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

Abstract

BackgroundGalectin-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.MethodsHybridomas 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.ResultsWe 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.ConclusionThe 1G3 monoclonal antibody will be a powerful tool to assess galectin-9 expression and distribution especially in diseases related to oncogenic viruses.

Impact of Exogenous Galectin-9 on Human T Cells

“…These results are representative of two independent experiments. ing a predominant immunosuppressive action of gal-9 in diseases like chronic active hepatitis or nasopharyngeal carcinomas related to the Epstein-Barr virus (16,(45)(46)(47). We believe that the activation of some CD4 ϩ Th1 cells by gal-9 is not likely to mitigate its overall immunosuppressive action for at least two reasons.…”

“…Most often, these di-saccharides are part of complex glycans carried by glycoproteins or glycolipids (1). The domains of galectins that directly interact with carbohydrate ligands are carbohydrate recognition domains (CRD) 4 (2). The CRDs are made of about 135 amino acids forming a groove in which the carbohydrate ligand can bind.…”

“…Gal-9 is sometimes detected at the surface of the plasma membrane. Finally, a fraction of gal-9 can be detected in the nucleus (4). 5 Like other galectins, gal-9 has no signal sequence.…”

Galectin‐9: Diverse roles in hepatic immune homeostasis and inflammation

“…Data on the role of Gal‐9 in hepatitis B virus (HBV) infection are relatively sparse. As with HCV, KCs in HBV‐infected liver biopsies express high levels of Gal‐9, and serum levels of this protein are significantly increased compared to uninfected controls . Moreover, TIM‐3 expression is relatively increased on HBV‐specific T cells and correlates with impaired effector function (IFN‐γ production) …”

Role of regulatory T-cells during hepatitis C infection: From the acute phase to post-transplantation recurrence

“…Indeed, high levels of galectin-9 are detected in the serum and liver of HCV-infected patients. Kupffer cells and infected hepatocytes are the principal sources of galectin-9 in the liver [43,44].…”