Experimental Study On Suppress Of High-risk Corneal Immune Rejection By Local CTLA4-Ig Expression

Experimental study on suppress of high-risk corneal immune rejection by local CTLA4-Ig expression Shaowei Li, Directed by Professor Lixin Xie.PurposeImmune rejection is the leading cause of corneal allograft failure,especially for the high-risk cases[l]. The critical role of T cells in allograft rejection is well established. CTLA4-Ig, a recombinant form of CTLA4 has been shown to inhitit T-Cell activation by blocking the costimulatory pathway, and thereby prolong graft survival and even produce donor-specific tolerance in some animal transplant models[2-7].However, there still remain many unresolved issues with respect to how best to use CTLA4-Ig in clinical setting. In of which, the relative shorter serum elimination half-life of the recombind CTLA4-Ig is concerned[3], and led to seeking of the potential of using gene therapy.Several studies were conducted in preventing corneal allograft by using CTLA4-Ig, but none of them demonstrated long-term graft survival[8-ll].It was noted that most of the studies using gene transfer were focused on endothelial layer. The reaseon is that it is believed that the major target of corneal graft rejection is the corneal endothelium. But numourus studies have shown that it was mainly the corneal stoma and epithelial layer act as the alloantigen[12].In this study, we explored the potential use of gene therapy in preventing of corneal rejection by transferring Ad-CTLA4-Ig to the different layers or sites of the donor or recipient eye in rabbit model.Materials and MethodsRecombinant adenovirusesAd-LacZ was the kind gift of Dr.Youhai Chen(Department of Molecular and Cellular engineering, University of Pennsylvania, School of Mecicine). Ad-GFP was the kind gift of Dr.Frank Liang(F.M. Kirby Center for Molecualr Ophthalmology, Department of Ophthalmology, University of Pennsylvania). Ad-CTLA4-Ig(human)was the kind of gift of Dr. Brend Ray-Sea Hsu(Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang-Gung Memorial hospital and Chan Gung University Medical College, Taiwan).Ad-CTLA4-Ig(mice) was a kind gift of Dr.Taosheng Li(First Department of Surgery,Yamaguchi University School of Medicine,Japan).The recombined adenovirus vectors were propagated in 293 cells and purified by centrifugation over caesium chloride gradients and titred following standard protocols[13-15].Detection of gene expressionGreen fluorescence protein expressed by Ad-GFP was observed under fluorescence microscope. LacZ gene expression was detected by X-Gal solution described previously[16]. CTLA4-Ig expression was detected by immunohistochemistry using goat antihuman IgG antibody(Sigma, 1-3382) as primary antibodies, and FITC-conjugated Rabbit Anti-Goat Ig, DAKO, F0250) as secondary antibodies.The results were observed under fluorescence microscope.Adenovirus-mediated Gene TransferThe Ad-CTLA4-Ig, Ad-LacZ, Ad-GFP were transferred to the corneal endothelium according to the method described before[ll,17]. Briefly, NZW rabbit corneas were incubated in 1ml 2% fetal calf serum(FCS) in DMEM containing 1.4X105 PFU/ml of Ad-CTLA4-Ig or AdD-LacZ or Ad-GFP for 3 hours at 37C in 5%CO2 After incubation, specimens were washed three times with Hanks' balanced salt solution. The corneas were translated to a healthy NZW rabbit eye. The gene expression was examined at different time point after penetrating keratoplasty(PKP).Genes were transfected into corneal stoma by direct injection of recombinant adenoviruses using a 1ml syringe with a 30 gage needle. The expression of transferred gene were examined at different time point.Gene in vivo transferring to rabbit anterior chamber was conducted according to the method described previously[18], in which the recombinant adenoviruses were injected into the anterior chamber.Penetrating keratplastyNew Zealand White (NZW) rabbits of both sexes weighing between 2.5-3.Okg were used as experimental model. Throughout this study, all animals were handled according to the ARVO Resolution on the Use of Animal...