Binding of divalent H-2Kd/IgG2aFc fusion protein to murine macrophage via Fc-FcR interaction

Peptide-MHC class I complex (pMHC) is a specific ligand for TCR recognition, and important for CD8(+) T cell activation. Here we described a genetically engineered divalent class I major histocompatibility complex (MHC) molecule, H-2K(d)/IgG2aFc, a fusion protein consisting of the extracellular domains of H-2K(d), a murine MHC class I molecule, and the Fc region of IgG2a. This fusion protein is expected to attach the H-2K(d) molecule to the surface of murine macrophage (MO) through its Fc portion binding to Fc receptor (FcR) of M phi. cDNAs coding for the extracellular domains of H-2Kd and the Fc region of IgG2a were cloned respectively, and then recombined into plasmid pcDNA3.1(+). The H-2K(d)/IgG2aFc protein was expressed by the plasmid-transfected cell line J558L, and purified from its supernatant with a Staphylococcal Protein A (SPA) column. The fusion protein showed a 58.4 kDa hand as revealed by SDS-PAGE and Western blotting with murine IgG-specific antibody, which consists with that expected for extracellular domains of H-2K(d) heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2K(d) indicated the fusion protein consists of both Fc portion and H-2K(d). Peritoneal M phi of C57BL/6 (H-2K(b)) can be stained with H-2K(d) specific monoclonal antibody (mAb) after incubated with the H-2K(d)/IgG2aFc fusion protein. These results demonstrate the fusion protein can be used to attach the H-2K(d) molecule to the surface of murine M phi, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine M phi, which can be applied to activate antigen-specific cytotoxic T lymphocyte (CTL).