Inhibition of EZH2 methyltransferase decreases immunoediting of mesothelioma cells by autologous macrophages through a PD-1–dependent mechanism
Malik Hamaidia, Hélène Gazon, Clotilde Hoyos, Gabriela Brunsting Hoffmann, Renaud Louis, Bernard Duysinx, Luc Willems
Malik Hamaidia, Hélène Gazon, Clotilde Hoyos, Gabriela Brunsting Hoffmann, Renaud Louis, Bernard Duysinx, Luc Willems
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Research Article Cell biology Immunology

Inhibition of EZH2 methyltransferase decreases immunoediting of mesothelioma cells by autologous macrophages through a PD-1–dependent mechanism

Abstract

The roles of macrophages in orchestrating innate immunity through phagocytosis and T lymphocyte activation have been extensively investigated. Much less understood is the unexpected role of macrophages in direct tumor regression. Tumoricidal macrophages can indeed manifest cancer immunoediting activity in the absence of adaptive immunity. We investigated direct macrophage cytotoxicity in malignant pleural mesothelioma, a lethal cancer that develops from mesothelial cells of the pleural cavity after occupational asbestos exposure. In particular, we analyzed the cytotoxic activity of mouse RAW264.7 macrophages upon cell-cell contact with autologous AB1/AB12 mesothelioma cells. We show that macrophages killed mesothelioma cells by oxeiptosis via a mechanism involving enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27–specific (H3K27-specific) methyltransferase of the polycomb repressive complex 2 (PRC2). A selective inhibitor of EZH2 indeed impaired RAW264.7-directed cytotoxicity and concomitantly stimulated the PD-1 immune checkpoint. In the immunocompetent BALB/c model, RAW264.7 macrophages pretreated with the EZH2 inhibitor failed to control tumor growth of AB1 and AB12 mesothelioma cells. Blockade of PD-1 engagement restored macrophage-dependent antitumor activity. We conclude that macrophages can be directly cytotoxic for mesothelioma cells independent of phagocytosis. Inhibition of the PRC2 EZH2 methyltransferase reduces this activity because of PD-1 overexpression. Combination of PD-1 blockade and EZH2 inhibition restores macrophage cytotoxicity.

Authors

Malik Hamaidia, Hélène Gazon, Clotilde Hoyos, Gabriela Brunsting Hoffmann, Renaud Louis, Bernard Duysinx, Luc Willems

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Figure 2

Direct cytotoxicity of RAW264.7 macrophages upon cell-to-cell contact with syngeneic mesothelioma AB1 cells.

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Direct cytotoxicity of RAW264.7 macrophages upon cell-to-cell contact wi...
(A) Experimental design. RAW264.7 macrophages were treated with L-NMMA or apocynin for 24 hours and then further cultivated in the presence or absence of LPS for 24 hours. After 3 washes in PBS, RAW264.7 macrophages were cocultivated with AB1 cells at a 10:1 ratio for 48 hours. (B) AB1 cells and CFSE-labeled RAW264.7 macrophages were monitored by time-lapse microscopy using an LSM 510 (Zeiss) equipped with an environmental chamber maintained at 37°C in a humidified 5% CO2 atmosphere. (C) Cells were fixed, permeabilized, and stained for F4/80 (shown in green) and nitrosylated tyrosine (N-Tyr; shown in blue). Images were acquired using a Zeiss LSM 510 confocal microscope equipped with a ×63-1.4 oil immersion objective. (D) Apoptotic rates of AB1 cells were determined by flow cytometry after staining with the annexin V-FITC kit (Becton Dickinson). Each bar represents the mean ± SD from 8 independent experiments performed in triplicate. (E) AB1 cells were transduced by lentivectors encoding PGAM5 shRNAs (#2 and #5) or a scramble control. The levels of PGAM5 transcripts were measured by reverse transcription quantitative PCR. (F) RAW264.7-induced apoptosis of shRNA-transduced AB1 cells was measured as described in D. Each bar represents the mean ± SD from 6 independent experiments. Statistical significance was evaluated using 1-way ANOVA followed by Tukey’s multiple-comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.