Establishment Of Cell Lines Transfected With Two Isoforms Of Human B7-H3 And Preparation Of A Monoclonal Antibody Against Human B7-H3 Molecule

Effective activation of the T cell requires the delivery of two separate but complementary signals."Signal 1"is delivered during the cognate interaction between the T cell receptor (TCR)/CD3 complex and an MHC-bound peptide on an antigen presenting cell (APC). The second requisite signal is an antigen nonspecific"positive"signal triggered by the interaction of a pair of cell surface costimulatory molecules expressed on the T cell and APC. As such, costimulatory molecules do not trigger T cell activation alone but rather serve to augment signals delivered via the TCR/CD3 complex. The most classical costimulatory signal is generated by the interaction between CD28 and B7 (B7-1 or B7-2). During the past 20 years, numerous T-cell co-stimulatory molecules (inducible co-stimulator [ICOS], programmed death-1 [PD-1], B and T lymphocyte attenuator [BTLA]) and their ligands (ICOS-L, PD-L1, PD-L2, HVEM, B7 homolog 3 [B7-H3], B7 homolog 4 [B7-H4]) have been identified, all of them are members of the CD28 and B7 families. Pathways in the B7:CD28 familys have key roles in regulating T cell activation and tolerance and are promising therapeutic targets. These pathways not only provide critical positive second signals that promote and sustain T cell responses, but they also contribute critical negative second signals that downregulate T cell responses. These negative signals function to limit, terminate, and/or attenuate T cell responses, and they appear to be especially important for regulating T cell tolerance and autoimmunity.Different splicing of human B7-H3 leads to a 4Ig domain (VCVC) transcript [also called B7-H3b and 4Ig-B7-H3] and a 2Ig domain (containing V1 and C2) transcript. Because we do not kown the functional diffence of the two isoforms, the further study about the biological function of B7-H3 and the existence of the two isoforms is valuable. So two cell lines carrying human 2IgB7-H3 gene and 4IgB7-H3 gene were established, and preliminary study of the difference of their costimulatory effects on T cell was done. Beside this, we prepared the monoclonal antibody specifically recognizes human B7-H3 expressed on the leukemia cell, which may provide a novel approach to analyze the biological and clinic function of B7-H3.PartI Establishment of cell lines transfected with two isoforms of human B7-H3 and preliminary study of the difference of their costimulatory effects on T cellObjective: To establish two cell lines carrying human 2IgB7-H3 gene and 4IgB7-H3 gene, which can be stably expressed, and to study their costimulatory effects on T lymphocytes.Methods: The gene encoding human 2IgB7-H3 gene and 4IgB7-H3 gene were amplified by RT-PCR from mature DC cells by induction, then the two target gene fragments were inserted into eukaryotic vector pIRES2-EGFP after being digested with EcoR I and BamH I. The two recombinant vectors were transfected into SHG44 cells with Lipfect AMINETM 2000, and the two cells were further screened with G418. Then the expression of human 2IgB7-H3 gene and 4IgB7-H3 gene were analysed by Immunofluorescence and flow cytometry. Effect of cell lines transfected with two human B7-H3 isoforms on T cells proliferation and cytokine production in vitro was studied by MTT and ELISA.Results: The stable expressions of human 2IgB7-H3 and 4IgB7-H3 on the transfected cell lines were identified by Flow Cytometry analysis. In vitro, SHG44/2IgB7-H3 and SHG44/4IgB7-H3 cells could both significantly down-regulate the proliferation of T cells stimulated by anti-CD3 mAb and inhabit the production of IFN-γ, which is shown by ELISA results. And there is no significant difference of their negative costimulatory effect on T cells.Conclusion: Two isoforms of human B7-H3 all have negative costimulatory effect on T cells, but there is no significant different effect.Part II Preparation and identification of a monoclonal antibody against human B7-H3 moleculeObjective: To prepare of a monoclonal antibody against human B7-H3 molecule, and characterize its specificity of epitope recognition.Methods: Female BALB/c mice of 6-8 weeks old were immunized with human 293T cell as immunogen. The spleen B cells of the mice were fused with SP2/0 and hybridoma cells were screened with SHG44/2IgB7-H3 and SHG44/4IgB7-H3 and SHG44/mock transfected cells by FCM. The expression patterns of 2IgB7-H3 and 4IgB7-H3 on different cell lines and subsets of immunocytes were analyzed by Flow Cytometry and RT-PCR.Results: One hybridoma (13F8) was obtained, which could specially recognize human 2IgB7-H3 and 4IgB7-H3.Conclusion: One novel specific mouse anti-human B7-H3 mAbs(13F8) was successfully got. B7-H3 is broadly express on various tumor cells to detect by anti-human B7-H3 mAbs(13F8), which may provide a novel approach to analyze the biological and clinic function of B7-H3.