| Human 4-1BBL(CD137L), a type II transmembrane glycoprotein, which is encoded by a gene located at 19p13.3, belongs to TNF Ligand superfamily . 4-1BBL is expressed on many types of cells, including IFN-activated macrophages, CD40 ligand-activated B cells, dendritic cells, monocytes, T cells or tumor cells. Its receptor, 4-1 BB(CD137), a member of the TNFR superfamily, is a 30 KDa type I transmembrane glycoprotein, which is encoded by a gene located at 1p36, and expressed on activated CD4 and CD8 T cells. Ligation of 4-1BB by anti-4-1BB Abs or 4-1BBL promotes T cells activation, proliferation, cytolytic effector function, cytokine secretion, and prevents activation-induced cell death. Morever CD28 and 4-1BB were found to synergize in the induction of IL-2 by human T cells. When given in conjunction with a strong signal through the TCR, 4-1BBL can endow T cells costimulation independently of the CD28 costimulatory pathway. CD28 is important for initial T cell expansion, whereas 4-1BB/4-1BBL signaling affects T cell numbers much later in the initial response and secondary response, and is essential for the survival and/or responsiveness of the memory CD8 T cell pool. Interestingly, human 4-1BBL may be involved in reverse signaling in APC, which can induce proliferation of monocytes and some tumor cells,and cytokine production. This reverse signal may play a critical role in immune response. For these reason, blocking 4-1BB signal or 4-1BBL reverse signal could result in the immune tolerance specific to T cells and contributes to a new way for intervention of autoimmune disease, hypersensitivity and allogenetic graft rejection, paradoxically, enhancing costimulatory signals is available for antitumor immunity. Therefore,transduction may have significant theoretic and clinical value.In this study, a multiple myeloma (MM) cell line transfected with human 4-1BBL gene XG1-4-1BBL was used to immunize Balb/c mice. The immunized spleencytes were fused with mouse myeloma cells (SP2/0) by using polyethylene glycol (PEG), and then they were cultured in HAT selection medium. With XG1-4-1BBL cells, the hybridomas secreting specific mAbs were screened by immunoflurescence assay. Through repeatedly cloning and screening, one hybridoma cell line(1F1) continuously and steadily secreting specific anti-human 4-1BBL was obtained. This hybridoma grew well after long-term culture in vitro and storage in liquid nitrogen.This mAb was produced by in-vivo procedures in mouse peritoneal cavity and the ascitic tker was over 1:2000 dilution by flow cytometry assay, in which every 1 X 106 cells required 0.25-2.00 microgram purified mAb. Fast-strip method analyses displayed that this mAb belonged to mouse IgG1 (1F1).To further elucidate the recognition ability of the mAb against 4-1BBL molecule, the phenotype analysis was utilized by flow cytometry assay. The result indicated that the mAb could recognize 4-1BBL molecule expressed on SupT, Jurkat, U266, HepG2, HL60, XG1, XG6, Daudi, Raji, monocytes. These results suggested that the antigen-antibody binding 4-1BBL in this particular circumstance to expressing cells had high specificity and affinity.The monocytes from PBMCs that naturally expressing tansmember molecule 4-1 BBL were cultured in the plates that precoated with the MAb 1F1. The effect of 1F1 on the monocytes was assessed by cell number and tritiated thymidine radioactivity counting. We demonstrated that, compared with rh4-1BB protein, the MAb 1F1 could more dramatically trigger the proliferation of monocytes from PBMCs , which in the context of GM-CSF+IL-4+TNF-a can be induced into mature dendritic cells(DCs). We also found that 1F1 could promoted proliferation of DCs. These results showed a new stratagy for obtaining a larger quantity of monocytes for inducing DCs in vitro. Very interestingly, 1F1 could replace GM-CSF, and induced monocytes into mature dendritic cells in conjugationof IL-4 and TNF-a. The mechanism is still in darkness, therefore 1F1 may become a new tool for studying the effect of 4-lBB...
|
PDF Full Text Request