Chimerism studies as an approach for the induction of tolerance to extremity allografts.

SUMMARY: Recent advances in the field of transplant immunology and reconstructive surgery have resulted in an increased interest in extremity allograft. Until now, more than 20 hand transplants have been performed in humans. Rejection is well controlled by currently available immunosuppressive drugs. The hand transplant, however, is not a life-supporting organ transplant and these drugs are unlikely to represent the final solution for hand transplantation due to serious adverse effects. The ultimate goal of extremity allograft is the induction of donor-specific immunotolerance. The major strategies for tolerance induction are: (1) T-cell costimulation blockade, (2) induction of mixed chimerism, (3) T-cell depletion, and (4) tolerance mediated by regulatory T cells. Amongst these, the establishment of a high level of chimerism may be the most stable strategy for donor-specific tolerance, and our laboratory has been investigating the induction of macrochimerism following extremity allotransplantation. Recently, some studies demonstrated that macrochimerism induces immunotolerance for extremity allograft in the rodent model. We made a new protocol using cyclophosphamide (CYP) and granulocyte colony-stimulation factor (G-CSF) to induce high-level chimerism following rat whole-limb allotransplantation. Limb allografting could function as a vascularised carrier for bone marrow transplantation, providing a continuous source of donor cells and contributing to a high level of chimerism in the recipient. Pretransplant CYP followed by G-CSF and FK506 treatment significantly prolong the survival of limb allografts, but frequently cause chronic graft-versus-host disease in the recipients. In this review, recent experimental chimerism studies are presented for tolerance induction and we review the prospect of clinical applicability in extremity allograft.

Donor cell repopulation of whole-limb allografts in the rat: detection with green fluorescent protein.

BACKGROUND: Although cell traffic between donor and recipient has previously been observed during allogeneic organ transplantation, little is known about cell traffic following whole-limb allografting. Whole-limb grafts are composed of composite tissues, and thus cell repopulations of recipients may be different for each component. This study was conducted using green fluorescent protein (GFP) transgenic rats to define cell repopulation of whole-limb allografts. METHODS: Twenty-four hind-limb allotransplants were performed across GFP-positive (Wistar background) and GFP-negative (Lewis) rats. Eighteen recipient animals were treated with continuous FK506 immunosuppression at a dose of 0.5 mg/kg/day up to 6 months after transplantation and assessed until 18 months posttransplantation. The expression of the GFP gene was examined under 489-nm excitation light and semiquantitatively assessed by polymerase chain reaction. RESULTS: Allografted limbs showed acute rejection in nontreated recipients, but no rejection episodes occurred in FK506-treated recipients until 18 months posttransplantation. Intense GFP expression was noted in allotransplanted GFP-negative limbs at 18 months posttransplant. GFP expression was especially marked at the interfollicular epidermis in the skin component and the endothelial cells. Polymerase chain reaction using GFP-specific primers confirmed the presence of the GFP gene in these tissues. Allotransplanted GFP-positive limbs retained marked GFP expression at the muscle fiber. CONCLUSIONS: The authors' results demonstrate that recipient-derived cells gradually migrate into grafted skin, endothelial cells, muscle, and bone marrow cells. Recipient-derived stem cells may contribute to this cell renewal within the graft. Repopulation of antigenic skin components in the graft with recipient cells may also help in avoiding rejection.

The role of cyclophosphamide and granulocyte colony-stimulation factor in achieving high-level chimerism in allotransplanted limbs.

The establishment of a high-level of chimerism may be the most stable strategy for donor-specific tolerance. The purpose of this study was to evaluate the efficacy of a new protocol using cyclophosphamide (CYP) and granulocyte colony-stimulation factor (G-CSF) to induce high-level chimerism following rat whole-limb allotransplantation. Seventy-three whole-limb allotransplants from LacZ transgenic rats to LEW rats were performed. CYP was injected at day 2, and G-CSF was given from day 0 to 3. Nontreated limb allografts were rejected after 4.2 days. In FK506-treated group for 28 days, the survival time was prolonged to 64 days. In the group treated with CYP/G-CSF, limb allografts were rejected after 5.4 days and 5 of 15 recipients showed acute lethal graft-versus-host disease (GVHD). Polymerase chain reaction (PCR) study showed a high level of chimerism even within 1 week after transplantation. Fourteen of 30 recipients given CYP/G-CSF/FK506 died within 2 weeks. The limb survival was significantly prolonged, however, with three grafts surviving more than 300 days. Seven recipients (24%) showed chronic GVHD. A high-level of chimerism was maintained when limb allografts were not rejected by recipients. Limb allografting could function as a vascularized carrier for bone marrow transplantation, provide a continuous source of donor cells and contribute to a high level of chimerism in the recipient. Pretransplant CYP followed by G-CSF and FK506 treatment significantly prolonged the survival of limb allografts but frequently caused chronic GVHD in the recipients.

Prolonged survival of rat whole-limb allografts treated with cyclophosphamide, granulocyte colony-stimulation factor and FK506.

