Preparation Of Mouse B7-H3 Transgenic Cell Line And Rat Anti-mouse B7-H3 Monoclonal Antibodies As Well As Analyse Of Costimulatory Effect Of Mouse B7-H3 On T Cell In Vitro

Since in 1970 it was proposed, the two-signal hypothesis for T lymphocyte activation has been widely accepted that T cells repuire two distinct signals for expansion. This model hypothesizes that peptides presented to antigen-specific T cells by the MHC molecule delivers the first signal, whereas costimulatory molecules (CMs) on T cell surface trigger the second signal. Costimulatory signals (positive or negative), play an important role in regulating the balance of immune responses.B7-H3 is a new member of the B7 superfamily. The function of B7-H3 is not clear and controversial till now. It was reported that mouse B7-H3 served as a positive or negative regulator of T cell activation. Some research showed that B7-H3 molecule could be used as a cancer marker and might be of great value for diagnosis and therapeutic interventions. The unknow receptor for B7-H3 is the most difficulty during studying B7-H3 molecule. further study of mAbs characteristics.Therefore, this study aims to illustrate the biological characteristice of mouse B7-H3 molecule as well as its function on T cells with generated monoclonal antibodies and fusion protein.Part I The construction of B7-H3 gene transfected cell line and the preparation of rat anti-mouse B7-H3 monoclonal antibodyThe complementary mouse B7-H3 gene coding region was cloned from dendritic cells derived from bone marrow by RT-PCR .The target gene fragment was inserted into vector pIRES2-EGFP and confirmed by sequencing. The recombinant plasmid was transfected into CHO and 293 cells with LipfectAMINETM2000 and the cells were further selected with G418. The transgenic cell lines CHO/B7-H3 and 293/B7-H3 stably expressing mouse B7-H3 molecule have been obtained and afford effective immungen for preparing rat anti-mouse B7-H3 monoclonal antibody.The splenocytes harvested from the immunized rats were fused with murine myeloma SP2/0 cells by means of cell fusion hybridoma technique. After repeated screening two cell strains of hybridoma secreting anti-B7-H3 mAbs continuously and steadily were obtained and designated as 18F9 and 19F6. Clonotyping beads rat isotyping analysis displayed that the isotype of two mAbs were IgG2b withκlight chain. The hybridoma cells grew well after long-term storage in liquid nitrogen and culture in vitro. We found mouse B7-H3 molecule was expressed on B cell,monocyte and dentritic cell, not on resting and activated T cell,NK cell. Mouse B7-H3 protein was not expressed on normal tissues except for cytoplasma and membrane of bladder epithelial cellson. Results suggested two mAbs could be used for FCM,Western blot and immunohistochemistry analysis.Part II Expression of mouse B7-H3-Fc fusion protein and characterization of its bioactivityThe genes coding extracellular domain of mouse B7-H3 and the Fc fragment of human IgG1 were amplified from pMD19-T/mouse B7-H3 and pMD19-T/human IgG1 vectors by PCR. The two genes were connected into mouse B7-H3-Fc fragment by overlap PCR and then inserted into eukaryotic vector pIRES2-EGFP to construct the recombinant vector pIRES2-EGFP/B7-H3-Fc.The recombinant vector was transfected into CHO cells as described above. The collected supernatant of transfected cell line after serum-free culture was condensed and purified by protein G affinity chromatography column, then was confirmed by Western blot. Effects of fusion protein on T cells proliferation and cytokine production in vitro was studied by methods of CCK8 and ELISA. The results showed that the transfected CHO cell line secreting fusion protein was constructed successfully. In vitro, fusion protein could obviously promote the proliferation of T cells and has dose-dependence. And antibody 18F9 could inhibit T cells producing cytokines. The fusion protein could not bind TLT2 transfected cells but recognize receptor on T cell. Therefore TLT2 may be not receptor for B7-H3.On the basis of above , the transgenic cell lines and two functional mAbs have been obtained. Studies showed fusion protein could promote T cells proliferation and the secretion of IL-2 and IFN-γ, but antibody could inhibit T cells producing cytokines. TLT2 molecule may be not receptor for B7-H3. We found B7-H3 was a positive costimulator during T cell activation.