Interleukin 6 Present in Inflammatory Ascites from Advanced Epithelial Ovarian Cancer Patients Promotes Tumor Necrosis Factor Receptor 2-Expressing Regulatory T Cells

BACKGROUND: Epithelial ovarian cancer (EOC) remains a highly lethal gynecological malignancy. Ascites, an accumulation of peritoneal fluid present in one-third of patients at presentation, is linked to poor prognosis. High levels of regulatory T cells (Tregs) in ascites are correlated with tumor progression and reduced survival. Malignant ascites harbors high levels of Tregs expressing the tumor necrosis factor receptor 2 (TNFR2), as well as pro-inflammatory factors such as interleukin 6 (IL-6) and tumor necrosis factor (TNF). IL-6 is also associated with poor prognosis. Herein, we study the effect of IL-6 and TNF present in ascites on the modulation of TNFR2 expression on T cells, and specifically Tregs. METHODS: Ascites and respective peripheral blood sera were collected from 18 patients with advanced EOC and soluble biomarkers, including IL-6, sTNFR2, IL-10, TGF-β, and TNF, were quantified using multiplexed bead-based immunoassay. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with cell-free ascites for 48 h (or media as a negative control). In some experiments, IL-6 or TNF within the ascites were neutralized by using monoclonal antibodies. The phenotype of TNFR2(+) Tregs and TNFR2(−) Tregs were characterized post incubation in ascites. In some experiments, cell sorted Tregs were utilized instead of PBMC. RESULTS: High levels of immunosuppressive (sTNFR2, IL-10, and TGF-β) and pro-inflammatory cytokines (IL-6 and TNF) were present in malignant ascites. TNFR2 expression on all T cell subsets was higher in post culture in ascites and highest on CD4(+)CD25(hi)FoxP3(+) Tregs, resulting in an increased TNFR2(+) Treg/effector T cell ratio. Furthermore, TNFR2(+) Tregs conditioned in ascites expressed higher levels of the functional immunosuppressive molecules programmed cell death ligand-1, CTLA-4, and GARP. Functionally, TNFR2(+) Treg frequency was inversely correlated with interferon-gamma (IFN-γ) production by effector T cells, and was uniquely able to suppress TNFR2(+) T effectors. Blockade of IL-6, but not TNF, within ascites decreased TNFR2(+) Treg frequency. Results indicating malignant ascites promotes TNFR2 expression, and increased suppressive Treg activity using PBMC were confirmed using purified Treg subsets. CONCLUSION: IL-6 present in malignant ovarian cancer ascites promotes increased TNFR2 expression and frequency of highly suppressive Tregs.