Study Of The Immune Intervention Effect And The Molecular Mechanism Of Anti-human B7-2 Chimeric Antibody On Lupus Nephritis

Systemic lupus erythematosus(SLE) is one of autoimmune disease(AID), producing a large number of autoantibodies and resulting in the damage of multiple systems and organ. Lupus nephritis(LN) is the most common and serious complications. The cause of the disease is not clear yet, and may be associated with genetic and environmental factors.B7-2 molecule is an important costimulatory molecule on the surface of antigen presenting cells(APC). It is combined with CD28 expressed on the surface of T cell, mediated T cell activation, proliferation, differentiation and immune effect. The lack of this signal, T cells will remain no response(Anergy) or immune tolerance(Tolerance) and even cause programmed cell death(Apoptosis). The study found that B7-CD28 costimulatory signals involved in the occurrence and development of SLE, and play an important role in it. Anti-human B7-2 chimeric antibody can block or weaken the B7-CD28 signal, thereby suppressing the immune response, so the application of Anti-human B7-2 chimeric antibody blocking or weakening the B7-CD28 pathway is expected to provide a new method for biological treatment of SLE.Application of traditional murine antibody to human body will produce human anti-mouse antibody(HAMA), so its use is limited. Anti-human chimeric antibody, which designed by genetic engineering techniques, has high specificity and affinity. It greatly reduces immunogenicity and avoids HAMA. It can also mediate ADCC effect, CDC effect and immune conditioning.This research will establish the chronic graft-versus-host disease(c GVHD) murine lupus nephritis model that similar to human SLE. Anti-human B7-2 chimeric antibody will be used to intervene this model, then we will examine the reversal effect and explore its possible molecular mechanism by immunology, serology, kidney tissue morphology and other aspects of the indicators.I Preparation and identification of Anti-human B7-2 chimeric antibodyObjective: To prepare anti-human B7-2 chimeric antibody and identify its biological characteristics. Methods: Utilizing the cell strain 4F4 which is successfully secreting stable anti-human B7-2 chimeric antibody. Using cell culture, concentrated by ultrafiltration and immunoaffinity chromatography technology to produce anti-human B7-2 chimeric antibody. Analyzing whether anti-human B7-2 chimeric antibody identity B7-1 molecule antigen epitope by Flow cytometry. Results: The yield of anti-human B7-2 chimeric antibody from 4F4 cell culture supernatants is approximately 4mg/L. the anti-human B7-2 chimeric antibody can recognize membrane B7-2 molecule on human B7-2 gene transfected mouse fibroblast cell lineL929-B7-2, Daudi and mice spleen cells with the positive of 99.0%, 93.7% and 63.0%. Conclusion: anti-human B7-2 chimeric antibody can specifically recognize membrane B7-2 molecule of different cell types.II Developing and charactering a c GVHD lupus nephritis disease model in mouseObjective: To explore and identify the pathogenic mechanism of a c GVHD lupus nephritis disease model in mice. Methods: Twenty 6-8 week-aged female(C57BL/6×BALB/c) F1 mice were devided into two groups randomly. The experimental group were injected with parental female BALB/c mice’s spleen cells 107/100μL/only on the 0、3、7 and 11 d while the control group with the same volume of 0.9% N.S. The activation of APCs and antibody-associated cells were measured by FCM at day 7 after the last injection, respectively by IFA and FCM. Antinuclear antibodies(ANA) and anti-double strand DNA antibodies(ds DNA) in serum were detected every two weeks after injection meanwhile proteinuria was checked mouthy. 3 months after injection, all mice were killed and kidneys were slided and stained with H&E or FITC-labeled Ig G to observe the evidence of glomerulonephritis histopathologically, and observe the change of the ultrastructural of glomerular by TEM. Result: After parental female BALB/c mice’s spleen cells injection, the activation of APCs and antibody-associated cells enhanced significantly(p<0.05). 2 weeks later, anti-ds DNA antibodies and ANA can be detected in 80% of the serum of mice, 4 weeks is 90%. 3 months later, the protein concentration of uria in 80% of the experimantal mice was ++ ~ ++++.HE staining and microscopic observation, the experiment mice appear glomerular swelling and lymphocyte infiltration. The basement membrane of the experimantal mice were changed to be more thick segmentally by TEM. Conclusion: Mouse model of c GVHD lupus nephritis disease in female mice is established successfully.III Study of the immune intervention effect and the molecular mechanism of anti-human B7-2 chimeric antibody on lupus nephritisObjective: To study of the immune intervention effect and the molecular mechanism of anti-human B7-2 chimeric antibody on lupus nephritis by blocking B7/CD28 signaling pathways. Methods: According to the above method to make the model, the F1 mice were randomly divided into 4 groups, namely to the experiment group, the group were intervented with anti-human B7-2 chimeric antibody,CTX group and the control group. The intervention group injected anti-human B7-2 chimeric antibody(4F4) 200μg/only on the 1, 3, 5, 8 and 15 d respectively and then 1 times a month for twice by tail intravenous after the last injection of lymphocyte cells. Analyzing the activation of APCs and antibody-associated cells in spleen, Analyzing the content of anti-ds DNA 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 activation of APCs and antibody-associated cells in the group intervented with anti-human B7-2 chimeric antibody were lower than the experiment group(p<0.05) by Immunofluorescence and flow cytometry analysis. 4 weeks later, anti-ds DNA antibodies and ANA can be detected in 90% of the serum of mice in the experiment group, but The intervention group is just 30%. And the concentration of the antibody is lower than the experiment group(p<0.05). 3 months later, the protein concentration of uria in 70% of the experimantal mice was ++~++++, the intervention group is just 20%, and the protein content is +~++. Microscopically observed after HE staining, the kidney tissue of the experiment group had the changes of glomerular swelling, lymphocyte’s infiltration and so on, but the intervention group just partly appear the changes of glomerular’s volume increases slightly, just a few lymphocytes infiltration and the volume of Capillary lumens and balloon lumen do not have change. The experiment group had IC deposition by IF and TEM, and the intervention group’s glomerular outline is clear, the deposition of IC is less than the experiment group.The basement membrane of the experimantal mice were changed to be more thick segmentally by TEM, but the basement membrane of the intervention group don’t have obvious pathological changes. These indicators of CTX group don’t have significant difference compared with the intervention group. Conclusion: anti-human B7-2 chimeric antibody can inhibit the abnormal activation of immune cells by inhibition of B7/CD28 signal, and the production of autoantibodies were reduced to alleviate renal function injury. Therefore, immune intervention of anti-human B7-2 chimeric antibody can reverse pathological injury of lupus nephritis, suggesting that it has a potential therapeutic effect on this disease.