Monoclonal antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells
Andrei Ivanov, … , Tim M. Illidge, Mark S. Cragg
Andrei Ivanov, … , Tim M. Illidge, Mark S. Cragg
Published July 20, 2009
Citation Information: J Clin Invest. 2009;119(8):2143-2159. https://doi.org/10.1172/JCI37884.
View: Text | PDF
Research Article

Monoclonal antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells

Abstract

mAbs are becoming increasingly utilized in the treatment of lymphoid disorders. Although Fc-FcγR interactions are thought to account for much of their therapeutic effect, this does not explain why certain mAb specificities are more potent than others. An additional effector mechanism underlying the action of some mAbs is the direct induction of cell death. Previously, we demonstrated that certain CD20-specific mAbs (which we termed type II mAbs) evoke a nonapoptotic mode of cell death that appears to be linked with the induction of homotypic adhesion. Here, we reveal that peripheral relocalization of actin is critical for the adhesion and cell death induced by both the type II CD20-specific mAb tositumomab and an HLA-DR–specific mAb in both human lymphoma cell lines and primary chronic lymphocytic leukemia cells. The cell death elicited was rapid, nonapoptotic, nonautophagic, and dependent on the integrity of plasma membrane cholesterol and activation of the V-type ATPase. This cytoplasmic cell death involved lysosomes, which swelled and then dispersed their contents, including cathepsin B, into the cytoplasm and surrounding environment. The resulting loss of plasma membrane integrity occurred independently of caspases and was not controlled by Bcl-2. These experiments provide what we believe to be new insights into the mechanisms by which 2 clinically relevant mAbs elicit cell death and show that this homotypic adhesion–related cell death occurs through a lysosome-dependent pathway.

Authors

Andrei Ivanov, Stephen A. Beers, Claire A. Walshe, Jamie Honeychurch, Waleed Alduaij, Kerry L. Cox, Kathleen N. Potter, Stephen Murray, Claude H.T. Chan, Tetyana Klymenko, Jekaterina Erenpreisa, Martin J. Glennie, Tim M. Illidge, Mark S. Cragg

×

Figure 4

Peripheral relocalization of cellular actin in cells undergoing HA after treatment with tositumomab.

Options: View larger image (or click on image) Download as PowerPoint
Peripheral relocalization of cellular actin in cells undergoing HA after...
(A) Raji cells were incubated with the anti-CD20 Ab tositumomab or L243 (10 μg/ml), washed, and sedimented onto poly-l-lysine–coated microscope slides. After fixation with 1% paraformaldehyde in PBS, cells were stained with Alexa Fluor 594–labeled phalloidin (red). DNA was counterstained with DAPI (blue). Lower panels represent higher-power images. Scale bars: 60 μm. (B) Time-lapse microscopy of Raji cells expressing AcGFP-labeled actin. After addition of tositumomab or L243 (10 μg/ml), cell suspensions were put into a glass-bottom Petri dish and assessed at 37°C under inverted time-lapse microscopy. Images were obtained every 2 minutes (see Supplemental Video 1). The interval between images displayed is 6 minutes, except for between the final 2 images (50 minutes). Scale bars: 15 μm. (C) The total amount of cellular actin was assessed by Western blotting. α-tubulin was assessed as a loading control. Samples were taken 24 hours after stimulation. (D) Image analysis of F-actin signal area following treatment with control mAbs (OKT3) or tositumomab. Cells were sedimented onto poly-l-lysine–coated slides and stained with phalloidin–Alexa Fluor 594; images were taken. The area of Alexa Fluor–positive signal was then measured using Image-Pro Plus software, version 6.3 (Media Cybernetics), and expressed graphically.