Experimental Study On Prolonging The Survival Of Heart Allografts With Adenovirus-mediated MHVEM Gene Transfection In Mice

Background:The major cause of graft failure for all types of organ transplantation is rejection. Despite an ever-growing armamentarium of immunosuppressive drugs to combat the recipient's immune response to the transplanted organ ,suppression of the allospecific T cell Response remains a major challenge. Co-receptor signaling is an important mechanism of coordinating and tightly regulating immune response. For instance, activation of naive T cells requires a second co-stimulatory signal in addition to stimulation of the T cell receptor by engagement with peptide-MHC complexes. Conversely, co-inhibitory signals are required to maintain T cell self-tolerance and prevent autoimmunity . The CD28-like family is one important class of co-receptors. These members of the immunoglobulin superfamily (IgSF)2 function as either co-stimulators (CD28 and inducible T cell costimulator) or co-inhibitors (CTLA-4, programmed death-1, and BTLA) in modulating immune cell activity.Recently the CD28 family member BTLA was unexpectedly shown to bind and be activated by the TNFRSF member herpes virus entry mediator. This is the first example of cross-talk between the CD28 family and the TNFRSF. Whereas HVEM has been previously described as a co-stimulator triggered by the TNF-like ligands lymphotoxin (LT) and LIGHT , recent results from HVEM knock-out mice as well as the interaction between BTLA and HVEM are consistent with HVEM playing a co-inhibitory roleIn my research, the recombinant mouse's HVEM ( mutated LIGHT binding site) protein and EGFP, which are believed to effectively activate of HVEM-BTLA co-inhibitory pathway in T cell activation, was chosen as the immunomodulator. In our previous research, the recombinant CTLA4Ig and PD1 adenovirus vector was successfully constructed by Doctor Huang Chibing and He Weifeng. Here, I focused on constructing mouse's HVEM gene-recombinant adenovirus . The effective way of transferring these target genes into the heart allografts with gene-recombinant adenovirus was explored. Furthermore, the effect of modification of heart allografts with target genes on the survival of heart allograft was investigated in mice. The main results and conclusions were as follows:Objective:1.Amplification and mutation of HVEM cDNA fragment from active T cell (by ConA) by nest RT-PCR.2.Construction and identification of HVEM and EGFP gene recombinant replication-deficient adenovirus.3. The effective way of recombinant adenovirus transfecting HVEM gene into hearts of donor mice.4. Built cervical heterotopic heart transplantation model.5. The effect of the survival of heart allografts transfected with HVEM geneMethods and Results:1. Amplification of HVEM full length domain cDNA fragment from tonsil by nest RT-PCRIn this study, with the help of computer-aided analysis, two pairs of PCR primers were designed based on the published HVEM cDNA in Genebank which encoding 32-859aa of the full length domain and a signal peptide. A 827bp cDNA fragment was amplified by reverse-transcription methods followed by two cycles of PCR from the total RNA of active T cell (by ConA). Moreover, the fragment mutated by LIGHT binding site (CDR3), end the fragment was then verified by sequencing analysis to make sure that the cDNA is just the one we expected.2. Construction and identification of HVEM full length domain gene recombinant replication-deficient adenovirus.In this research, we use AdEasy system from Stratagene Company to construct HVEM recombinant replication-deficient adenovirus. Firstly, we clone HVEM full length domain gene into an AdEasy transfer vector pAdtrack-cmv plasmid; Secondly, the resulting plasmid pAdtrack-cmv-HVEM was then linearized with PmeI and co-transformed into the E.coli strain BJ5183 together with pAdEasy-1, the viral DNA plasmid. The pAdEasy-1 is E1 and E3 deleted; its E1 functions can be complemented in 293 cells. Recombinants are selected with kanamycin and screened by restriction enzyme analysis. The recombinant adenoviral construct is then cleaved with PacI to expose its ITR (Inverted Terminal Repeats) and transfected into 293 cells to produce viral particles. The resulting recombinant adenovirus was identified by PCR and expression of HVEM full length domain protein by immunohistochemisty. Furthermore, the biological activity of HVEM full length domain protein produced by recombinant adenovirus was determined by MLR.3. Simultaneous expression of both HVEM full length domain and EGFP gene in PK15 cell line.Transduction efficiency varies greatly from one cell line to another. So, after we have harvest the recombinant adenovirus, it is very important for us to determine if an Ad particle can transduce its DNA into histiocytes. In our study, we used EGFP which