| GL50 is a newly identified member of the B7 superfamily. The interaction of GL50 with ICOS, the specific receptor for GL50, is critically involved in the activation, proliferation, differentiation and cytokine production of T cells as well as in the antibody secretion from B cells during the secondary immune responses. It is well-accepted that ICOS /GL50 signal pathway plays a pivotal role in the regulation of inflammatory responses, in the prevention of transplantation rejection and tumor development, as well as in the modulation of autoimmune diseases. Therefore, the specific mAb against GL50 molecule, a valuable tool for modulation of ICOS/GL50 signal pathway, not only provides a new perspective for a thorough understanding of the delicate regulation of the immune system, but also plays a potential role in clinical application.Objective: To understand the role of ICOS /GL50 signal in human immune responses and to elucidate the mechanisms by which this signal function, as well as to explore the possible application of ICOS /GL50 signal to clinical diagnosis and therapy, this study managed to prepare the specific monoclonal antibody against human GL50 molecule and to explore their role in humoral and tumor immunity.Methods and results: 6-8 week-old Balb/C mice were immunized with Daudi cells, a B lymphoma cell line. Fusion was accomplished by co-centrifuging freshly harvested spleen cells from immunized mice and murine myeloma cells SP2/0 in polyethylene glycol (PEG). GL50-L929 and VK-L929 transfectants were used as screening cells for antigen binding analysis of the hybridoma culture supernatants. After repeated "limiting dilutions" and subclonings were performed on the indicated hybridoma cells, two strains of hybridoma (named 11C4 and 12B11 respectively), secreting GL50 mAb specifically and stably were obtained. The hybridoma grew well after long-term storage in liquid nitrogen.For analysis of the characteristic of the mAbs (11C4, 12B11), (DProtein G affinity chromatography aimed to purify mAbs from ascites, (2)fast-strip method analysis used to determine the subclass of mAb, (3)competitive inhibition test designed to understand the epitope recognition of mAbs with the GL50 antigen, (4)Western-Blotting analysis conducted to discern the specific recognition of mAbs with GL50 membrane proteins and (5)immunohistochemistry staining analysis targeted to test the recognition of mAbs with GL50 molecule in indicated tissues, were performed in this study. The results indicated that the purified antibody concentration of 11C4 and 12B11 reached 4.05mg/ml and 1.56mg/ml respectively, the subclasses of 11C4 and 12B11 were both mouse IgG2a, mAbllC4 recognized different epitope from mAb 12B11 and both the mAbs could specifically recognize the membrane GL50 protein on Daudi and GL50-L929 cells. Indirect irnmunofluorescence staining and Flow Cytometry analysis suggested that GL50 was highly expressed on Daudi, HL-60, U266, moderately expressed on XG-2 and tonsilar cells and lowly expressed on 8266,Raji, Jurkat, XG-1, XG-4, XG-6, XG-7 and PBMC. Immunohistochemistry analysis indicated that some of B lymphoma samples were positively stained by mAb 11C4, but not mAb 12B11.3[H] thymidine incorporation and ELISA assay that were conducted to explore the role of ICOS/GL50 signal in the proliferation of and cytokine secretion from T cells and antibody production of B cells in vitro manifested that the interaction of GL50 with ICOS enhanced the proliferation and IL-10 secretion of T cells and regulated the antibody secretion from PWM-driven B cells. While mAb 11C4 could, to some extent, inhibit such biological effects. Trypan blue staining and MTT analysis showed an obvious inhibition of the proliferation of Daudi cells in vitro in the presence of mAb 11C4. PI staining and AnnexinV- PI double staining that were then utilized to elucidate the underling mechanism by which mAb 11C4 induced such an inhibition effect suggested that mAb 11C4 prompted an apoptosis of Daudi in vitro.Conclusion: Two strains of hybridoma (11C4, 12B11) that...
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