Anti mouse cd40

For mice, splenic B cells were purified from WT, Ifnar^−/−, Tbx21^−/−, and Ifngr^−/− mice by CD43 microbead depletion (Miltenyi Biotec). Purified B cells were cultured in RPMI at 37°C for 48 h at 10⁶ cells/well in a 96-well plate with or without 5 ng/ml R848, 1 µg/ml anti–mouse IgM F(ab')₂ fragment (Jackson ImmunoResearch Laboratories, Inc.), 1 µg/ml anti–mouse CD40 (SouthernBiotech), recombinant mouse IFN-γ or IFN-β (200 and 300 U/ml, respectively; BioLegend), and 500 nM ruxolitinib or 500 nM tofacitinib. For co-culture experiments, congenically marked CD45.1 WT and CD45.2 Ifngr^−/− B cells were stimulated together in 96-well plates (10⁶ total cells/well). B cell surface marker and transcription factor expression was evaluated by flow cytometry. Cell proliferation was evaluated by Cell Trace violet (Invitrogen) dilution.
Total human B cells were purified by the Human B Cell Isolation kit II (Miltenyi Biotec). Total B cells were plated at 5 × 10⁴ in a 96-well plate for 24 or 72 h with 10 µg/ml anti-IgM (Jackson ImmunoResearch Laboratories, Inc.), 5 µg/ml CD40L (PeproTech), or 3.75 µg/ml R848 (InvivoGen) with or without 10 µg/ml IFN-γ (R&D Systems). For JAK inhibitor experiments, ruxolitinib or tofacitinib was added at 100 nm or 500 nm at the initiation of culture. Cells were analyzed at 24- and 72-h time points.

Splenic B cells were purified from WT, Ifngr^−/−, Tbx21^−/−, and Stat1^−/− mice by CD43-microbead depletion (Miltenyi Biotec). Purified B cells were cultured in RPMI-1640 medium (supplemented with 10% FCS, 1% penicillin-streptomycin, sodium pyruvate, Hepes, glutaMAX, and 0.1% β-ME) at 37°C for 48 h. B cells were seeded at a density of 10⁶ cells/well in a 96-well plate with or without R848 (5 ng/ml); anti–mouse IgM F(ab′)₂ fragment (1 µg/ml, Jackson ImmunoResearch, Inc.); anti–mouse CD40 (1 µg/ml, SouthernBiotech); recombinant mouse IFN-γ (200 U/ml, BioLegend); and ruxolitinib (500 nM) or tofacitinib (500 nM). B cell surface markers were evaluated by flow cytometry. Cell proliferation was evaluated by Cell Trace Violet (Thermo Fisher Scientific) dilution. IL-6 in the supernatants and serum was measured by mouse IL-6 ELISA (eBioscience).