Functional Study Of Anti-human B7-H4 Monoclonal Antibody And Preparation Of Mouse Anti-human B7-H4 Receptor Monoclonal Antibody

The B7 family is the most important costimulatory molecule, which plays an important role in regulating the activation of T cells and the dynamic balance of immune tolerance. B7-H4(B7X, B7S1), is one of the new members of the B7 family.The gene of human B7-H4 contains 5 introns and 6 exons, which is located on the1p11.1 of human chromosome. B7-H4 and B7 family molecules, are highly expressed in many tumors, such as lung cancer, breast cancer, ovarian cancer, gastric cancer and others, but no expression in normal human tissues, which makes the B7-H4 expected to become a new tumor marker. As a negative stimulating factor, B7-H4 can inhibit T cell activation, proliferation and cytokine secretion when binding to its receptor. We hypothesize that B7-H4 plays an important role in tumor cells escape from the immune surveillance and maintenance of immune tolerance. However, B7-H4 receptor, which has important effect on the T cell signaling pathway as B7-H4, has not been cloned. The research of receptors will preferably help us to understand the role of B7-H4 in tumor tissue, and expect to be the key molecule of tumor immunotherapy.In this study, we chose the breast cancer cell MCF-7 and acute lymphoblastic leukemia cell Jurkat to construct a co-culture system, which simulates conditions of tumors in vivo. The anti human B7-H4 monoclonal antibody 5C8(prepared by our laboratory) was used to block the B7-H4 molecules on MCF-7 cells and to inhibit the negative regulation of B7-H4 molecules. The biological characteristics of Jurkat cells was examined after co-culture.We detected the expression of B7-H4 receptor in the inflammatory infiltrate of human lymphocytes. So we used this as immunogen to prepare monoclonal antibodies against human B7-H4 receptor.Partâ… . Functional study of anti-human B7-H4 monoclonal antibody 5C8.Objective : To identify the characterization of Jurkat cells by establishing an coculture system in vitro.Methods:Flow cytometry was used to detect the expression of B7-H4 in MCF-7cells and the expression of B7-H4 receptor on Jurkat cells. After 72 h of co-culture,Jurkat cells that adhered to MCF-7 cells were collected. The expression of CD3 and CD69 on the surface of Jurkat cells was detected using flow cytometry, and the proliferation of Jurkat was tested by the method of CCK8.Results:B7-H4 molecules on the surface of MCF-7 cells could be bound to B7-H4 receptors on Jurkat cells, which induced the down-regulation of CD3 and CD69 molecules on the cell surface and inhibited the proliferation of Jurkat cells. When 5C8 was added, the expression and proliferation of Jurkat cells were recovered.Conclusion : B7-H4 is a kind of negative costimulatory molecules, which can inhibit the proliferation of T cells and membrane molecule expression.Partâ…¡. Preparation of mouse anti-human B7-H4 receptor monoclonal antibodyObjective : To prepare the monoclonal antibody of mouse anti-human B7-H4 receptor and identify its biological functions.Method : We used the inflammatory infiltrate of the cells of the amygdala as immunogen to immunize the BALB/c mice of 6-8 weeks of age. The spleen cells were fused with mouse myeloma cells SP2.0. The supernatant of the hybridoma was screened by flow cytometry. The positive hybridoma strains were further studied through the site competition inhibition test and ELISA.Results : We found two strains secreting monoclonal antibody against B7-H4 receptor, which named 3B2 and 2E2. The results of site competition inhibition test and ELISA showed that, 3B2 had the better effect than 2E2. The two strains of antibody subtypes were all determined as IgG2 a.Conclusion : Two hybridoma strains of B7-H4 receptor monoclonal antibodies were were obtained. The function of them remains to be studied. And this provide thebasis for further research on the molecular mechanism of B7-H4 and B7-H4 receptor.