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 2

Direct involvement of HA in cell death evoked by CD20 and HLA-DR mAbs.

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Direct involvement of HA in cell death evoked by CD20 and HLA-DR mAbs. (...
(A) Cells were treated with tositumomab or L243 mAbs, plated as usual or embedded either immediately or 1 hour later in low–melting point agarose containing SYTOX Green nuclear stain to prevent cell-cell association, and assessed by fluorescence microscopy 4 hours after treatment. Scale bars: 100 μm. (B) CLL cells were treated or not with the actin inhibitor latrunculin B (10 μM) for 45 minutes prior to the addition of various mAbs (10 μg/ml) for 18 hours, when HA was assessed by light microscopy. A typical example of the results is demonstrated. Scale bar: 40 μm. (C) The same samples were then assessed for the extent of cell death following staining with AnV-FITC and PI and flow cytometry. Bars represent the mean cell death (AnV- and PI-positive cells) above cell death observed in the untreated samples + SEM from 7 different CLL samples. Due to heterogeneous levels of basal apoptosis in the CLL samples, these data were expressed as percentage of cell death above control. These data clearly demonstrate that adhesion and death evoked by both anti-CD20 and HLA-DR mAbs in CLL samples are dependent upon actin.