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

Nirmala Chandralega Kampan; Mutsa Tatenda Madondo; Orla M McNally; Andrew N Stephens; Michael A Quinn; Magdalena Plebanski

doi:10.3389/fimmu.2017.01482

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

Front Immunol. 2017 Nov 6:8:1482. doi: 10.3389/fimmu.2017.01482. eCollection 2017.

Authors

Nirmala Chandralega Kampan^{1

2

3}, Mutsa Tatenda Madondo¹, Orla M McNally², Andrew N Stephens^{4

5

6}, Michael A Quinn², Magdalena Plebanski^{1

7}

Affiliations

¹ Department of Immunology and Pathology, Monash University, Melbourne, VIC, Australia.
² Oncology Unit, Royal Women's Hospital, Melbourne, VIC, Australia.
³ Department of Obstetrics and Gynaecology, Pusat Perubatan Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
⁴ Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, VIC, Australia.
⁵ Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia.
⁶ Epworth Research Institute, Epworth Healthcare, Richmond, VIC, Australia.
⁷ School of Health and Biomedical Sciences, RMIT University, Melbourne, VIC, Australia.

Abstract

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^hiFoxP3⁺ 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.

Keywords: FoxP3; effector T cells; epithelial ovarian cancer; inflammation; interleukin 6; malignant ascites; regulatory T cells; tumour necrosis factor 2.