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 3

Involvement of microvilli and plasma membrane cholesterol in mAb-induced HA.

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Involvement of microvilli and plasma membrane cholesterol in mAb-induced...
(A) Raji cells were incubated with tositumomab or L243 Abs (10 μg/ml) for 1 hour. Then samples were collected and processed for TEM as described in Methods. Left panel shows the early stage of intercellular adhesion of Raji cells. Scale bar: 2 μm. Right panel shows high-power view of the area shown in the square demonstrating the involvement of microvilli in HA of Raji cells. Scale bar: 0.5 μm. (B) Assessment of plasma membrane cholesterol in MCD-pretreated Raji cells. MCD-treated cells were harvested and stained with FITC-labeled CTB and assessed by flow cytometry. The prominent reduction of plasma membrane cholesterol observed in MCD-treated cells (MCD) could be reversed by coincubation of cells with cholesterol (MCD/cholesterol). CTB unlabeled cells are shown as a background control (background). (C) Raji cells were pretreated with 10 μM MCD for 30 minutes, then washed and incubated with tositumomab or L243 Abs (10 μg/ml) for 4 hours. Then aggregation of cells was assessed by inverted phase contrast microscopy. Scale bar: 150 μm. (D) Involvement of plasma membrane cholesterol in anti-CD20– and anti–HLA-DR–induced cell death. Plasma membrane cholesterol was modulated as described in B and C and then cells were incubated with Abs (10 μg/ml) or in HBSS. Cell death was assessed 4 hours later. Data represent the average of at least 2 independent experiments + SEM. *P < 0.01; **P < 0.003.