Preparation Of Two Anti-human CD40 Iigand Monoclonal Antibodies And Analysif Of Their Biological Functions

Preparation of tWo anti-human CD40 ligandmonoclonal antibodies and analysis of theirbiological functionsPostgraguate Fan yisunSupervisor Zhang xueguangABSTRACTCD40 ligand (CD40L, gP39, CDl54) is a 39 Kda glycoproteinexPressed as a tyPe II intergrel membrane protein on the suffoce ofactivated CD4ty cells and mast cells. CD40L, encoded by a gene locatedat Xq26.3-Xq27. 1, belongs to tumor necrosis faCtor (TN'F) suPerfamily.The demonstraion of the critical role of CD40/CD40L interactions in.vivo came frOm the discovery that the hyper IgM syndrome, an X-linked. fLmmunodeficiency is due tO a genetic alteration of the CD40L gene. Thisdisease is characterized by a server impairment of T cell dependentanibody responses with no B cell memory deficient induction of somaticmutations, and little or no circulating IgG, IgA or IgE antibodies. The roleof CD40L for B cell proliferation and germinal center formation wasdemonstrated in vivo by simultaneous tracking of anigen-specific T and Bcells after irnmunhation.The contribution of CD40/CD40L interactions to the process of T cell.. jpnming, differentiation, and effector functions has been exensivelyreviewed. The notion that CD40 is exPressed on APC has been abreNugh in this field. Activation of CD40 has iInPortan effectS on the4exPression of costimulatOry molecules and the Production of IL-l2,thereby Thl vs. Th2 responses. Priming of CD8+ cytotOxic T lymphocytes(CTh) generally requires the participation of CD4+ Th cells and APC.Recent theory is that, rather than being directly supplied to the CTL by theTh cell, help is delivered through activation of the APC, Which can thenprime the CTL directly Blockade of CD40L inhibitS CTh priming; thisinhibition is overcome by signal through CD40/CD40L, Which can rePlaceTh cells in priing ofhelperdependent CTL response.The availability of either blocking anti-mouse CD40L anibodies andboth CD40 and CD40L knockout mice have offered the possibility to testthe role of CD40/CD40L interactions in several disease models.AdIninistration of blocking anti-CD40L has been demonstrated to bebeneficial in several models of autoinununity including spontaneousdiseases lite IuPus nephritis in SNFl mice or diabetes in NOD mice or inexPerimentally induced fOrms of disease lilie EAE. CD40/CD40Linteractions play a critical role in T cell priming and have been suggestedto prohibjit tolerance induction. Therefore, interference with CD40ativation has been extensively investigated to prolong the survivl ofexPerimentally transPlaned organs. The treatment with ani-CD40Lprolong survival of transplaned organs in some murine models includingalIografts of heart, skin, aorta, and pancreatic isletS. It is imPortan that,also in rhesus monkey modeI of kidney tuspIantaion, beneficial effectSof ani-CD40L treatment have been observed.In this stUdy, human CD40L bosfected cells(CD40L-TC), whichexPressed CD40L strongly, were used to inununize Balb/C mice. The..inununixed murine splenocytes were fiIsed with SP2/0 murine myeloma5ABSTRACTcells. Monoclonal antibody screening, immunofluorescence, western blot and competition tests were used and two hybridoma cell lines, lB 1 and 4F 1 respectively, which secrete specific anti-human CD4OL mAbs were obtained. They recognized different antigen epitopes. The subclass of the mAbs were mouse IgG 1.The biological functions of mAb lB 1 and 4F 1 were investigated byh Daudi cell apoptosis inhibition test and mixed lymphocyte reaction (MLR). MAb 1B1 and mAb 4F1 could combine the CD4OL on the CD4OL-TC, then interrupted CD4O/CD4OL interactions between Daudi cells and CD4OL-TC respectively. MAb IBI and mAb 4F1 also could block the CD4O/CD4OL interactions between the donor and receptor PBMC, and inhibit MLR. But the blockade effects of mAb lB 1 and mAb 4F 1 in two tests were different from each other, in that they combine different antigen epitopes. Although several developments are...