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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 Article has an altmetric score of 9

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

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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 1

Actin-dependent HA is involved in cell death evoked by mAbs directed against CD20 or HLA-DR.

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Actin-dependent HA is involved in cell death evoked by mAbs directed aga...
(A) Raji cells were incubated with various mAbs (10 μg/ml) for 4 to 6 hours, at which point HA was assessed by light microscopy. The number of plus signs indicates the strength/extent of adhesion as assessed semiquantitatively by microscopic visualization. 20 hours later, samples were assessed for the extent of cell death following staining with AnV-FITC (AnV) and propidium iodide (PI) and flow cytometry. Bars represent the mean cell death (AnV- and PI-positive cells) + SEM from 3 to 7 independent experiments. Representative data are shown in B. A typical “apoptotic” plot is shown for reference following treatment of Raji cells with 4 Gy irradiation. Tos, tositumomab; Ritux, rituximab; NT, no treatment. (C) Raji cells were treated with various actin inhibitors prior to the addition of anti-CD20 or HLA-DR mAbs, and cell death was assessed 4 hours later as previously described. (D) Inhibitor of actin cytoskeleton at noncytotoxic concentrations protects cells from long-term cytotoxicity evoked by both anti-CD20 and HLA-DR mAbs. Prior to the addition of various mAbs (5 μg/ml), Raji cells were treated for 45 minutes with cytochalasin D (CytoD; 0.1 μM). Cell viability was assessed 24, 48, and 72 hours later using Cell Proliferation Kit II (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate [XTT] assay; Roche). Plot represents mean metabolic activity relative to nontreated control from 2 independent experiments run in 3 technical replicates. Data shown represent mean + SEM. (E) Raji cells were treated with DMSO or the actin inhibitor latrunculin B (LatB; 10 μM) prior to the addition of Tos or L243 (10 μg/ml) and assessed for HA 4 to 6 hours later. Original magnification, ×20. Together, these data clearly demonstrate that cell death evoked by both anti-CD20 and HLA-DR mAbs is dependent upon adhesion and that both these processes are dependent upon actin.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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