Supplementary Figure S3 - PDF file 9497K, A: Immunochemical analysis of IL-18BP expression in cells from EOC ascites. Double staining with anti-IL-18BP (red, rabbit mAb clone EP1088Y, Epitomics) and anti-macrophage (brown, mAb HAM-56, Ventana Medical Systems) antibodies is shown. Biotin-labeled goat anti-rabbit followed by alkaline phosphatase-conjugated streptavidin and peroxidase-conjugated anti-mouse (BioSpa) were used as secondary antibodies. Fast Red and DAB (Sigma) served as substrates. Bar=100�m. Some of the cells stained by the anti-macrophage Ab were also stained by anti-IL-18BP Ab (see enlarged inset). However, the brightest IL-18BP positive cells were negative for the anti-macrophage Ab. B: Two-color immunofluorescence analysis of IL-18BP protein versus leukocyte surface markers expression in cells from EOC ascites. Numbers indicate the % of cells in each quadrant. IL-18BP positive cells were CD13 positive and CD14 low or negative (as all the CD14 positive cells were within the CD13 positive population: lower right panel). FITC: fluorescein isothiocyanate; APC: allophycocyanin; PE: phycoerythrin. CD14APC and CD14PE were from Miltenyi Biotec, CD13PE from BD Pharmingen and NKp46PE from Beckman Coulter. Cells were analyzed on a FACSCalibur (Becton Dickinson) flow cytometer
ARTICLE ABSTRACT
Purpose: Interleukin (IL)-18 is an immune-enhancing cytokine, which induces IFN-γ production, T-helper 1 responses, and antitumor effects. In turn, IFN-γ stimulates IL-18–binding protein production, which blocks IL-18 activity. In view of the potential use of IL-18 in epithelial ovarian cancer (EOC) immunotherapy, here, we studied IL-18BP expression and its regulation by cytokines in EOC cells in vitro and in vivo.Experimental Design: Expression and production of IL-18BP in EOC cell lines, primary ovarian carcinomas, and the corresponding normal tissues, patients' serum, and ascites were investigated by immunochemistry, ELISA, screening of gene expression profiles, and reverse-transcription PCR.Results: Analysis of gene expression profiles revealed that IL18BP mRNA is increased in EOC tumors compared with normal ovary cells. Release of IL-18BP was detectable in EOC sera and to a greater extent in the ascites, indicating production at the tumor site. Indeed, immunochemical analyses on cells isolated from the ascites and on tumor sections indicated that IL-18BP is expressed in both tumor cells and tumor-associated leukocytes, which displayed a CD3−CD20−NKp46−CD13+CD14low phenotype. EOC cell lines do not constitutively express IL-18BP. However, its release is inducible both by IFN-γ stimulation in vitro and by xenotransplantation of EOC cells in immune-deficient mice, suggesting a role for the microenvironment. In vitro experiments and immunochemistry indicated that IL-27 is also involved in IL-18BP upregulation in EOC cell lines and primary cells through STAT1 activation. Together, these data indicate that IL-18BP, which is produced in EOC in response to microenvironmental factors, may inhibit endogenous or exogenous IL-18 activity. Clin Cancer Res; 19(17); 4611–20. ©2013 AACR.
