Supplementary Figure 1
(A) Raji cells pre-incubated for 48h with increasing concentrations of R406, ibrutinib or CAL-101 were washed and incubated for 1h with ofatumumab (1-100 μg/ml) and 10% human AB serum. Cell viability in CDC assay was measured with PI assay using BD FACScan flow cytometer. The survival of cells is calculated as a percentage of corresponding control cultures incubated with medium and serum without antibody and expressed as a relative viability of cells +/- s.d. (B) Primary cells from MCL patient were incubated for 48h with 1 µM R406, ibrutinib or CAL-101. CD20 levels were determined with flow cytometry as described earlier. For CDC assay cells were incubated for 1 h with rituximab or ofatumumab (1-100 μg/ml) and 10% human AB serum. Cell viability was measured with MTT assay. (C) Ofatumumab-mediated ADCC assays were performed with CFSE/PI staining using flow cytometry as described previously for rituximab. (D) NK cells positively isolated from PBMC were incubated with increasing concentrations of R406, ibrutinib or CAL-101 for 48h. Viability of NK cells was evaluated with PI staining using flow cytometry. Results are presented as a percentage of untreated cells.
Supplementary Figure 2
Raji cells pre-incubated with 1 µM R406, ibrutinib or CAL-101 for 48h were stained with saturating amounts of FITC-conjugated anti-CD46, anti-CD55, anti-CD38, anti-CD19, anti-CD21 or anti-CD22 mAbs for 30 min at RT in the dark. Afterwards, cells were washed with PBS and resuspended in PBS with 4 µg/ml of PI. Results are presented as a percentage from 3 independent experiments of control MFI +/- s.d. of PI-negative cells.