Construction Of Human CD160 Transgenic Cells And Preparation Of Anti-human CD160 Monoclonal Antibody

Glycosylphosphatidylinositol(GPI)-anchor protein CD160 is an 27 kDa glycoprotein that belongs to IgSF(Immunoglobulin superfamily). Its gene locates on chromosome 1q21.1 of Homo sapiens. It is found expressed on cytotoxic cells with a single IgV-like domain. Because of different alternative splicing, there are four isoforms of CD160 in NK cells. All previous studies have been on the GPI-anchored CD160 isoform. CD160 mRNA and protein are highly expressed on human CD56_dimCD16⁺ NK cells, NKT cells, and all intestinal intraepithelial T cells (IEL) and is not found on B or myeloid cells. The tissue distribution of CD160 mRNA in human shows high expression in spleen, small intestine and peripheral blood lymphocytes, and low expression in brain, liver, heart and thymus. CD160 recognize a broad range of classical and non-classical human MHC-I molecules on NK cells, and activate NK cells in immune response. In addition, CD160 that bound across to HVEM of TNFR superfamily can inhibit the proliferation of CD4⁺ T cells and the secretion of cytokine, reduce the phosphorylation of CD3ζchain and generate a new co-inhibition signaling pathway, which plays a negative regulation in immune response. Because it is late to discover this new signaling pathway, the functions of CD160 among many diseases are unknown. Here our aim is to clone the gene of CD160, establish the transgenic cell lines of CD160 and prepare the monoclonal antibody of CD160, which will provide useful tool in future work.Partâ… Cloning of human 160 gene, establishment of transgenic cell lines and preliminary study of the biological function of the transfectantObjective: To clone human CD160 gene, construct recombinant vectors carrying human CD160 gene, which can stably expressed in L929 cell line, and examine and certify whether it is functional or not.Methods: The CD160 gene was amplified by RT-PCR from human peripheral blood mononuclear cells, and cloned into pMD19-T vector. Besides, we amplified transmembrane region gene by PCR using the template from the whole cDNA of VSIG4 gene. After double enzymatic digestion of pMD19-T vector and transmembrane region gene, we recovered two genes and inserted them into eukaryotic expressing vector pIRES2-EGFP. The recombinant vector was then transfected into L929 cells. After being selected by G418, transgenic cell line expressing stably human CD160 was established and named L929/CD160TMV. Then the expression of CD160 on transfected cell line and co-culture of this transfectant with L929/HVEM were analyzed by FCM.Results: The gene of CD160 was successfully cloned, and its recominant vector was constructed. After screening by FCM, the transfectant stably expressing human CD160 protein on the cells membrane was established successfully. This transfectant can bind to another transgenic cell line L929/HVEM through co-culture.Conclusion: The transgenic cell lines stably expressing human CD160 molecule on the cells surface have been obtained and afford effective immunogen for preparing mouse anti-human CD160 monoclonal antibody.Partâ…¡Preparation of the monoclonal antibodies against human CD160Objective: To prepare mouse anti-human CD160 monoclonal antibodies and provide useful means for study the effect of CD160/HVEM interaction on T cells.Methods: BALB/c mice were immunized with human CD160 transfectant as immunogen. Mouse spleen B cells were fused with mouse plasmocytoma cells SP2/0. Hybridoma cells were screened with human CD160 transgenic cell lines. Fast-strip analysis was performed to identify Ig subclass of the generated monoclonal antibodies. The methods of indirect immunofluorescence and Dot-blot were employed to identify the specificity of generated mAb. The epitopes recognized by mAbs were analyzed by competition binding assay.Results: After multiple screening and subcloning, one monoclonal antibody named 3E2 was obtained. The isotype of the mAb was shown to be IgG1 withκlight chain. Indirect immunofluorescence and Dot-blot showed that mAb 3E2 could bind to CD160 transfectant specifically. The competition binding test conducted by 3E2 and commercialized IgM mAb of CD160 was carried and showed that they bound to different epitopes of CD160.Conclusion: One anti-human CD160 monoclonal antibody was generated, and provide useful tool for studying the inhibitory role of CD160/HVEM interaction in T cell.In summary, the transgenic cell lines stably expressing human CD160 molecule on the cells surface have been obtained and afford effective immunogen for preparing mouse anti-human CD160 monoclonal antibody. One anti-human CD160 monoclonal antibody was generated, and provide useful means for studying CD160/HVEM co-inhibitory signal on T cell immune response.