Plasmablast Formation from B Cells

B cell stimulation with a minor cocktail of stimuli was performed to study the effect of IL-21, co-stimulation, and BCR activation on plasmablast formation. CD19⁺ B cells were isolated via CD43 negative selection with CD43 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) (purities ≥85%). B cells were incubated with anti-IL-21R antibody ATR-107 (10 μg/ml, Pfizer) or isotype-matched control (10 μg/ml IgG1-Fc, R&D systems). Next, cells were stimulated with 5 μg/ml soluble anti-CD40 (Bioceros, Utrecht, The Netherlands), 10 μg/ml goat-anti-human IgM (Jackson Immunoresearch, West Grove, PA, USA) and human recombinant IL-21 (100 ng/ml, eBioscience). Subsequently, the presence of plasmablasts on day 0 and the differentiation of memory B cells into plasmablasts on day 8 were determined with flow cytometry. Plasmablasts were defined as CD19^posCD27^highCD38^high cells (16 (link)). The following MoAbs were used: CD19 BV510 (Biolegend), CD27 Pe-Cy7 (eBioscience), IgD APC-Cy7 (Biolegend), and CD38 BV421 (BD Biosciences). In addition, viability staining with 7-AAD PerCP was performed (BD Biosciences).