Fig 2: WIP Binding to Actin Influences CD19 Mobility and CD81 Expression on the B Cell Surface(A) Single-particle tracking (SPT) of CD19 on WT and WIPΔABD B cells settled on nonstimulatory coverslips. Shown is the diffusion coefficient (D) in square micrometers per second. The median of 300 representative tracks is given as a value and indicated by black bars. 1,000–2,000 tracks were analyzed with a minimum of 20 cells from 2 experiments.(B) Trajectories of CD19 in WT (left) and WIPΔABD B cells (right) showing diffusion of single particles over 10 s and magnified regions from white rectangles. Scale bar, 2 μm.(C) Analysis of the CD19 diffusion coefficient plotted in a logarithmic histogram and fitted to two distinct populations exhibiting slower (dark gray) and faster (light gray) diffusion on WT and WIPΔABD B cells.(D) The percentage of the fast-diffusing population calculated from the histograms.(E and F) Flow cytometric analysis of CD19 (E) and CD81 (F) on the surface of WT and WIPΔABD B cells. Quantification shows the gMFI (mean ± SEM).See also Video S1.

Fig 3: CD47/CD19 co-engagement inhibits BCR-induced cytokines production by human B cells.Purified human B cells were pretreated with 10 μg/mL hIgG1 isotype control, anti-CD47xCD19 biAb, bivalent anti-CD19 or 10 nM ibrutinib and then stimulated with 5 μg/mL anti-BCR and 1 μg/mL anti-CD40 antibodies. As controls, B cells pretreated with 10 μg/mL hIgG1 isotype control were left unstimulated. After 5 days of incubation at 37°C, supernatants were harvested and IL-6 (a) and TNF (b) levels were measured by a multiplexing assay. Dashed lines represent the lower limit of cytokine detection. Data represent the mean values ± SEM of a minimum of 6 independent donors. Statistical analysis was performed using the one way ANOVA test: **p < 0.01, ***p < 0.001.

Fig 4: BCR-induced gene expression changes in response to CD47xCD19 co-engagement.Purified human primary B cells were pretreated (15 min, RT) with 10 μg/mL hIgG1 isotype control, anti-CD47xCD19 biAb or bivalent anti-CD19 and then stimulated with 5 μg/mL anti-BCR and 1 μg/mL anti-CD40 antibodies. After incubation (4 hours, 37°C), total RNA were isolated for subsequent investigation by RNA sequencing. (a). Number of differentially expressed features and their functional classification identified in response to BCR stimulation. (b) Heatmap of the genes differentially expressed between the hIgG1-treated group and anti-CD47xCD19 biAb-treated group. The color key represents fold of induction of each gene over the unstimulated control. Genes are classified according to their biological function. (c) Relative expression levels of the genes differentially expressed between the hIgG1-treated group and the anti-CD47xCD19 biAb-treated group or (d) the bivalent anti-CD19 mAb-treated group. The values were normalized to the mean expression level in the hIgG1-treated group, which was arbitrarily set as 1. Data shown are the mean values ± SEM of each gene from three B cell donors. Statistical analysis was performed using the paired T test: *p < 0.05, **p < 0.01, ***p < 0.05.

Fig 5: 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.