Abstract 458: Immune-inhibitory HLA-G is expressed in the tumor microenvironment of Ewing Sarcomas
Authors: Spurny Christian , Altvater Bianca , Kailayangiri Sareetha , Landmeier Silke , Ahlmann Martina , Dirksen Uta , Ranft Andreas , Wiendl Heinz , Hartmann Wolfgang , Wardelmann Eva , Rossig Claudia 
Published: August 2015

Abstract
Ewing Sarcoma (EwS) is an aggressive malignancy of bone and soft tissue which still lacks efficient treatment in case of metastases and relapse. Cellular immunotherapies for EwS are under development, but inhibitory molecules in the tumor microenvironment may counteract antitumor immune responses by preexisting or therapeutic immune effector cells. Here we hypothesized that the non-classical HLA-molecule HLA-G may contribute to immune escape of EwS. HLA-G is a potent inhibitor of both T cells and NK cells. It is naturally expressed on trophoblast cells during pregnancy as well as on mesenchymal stem cells from which EwS cells are thought to originate. We analyzed expression of membrane-bound HLA-G1 by flow cytometry and expression of shedded HLA-G1 and soluble HLA-G5 by ELISA in 14 EwS cell lines with and without stimulation with interferon-γ (IFN-γ). Whereas all cell lines failed to express HLA-G1 both before and after IFN-γ stimulation, and none secreted HLA-G without stimulation, 1 of 14 cell lines (TC-32) responded to IFN-γ stimulation by significant upregulation of soluble HLA-G (p = 0.004). To study HLA-G expression in EwS within their tumor microenvironment, we analyzed paraffin-embedded pretherapeutic tumor biopsies from 35 patients by IHC using the HLA-G specific antibody clone 4H84 and detected HLA-G expression in 12 cases (34%), either on the tumor cells (10/35) and/or on infiltrating lymphocytes (7/35). We further studied the presence of soluble HLA-G and HLA-G+ T cells in the peripheral blood of 19 EwS patients and 15 healthy donors. Serum HLA-G was not increased in the EwS patients compared to healthy controls. Moreover, no significant difference in the proportions of naturally occurring HLA-G+CD4+ (Mean 0.9±0.8% vs. 0.9±0.6%, p = 0.627) or HLA-G+CD8+ (Mean 1.2±1.2% vs. 1.7±1.0%, p = 0.134) suppressor T cells among peripheral blood lymphocytes was found between EwS patients and healthy donors by flow cytometry. Thus, systemic HLA-G secretion and expression is unlikely to have a major role in EwS, but the presence of HLA-G+ cells found in EwS biopsies in a substantial proportion of patients deserves further exploration. To address the potential functional relevance of HLA-G+ cells in the tumor microenvironment, we expressed HLA-G1 in 2 EwS cell lines by retroviral gene transfer. Coincubation of HLA-G-expressing EwS cells with freshly isolated allogeneic NK-cells resulted in suppression of EwS cell lysis by NK cells in 3 of 6 NK cell donors in a flow cytometry based cytotoxicity assay. In detail, HLA-G+ EwS cells suppressed NK-cell cytotoxicity up to 47.0±14.8%, (VH-64, p = 0.001) and up to 87.0±16.0% (WE-68, p = 0.002) compared to mock transduced control.
We conclude that local expression of HLA-G within the tumor microenvironment in EwS is a candidate mediator of immune escape and a potential barrier to cellular immunotherapeutics. Strategies that modulate HLA-G expression may be effective to overcome local immune suppression in this cancer.
Citation Format: Christian Spurny, Bianca Altvater, Sareetha Kailayangiri, Silke Landmeier, Martina Ahlmann, Uta Dirksen, Andreas Ranft, Heinz Wiendl, Wolfgang Hartmann, Eva Wardelmann, Claudia Rossig. Immune-inhibitory HLA-G is expressed in the tumor microenvironment of Ewing Sarcomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 458. doi:10.1158/1538-7445.AM2015-458