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 6

Effect of EPZ on PD-1 expression and activity of anti–PD-1 blockade in AB1/RAW264.7 cocultures.

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Effect of EPZ on PD-1 expression and activity of anti–PD-1 blockade in A...
(A) RAW264.7 macrophages were cultured in the presence of LPS and/or EPZ as described in Figure 3. After labeling with an anti–mouse PD-1 antibody, fluorescence emission was analyzed by flow cytometry using a BD FACSAria. Representative dot plots of FSC-H (x axis) and percentage of PD-1 cells (y axis) are shown. (B) Percentages of RAW264.7 macrophages expressing PD-1 were deduced from 4 independent experiments. Each bar represents the mean ± SD. **P < 0.01, and ***P < 0.001, calculated using 1-way ANOVA followed by Tukey’s multiple-comparisons test. (C) Time-lapse analysis of CFSE-labeled AB1 cells cocultured with RAW264.7 macrophages for 24 hours at a 1:1 ratio in the presence of PE/Cy7–conjugated anti–mouse PD-1 antibody (red). (D) RAW264.7 macrophages were incubated with anti–PD-1 antibody (10 μg/mL; InVivoMAb, BioXcell) or rat IgG2a isotype control for 6 hours and then cocultivated with CFSE-labeled AB1 cells in the presence of annexin V-APC. 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. The number of annexin V+ AB1 cells (percentage) was quantified every 10 minutes for 24 hours. Statistical significance was evaluated using 2-way ANOVA with Bonferroni’s post test. ***P < 0.001. (E) RAW264.7 macrophages were cultured with the EZH2 inhibitor (10 μM EPZ5687) for 24 hours and with anti–PD-1 antibody (10 μg/mL; InVivoMAb, BioXcell) or Rat IgG2a isotype control (10 μg/mL, BD) for 6 hours before inoculation. After 3 washes, RAW264.7 macrophages were coimplanted SC into BALB/c mice with 2 × 106 AB1 or AB12 cells at a 1:3 ratio. Tumor volumes (in mm3) were calculated weekly using the following formula: 4/3 × π × (diameter/2)3. (F) The corresponding survival curve for E. Groups of at least 6 mice were tested in each experimental condition. All data are plotted as mean ± SEM (n = 6). Statistical significance was evaluated using 2-way ANOVA with Bonferroni’s post test (E) and log-rank χ2 test (F). *P < 0.05; **P < 0.01; ***P < 0.001.