A novel monoclonal antibody for detection of galectin-9 in tissue sections: application to human tissues infected by oncogenic viruses

Background: Galectin-9 is a mammalian lectin which possesses immunosuppressive properties. Excessive production of galectin-9 has been reported in two types of human virus-associated diseases chronic hepatitis C and nasopharyngeal carcinoma associated to the Epstein-Barr virus. The objective of this study was to produce new monoclonal antibodies targeting galectin-9 in order to improve its detection in clinical samples, especially on tissue sections analysed by immunohistochemistry. Methods: Hybridomas were produced through immunization of mice with the recombinant c-terminus part of galectin-9 (residues 191 to 355 of the long isoform) and semi-solid fusion of spleen cells with Sp2/0 cells. Monoclonal antibodies were characterized using ELISA, epitope mapping, western blot and immunohistochemistry. Results: We selected seven hybridomas producing antibodies reacting with our recombinant c-terminus galectin-9 in ELISA. Five of them reacted with the epitope “TPAIPPMMYPHPA” (common to all isoforms, residues 210 to 222 of the long isoform) and stained all three isoforms of galectin-9 analysed by western blot. One of them, 1G3, demonstrated very good sensitivity and specificity when used for immunohistochemistry. Using 1G3, we could confirm the intense and constant expression of galectin-9 by Epstein-Barr virus positive malignant cells from nasopharyngeal carcinomas. In most samples, specific staining was detected in both cytoplasm and nuclei. Galectin-9 was also detected in liver biopsies from patients infected by the human hepatitis C or B viruses with expression not only in inflammatory leucocytes and Kupffer cells, but also in hepatocytes. In contrast, galectin-9 was virtually absent in non-infected liver specimens. Conclusion: The 1G3 monoclonal antibody will be a powerful tool to assess galectin-9 expression and distribution especially in diseases related to oncogenic viruses. Background Galectin-9 is a β-galactoside binding lectin of mammalian origin which possesses two distinct carbohydrates domains linked together by a peptide sequence of 14, 26 or 58 aminoacids depending on the isoform, respectively S, M or L isoform. Galectin-9 holds multiple immunomodulatory properties and an overall predominantly immunosuppressive function. In the context of murine immunity, galectin-9 has been shown to play a key role in a regulatory feed-back essential for a physiological