Preparation Of Anti-human 4-1BBL Monoclonal Antibodies And Analysis Of Their Biological Functions

Human 4-1BBL(CD 137L),a typeⅡtransmembrane 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,4-1BB(CD137),a member of the TNFR superfamily,is a 30 KDa typeⅠtransmembrane glycoprotein,which 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 reasons,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 eostimulatory signals is available for antitumor immunity.Therefore,the obtaining of anti-4-1BBL mAbs and investigation of their effects on 4-1BB/4-1BBL signal transduction may have significant theoretic and clinical value.In this study,L929 transfected with human 4-1BBL gene L929/4-1BBL 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 L929/4-1BBL cells,the hybridomas secreting specific mAbs were screened by immunoflurescence assay.Through repeatedly cloning and screening,one hybridoma cell line(3E7)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.The ascitic titer was over 1:1000 dilution by flow cytometry assay,in which every 1×10~6 cells required 0.25～2.00 microgram purified mAb.Fast-strip method analyses displayed that this mAb belonged to mouse IgG1.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 U937,SHI-1,HL-60,K562,Daudi and 8226.These results suggested that the antigen-antibody binding 4-1BBL in this particular circumstance to expressing cells had high specificity and affinity.The effect of mAb 3E7 on the monocytes was assessed by cell number.We demonstrated that the mAb 3E7 could effectively enhance the growth of monocytes and the secretion of IL-6 and TNF-α.4-1BBL was detected on the HL-60 and U937 by FCM and RT-PCR.We also found that mAb 3E7 could promote growth of HL-60 cell and U937 cell.Meanwhile,mAb 3E7 could induce HL-60 cell entering the cell cycle and secreting IL-6.In addition,colocalization of 4-1BBL and TLR-4 on the surface of HL-60 was observed by confocal microscopy.Then 4-1BBL and TLR-4 coendocytosis was monitored when incubated at 37℃,which provided evidence that 4-1BBL and TLR-4 functionally associate.In conclusion,one hybridoma continuously and steadily secreting specific anti-4-1BBL mAbs was obtained successfully.We substantiated firstly that mAb 3E7 could deliver the reverse signal,and promote growth of monocytes effectively through a reverse signal.We also found that mAb 3E7 could enhance growth of HL-60 cell and U937 and stimulate cytokine secretion.In addition,we monitored colocalization and coendocytosis of 4-1BBL and TLR-4 first time.Therefore all the results laid material basis for further studying of 4-1BBL and its biological function,meanwile the mAb 3E7 has potential clinical value.