carried by recombinant adenovirus to perform the infectivity test.4. Cytotoxicity of Adv-HVEMThe cell lines PK-15 and ECV-304 were transfected by Adv-HVEM with the tite of 109,108 and 107. Significant difference of the survival rates of the cells were not observed between the transfected cells and the controls. The results suggest that Adv-HVEM have little cytotoxic effect on PK-15 and ECV-304 cell line. The expression of EGFP in the two kinds of transfected cell lines was determined by fluorescence microscope. The results showed that the expression rates of EGFP in both cell lines transfected at 37℃were over 90% while the expression rates and tensities in both cell lines transfected at 4℃were decreased significantly, which suggest that the transfection temperature be the important factor influencing the tranfection efficiency and the expression of target gene. The expression of HVEM full length domain protein in the two kinds of transfected cell lines was determined by immunohistochemistry. These results suggest that Adv-HVEM has the ability mediating non-packing cells to express HVEM protein, thus sets the functional foundation of further gene manipulation in organ grafts.5. The effective way of recombinant adenovirus transfecting HVEM gene into hearts of donor mice.The transfection efficiency of Adv-HVEM in heart of BALB/c mice was determined by observing the expression of EGFP in heart with fluorescence microscope. The results showed that green fluorescence was not observed in section of heart after perfusion of Adv-HVEM into heart at 37℃for 3d. However, evident green fluorescence was observed in sections of kidneys when the vessel of liver and the aorta and vena cava below the renal vessel were ligated and Adv-HVEM was then perfusion in vivo via the renal artery for 3d and the green fluorescence was ever increased when the renal vessel was clamped intermittently during the transfection. The results suggested that the mouse heart cell could be transfected efficiently by high titer Adv-HVEM in circulation at body temperature may increase the transfection efficiency. The results showed that the expression had occurred on the 3^rd postoperative day, which suggest that HVEM protein can be expressed for a short time(2 weeks) in HVEM gene-transfected heart after transplantation.6. Establishment of new model for cervical heterotopic heart transplantation in mice.The surgical technique of heterotopic cervical heart transplantation in mice, a standard model for transplantation immunology research, is very challenging restricting the widespread use of this model. We connect the recipient's external jugular vein with aorta by using a 24GA Cuff and the recipient's common carotid artery whit the pulmonary aorta by using self-made Cuff. Findings: we describe the modified methods with a shortened transplant ischemic time( less than 15mins ) and higher success rate( >90% ).7. The effect of HVEM gene transfection in heart on the survival of heart allograftsThe effect of HVEM gene transfection in kidney on the survival of renal allografts was observed in BALB/c (H-2d)→C57BL/6 (B6; H-2b) cervical heterotopic heart transplantation manner. The results showed that the survival time of HVEM gene-transfected HVEM allograft was prolonged significantly compared with that in controls (32±8.0d vs 8.5±1.4d , p<0.01) and that the function of HVEM gene transfected heart on the 2nd and 20th postoperative days were superior to that of controls on the 8th postoperative day significantly and that at the same time the inflammation in gene-transfected grafts was less sever than that in controls. The results suggest that HVEM gene transfection in heart can reduce immune injury to heart allografts and prolong the survival time significantly but can't induce indefinite survival of the grafts. The immune status of recipients with gene-transfected kidney were assessed by determining the reaction of recipients splenic cells to donors and the third party's splenic cells. The results showed that a hyporesponsiveness to donor occurred while the normal reaction to the third party was retained, which suggest that a donor-specific hypo-responsive status be induced in the recipients. Conclusion:1. The HVEM full length cDNA fragment was successfully Amplified and mutated by nest RT-PCR from mouse active T cell (by ConA).2. The HVEM full length gene recombinant replication-deficient adenovirus was successfully constructed.3. The HVEM full length gene recombinant replication-deficient adenovirus have the qualities of high efficiency, stable expression of HVEM and safety in cultured cells.4. Warm perfusion was one of the major factor that influence on Adv-HVEM mediated HVEM gene transfection in hearts of mice.5. HVEM gene transfecting heart significantly prolonged the survival of recipient in mice cervical heterotopic heart transplantation model.