Figure 3.

CD47/CD19 co-engagement does not induce receptor clustering or internalization.

(a) Targets clustering were quantified on living Raji B cells using a fluorescence resonance energy transfer applied in flow cytometry (FCET) approach. Anti-CD20 mAb, bivalent or monovalent anti-CD19 antibodies or anti-CD47xCD19 biAb were conjugated to the FRET pair Cy3/Cy5 (donor/acceptor molecules respectively), and energy transfer values were obtained by combining equimolar Cy3- and Cy5-labeled antibodies on Raji B cells after 1 to 4 hours at 37°C. The FRET value was calculated and presented as normalized FCET where the FCET values obtained for each pair Cy3/Cy5 antibodies incubated at 4°C were set at 1 defining a threshold (dashed line) above which a sample is considered positive for targets clustering. The average normalized FCET ± SD were calculated from 3 independent experiments. (b) Images obtained by confocal microscopy of Raji B cells incubated with 10 μg/mL Alexa Fluor 488 (AF488)-conjugated anti-CD20 mAb or anti-CD47xCD19 biAb for 15 min at 4°C or 4 hours at 37°C. The nuclei were counterstained with DAPI. Scale bars represent 10 μm. The white arrows show anti-CD20 mAb clustering. (c) Raji B cells were incubated at 37°C with AF488-conjugated anti-CD20 mAb, anti-CD47xCD19 biAb or bivalent or monovalent anti-CD19 antibodies. Internalization fluorescence ((quenched fluorescence/unquenched fluorescence) x 100) was analyzed over time using flow cytometry. AF488-conjugated hIgG1 isotype control was used as a negative control to assess baseline internalization. The results are depicted as the mean internalized fluorescence ± SEM of 4 independent experiments. The hIgG1 control is used as a negative control to assess baseline internalization. (d) Raji B cells were incubated (4 hours, 37°C) with AF488-conjugated anti-CD20 or anti-CD47xCD19 biAb then with an anti-CD45-APC.Cy7 to delimit B cell membrane. Cells were analyzed using a FlowSight instrument. AF488 fluorescence signals were captured without quenching (nQ) to assess total cell fluorescence and with quenching (Q) to visualize the fluorescence signal from the cytoplasmic compartment. Scale bars represent 10 μm. Statistical analysis was performed using the one way ANOVA test: *p < 0.05, **p < 0.01, ***p < 0.001.