Abstract
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. 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. 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+CD25hiFoxP3+ 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. IL-6 present in malignant ovarian cancer ascites promotes increased TNFR2 expression and frequency of highly suppressive Tregs.
Highlights
Ovarian cancer is one of the most lethal types of cancer in women globally [1, 2]
Our results suggest a critical role for interleukin 6 (IL-6), present in ovarian cancer ascites, in promoting highly functional tumor necrosis factor receptor 2 (TNFR2)+ Tregs, which are shown to be the only Treg subset capable of suppressing TNFR2+ T effector (Teff) in ovarian cancer ascites cultures
Within the CD4+ gate, Tregs were further identified as CD25hiFoxP3+, while effector T cells (Teff) were identified as CD25−FoxP3− (Figure 1C)
Summary
Ovarian cancer is one of the most lethal types of cancer in women globally [1, 2]. This is because the majority of ovarian cancer patients are diagnosed in late stages, with up to one-third of patients presenting with a prominent peritoneal accumulation of fluid called “ascites.” Ascites development is associated with chemo-resistance, disease recurrence [3, 4], and poorer survival in ovarian cancer patients [5,6,7,8,9]. Ovarian cancer ascites further contains a complex reservoir of immune cells and cytokines, harboring immunosuppressive cells as well as inflammatory soluble factors [7, 10, 11]. This unique milleau has been proposed to help tumor cells evade host immunosurveillance, so that tumor cells can continue growing without restriction [3, 7, 9, 10]. Decreasing TNFR2+ Treg levels using cyclophosphamide in mice [21] or panobinostat and azacitidine in humans [19] is associated with improved antitumor immune responses and prolonged 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). We study the effect of IL-6 and TNF present in ascites on the modulation of TNFR2 expression on T cells, and Tregs
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