In Vitro B Cell Differentiation Assays

In vitro naive and memory B cell differentiation assays were performed as reported previously (8 (link), 13 (link)). In short, irradiated 3T3 fibroblasts expressing human CD40L were cocultured with purified naive mature (CD19⁺CD38^–/dimCD27^–; primary response) or memory (CD19⁺CD38^–/dimCD27⁺; recall response) B cells in the presence of rhIL-21 (50 ng/ml; Thermo Fisher Scientific). For naive B cell cultures, rhIFN-γ (50 ng/ml; Peprotech) was added with and without CpG-ODN (10 μg/ml; Invivogen) to induce class switching and ASC development, respectively (8 (link)). After 6 (recall response) or 11 (primary response) days of culturing, viable (live/dead^–) CD19⁺ cells were examined using flow cytometry. The supernatants were collected and stored at –80°C until further use.