Fig. 6.

R406 and GS-9973 impair rituximab (Rx)-coated CLL cells phagocytosis by human macrophages. a Human macrophages were obtained by culturing monocytes from healthy donor’s peripheral blood for 5 days in RPMI 1640 supplemented with 10% FCS and M-CSF (50 ng/mL) in a 48 well-plate. At day five, human macrophages were treated for 30 min with R406, GS-9973 or DMSO (vehicle) and then CLL cells, that were previously labeled with CFSE (1 µM) and coated or not with Rx (50 µg/mL), were added to the culture. After 2 h, macrophages were trypsinized and evaluated by flow cytometry and phagocytosis was calculated as the percentage of CFSE⁺ macrophages. Results are shown as the mean ± SEM, n = 10. *p < 0.05, **p < 0.01, ***p < 0.001, Friedman test followed by Dunn’s multiple comparison post test. b Macrophages were stained with anti-CD14-PE mAb, and preparations were analyzed by confocal microscopy. 3 × magnification from the original images is shown in the insert. Representative images of the experiment are shown. c The same phagocytosis experiment was performed using macrophages differentiated from CLL patients’ monocytes. Representative dot plots are shown