| According to the two-signal model, naive T cells require two necessary signals to become fully activated. Costimulatory signals play important roles in the regulation of immune response during different steps of initiation, effection and termination.ICOSL, which is a B7 family molecule, with its receptor ICOS, plays a key role in diverse immunological processes, including the survival, activation and function of T cells, such as secretion of cytokines and so on.In addition,they are essential for the development of T follicular helper(Tfh) cells and maintenance of GC reaction and the formation of antibodies. It has been reported that ICOS/ICOSL pathway is critical for transplantation, cancer, infectious immunity diseases and especially the development of autoimmune diseases.Inducible Costimulator Ligand(ICOSL) is a costimulatory molecule, existing as membrane or soluble factors. It’s expressed on a wide variety of cell types, including B cells, macrophages, dendritic cells(DCs) and a subset of T cells.It can be induced on epithelial cells,vascular endothelial cells(ECs) and fibroblasts, as well as many primary tumors and tumor cell lines.By binding with its receptor ICOS,ICOSL can adjust the function of T cells. The intracellular domain of ICOSL contains a signaling motif “YMFM”, which can bind with PI3 K.Deleting the cytoplasm domain of ICOSL will attenuate its binding ability with PI3 K and the kinase activity. ICOS/ICOSL interaction can activate PI3 K dependant pathway of T cells. Positive costimulatory molecules assume two forms of expression:membrane-bound forms and soluble forms.The soluble protein is either produced by the special m RNA byimmune cells or shed from the membrane with the help of proteinase(such as MMPI).Many soluble proteins are valuable for clinical diagnosis. However, the functions and biological significance of s ICOSL remain unknown.Thus, in view of its critical role in regulating immune homostasis,ICOS/ICOSL signal may be a potential way for immunodiagnosis and therapy.In this study, we generated monoclonal antibodies against human ICOSL. Then the m Ab were used to develope the sandwich enzyme-linked immunosorbent assay(ELISA) system for the detection and quantification of s ICOSL. The levels of s ICOSL were determined by the established ELISA. The ELISA system was used to analyze the expression levels of s ICOSL in human serum from SLE patients and healthy donors.The expression mechanisms of the soluble factors were also studied in this work.Moreover, we focused on the role of ICOSL in the regulation of ICOS/ICOSL pathway and their biological significance for SLE or other autoimmune diseases.Part ? Generation and characterization of monoclonal antibodies against human ICOSLObjective: To prepare mouse anti-human ICOSL monoclonal antibody to study ICOS/ICOSL pathway.Methods: BALB/c mice were immunized with L929/ICOSL cells. The splenocytes of immunized mice and the SP2/0 cells were fused with PEG1500. The monoclonal antibodies produced by the hybridomas were screened by flow cytometry.After multiple screening and subcloning, several monoclonal antibodies against human ICOSL were generated. Fast-strip analysis and CBA method were performed to identify Ig subclass of the antibodies. The specificities of the antibodies were measured by immunostaining,Western blot,Dot blot and ELISA. The antigen epitopes recognized by the m Abs were analyzed by competition assays. The expression patterns of ICOSL on different human cell lines were determined with the obtained m Abs by immunostaining and flow cytometry. The antibody-mediated functions on humanT cells were studied in vitro.Results: Two novel antibodies against human ICOSL,20B10 and 13D11, were generated and characterized successfully. Flow cytometry assay showed that the m Abs could bind specifically to human ICOSL protein. The m Abs could be applied to FACS, immunostaining, Western blot, Dot blot and ELISA. The antibody competition assays indicated that the two m Abs recognized different epitopes of ICOSL. It was found that they could completely block the interaction of ICOS/ICOSL.The functional 13D11 m Ab could block the signal of ICOS/ICOSL and inhibit T cell proliferation with a dose-dependent manner.Conclusion: The generation of monoclonal antibodies with distinct characteristics against human ICOSL provide essential reagents for the study of ICOS/ICOSL pathway.Part II Development of a sandwich ELISA system for measuring human soluble ICOSLObjective: To establish a specific ELISA system with high sensitivity to detect human soluble ICOSL protein.Methods: Mouse anti-human ICOSL m Ab(20B10), used as coating antibody,was precoated in the ELISA plate with the CBS. Another biotin-anti-ICOSL m Ab(2B4) was used as detecting antibody to recognize the 20B10-bound soluble ICOSL protein. Then the Streptavidin-HRP was added to the reaction system. Finally, TMB substrate was added for colorable reaction. The absorbance was measured by the microplate reader.Results: The results showed that the ELISA system for detecting human s ICOSL was established successfully. It could be used to detect human s ICOSL protein specifically without any cross-reaction with other proteins. Standard curve of the system displayed favorable linear correlation when the concentration of s ICOSL was0.15625-10ng/ml. The ELISA system had favorable stability, precision andspecificity, and the variations(CV%) were <10.9%'<3.65% respectively.Conclusion: The ELISA system with high specificity and sensitivity for detecting human s ICOSL provided an useful method to study s ICOSL in the regulation of ICOS/ICOSL pathway.Part III Expressions and clinical significance of ICOS and ICOSL in peripheral blood of Systemic Lupus Erythematosus patientsObjective: To investigate the expression of inducible costimulatory(ICOS) and inducible costimulatory ligand(ICOSL) on peripheral blood mononuclear cells(PBMCs), the plasma concentrations of soluble forms of ICOSL and their clinical correlation with Systemic Lupus Erythematosus(SLE) patients.Methods: Peripheral blood samples were colected from 45 SLE patients and 39 healthy subjects(HC).The expressions of ICOS and ICOSL on PBMC were detected by immunofluorescence and flow cytometry.The concentrations of soluble ICOSL were assessed by ELISA. And the correlation between their expression level and patients’ clinical manifestations was analysed.Results: The expression levels of ICOS on CD4+and CD8+T cells in SLE patients were significantly higher than those in the HC [(19.07±1.74)%vs(13.99±1.54)%,(P=0.035)],[(10.04±0.99)%vs(6.36±0.99)%,(P=0.012)].The expression of ICOSL on CD14+mononuclear cells in SLE patients was significantly higher than that in the HC group[(2.94±0.88)%vs(0.89±0.21)%,(P=0.04)].Plasma ICOSL concentrations in patients with active SLE were significantly higher than those of patients with inactive SLE[(361.5±25.28)(ng/ml)vs(278±14.75)(ng/ml),We also found a significant inverse correlation between the soluble ICOSL expression and the surface ICOSL expression on both mononuclear cells and B cells[(r=-0.4243,P=0.022)、(r=-0.4099,P=0.025)].MMPI caused a clear reduction of s ICOSL levels released from the cells, which was statistically significant incomparison with untreated cells(P < 0.05).Conclusion: The upregulated expressions of ICOS and ICOSL on peripheral lymphocytes and the high levels of plasma concentration of soluble ICOSL were closely correlated with the degree of disease,suggesting that ICOS/ICOSL pathway may play a critical role in pathogenesis of SLE.In summary, we carried out the analysis as follows: Two novel m Abs against human ICOSL were generated after being screened by flow cytometry. A ELISA system was also developed successfully to detect human s ICOSL. The results of ELISA showed that s ICOSL existed in human serum. MMPI caused a clear reduction of s ICOSL levels released from the cells, which was statistically significant in comparison with untreated cells. The upregulated expressions of ICOS and ICOSL on peripheral lymphocytes and the high levels of plasma concentration of soluble ICOSL were closely correlated with the degree of disease, suggesting that ICOS/ICOSL pathway may play a critical role in pathogenesis of SLE. |
PDF Full Text Request