Abstract A55: Human endogenous retroviruses differentially regulate immune checkpoints and modulation of immune responses in serous ovarian carcinoma.
Authors: Strissel Pamela L. , Chiappinelli Katherine B. , Strehl Johanna , Würfel Franziska MB , Beckmann Matthias W. , Baylin Stephen B. , Strick Reiner 
Published: March 2017

Abstract
Purpose: The first aim is to investigate the activation of 7 immune checkpoints at different time points following transfection of three diverse ERV envelope (env) genes in serous ovarian carcinoma cells. The second aim is to determine if the ERV-W1 Syncytin-1 env and ERV-3 protein are expressed in primary serous OvCa tumors and ascites fluids to explore a possible role in the tumor microenvironment.
Introduction: The overall survival of serous OvCa has not greatly improved. We and others have demonstrated up-regulation of interferon (IFN) type I signaling pathway viral defense genes including IFN stimulated genes (ISGs), in a variety of human tumors (ovary, breast, lung) but also in ovarian and colon carcinoma cell lines treated with the DNA methyltransferase inhibitor Aza-C. Aza-C treatment of serous OvCa cell lines leads to an induction of PD-L1 expression as mediated downstream signaling via IFNg. We recently demonstrated that activated ERVs function at the RNA level to ignite the IFN response. Exogenous retroviruses like HIV have evolved to suppress the immune response by up-regulation of PD-L1 on target cells. Tumors mimic regulatory mechanisms involving immunosuppression and inflammation, which we propose involves ERVs.
Experimental Procedures: ERV-3, Syncytin-1 and ERV-W2 env CMV overexpression vectors and a control EGFP-vector were transfected into OvCa serous cell lines (TykNu, A2780 and Hey) and RNA expression of 7 immune checkpoint molecules (LGALS9, PDL-2, CD80, CD276, PD-L1, VTCN1 and PD1) were monitored at days 3 and 7 post transfection using real time PCR. Immunohistochemistry (IHC) and Western analyses using antibodies for Syncytin-1 and ERV-3 were performed with primary serous OvCa tumors. Western and ELISA analyses of serous OvCa ascites were executed to detect Syncytin-1 protein levels. Syncytin-1 protein concentrations were quantified in ascites with two different antibodies using ELISA and correlated to a standard curve based on purified Syncytin-1 levels.
Results: Three serous OvCa cell lines following ERV env transfection showed differences of immune checkpoint activation. TykNu demonstrated the highest fold-levels of induction for LGALS9 (73.1x), PD-L1 (15.2x), PD-L2 (9.8x) and PD1 (6.1x) at day 3 for all ERV expression vectors, which decreased by day 7 except for PD1. A2780 showed increased levels for LGALS9 (7.2x) and PD1 (5.2x) with all ERV expression vectors at day 3, which decreased by day 7. In contrast, Hey cells showed no significant increase of the analyzed immune checkpoint genes. Syncytin-1 and ERV-3 protein was highly expressed in primary OvCa epithelial cells using IHC, compared to patient matched control ovarian tissues. Western analyses of ascites from OvCa patients showed high levels of Syncytin-1 in the supernatant, supporting protein secretion. Absolute quantification using real time PCR of Syncytin-1 and ERV-3 RNA with fractionated OvCa ascites cells demonstrated high amounts of molecules. ELISA results confirmed high levels of Syncytin-1 (98.6 - 525.8 microg/ml) in ascites of primary OvCa and in one relapse patient (413.4 microg/ml), but were virtually undetectable in an ascites from a gastro-intestinal carcinoma and control peritoneal fluids (2.7 microg/ml).
Conclusions: Our findings that various immune checkpoints are differentially regulated following ERV activation in serous OvCa cell lines endorses that analyses of immune checkpoints prior to treatment could help stratify patient therapy. ERVs like exogenous retroviruses not only play an essential functional role at the RNA level via TLR3, but also possibly at the protein level via TLR4 modulation of immune / inflammatory responses.
Citation Format: Pamela L. Strissel, Katherine B. Chiappinelli, Johanna Strehl, Franziska MB Würfel, Matthias W. Beckmann, Stephen B. Baylin, Reiner Strick. Human endogenous retroviruses differentially regulate immune checkpoints and modulation of immune responses in serous ovarian carcinoma.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A55.