Study Of The Immune Intervention Effect And The Molecular Mechanism Of Anti-human B7-1Antibody On Lupus Nephritis

B7-1molecule is a member of immunoglobulin superfamily, and its combining toreceptors CD28/CTLA-4can generate costimulatory signals, which can mediate T cellimmune response. If there is no B7/CD28cosimulatory signal, T cells will enter to astate of anergy or tolerance, or even death.Autoimmune diseases (AID) is an kind of severe illness which resulted from theover activation of T and B cells, producing a large number of autoantibodies andresulting in the damage of multiple systems and organ. systemic lupus erythematosus(SLE) is the typical representative.At present, studies show that B7-CD28costimulatory signal were involved in the development of SLE, blocking or weakeningthe signal can reduce the pathological damage of these disorders. That is the importantmeans to study the occurrence,development,outcomes of diseases and find new ways todiagnosis and treatment by establish model of animal disease to simulate human diseaseand clinical manifestation. This project which based on establishing chronic graft versushost disease (cGVHD) lupus nephritis model in mice and conducting biological assayused B7-1monoclonal antibody which is developed by ourselves to intervene thedisease model. By immunology, serology and renal pathological damage evaluationindex examining the reversal effect by the antibody towards to the disease models andthe molecular mechanism of disease models. In order to find new, high efficiency andlow toxicity of biological agents which have the prevention and cure function to SLEand related disorders.I Preparation and identification of B7-1monoclonal antibodyObjective: To prepare mouse anti B7-1monoclonal antibody and identify itsbiological characteristics. Methods: preparing monoclonal antibody by inducing ascitesin vivo;Purifying antibody by Protein G immune affinity chromatography; Analyzingwhether the monoclonal antibody identity B7-1molecule antigen epitope by Flow cytometry. Results: the positive rate of ascites formation in mice was about80%.Ascites were induced to produce the mAb and3.5ml ascites was obtained from eachBALB/c mouse on average. The purified4E5from ascites was about3.2mg per mlascites with Protein G affinity chromatography method. The4E5could specificallyrecognize membrane B7-1on cells of L929、spleen cells of mouse with the positive of99.3%、65.4%, respectively. Conclusion: Mouse anti-human B7-1antibody couldspecifically recognize membrane B7-1expressed on cells line L929and spleen cells inmice.II Developing and charactering a cGVHD lupus nephritis disease model inmouseObjective: To explore and identify the pathogenic mechanism of a cGVHD lupusnephritis disease model in mice. Methods: Thirty8-week-aged female (C57BL/6×BALB/c) F1mice were devided into two groups randomly. The experimental groupwere injected with parental female BALB/c mice’s spleen cells107/100μL/only on the0、3、7and11d while the control group with the same volume of0.9%N.S.. Theactivation of macrophage, dendritic cells and granular cells in spleens and markers on Bcells were measured by FCM at day7after the last injection, respectively by IFA andFCM. Antinuclear antibodies(ANA) and anti-double strand DNA antibodies (dsDNA)in serum were detected every two weeks after injection meanwhile proteinuria waschecked mouthy.3months after injection, all mice were killed and kidneys were slidedand stained with H&E or FITC-labeled IgG to observe the evidence ofglomerulonephritis histopathologically, and observe the change of the ultrastructural ofglomerular by TEM. Result: After parental female BALB/c mice’s spleen cellsinjection, the rate of CD11b+、CD11c+and Gr1+were increased significantly comparedto the control ones (p<0.05). The expressions of CD21+、CD23+、CD80+and CD86+on B cells were also increased (p<0.05).2weeks later, anti-dsDNA antibodies and ANAcan be detected in70%of the serum of mice,4weeks is90%.3months later, theprotein concentration of uria in70%of the experimantal mice was++~++++.HEstaining and microscopic observation, the experiment mice appear glomerular swellingand lymphocyte infiltration. The basement membrane of the experimantal mice werechanged to be more thick segmentally by TEM. Conclusion: Mouse model of cGVHD lupus nephritis disease in female mice is established successfully.III Study of the immune intervention effect and the molecular mechanism ofanti-human B7-1antibody on lupus nephritisObjective: To study of the immune intervention effect and the molecularmechanism of anti-human B7-1antibody on lupus nephritis by blocking B7-1/CD28signaling pathways. Methods: According to the above method to make the model, theF1mice were randomly divided into3groups, namely to the experiment group, thegroup were intervented with mouse anti-human B7-1antibody and the control group,Each group had15mice. The intervention group injected B7-1antibody (4E5)200μg/only on the1,3,5,8and15d respectively and then1times a month for twice bytail intravenous after the last injection of lymphocyte cells. The experiment group weregiven the isodose isotype IgG at the same time. Analyzing the positive rate of themacrophages (CD11b+), the dendritic cells (CD11c+), the neutrophils (Gr1+) cells andthe cells producing antibody(CD21+,etc.) in spleen, Analyzing the content ofanti-dsDNA antibodies and ANA, the protein in the urine and immune complex (IC),Analyzing the ultrastructure of glomerular according to the above time points. Result:the expression of CD11b+、CD11c+and GR1+in spleen cells and CD21+、CD23+、CD80+and CD86+in B cells in the group intervented with mouse anti-human B7-1antibody were lower than the experiment group(p <0.05) by Immunofluorescence andflow cytometry analysis.4weeks later, anti-dsDNA antibodies and ANA can bedetected in90%of the serum of mice in the experiment group, but The interventiongroup is just40%. And the concentration of the antibody is lower than the experimentgroup (p <0.05).3months later, the protein concentration of uria in70%of theexperimantal mice was++~++++, the intervention group is just20%,and the proteincontent is+~++. Microscopically observed after HE staining, the kidney tissue of theexperiment group had the changes of glomerular swelling, lymphocyte’s infiltration andso on, but the intervention group just partly appear the changes of glomerular’s volumeincreases slightly, just a few lymphocytes infiltration and the volume of Capillarylumens and balloon lumen do not have change. The experiment group had IC depositionby IF and TEM, and the intervention group’s glomerular outline is clear, the depositionof IC is less than the experiment group.The basement membrane of the experimantalmice were changed to be more thick segmentally by TEM, but the basement membrane of the intervention group don’t have obvious pathological changes. Conclusion: B7-1monoclonal antibody can inhibit the activation of immune cells, reverse the pathologicaldamage of lupus nephritis by combining the corresponding antigen molecules,suggesting that these specific antibody can prevent and control the SLE and cGVHD.