Combination Of CTLA4-FasL Gene Transfer And Allogeneic Bone Marrow Transplantation Led To Durable Macrochimerism And Donor-specific Tolerance

[Objective] Mixed hemopoietic chimerism has the potential to induce donor specific tolerance and eliminate chronic immunosuppressive therapy following organ transplantation. To date, clinical implementation of a safe and effective tolerance protocol has not yet been achieved. New experimental protocols for inducing tolerance through bone marrow transplantation(BMT) without toxic conditioning might be developed. Our study aimed to analyse whether an alternative, nonmyeloablative based strategy using a single injection of recombination adenovirus vector encoding CTLA4-FasL fusing gene and donor bone marrow can induce durable mixed macrochimerism and skin allograft tolerance, and to analyse the effects of conventional immunosuppressive drugs on chimerism and tolerance induced by this regime.[Methods] 1. Recombinant fusion gene CTLA4-FasL from plasmid pcDNA3.1 CTLA4-FasL digested with KpnI and XholI restriction enzyme inserted to adenovirus shuttle plasmid pAdTrack-CMV, then cotransfected with Ad backbone plasmid named pAdeasy-1 into E coli. strain BJ5183 for preparing recombinant adenovirus plasmid pAd-CTLA4-FasL. After that it was transfected into 293 cells to produce intact Adenovirus Ad-CTLA4-FasL. 2.C57BL/6(H-2~b,B6) mice received BALB/c(H-2~d) mice skin transplantation, followed by both adenovirus vector encoding CTLA4-FasL gene and bone marrow cells(BMCs) of BALB/c mice administrated via tail vein. Then, the serum level of CTLA4-FasL fusion protein was detected by enzyme-linked immunosorbent assay (ELISA), morphological analysis of liver tissue was performed, and the degree of chimerism were analyzed by flowcytometry(FCM), and skin grafts survival were observed, and the frequency of HTLp and CTLp was quantitatively analysed by limiting dilution mixed lymphocyte culture (LD-MLC). 3. C57BL/6(H-2~b,B6) mice received BALB/c(H-2~d) mice skin transplantation, followed by both adenovirus vector encoding CTLA4-FasL gene and bone marrow cells(BMC) of BALB/c mice injected via tail vein. Cyclosporin A(CsA) or mycophenolate mofetil (MMF) or both was administered daily from day 0 through 28 after donor skin transplantation.Then, skin grafts survival were observed, the level of V β 11+T cell and donor chimerism were detected by flow cytometry, tolerance to donor antigen was evaluated through one-way MLR.[Results] 1. AdCTLA4-FasL DNA was correct production of homologous recombination between adenovirus shuttle plasmid pAdTrack-CMV and backbone plasmid named pAdeasy-1,indicated by digestation with PacI restriction enzyme. PCR analyse shew that this recombinant virus include exogenous CTLA4-FasL gene. Moreover Western-Blot analyses proved that secretory fusing protein CTLA4-FasL could be expressed in culture supernatant after transfectation of these viruses into 293 cells. 2. In vivo, the expression level of fusing protein CTLA4-FasL reached culmination two weeks after injection of adenovirus vector encoding CTLA4-FasL gene into B6 mice through caudal vein ,and the expression of this protein lasted more than 56 days. Furthermore, AdCTLA4-FasL led to less hepatotoxicity morphological analysis of liver tissue. The alternative nonmyeloablative based strategy of using a single injection of recombination adenovirus vector encoding CTLA4-FasL fusing gene and donor bone marrow cells to promote durable mixed macrochimerism (>20% on 140d). Chimeras exhibited robust donor-specific tolerance, as evidenced by acceptance of fully allogeneic skin grafts (the mean survival time (MST)>200 d) and rejection of third-party skin grafts in a normal manner (MST<10 d). In this model, the frequencies of helper T lymphocyte precursor (HTLp) and cytotoxic T lymphocyte precursor (CTLp) were greatly reduced on day 14 after transplantation, suggesting that CTLA4-FasL led to rapid systemic peripheral tolerance to facilitate the bone marrow engraftment, while both HTLp and CTLp remained at low level only in recipient mice with mixed chimerism on day 140 after transplantation, demonstrating that long-term skin grafts tolerance was associated with stable mixed chimerism, and central deletion of donor specific T cell may be the main mechanism for tolerance maintenance. 3. In our non-cytoreductive model using BMT and CTLA4-FasL gene transfer, short-couse immunosuppression facilitated early engraftment of donor bone marrow, while the mixed chimerism in peripheral blood of B6 recipients treated with CsA or both CsA and MMF were reduced very low level on day 140 after skin transplantation; when compared with B6 recipients treated with MMF or no immunosuppressive drugs, the mean survival time(MST) of skin allografts of those mice treated with CsA or both CsA and MMF was significantly reduced, the levels of V β 11+T cell was more higher; furthermore, B6 recipients treated with CsA or both CsA and MMF show no tolerance to donor antigen.[Conclusions] 1. recombination adenovirus vector encoding CTLA4-FasL fusing gene was successful generated using AdEasy vector system. 2.Combined protocol of AdCTLA4-FasL and donor BMT established stable mixed macrochimerism and prolonged mouse skin allografts survival. Peripheral and central elimination of alloreactive T cells were involved in the induction and maintenance of tolerance, respectively. 3.Immunosupression regime containing CsA could abrogate long-lasting mixed chimerism induction and tolerance development in non-cytoreductive model using BMT and CTLA4-FasL gene transfer, owing to negative effect of CsA on peripheral deletion of donor-specific T cell. However, MMF was compatible with our non-cytoreductive protocol.