Viral Vector-mediated B7-H1 Prolong The Survival Period Of The Skin Allograft In Mice

It can be ultimately overcome the immune rejection in clinical organ transplantation by inducing donor-specific tolerance. Persistent using high dosage immunosuppressive drugs, though it could be efficacious for preventing the grafts from the attack of host's immune system, it can also increase the probability for patients to have severe opportunistic infections or tumors too,which are not suitable for all patients. For many years, researchers have been trying to achieve this goal, although some of them have succeeded in animal models, but there is no protocol available for clinical application up to now. Among all of these protocols we know, combining blockade of co-stimulatory signals with donor specific bone marrow transplantation to establish allogeneic mixed chimerism is the one that has the best prospect to be used in the clinic. Recently years, protocols of transferring CTLA4Ig gene to block B7/CD28 costimulatory signal have been proved to be able to induce organ allograft tolerance in many animal models. In our study, a recombinant lentivirus vector that can express B7-H1 was constructed to evaluate the effects of lentivirus mediated B7-H1 gene transfer on induction of skin allograft tolerance between distinguishing strains of mouse.B7-H1, also called PD-1L, a recently identified B7 family molecule, widely expressed in lymphoid and non-lymphoid tissue, is the key co-stimulatory signal which regulate the effector T cell and memory T cell. Its receptor PD-1 is an immunoreceptor that belongs to the immunoglobulin (Ig) superfamily and containing the immunoreceptor tyrosine-based inhbitory motif (ITIM). PD-1, like CD28, CTLA-4 and ICOS, is a member of the CD28 superfamily. It has 24% homology to CTLA-4 at the extracelluar region,and it also delivers an inhibitory signal to T cells. The PD-1 receptor is a type I transmembrane protein mainly expressed on activated CD4+,CD8+T cells and B cells as a monomer. In vitro studies show that B7-H1 can inhibit the proliferation of activated T cells and reduce cytokine production,especially suppress the function of effector CD8+ T cell after the engagement of binding PD-1. B7-H1/PD-1 pathway can antagonize a stronger CD28-B7 signal after the antigen exposure. Thus,it suggests that the negative role of B7-H1/PD-1 pathway in regulating T cell responses. The expression of B7-H1/PD-1 in non-lymphoid tissues indicate that they can down-regulate the response of auto-reactive T,B cells in peripheral tissues. Previously studies show that engagement of B7-H1 can downregulate the immune response of activated T,B cells,and its biological significance in sustaining peripheral tolerance. For its key role in negative modulation function and memory T cell signal,many researchers focus their interest on B7-H1.To investigate the possible mechanism of B7-H1 in allogeneic skin transplantation and its biological function, we were supposed to proceeding the following study.1. Cloning and sequencing identification of mB7-H1. Mouse liver tissue was taken and its total RNA was extracted. Using RT-PCR cDNA was reversed and mouse B7-H1 encoding sequence was amplified,gel purified.The sequence of m B7-H1 was verified by PCR amplification,restriction enzyme double digestion and sequencing.2. Construction the Lentivirus expression vector of B7-H1.The mB7-H1 recombinant lentivirus (LV/B7-H1)and control lentivirus (LV/B7-H0) were constructed using the plenti6/V5-D-TOPO vector system by homologous recombination. After the B7-H1 recombinant lentivirus infection to B16F10 cells line in selective medium,the expression of B7-H1 and V5 was verified by immunofluorescence stain using the FITC labeled specified monoclonal antibody,and the fusion protein of B7-H1 and V5 detected by flow cytometry using the FITC labeled specified monoclonal antibody. LV/B7-H1 based further investigation of the role of B7-H1 in allogeneic skin transplantation.B16F10 cells were transduced with 10-fold serial dilutions of the lentiviral supernatant(10-1 to 10-9dilutions),including recombinant lentivirus or control lentivirus. At 48 hours post-transduction, the cells were placed under Blasticidin selection medium. After 4 days of selection, the cells were stained with crystal violet, and colonies were counted. The titer of LV/B7-H1 and LV/B7-H0 were detected by microscope counting and it comes up to 3.4×108 TU/ml and 8.5×108 TU/ml respectively.3. Construction the adenovirus expression vector of B7-H1.The mB7-H1 recombinant adenovirus (AD/B7-H1) and control adenovirus (AD/B7-H0) were constructed by Wenyuan Duan. It was identified that vector had been constructed successfully. The titer of AD/B7-H1 and AD/B7-H0 were detected by GFPcounting and it comes up to 1×1012 TU/ml and 3×1012 TU/ml respectively.4. Viral vector-mediated B7-H1 skin transplantationTo investigate the effect of LV/B7-H1 in allogeneic skin transplantation in mice. C57BL/6 and BALB/c mice were used as skin transplant donors and recipients respectively. Seventy BALB/c mice were randomly divided into A (normal control),B (Ciclosporin A,CsA),C (B7-H1 recombinatant lentivirus,LV/B7-H1),D (empty B7-H1 lentivirus vector,LV/B7-H0),E(B7-H1 recombinatant adenovirus,AD/B7-H1),F(empty B7-H1 adenovirus vector,AD/B7-H0),G(B7-H1 recombinatant lentivirus and adenovirus,LV/B7-H1+ AD/B7-H1) groups,with 10 mice each group. All mice were preconditioned different factor according to their groups before skin transplantation,and the mice in C,D,E,F,G groups were again administered viral suspension correspondingly on 7th day after operation. Median survival time (MST) of skin graft was recorded,and biopsies of grafts on 5th and 10th day were harvested for histologic examination. The clear epithelial structure and infiltration of inflammatory cells were observed in specimens.The MST of all groups from A to G is 10.80±1.87,14.80±3.43, 20.5±4.30,11.1±2.42,15.6±2.32,10.7±2.06 and 16.1±2.73 respectively.When compared to A group,the MST of skin graft in D and F groups were not significantly difference (P>0.05), the MST of group B,C,E and G is significantly prolonged compared with the group A,D and F(P<0.01); The differences among E and G compared with group B is significant (P<0.05);The MST differences between group E and G is not significan(P>0.05)t;the MST of group C is significantly prolonged compared with group E and G (P<0.01),these results showed that the strategy adopted in this experiment LV/B7-H1 could successfully induce the establishment of specific transplant tolerance in murine,and significantly improve the survival of donor-specific skin grafts.The date from our test above show that it might be a prosperous strategy for immune tolerance inducing in skin transplantation, may have potential application in clinical .