Anti cd40

Primary peripheral blood mononuclear cells (PBMC) were isolated by Ficoll separation (Ficoll-Paque, GE Healthcare). Normal CD19 + B cells and CD4 + T cells were prepared from human PBMC by B Cell Isolation Kit II and CD4 + T Cell Isolation Kit, respectively (Miltenyi Biotec). The immunopurified cells were confirmed by flow cytometry. DLBCL cell lines SUDHL4, SUDHL6, and SUDHL8 were purchased from DSMZ. Adult T cell leukemia (ATL) patient-derived TL-Om1 cell line was a gift from Dr. K. Sugamura, Tohoku University, Japan. B95.8 EBV-transformed lymphoblastoid cell lines (LCLs) were previously established by infection of lymphocytes from four independent healthy donors with culture supernatants of the virus producer B95.8 line56 (link). All lymphoid cell lines and primary lymphocytes were cultured in RPMI1640 (GIBCO) supplemented with 10% of FBS (GIBCO) and antibiotics (GIBCO). 293T, 293FT, and MDA-MB-231 cells were maintained in DMEM (Nissui, Japan) with 10% of FBS and antibiotics.
B cell activation was performed by anti-IgM (Jackson Immunoresearch Laboratories), BAFF (R&D Systems), anti-CD40 (R&D Systems), and/or IL-4 (R&D Systems). T cell activation was performed by anti-CD3/CD28 antibodies (Miltenyi Biotec).

Human blood B cells were isolated from human peripheral blood mononuclear cells of healthy donors obtained from the Taipei Blood Donation Center and were cultured as described42 (link). The research involving human subjects was approved by the Institutional Review Board of Academia Sinica and informed consent was obtained from all subjects. The experiments were performed in accordance with the approved guidelines. CD27⁻CD19⁺ and CD19⁺ B cells were purified by RosetteSep (StemCell). Purified human B cells cultured at a density of 1 × 10⁶ cells/mL were stimulated with IL-21 (100 ng/mL; Invitrogen) and anti-CD40 (1 μg/mL; R&D Systems). NCI-H929 (H929) human multiple myeloma cells and SKW6.4 human lymphoblastoid cells were maintained in RPMI 1640 (Life Technologies) containing 10% FBS (Life Technologies), and 2-ME (50 μM; Life Technologies). FLAG-PRDM1-ERD WI-L2 cells were maintained in phenol red-free RPMI 1640 containing 10% C/D FBS (Life Technologies) and Hygromycin B (0.5 mg/mL; invitrogen). All cells were maintained in medium containing penicillin/streptomycin (100 units/mL; Life Technologies).

Human peripheral B cells were treated with KZR-616 as described above for PBMCs and stimulated for 6 days at 0.5x10⁶ cells/mL with 100 ng/mL IL-21 (R&D Systems), 1 µg/mL anti-CD40 (R&D systems), and 5 ug/mL anti-IgM (Jackson ImmunoResearch) to induce plasmablast differentiation. Cells were analyzed by flow cytometry for surface markers including CD19, CD20, and CD38 (BD Biosciences). Supernatants were analyzed for IgG by MSD immunoassay (Meso Scale Diagnostics).

Sort purified SLE naive peripheral blood B cells were cultured at a density of 0.5–1 × 10⁵ B cells per well and healthy donor naive peripheral blood B cells were cultured at a density of 1.5 × 10⁵ B cells per well in 96-well round-bottom plates in a final volume of 200 µl complete medium. Culture medium for all experiments was RPMI 1640 (Invitrogen) supplemented with 10% FCS, penicillin-streptomycin (100 units/ml penicillin, 100 mg/ml streptomycin), 2-mercaptoethanol (55 mM), L-glutamine (2 mM), and HEPES (5 mM). At initiation of culture, B cells were stimulated with a combination of IL-21 (40 ng/ml, MedImmune, LLC), anti-CD40 (0.1 µg/ml, goat IgG, R&D Systems), and anti-IgM F(ab’)₂ (5.0 µg/ml, Jackson ImmunoResearch Laboratories). The concentration of antibodies used in these experiments was determined based on maximal inhibition achieved in extensive titration studies. B cells were cultured for 5 days prior to examination. The healthy donor naive B cells were also stimulated with CpG-B (1 µg/ml, Invivogen). In order to calculate the total number of CD11c^hi B cells present after 5 days of culture, the total number of events within the CD11c^hi gate that was collected in 70 µl of FACS buffer on the BD LSR II flow cytometer (BD Biosciences) was back calculated to the total volume of 100 µl.