Background

Membrane proteins such as cytokine receptors and G protein-coupled receptors have been regarded as medicinal drug targets, and generation of antibodies reactive with the native form of such membrane proteins would lead to antibody-drug development. However, most antibodies generated so far by immunization with a recombinant protein produced in Escherichia coli react only with the immunizing recombinant proteins, but not with the native proteins on cell surfaces. The same problems have often been experienced with immunization with peptides as antigens. In order to generate mAbs which could recognize the native proteins, we have developed the original DNA-immunization method in which plasmid DNA is directly transferred into mouse skeletal muscle in vivo utilizing Escherichia coli (E. coli) GroEL (Fujimoto et al., 2009; Takatsuka et al., 2011) and hyaluronidase. E. coli GroEL, is a molecular chaperone that is responsible for the transportation and refolding of proteins and GroEL fusion proteins are highly expressed in the soluble fraction (Furutani et al., 2005). Further, E. coli GroEL also acts as an adjuvant via TLR4 (Fujimoto et al., 2012). Pre-treatment with hyaluronidase enhances gene expression by prevention of tissue damage (McMahon et al., 2001). Furthermore, low molecular weight hyaluronan produced by hyaluronidase in vivo provides adjuvant effects via TLR2 and TLR4 (Scheibner et al., 2006, Gariboldi et al., 2008). The striated muscle is an excellent tissue for taking up and expressing genes that are transferred in the form of plasmid DNA (Wolff et al., 1990 and 1992, Davis et al., 1993; Ulmer et al., 1993; Aihara and Miyazaki, 1998). The longevity of myofibers allows the stable expression of transfected exogenous genes. Once induced, the expression of the transduced gene can last for at least 70 days thereafter (Wolff et al., 1990 and 1992; Davis et al., 1993; Ulmer et al., 1993; Yamazaki et al., 2011) and the expressed antigens are continuously exposed to the host immune system.

In the present paper, we report an application of the mouse DNA-immunization method in rats in order to generate anti-mouse IL-9R mAbs. IL-9 receptor (IL-9R) is composed of the IL-9Rα and common γ chains and is expressed on mast cells, basophils, ILC2 cells and others (Renauld et al., 1992, Bauer et al., 1998; Noelle and Nowak, 2010; Wilhelm et al., 2011; Licona-Limón et al., 2013). IL-9 is produced by various immune cells, such as activated T cells (exemplified by T_H9 cells), mast cells, eosinophils, and type 2 innate lymphoid cells (ILC2 cells), and induces various functions depending on the target-cell types expressing its receptor (IL-9R) (Gounni et al., 2000; Stassen et al., 2000; Noelle and Nowak, 2010; Wilhelm et al., 2011; Licona-Limón et al., 2013). Thus, IL-9/IL-9R signaling is mainly involved in immune responses such as allergic inflammations. We successfully generated four rat-mouse hybridomas producing mAbs that react with the native form of mouse IL-9R on cell surfaces. Using these mAbs, we found that IL-9R is selectively expressed on memory B cells among B-lineage cells in the T-cell-dependent immune response. We also found that IL-9R is induced on B cells by stimulation through CD40, but suppressed by IL-4 or IL-21, which may explain the minimum expression of IL-9R on germinal center B cells (Takatsuka et al., 2018). Thus, rat mAbs generated by the DNA-immunization method turned out to be quite reliable tools in staining cell surface proteins, and this method can be extended to immunization for various cell surface antigens against which specific mAbs are yet to be obtained.