Expansion of Naive B Cells with CD40L

B cell cultures were carried out as described before [36 (link)] with a few adjustments. In short, 3T3 ‐CD40L⁺ were harvested, irradiated with 30 Gy and seeded in B cell medium (RPMI 1640 (Gibco) without phenol red containing 5% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l‐glutamine, 50 μM β‐mercaptoethanol, and 20 μg/ml human apo‐transferrin (Sigma–Aldrich; depleted for human IgG with protein G sepharose (GE Healthcare)) on 96‐well flat‐bottom plates (Nunc) to allow adherence overnight. 3T3 ‐CD40L⁺ were seeded at 10.000 cells per 96‐well. The next day, CD19⁺ B cells were thawed after cryopreservation and CD19⁺CD27⁻IgG⁻ naive B cells were sorted on a FACSAria II. 2.5 × 10⁴ naive B cells were cultured on the irradiated CD40L‐expressing 3T3 fibroblasts in the presence of IL‐21 (50 ng/mL; Peprotech, London W6 8LL, UK) for 6 days. After 48 h or at indicated timepoints either medium or anti‐CD40L clone 5C8 antibodies (13 μg/ml) were added to the cultures.