Clone, Expression Of Human B7-H3 And The Study Of Its Biological Functions In Vitro

B7-H3, as a new member of the B7 family, is a type I membrane protein of about 45kD encoding 316 aa. The molecule was identified upon database searches for sequences with homologies to B7 molecules. Human and mice B7-H3 share ~88% amino acid sequence identify. Similar to other B7 homologs, the mRNA of B7-H3 was detected on a broad spectrum of tissues. However, its expression at the protein level remains largely unknown. The receptor for B7-H3 was unknown until now, but the counter-receptor that is distinct from CD28, CTLA-4, ICOS, and PD-1 was observed to be rapidly induced following T lymphocytes activation. The B7-H3 was first reported as a positive costimulatory molecule to stimulate the proliferation of T cells and selectively enhanced IFN- γ secretion with modest effects on TNF-α. However, other studies also demonstrated an inhibitory function for mouse and human B7-H3, whereas different groups observed enhancing effect. Therefore, a lot of study was maintained to identify the expression of B7-H3 protein and explore its biofunction in the immunology responses.Part 1: Human recombinant B7-H3 expressed in E.coli enhances Tlymphocyte proliferation and IL-10 secretion in vitroObject: To explore the biofunctions of human B7-H3 to activated T lymphocyte. Methods: the gene of human B7-H3 encoding the extracellular region (IgV-like and IgC-like domains) was obtained by RT-PCR from human lung cells and subcloned into the prokaryotic expression vector pGEX-5X-3 expressing glutathione S-transferase (GST) fusion protein. The fusion protein was induced by IPTG and purified by standard methods reported in prokaryotic system. In the presence of the first signal imitated by anti-CD3 monoclonal antibody, T lymphocyte proliferation was analyzed by MTT. The concentrations of IL-10 and IFN-γ in the supernatants of T cells were determined by ELISA. Results: A 49 KD fusion protein was obtained and purified through Protein Gcolumn. T cells proliferation was observed by incubating purified T cells with soluble GST/B7-H3 fusion protein in the presence of anti-CD3 mAb. And GST/B7-H3 protein could enhance the secretion of IL-10 and IFN-y. Conclusion: The GST/B7-H3 protein produced in bacteria had obvious biological activity to proliferate the T lymphocyte and enhance IFN-y as well as IL-10 secretion.Part 2: Preparation and application of two novel monoclonal antibodies against B7-H3: Expression analysis of B7-H3 on Dendritic cells andtumor cellsObject: To prepare monoclonal antibodies against human B7-H3; on the base to detect the expression of B7-H3 on DCs and tumor cells. Methods: The Balb/c mice were immunized with B7-H3/L929 transfected cells. The splenocytes of the immunized mice were fused with SP2/0 cells according to the routine method. Flow Cytometry was performed to identify the mAbs and the mAbs were purified from ascites of BALB/c mouse using protein G affinity column. The specific recognition of mAbs with the membrane protein B7-H3 was further determined by Western-Blotting analysis. Firstly, we detect the expression of B7-H3 on tumor cell lines through Flow Cytometry and western blotting with the two mAbs. Immunohistochemistry was performed to analyze the expression of B7-H3 on fresh isolated tumor tissues. Following, the expression pattern of B7-H3 molecule on DCs was explored by means of Flow Cytometry. Results: Two hybridoma cell lines (4H7 and 21D4) specifically secreting mAb against B7-H3 were obtained both belonging to IgGl subclass. Contrast to the opinion that the protein level expression of B7-H3 by these cell lines was tightly regulated, using the mAbs against B7-H3 through cytometry flow and western blotting, we found that B7-H3 protein also could be constitutively expressed by a series of tumor cell lines from epithelium, but not by those from other tissues. Interestingly, immunohistochemistry showed notwithstanding the staining was found in the sections, the positive cells almost difiused in the surrounding of tumor cells. Additionally, B7-H3 was predominately expressed on DCs. Conclusion:MAbs 4H7 and 21D4, recognized different epitopes, were specific anti-B7-H3 mAbs. B7-H3 was constitutively expressed on DCs and slightly up-regulated costimulatory molecules on the surface of DCs. The studies described above demonstrate that the B7-H3 protein could be broadly expressed by tumor cell lines from epithelial tissue, however, the cancer cells isolated from fresh tissues seldom could detected the B7-H3 expression. The aberrant expression of B7-H3 by human cancers was unclear and the biofunctions ofB7-H3 on tumor immunity remained to be elucidated in the further study.Part 3: Anti-B7-H3 monoclonal antibody enhance the maturation ofDendritic cells and the proliferation of T cells by up-regulating the expression of CD80 and CD86 through reverse Signaling.Object: To explore the biofunction of B7-H3 on DCs. Methods: The effect of B7-H3 mAb on the differentiation and maturation of Mo-DCs were measured by FCM and potomicroscopy. In MLR experiment, 3H-TdR incorporation test and ELISA were performed to analyze the costimulatory ability of DCs mediated by B7-H3 mAb through reverse signaling. Results: B7-H3 mAb could up-regulate the expression of costimulatory molecules CD80 and CD86 in the different stage of the maturation of Mo-DCs. In the presence of TNF-a, B7-H3 mAb transferred the reverse signals to enhance Mo-DCs maturation obviously. The combination of B7-H3 mAb with TNF-a could increase the costimulatory ability of Mo-DCs to proliferate T cells and enhance the secretion of IL-10. Conculusions: Taken together, our data suggested that B7-H3 mAb might be one of important factors to induce Mo-DCs differentiation and maturation and enhance the costimulatory ability o through reverse signal pathway.