"Obra Social" La Caixa, Cellex Foundation, and the Health and Science Departments of the Generalitat de Catalunya.

Introduction: A comprehensive analysis of the immune cell infiltrate collected from pleural fluid and from biopsy specimens of malignant pleural mesothelioma (MPM) may contribute to understanding the immune-evasion mechanisms related to tumor progression, aiding in differential diagnosis and potential prognostic stratification. Until now such approach has not routinely been verified. Methods:We enrolled 275 patients with an initial clinical diagnosis of pleural effusion. Specimens of pleural fluids and pleural biopsy samples used for the pathologic diagnosis and the immune phenotype analyses were blindly investigated by multiparametric flow cytometry. The results were analyzed using the Kruskal-Wallis test. The Kaplan-Meier and log-rank tests were used to correlate immune phenotype data with patients' outcome.Results: The cutoffs of intratumor T-regulatory (>1.1%) cells, M2-macrophages (>36%), granulocytic and monocytic myeloid-derived suppressor cells (MDSC; >5.1% and 4.2%, respectively), CD4 molecule-positive (CD4 þ ) programmed death 1-positive (PD-1 þ ) (>5.2%) and CD8 þ PD-1 þ (6.4%) cells, CD4 þ lymphocyte activating 3-positive (LAG-3 þ ) (>2.8% ) and CD8 þ LAG-3 þ (>2.8%) cells, CD4 þ T cell immunoglobulin and mucin domain 3positive (TIM-3 þ ) (>2.5%), and CD8 þ TIM-3 þ (>2.6%) cells discriminated MPM from pleuritis with 100% sensitivity and 89% specificity. The presence of intratumor MDSC contributed to the anergy of tumor-infiltrating lymphocytes.

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Systemic treatment of malignant pleural mesothelioma (MPM) is moderately active for the intrinsic pharmacological resistance of MPM cell and its ability to induce an immune suppressive environment. Here we showed that the expression of bromodomain (BRD) proteins and was significantly higher in human primary MPM cells compared to normal mesothelial cells (HMC). Nanomolar concentrations of bromodomain inhibitors (BBIs) JQ1 or OTX015 impaired patient-derived MPM cell proliferation and induced cell-cycle arrest without affecting apoptosis. Importantly, BBIs primed MPM cells for immunogenic cell death, by increasing extracellular release of ATP and HMGB1, and by promoting membrane exposure of calreticulin and ERp57. Accordingly, BBIs activated dendritic cell (DC)-mediated phagocytosis and expansion of CD8 T-lymphocyte clones endorsed with antitumor cytotoxic activity. BBIs reduced the expression of the immune checkpoint ligand PD-L1 in MPM cells; while both CD8 and CD4 T-lymphocytes co-cultured with JQ1-treated MPM cells decreased PD-1 expression, suggesting a disruption of the immune-suppressive PD-L1/PD-1 axis. Additionally, BBIs reduced the expansion of myeloid-derived suppressor cells (MDSC) induced by MPM cells. Finally, a preclinical model of MPM confirmed that the anti-tumor efficacy of JQ1 was largely due to its ability to restore an immune-active environment, by increasing intra-tumor DC and CD8 T-lymphocytes, and decreasing MDSC. Thereby, we propose that, among novel drugs, BBIs should be investigated for MPM treatment for their combined activity on both tumor cells and surrounding immune-environment.

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