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
View: Text | PDF
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

×

Figure 3

Effect of EZH2 inhibition by EPZ on RAW264.7-mediated killing of AB1 cells.

Options: View larger image (or click on image) Download as PowerPoint
Effect of EZH2 inhibition by EPZ on RAW264.7-mediated killing of AB1 cel...
RAW264.7 macrophages were incubated with the EZH2 inhibitor (10 μM EPZ5687) for 48 hours and treated or not with LPS for 24 hours. (A) RAW264.7 macrophages were labeled with an anti-H3K27me3 antibody, stained with Draq5, and analyzed by confocal microscopy. Magnification 40×. (B) The rMFI corresponds to the ratio of fluorescence intensities associated with H3K27me3 and Draq5. (C) RAW264.7 macrophages were fluorescently labeled with antibodies against pan-Histone 3 or H3K27me3 and analyzed by flow cytometry using a FACSaria flow cytometer (Becton Dickinson). The rMFI represents the ratio of fluorescence intensities associated with H3K27me3 and pan-H3. (D) Intracellular ROS were measured by flow cytometry using the cell-permeant H2DCFDA probe. MFI, mean fluorescence intensity. (E) Nitrites (in μM) in the cell SNs were quantified by the Griess reaction assay. (F) RAW264.7 macrophage–conditioned SNs were added to AB1 cell cultures as described in Figure 1A. Apoptosis was determined by flow cytometry after labeling with annexin V-FITC. (G) CFSE-labeled RAW264.7 macrophages were cocultivated for 48 hours with AB1 cells at a 10:1 ratio. Apoptotic rates of CFSE– AB1 cells were determined by flow cytometry after staining with the annexin V-FITC kit (Becton Dickinson). Each bar represents the mean ± SD. Statistical significance was evaluated using the paired Student’s t test (B and C) and 1-way ANOVA followed by Tukey’s multiple-comparisons test (D–G). *P < 0.05; **P < 0.01; ***P < 0.001. (H) CFSE-labeled RAW264.7 macrophages were cocultivated for 24 hours with AB1 cells at a 1:1 ratio in the presence of annexin V-APC (red fluorescence). The cells were monitored by the IncuCyte S3 Live-Cell imaging system (Essen Bioscience) placed in an incubator maintained at 37°C in a humidified 5% CO2 atmosphere. (I) RAW264.7 macrophages pretreated with EPZ and/or LPS were cocultured with CFSE-labeled AB1 cells. The number of CFSE+ (AB1) annexin V+ events was determined every 10 minutes for 24 hours using the IncuCyte S3 Live-Cell imaging system. Each curve is the average of 5 independent experiments performed in triplicate.