PBMC were prepared by Ficoll-Paque density centrifugation. Isolated B cells were prepared from PBMC using the Dynabeads Untouched Human B Cells Kit (11351D, Thermo Fisher Scientific) according to the manufacturer’s recommended protocol. Purity of the B cell suspension was assessed by flow cytometry, indicating a 99% CD19+ cell population consistent with high purity of B cells. B cells were resuspended 0.5 million/ml ml in RPMI 1640 containing 10% heat-inactivated human serum, 2 mM l-glutamine, 1 mM Na-pyruvate, 0.1 mM nonessential amino acids, 50 μM β-mercaptoethanol, streptomycin (100 μg/ml), and penicillin (100 U/ml), alone, or supplemented with IL-4 (20 ng/ml; PeproTech, Rocky Hill, NJ) plus anti-human IgA + IgG + IgM (30 μg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) plus sCD40L (1000 ng/ml; Enzo Life Sciences, Farmingdale, NY). B cells (0.1 million) were added in 0.2 ml per well in 96-well U-bottom tissue culture plates and incubated at 37°C in 5% CO2. After 24 hours, supernatants were harvested and frozen until evaluation.

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