We evaluated the efficacy of a new protocol using cyclophosphamide (CYP), granulocyte colony-stimulation factor (G-CSF) and FK506 to induce high level chimerism following rat whole-limb allotransplantation. The present study investigated the dose requirement and toxicity of CYP monotherapy in inducing stable bone marrow chimerism. Fifty-six whole-limb allotransplants from LacZ transgenic rats to LEW rats were performed. CYP at a dose of 100 mg to 200 mg/kg was injected 2 days before transplantation and G-CSF of 25 microg/kg/day was given for 4 days. FK506 was used for 28 days at 1 mg/kg/day. The level of chimerism was evaluated by semi-quantitative polymerase chain reaction. The survival of limb allografts in recipients treated with CYP of 150 mg/kg was significantly prolonged to 107 days. The onset of rejection was more prolonged to 158 days in recipients with CYP of 200 mg/kg, with two of eight grafts surviving >1 year and three recipients (38%) showed chronic, nonlethal GVHD with a high level of bone marrow chimerism. Limb allografting could contribute to chimerism in the recipient. Pretreatment with CYP had the dose-dependent effects of prolonging the survival of limb allografts. A CYP dose of 200 mg/kg appears to significantly prolong limb graft survival but frequently causes chronic nonlethal GVHD in the longer surviving recipients.

Donor cell engraftment in recipient lymphoid tissues after rat limb allograft.

BACKGROUND: The movement of cells from a transplanted tissue into the host organs, the so-called systemic chimerism, is a phenomenon known to occur and be associated with the development of immunologic tolerance in allotransplantation cases. The purpose of this study was to identify donor cell engraftment in recipient lymphoid tissues after performing rat hind limb allograft. MATERIALS AND METHODS: Fifty-five whole-limb allotransplantations were performed in sex-mismatched pairs of rats. Syngeneic male Lewis and allogeneic Dark Agouti donors were transplanted to female Lewis recipients. FK506 was used for immunosuppression. Donor male cells could be identified in the recipient female tissues by semiquantitative polymerase chain reaction analysis for a Y chromosome-specific DNA sequence. Chimerism was assessed at 1, 24, and 48 weeks after transplantation. RESULTS: There was no rejection episode in any of the limb grafts. Although levels of chimerism were highly variable in each lymphoid tissue, a gradual increase was noticed in all during the course of time. At 1 week after the transplant period, only intrasplenic chimerism was at high level (1%) in three groups. At 48 weeks after the transplant, all recipients with allografts showed very high level (10%) of chimerism in the bone marrow. Two, two, and two of six recipients showed very high levels in the spleen, lymph node, and liver, respectively, at 48 weeks. Intrathymic chimerism was higher at 24 weeks after transplant rather than at 48 weeks. CONCLUSION: We demonstrated donor cell engraftment into recipient lymphoid tissues after successful whole limb transplantation. We conclude that limb allograft can work as a vascularized carrier for the bone marrow transplantation, provide a continuous source of donor cells and contribute to chimerism in the recipient.

Cell traffic between donor and recipient following rat limb allograft.

Although cell traffic from the graft into the recipient and from the recipient into the graft had been noticed in allogeneic organ transplantation, little is known following whole-limb allografting. This study was conducted to define cell migration between donor and recipient. Sixty-seven vascularized hind limb allotransplantations were performed in rat sex-mismatched pairs and the recipient animals were treated with FK506 immunosuppression. The ratio of donor and recipient cells was evaluated by semi-quantitative PCR using the specific primers of the Y-chromosome. Allografted limbs had no rejection episode until the final assessment. The male recipient cells were detected in female limb grafts not at 1 week but at 48 weeks after transplantation. The male donor cells were detected in the humerus and tibia in the female recipient but not in the gastrocnemius muscle and leg skin. Our results demonstrated that recipient-derived cells gradually migrated into the grafted bone, muscle and skin cells with the duration of time. Donor-derived cells migrated into the healthy bones but not into the healthy muscle and skin. Because active regeneration occurs in the grafted limb to compensate graft damage secondary to ischemia and operative intervention, recipient-derived cells may mediate a muscular and dermo-epidermal renewal.

Behavior of male-specific minor histocompatibility antigen in skin and limb transplantation.

BACKGROUND: Although the role of male-specific minor histocompatibility antigen H-Y has been increasingly understood in both experimental and clinical organ transplantation, little has been investigated on musculoskeletal tissue transplantation. This study was performed to describe the behavior of male-specific minor histocompatibility H-Y antigen in rat skin and whole limb transplantation. MATERIALS AND METHODS: Using three different strains of inbred rats (Lewis, F344, and Dark Agouti), 75 donor hindlimbs and eighteen skin grafts were isogenically transplanted to the sex-mismatched recipients. Recipients were observed up to 48 weeks postoperatively. Rejection was monitored by the appearance of the skin of the grafted limb and histology. Systemic microchimerism was assessed by polymerase chain reaction using Y-chromosome specific primers. RESULTS: Skin rejection didn't occur in all limb transplant recipients and histology did not show any rejection findings in all components of the limb graft through 48 weeks. Successful functional recovery was expected. Stable and high level of chimerism (>1%) was detected in the lymphoid tissues in nontreated female recipients. Male skin grafts were rejected by Lewis and F344 female recipients within 6 weeks postoperatively. All female skin grafts survived in male recipients. CONCLUSION: Our results suggest that H-Y antigen can induce graft rejection in rat skin graft but causes no rejection reaction in whole limb transplantation. Systemic chimerism may play an important role for acceptance of sex-mismatched limb graft.