In vitro generation of monocyte-derived dendritic cells with granulocyte–macrophage colony-stimulating factor and interleukin-4 was performed as previously described.15 B cells were then cultured as 1 × 106 cells/well in a 24-well flat-bottom culture dish in X-Vivo 15 serum-free cell culture medium (Cambrex, Charles City, IA). To test the shaving reaction, RTX

antibody (MabThera® from Roche, Basel, Switzerland) was added in a final concentration of 5 μg/ml. Syngeneic monocytes were added in titrated numbers up to 1 × 106 cells/well. After 18 hr, cells were harvested and labelled with relevant antibodies for flow cytometry. AZD9668 Based on flow cytometry data, mean fluorescence intensity (MFI), % shaving was defined as (1 − [MFI of monocyte containing RTX sample − MFI of monocyte containing isotype control/MFI of RTX sample − MFI of isotype control]) × 100. Cells from co-cultures were labelled with FITC-conjugated anti-RTX antibody (Clone MB2 A4 from AbD Serotec, Dusseldorf, Germany) or a relevant isotype control (IgG2a) and the effect of RTX was tested. Related antibody combinations where used when testing alternative anti-CD20 antibodies.

After labelling and washing, Regorafenib cells were resuspended in PBS containing 1% BSA as running buffer and directly analysed on a FACSCalibur (San Jose, CA) using bd cell quest pro software (Becton Dickinson, Franklin Lane, NJ). Analyses of monocytes and B cells were separated using gates defined by forward and side scatter. In separate experiments, cell viability including the effect of CDC was tested

with a commercial kit from BD (Becton Dickinson) using FITC-annexin V and propidium iodide. Human IgG was from Sigma (St Louis, MO), anti-CD14 and anti-CD64 was from BD. Type I and type II anti-CD20 antibodies were a kind gift from Mark S. Cragg and Claude H.T. Chan. In experiments, testing the effect of hyperosmolar sucrosis, 0·4 m sucrosis (Sigma) was added to the monocyte–B-cell co-culture. Similarly, 80% active human AB serum was used when testing CDC. In experiments with blockade of protease activity, 10 mm EDTA (Sigma) was used and 3 mm PMSF, 2·8 mg/ml aprotinin, 20 nm bestatin hydrochloride, 5 mm 3-mercaptopyruvate sulfurtransferase 1,10-phenanthroline monohydrate, 5 μm phosphoramidon disodium salt and 5 μg/ml α2-macroglobulin (all from Sigma) were used for inhibition of specific classes of proteases. Monocyte-mediated shaving of RTX/CD20 complexes from the surface of CD20+ cells has recently been reported as a major obstacle for RTX treatment in haematological malignancies.13 Our results here confirm the shaving reaction and demonstrated monocyte-mediated shaving of RTX antibody from the surface of CD19+ B cells. This phenomenon was monocyte number-dependent, not evident with 1 × 104 monocytes, increased at 1 × 105 and almost complete after the addition of 4 × 105 to 5 × 105 monocytes (Fig. 1).