Interleukin 5

Splenocytes were enriched for B cells by negative selection using the VarioMACS magnet, LD depletion columns, streptavidin microbeads (Miltenyi Biotec, Germany) and biotin-conjugated mouse anti-CD43 (S7). Enriched B cells were stained using the CellTrace Violet Cell Proliferation Kit (ThermoFisher) at a concentration of 1.5 μM. Enriched B cells or murine 38B9 cells were cultured in were cultured in complete RPMI-1640 containing 10% fetal bovine serum (Wisent, St. Bruno, QC, Canada), 5 × 10^–5 M β-mercaptoenthanol (Sigma-Aldrich, St. Louis, MO, United States), 0.01 M HEPES (Sigma-Aldrich), and 1X penicillin/streptomycin/L-glutamine (Wisent). WEHI-279 cells were cultured in complete DMEM medium containing 4.5 g/L glucose (Wisent). Cells were maintained in 5% CO₂ at 37°C. B cells were stimulated with LPS 0111:B4 (10 μg/ml, List Biological Laboratories, Campbell, CA, United States), 100 ng/ml CD40L (R&D Systems, Minneapolis, MI, United States), 10 ng/ml Interleukin-4 (R&D Systems), and/or 10 ng/ml Interleukin-5 (R&D Systems). Cultured plasmablasts were analyzed by flow cytometry on day 3, 4, or 5 of culture.

After purification, the two cell types were cultured in a 96 round bottom wells (Costar, Corning Incorporated, NY, USA) at different ratio in complete RPMI in the presence of interleukin-5 (20 ng/ml) (R&D Systems, Minneapolis, MN, USA): NK cells alone (shown as ratio 1∶0), eosinophils alone (shown as ratio 0∶1) and NK cells: eosinophils at the ratios 1∶1; 5∶1; 10∶1. Cultures were performed for 3 or 12 hours as indicated in the figures.