Figure 1.

CD47/CD19 co-engagement inhibits B-cell proliferation triggered by BCR cross-linking.

(a) CFSE-labeled purified human primary B cells were incubated (15 min, RT) with either 10 μg/mL of hIgG1 isotype control, bivalent or monovalent anti-CD19 antibodies, the CD47xCD19 biAb, bivalent or monovalent anti-CD47 antibodies or a combination of monovalent anti-CD19 and anti-CD47 antibodies. Cells were then stimulated with 5 μg/mL anti-BCR (e.g. anti-IgM/IgG) and 1 μg/mL anti-CD40 antibodies for 5 days at 37°C. As controls, B cells were incubated for 5 days with 10 μg/mL hIgG1 isotype control in absence of BCR stimulation. (b) CFSE-labeled primary B cells were incubated (15 min, RT) with either 66.6 nM of hIgG1 isotype control, anti-CD47xCD19 biAb full-length IgG or F(ab)’2 before being stimulated with 5 μg/mL anti-BCR and 1 μg/mL anti-CD40 antibodies for 5 days. As controls, B cells were incubated for 5 days with 10 μg/mL hIgG1 isotype control alone. (a, b) CFSE staining was analyzed by flow cytometry and data presented as percentage of dividing B cells. (C) Human B cells were incubated with 10 μg/mL hIgG1 isotype control or 10 nM ibrutinib (5 days, 37°C); or pretreated with 10 μg/mL of hIgG1 control, anti-CD47xCD19 biAb or anti-CD19 mAb (15 min, RT) before being stimulated with 5 μg/mL anti-BCR (e.g. anti-IgM/IgG) and 1 μg/mL anti-CD40 antibodies (5 days, 37°C). Cells were then stained with a viability marker (BD Horizon 620) to detect live cells by flow cytometry. Graph represents the percentage of viable B cells. Each dot represents one unique donor as a source of B cells and the horizontal bars on each graph show the mean values ± SEM. Statistical analysis was performed using the one way ANOVA test: *p < 0.05, ***p < 0.001, ns = non-significant.