC isolation, B cells were enriched by positive selection using magnetic columns according to the manufacturer’s instructions (EasySepTM, StemCell Technology, Vancouver, BC, Canada). The cells were stained with anti-CD19-PerCP-Cy5.5, anti-CD27-PE, anti-IgM-APC, and anti-IgD-FITC (all antibodies from BD Biosciences, San Diego, CA, USA). The B cells, defined as CD19⁺CD27⁻IgD⁺IgM⁻, were sorted with FACSAria II cell sorter (Beckton-Dickinson, San Jose, CA, USA). After sorting, the cells were labeled with 5 µM of CFSE dye (Invitrogen, Eugene, OR, USA) before the culture, according to manufacturer’s instructions. The cells were cultured in AIM-V serum-free medium in the presence of CpG-ODN2006 (InvivoGen, San Diego, CA, USA) (2.5 µg/mL) and anti-CD40 (BD Bioscience, San Diego, CA, USA) (2 µg/mL) in a 96-well U bottom plate at 37 °C in a humidified 5% CO₂ incubator. On day 5, the cells were harvested and acquired using FACSCelesta (Beckton-Dickinson, San Jose, CA, USA).

Killing of cell lines was tested in standard ⁵¹chromium release assays as described previously [9 (link)]. Killing of primary material was analyzed in FACS-based cytotoxicity assays. 50,000 target cells were co-cultured with T cells in effector/target (E/T) ratio 3:1 overnight. After overnight culture cells were stained with specific antibody panels and SYTOX Blue Dead Cell Stain (Invitrogen by Thermo Fisher Scientific) was added prior to acquisition to identify living cells. Samples were acquired by FACS using fixed flow rates and stable acquisition was validated using Flow-Count Fluorospheres (Beckman Coulter). Co-cultures with activated B cells were stained using anti-CD3 (Alexa Fluor 700, BD Pharmingen), anti-CD19 (PE-Cy7, BD Pharmingen), anti-IgA (PE, Miltenyi), anti-IgG (FITC, DAKO) and anti-IgM (APC, BD). For primary MM, 50,000 patient-derived BM mononuclear cells were used as target cells. Co-cultures were stained with anti-CD3, anti-CD19 (APC, BD Pharmingen), anti-CD45 (FITC, BD), anti-CD38 (PE-Texas Red, Invitrogen), and anti-CD56 (PE-Cy7, BD Pharmingen). MM cells were defined as CD3^neg, CD19^neg, CD45^neg−int, CD38^pos, CD56^pos.

B-cell subsets were analyzed on whole blood by incubating various combinations of monoclonal antibodies, including anti-human CD19 PerCP, anti-IgM APC, CD27 FITC, CD38 FITC, anti-IgD PE, CD21 PE, CD70 PE, CD27 APC, CD24 FITC, CD38 PE, CD183 PE (BD Pharmingen) and CD43 APC (Biolegend, San Diego, CA, USA) monoclonal antibodies, and corresponding isotypes. After staining, blood was lysed by 1 × lysing solution (BD Pharmingen) and washed with phosphate-buffered saline and analyzed. Flow cytometry was performed using FACSCalibur (Becton-Dickenson, San Jose, CA, USA) equipped with argon ion laser emitting at 488 nm (for FITC, PE and APC excitation). Forward and side scatters were used to gate and exclude cellular debris. Ten thousand cells were acquired and analyzed using the Flowjo software (Tree star Inc., Ashland, OR, USA).