Establishment Of Human HVEM Transfected Cell Line And Preparation Of Mouse Anti-human HVEM Monoclonal Antibody

HVEM (Herpesvirus-entry mediator) is the only one signaling molecule that can bind with tumor necrosis factor superfamily member LIGHT ,and aslo combind with immunoglobulin family member BTLA.It is known as a bidirectional switch regulating the immune response such as T-cell activation in a costimulatory or coinhibitory fashion .HVEM is the media of the herpes simplex virus entrying into the body ,and also a member of tumor necrosis factor (TNF) receptor superfamily and co-stimulatory molecules. HVEM is the receptor of LIGHT and LTα3 , both of which belongs to TNFR and can interact with herpes simplex virus gD glycoprotein (HSV1-gD),LTα3,BTLA and newly discovered ligand moleucle CD160. Combiding with LIGHT, LTα3 ,HVEM can produce positive signals for T cell activation and proliferation,but also contact with BTLA, CD160 passing negative signals to inhibit T cell activation and proliferation. Thus, HVEM can act as a mediating positive and negative signaling roles in signal pathway. HVEM and TNFR / TNF superfamily and CD28/B7 superfamily formed between the micro-cross network across the costimulatory molecule interaction within the family, the co-stimulatory signals and inhibitory signals coordinated collaborative linked together to form the one pair of T cell immune response regulatory network. With the deep research of HVEM function HVEM plays an increasingly important role at the cellular and humoral immunity involved in regulating the immune response. In the future it may be widely applied to autoimmune diseases, transplant rejection, cardiovascular and cerebrovascular diseases and malignant tumors and other clinical areas. Therefore, the expression of HVEM features and reveal their role in the regulation of immune responses has important biological significance, can the special total depth HVEM signaling molecule important biological role in laying the theoretical foundation.This study aims to clone human HVEM gene, establish transgenic cell lines as an immungen to prepare mouse anti-human HVEM monoclonal antibody, and illustrate the biological characteristics of human HVEM molecule as well as its cosimulatory function on T cells with generated monoclonal antibodies.PartⅠCloning of human HVEM gene, establishment of transgenic cell lines and preliminary studying of the biological function of the transfectantsObjective: To clone human complementary HVEM gene, establish transgenic cell lines expressing human BTLA molecule, and study the transfectant function on T cells.Methods: The total length gene encoding human HVEM was amplified from constructed pMD18-T/HVEM, then digested with restriction endonuclease EcoR I and BamH I and inserted into eukaryotic vector pIRES2-EGFP. The recombinant vector was transfected into mouse fibroblast cell line L929 with LipfectAMINETM 2000 and and the cells were further selected with G418. Zeocin-resisting cells were harvested for immunofluorescence and flow cytometry analysis to identify the expression of transfected L929/HVEM cell. Effect of L929/HVEM cells on T cells proliferation and cytokine secretion in vitro was studied by methods of MTT and ELISA.Results: The sequencing results show that the obtained full-length gene encoding HVEM, HVEM full-length gene for the recombinant vector was digested to release the target gene fragment of about 852bp, DNA sequencing further proved that the gene fragments into vector and were identical with HVEM sequences, The stable expression of human HVEM on the transfected cell line was identified by flow cytometry analysis. The cell co-culture experiment in vitro manifested that L929/HVEM had a positive effect on the proliferation of T cells stimulated by anti-CD3 mAb while prominently up-regulating the secretion of IL-2, IFN-γ, IL-10 and TNF-α.Conclusion: The transgenic cell lines L929/HVEM stably expressing human HVEM molecule have been obtained and afford effective immungen for preparing mouse anti-human HVEM monoclonal antibody. The transfectant L929/HVEM can partially enchance the proliferation and activation of T cells in vitro. PartⅡPreparation of mouse anti-human HVEM monoclonal antibodies and analysis of their biological characteristicsObjective: To prepare mouse anti-human HVEM monoclonal antibodies applied to illuminate the regulatory expression and cosimulatory function of human HVEM on T cells.Methods: BALB/c mice were immunized with human HVEM transfectant L929/HVEM as an immungen. Mouse spleen B cells were fused with mouse plasmocytoma cells SP2/0. L929/HVEM was ued as positive screening cell line. Hybridoma cells were eventually obtained through repeated sub-cloing and screening. Fast-strip analysis was performed to identify Ig subclass of the generated monoclonal antibodies. The methods of immunophenotyping and western blot were designed to identify the specificity of generated mAbs. The epitopes recognized by mAbs were analyzed by competition assay. The expression of HVEM on different human tumor cell lines, na?ve and PHA activated human T cells was detected by FCM.Results: After multiple screening and subcloning,one monoclonal antibodies named 2B11 were obtained. Immunophenotyping showed that the mAb could bind to L929/HVEM transfected cells, but not to L929/mock, L929/LIGHT and L929/PD-1 cells, indicating that the antibodies are specific for human HVEM. The mAb 2B11were proved to be IgG1 withκlight chain. After primed with pristine, ascite was induced in BALB/c mice by intraperitoneal injection of well-grown hybridoma and the output is average to 5ml each mouse. ( NH4)2SO4 salting method was used to purify mAb, and the concentration of protein is 1.5~5.0mg/ml. Indirect immunofluorescence assay suggested the engagement of purified HVEM mAb to cells was 0.5-1μg/1×106 cells. Western blot showed mAb 2B11 specifically bind to HVEM-Fc fusion protein. The mAb was revealed to bind to not only na?ve but also activated T lymphocytes and could recognize human tumor cell lines THP-1.Conclusion: A specific mouse anti-human HVEM monoclonal antibody was successfully prepared. The regulatory expression of HVEM on human kinds of immunocytes was successfully analyzed by generated mAbs. The antibody also provided materials to reveal the expression characteristics of human HVEM molecule on immunocytes and its functional mechanism on T cells.In summary, this paper has successfully accomplished the investigations as follows: The human HVEM complementary gene has been cloned, and the transgenic cell lines L929/HVEM stably expressing human HVEM molecule have been obtained. The transfected L929/HVEM was revealed to partially influence on the proliferation and activation of T cells to a certain extent in vitro. Using the transfected L929/HVEM as immungen, The specific anti-human HVEM monoclonal antibodies were generated. The expression of HVEM on many kinds of human immunocytes was revealed to be regulated along with stimulation. Specially, therefore all the results laid material basis for the further study of HVEM and its biological function , meanwhile the mAb 2B11 may has potential clinical value.