Establishment of drug-induced tolerance for heart transplantation in large animals

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Recently, we have described a drug (cyclophosphamide (CP) plus busulfan (BU))-induced skin allograft tolerance in mice that cagularly overcome fully H-2 mismatched barriers. Using this method, we have investigated whether or not this regimen can prolong survival of heart allografts and inhibit the development of post-transplant cardiac allograft vasculopathy (CAV).Method. The components of the method are intravenous administration of 1x108 allogeneic spleen cells on day 0, intraperitoneaction of 200 mg/kg CP and 30 mg/kg BU on day 2, and intravenous injection of T cell-depleted 1x107 allogeneic bone marrow cells in the same strain of mice on day 3. Heart grafting (HG) was performed on day 28. Chimerism in peripheral blood was followed by cytometric (FCM) analysis, and the transplantation tolerance was assessed by specific acceptance of heart grafting followed by set-skin grafting. The frequency of certain Vb families was determined by FCM to assess deletion of donor-reactive T cells. H … More i al analysis (elastica van Gieson) was performed at various timings after grafting. Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytopressions in the heart grafts were analyzed with RT-PCR.Results. In a fully MHC mismatched combination of B10.D2 (H-2d, IE+)→B10 (H-2b, IE-), stable multilineage mixed chimerism was observed permanently, and IE-reactive Vb11 + T cells were specifically reduced in the periphery in the recipient B10 mice. B10.D2 grafts were accepted permanently in a donor specific manner, and post-transplant CAV did not develop. Induction of the transplan tolerance was confirmed by specific acceptance of second set-skin grafting from donor strain. In the donor B10.D2 heart grafter Th1 (IL-2, IFN-γ) nor Th2 (IL-4, IL-10) cytokine was not accumulated.Conclusions. These results demonstrated that the drug-induced tolerance recently established by us can regularly induce a long-g heart allograft tolerance without development CAV or intragraft mRNA accumulation of Th1 or Th2.Recently, we have described a drug (cyclophosphamide (CP) plus busulfan (BU))-induced skin allograft tolerance in mice that can ularly overcome fully H-2 mismatched barriers. Using this method, we have investigated whether or not this regimen can prolong urvival of heart allografts and inhibit the development of post-transplant cardiac allograft vasculopathy (CAV).Methods. The components of the method are intravenous administration of 1x108 allogeneic spleen cells on day 0, intraperitoneaction of 200 mg/kg CP and 30 mg/kg BU on day 2, and intravenous injection of T cell-depleted 1x107 allogeneic bone marrow cells in the same strain of mice on day 3. Heart grafting (HG) was performed on day 28. Chimerism in peripheral blood was followed by cytometric (FCM) analysis, and the transplantation tolerance was assessed by specific acceptance of heart grafting followed by set-skin grafting. The frequency of certain Vb families was determined by FCM to assess deletion of donor-reactive T cells. Hi al analysis (elastica van Gieson) was performed at various timings after grafting. Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytopressions in the heart grafts were analyzed with RT-PCR.Results. In a fully MHC mismatched combination of B10.D2 (H-2d, IE+)→B10 (H-2b, IE-), stable multilineage mixed chimerism was observed permanently, and IE-reactive Vb11+ T cells were specifically reduced in the periphery in the recipient B10 mice. B10.D2 grafts were accepted permanently in a donor specific manner, and post-transplant CAV did not develop. Induction of the transplan tolerance was confirmed by specific acceptance of second set-skin grafting from donor strain. In the donor B10.D2 heart grafter Th1 (IL-2, IFN-γ) nor Th2 (IL-4, IL-10) cytokine was not accumulated.Conclusions. These results demonstrated that the drug-induced tolerance recently established by us can regularly induce a long-g heart allograft tolerance without development CAV or intragraft mRNA accumulation of Th1 or Th2. Less