Anti cd20 rituximab

J774A.1 macrophages were cultivated in 96-well plates at 1 × 10⁴ cells per well. After 4 h of incubation at 37 °C, 1.5 × 10⁵ hMB “double-hit” lymphoma or CD19⁺ B-cells derived from CLL patients per well were co-cultured with respective macrophages. Subsequently, this co-culture was treated for 17 h with tyrosine kinase inhibitors and monoclonal antibodies in combination or as mono treatment. To stimulate the ADCP of human cells the human specific anti-CD52 monoclonal antibody alemtuzumab (Genzyme, Cambridge, MA, USA; 10 µg/mL), anti-CD20 obinutuzumab (GA101, Roche, Basel, Switzerland; 1 µg/mL), and anti-CD20 rituximab (Roche, Basel, Switzerland; 20 µg/mL) antibodies were used. Primary human CLL cells were stained with CD19 (fluorescein isothiocyanate (FITC), #363008, BioLegend, San Diego, CA, USA) fluorescent antibody for 15 min at 4 °C before measurement by flow cytometry. Each condition was performed with five replicates. For determination of ADCP GFP⁺, target cells were analyzed using a MACSQuant VYB flow cytometer (Miltenyi Biotec, Berg. Gladbach, Germany). The percentage of ADCP was calculated as follows:

NK cells were assayed for antibody-dependent cellular cytotoxicity (ADCC) against cells of the human B lymphoblastoid cell line 721.221 (221) [44] (link) coated with anti-CD20 (rituximab, Roche, Basel, Switzerland). Purified NK cells were first preincubated with either PD98059 (25 µM) or DMSO (0.05%) for 1 hour and subsequently co-incubated with autologous monocytes at monocyte:NK cell ratios of 0.25∶1, 0.5∶1 and 1∶1 respectively. After 16 hours, rituximab 10 μg/ml and CFSE-labeled 221 cells were added at an effector to target cell ratio of 1∶1. After another four hours of incubation the plates were centrifuged at 500G for 5 minutes, the supernatant discarded, and the cells stained with the Live/Dead Fixable Far Red Dead Cell Stain kit (Invitrogen, Carlsbad, USA). Cell death in target cells and NK cells was assessed using a BD LSRFortessa flow cytometer and BD FACS Diva software as described above.