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.
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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

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Figure 5

Lateral mobility of cellular actin, formation of syncytium, and polarization of mitochondria in cells undergoing HA.

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Lateral mobility of cellular actin, formation of syncytium, and polariza...
(A) AcGFP-actin–expressing Raji cells were treated with either control mAbs (OKT3), tositumomab, or L243 (all at 10 μg/ml) for 4 hours, then studied by FRAP, with flurorescence recovery assessed for 100 seconds. Typical image data is shown for control- and tositumomab-treated cells. Upper panels: virtually no recovery was observed in control mAb–treated cells. Lower panels (Tos): recovery of junctional actin within the cell-to-cell contact area was observed after approximately 80 seconds. Scale bars: 20 μm. Squares indicate regions targeted for photobleaching. (B) Typical fluorescence recovery pattern of AcGFP-actin after photobleaching. Control cells showed no recovery within 100 seconds (left). Recovery of junctional actin fluorescence was shown within 80 seconds after photobleaching in cells treated with anti-CD20 (Tos; center) and anti–HLA-DR (L243; right). (C) Transient cytoplasmic bridges between cells undergoing HA. Phase contrast time-lapse microscopy of Raji cells treated with tositumomab (10 μg/ml). Intervals between images are 5 minutes. Arrows indicate location of bridge formation. Scale bar: 20 μm. (D and E) Peripheral relocalization of mitochondria toward cell-to-cell contact area as assessed by TEM (D) and JC-1 staining (E). Arrows indicate localized mitochondria. Scale bar: 10 μm. (E) JC-1–labeled cells were treated with tositumomab or L243 (10 μg/ml). After that, images were captured detecting monomeric (green) and J-aggregate (red) forms of JC-1. Arrows indicate J-aggregate mitochondria at cell-cell junctions. Scale bars: 10 μm (D); 20 μm (E). (F) Time-lapse video microscopy of JC-1–labeled cells. Red mitochondria represent mitochondria with high membrane potential, migrating toward cell-to-cell contact areas. Interval between images is 1 hour. Scale bar: 20 μm.