Anti cd40 hm40 3

Proliferation of B cells was measured by dye dilution assays. Splenocytes were isolated and re-suspended in pre-warmed cell culture media (RPMI 1640 [Cellgro] containing 10% FBS, 1% L-glutamine, 1% HEPES, 0.2% gentamicin, and 0.1% 2-ME; Life Technologies). Cells were stained with CellTrace™ Violet (CTV) dye (Invitrogen) per manufacturer’s instructions (5mM stock solution of CTV was prepared by adding 20μL of DMSO), and 1μL of CTV was added per 1 × 10⁶ cells. Labeled cells were incubated with LPS, anti-IgM (AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgM, μ chain specific; Jackson ImmunoResearch Laboratories, Inc), or anti-CD40 (HM40–3; BD Biosciences) with and without IL-4 and incubated at 37°C in 5% CO₂ for 3.5 days. After incubation, cells were stained for flow cytometry as described above. Data was analyzed and proliferation index calculations were made with FlowJo software (Tree Star, Inc).

Sort-purified splenic B-lymphocytes isolated from NOD mice treated with BAFFR-Fc or ctrl mAb were stimulated with 10 μg/ml LPS (Sigma-Aldrich) or 10 μg/ml goat anti-mouse IgG + IgM (H+L) (Jackson ImmunoResearch) plus 1 μg/ml anti-CD40 (HM40-3, BD Biosciences) for 3 days. In another experiment, TACI^high or TACI^low B-lymphocytes isolated from NOD mice were sorted and stimulated with 10 μg/ml LPS (Sigma-Aldrich) for 3 days. IL-10 concentration in culture supernatants was determined using the Mouse IL-10 ELISA MAX kit (Biolegend) according to the manufacturer’s recommended protocol.