Binding Of Divalent H-2K~d/IgG2aFc Fusion Protein To Murine Macrophage Via Fc-FcR Interaction

The MHC classⅠmolecule contains a light chain (β2 microgloblin,β2m) and a heavy chain ,which includedα1,α2 andα3 regions, the antigen-binding groove composes ofα1 andα2 regions with antigenic peptide fragment resident in it. The mouse MHC is H-2, the class I of it comprises H-2K, H-2D and H-2L loci. The MHC classⅠmolecule plays the central role in the antigen presentation and T cell activation.Recently, the soluble MHC-Ig dimer is a new technology developed to detect antigen-specific T cells. This kind of divalent protein composes 2 ligands for specific T cell receptors linked via covalent disulfide of Ig hinge region. Compared to monomeric molecules, the dimeric protein has higher stability to enhance the interaction between the ligand and TCR. The antigen-binding groove of the soluble MHC-Ig dimer can be loaded with a MHC restricted peptide, forming a pMHC dimer, which can be applied to identify antigen-specific T cell. In additon, it has been reported that the activation of antigen-specific T cells is achieved by pMHC dimer bound to non-cell carrier.In this study, we have successfully constructed and expressed the divalent H-2K~d/IgG2aFc fusion protein, which consists of the extracellular domains of H-2K~d and the Fc region of IgG2a. Furthermore, peritoneal Mφof C57BL/6 (H-2Kb) can be stained with H-2K~d specific monoclonal antibody (mAb) after incubated with the H-2K~d/IgG2aFc fusion protein in vitro. This result demonstrates the fusion protein can be used to attach the H-2K~d molecule via Fc-FcR interaction to the surface of murine Mφ, and provides a novel means to manipulate the T-cell recognized peptide on the surface of murine Mφ, which can be applied to activate antigen-specific cytotoxic T lymphocyte (CTL). Compared to the previous means, this method is much more similar to the na?ve activation of T cell, which is made to be more valuable.1. Construction of H-2K~d /IgG2aFc hybrid geneMethods The H-2K~d/IgG2aFc hybrid gene was constructed by recombinant PCR and the recombinant plasmids pBS-T-H-2K~d(extra) and pGEM-T-IgG2aFc were used as stencil-plates. The PCR products were purified and digested by restriction endonucleases HindⅢand Xbal, and ligated into eukaryotic expression vector pcDNA 3.1(+) which was digested by the same restriction endonucleases. Result DNA sequencing indicated that fragment was according with the sequence of GeneBank. The recombinant plasmid pcDNA 3.1(+)-H-2K~d/IgG2aFc was constructed successfully by double digestion of restriction endonucleases analysis.2. Expression and functional test of the divalent H-2K~d/IgG2aFc fusion proteinMethods To generate stable transfectants, the gene of H-2K~d /IgG2aFc was transfected into J558L cell line by electroporation. High expressing clones were selected under G418 and adapted to multiply in serum-free media for collection. The cultural supernatant was passed over a Staphylococcal Protein A (SPA) column to purify the dimeric H-2K~d/IgG2aFc fusion protein. The ability of the fusion protein to bind with Fc receptors on murine macrophages was confirmed by flow cytometry (FCM).Result A 1602bp fragment was obtained from cell RT-PCR and it confirmed that the H-2K~d/IgG2aFc hybrid gene expressed in J558L cells. The fusion protein showed a 58.4kD band 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. The binding test by FCM showed that peritoneal Mφof C57BL/6 (H-2Kb) can be stained with H-2K~d specific monoclonal antibody (mAb) after incubated with the H-2K~d/IgG2aFc fusion protein. In this study, the divalent H-2K~d/IgG2aFc fusion protein binds to murine macrophage via Fc-FcR interaction to form a single-epitope presemting cell which can be used to activate and expand antigen-specific T cells for adoptive immunotherapy, such as treatment for cancer and virally infected diseases.