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.
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.