Induction Of Donor-specific Tolerance Research Articles

MIXED CHIMERISM WITH COSTIMULATORY BLOCKADE PROLONGS ALLOGENEIC GRAFT SURVIVAL IN MURINE SMALL BOWEL TRANSPLANTATION.

P1032 Aims: Mixed chimerism is known as one of the most powerful method to induce donor specific tolerance. Here we evaluate the potential advantage of mixed chimerism with costimulatory blockade in murine allogeneic small bowel transplantation. Methods: C57BL/6 received various combination of anti-CD8 (2.43; 1.4mg, day -2) and anti-CD154 (MR1; 2mg, day -1)mAbs with/without 3Gy total body irradiation (TBI, day -1), and 20×106 fully MHC-mismatched B10.A bone marrow cells (BMC, day -1). Heterotopic small bowel transplantation was performed on day 0 to assess induction of donor specific tolerance. Chimerism in peripheral blood was followed by FCM analysis and the frequency of TCR Vβ usage was determined by FCM to assess deletion of donor-reactive T cells. Stoma was daily observed, and acute rejection was evaluated with a microscope on POD14. Results: All animals without any treatment (n=6) showed acute rejection within 18 days after transplantation, and microscope examination presented the findings of acute rejection such as flatttend villous epithelium with the infiltration of inflammatory cells. Mice receiving anti-CD8 and anti-CD154mAbs were not induced mixed chimerism, and 4 of 8 mice rejected small bowel allograft by 79 days. All animals treated with anti-CD8mAb, anti-CD154, TBI, and BMC was induced long term multi-lineage mixed chimerism and accepted small bowel allografts (>60 days). Microscope examination presented the no findings of rejection. These chimeric animals showed specific deletion of donor reactive CD4+ peripheral blood lymphocytes (Vβ5 and Vβ11 cells). Conclusions: Prolongation of allogeneic small bowel graft survival was achieved using mixed chimerism with costimulatory blockade. Mixed chimerism with costimulatory blockade is the reliable regimen toward to the small bowel transplantation.

DIFFERENT ROUTES OF DONOR DERIVED HEMATOPOIETIC STEM CELLS TRANSPLANTATION FOR DONOR SPECIFIC CHIMERISM INDUCTION ACROSS MHC BARRIER

P940 Aims: Transplantation of donor–derived stem cells can improve organ allograft survival in animal models. This study was designed to investigate the effect of different routes of stem cell transplantation (SCT) on donor specific tolerance induction across MHC barrier under short-term CsA monotherapy and αβTCR/CsA treatment protocols. Methods: Forty-eight SCT were performed between BN (RT1n) donors and Lewis (RT1l) recipients. Intraosseous and intravenous SCT was studied in 6 groups of 8 animals each receiving 35x106 (n=4) and 70x106 (n=4) SCT. Groups I and II served as controls and received SCT but no treatment. Groups III and IV received CsA monotherapy for 7-days, Groups V and VI were under αβTCR/CsA protocol for 7-day. Flow cytometry analysis was used for evaluation of immunodepletion of T-lymphocytes and multilineage donor specific chimerism for MHC class-I (RT1n) for RT1n/CD4, RT1n/CD8 and RT1n/CD45RA antigens in peripheral blood of recipients at post-transplant days 7, 21, 35 and 63. Results: All animals survived without sign of graft versus host disease. At day 63 non-treatment groups receiving 70x106 SCT developed 4.3% of multilineage donor-specific chimerism following intraosseous transplantation and 1.0% following intravenous transplantation. At day 7 under CsA monotherapy and 70x106 SCT, chimerism level for 1.5% RT1n/CD4 was induced in intraosseous and 10.5% in intravenous transplanted groups. At day 63 following intraosseous transplantation chimerism for RT1n/CD4 was stable, but following intravenous transplantation significantly declined to 0.8%. Under αβTCR/CsA protocol T-lymphocyte subpopulation was significantly depleted at day 7 and repopulated to pre-transplant level at day 63. After intraosseous transplantation of 70x106 SCT at day 7 chimerism was 5.7% for RT1n/CD4, and increased up to 6.5% at day 63. In contrast in intravenously transplanted groups at day 7 chimerism was higher and showed 23.7% for RT1n/CD4 antigens, however declined to 0.9% at day 63. Multilineage chimerism was 75% higher in intraosseous (9.9%) when compared with intravenous (3.4%) transplant groups receiving 70x106 SCT at day 63. Following intraosseous SCT under αβTCR/CsA protocol, multilineage chimerism was 50% higher in group receiving 70x106 stem cells (9.9%) compared to 35x106 stem cells (4.9%). Conclusions: Stem cell transplantation under αβTCR/CsA protocol showed better engraftment of donor cells following intraosseous delivery leading to development of higher level (75%) of multilineage donor-specific chimerism when compared with intravenous transplantation. High level of chimerism in intravenously transplanted groups was transient, whereas intraosseous transplantation induced stable chimerism, which was maintained up to 63 days post-transplantation. This strategy of intraosseous stem cell transplantation under αβTCR/CsA protocol could be applied for chimerism and tolerance induction during transplantation of composite and solid organ allografts.

Functional antigen-presenting leucocytes derived from human embryonic stem cells in vitro

Differentiated cells derived from pluripotent human embryonic stem (hES) cells offer the opportunity for new transplantation therapies. However, hES cells and their differentiated progeny express highly polymorphic MHC molecules that serve as major graft rejection antigens to the immune system of allogeneic hosts. To achieve sustained engraftment of donor cells, strategies must be developed to overcome graft rejection without broadly suppressing host immunity. One approach entails induction of donor-specific immune tolerance by establishing chimeric engraftment in hosts with haemopoietic cells derived from an existing hES cell line. We aimed to develop methods to efficiently differentiate hES cells to haemopoietic cells, including immune-modulating leucocytes, a prerequisite of the tolerance induction strategies applying to hES cell-mediated transplantation. We developed a method to generate a broad range of haemopoietic cells from hES-generated embryonic bodies in the absence of murine stromal feeder cells. Embryonic bodies were further cultured in the presence of haemopoietic cytokines. In addition to flow cytometric analyses of haemopoietic cell markers, we analysed the hES cell-derived haemopoietic cells by colony-forming assays (for erythroid and myeloid progenitor cells), cytochemical staining, and mixed leucocyte reactions to determine the functional capacity of the generated antigen-presenting cells. 12 independent experiments were done. When selected growth factors were added, leucocytes expressing CD45 were generated and released into culture media for 6-7 weeks. Under the condition used, both erythroid and myeloid progenitor cells were generated. About 25% of the generated leucocytes acquired MHC class II and costimulatory molecule expression. These hES-derived, MHC class II+ leucocytes resembled dendritic cells and macrophages, and they functioned as antigen-presenting cells capable of eliciting allogeneic CD4 and CD8 T-cell responses in culture. The hES cell-derived antigen-presenting cells could be used to regulate alloreactive T cells and induce immune tolerance for improvement of the transplant acceptance of hES-cell derivatives.

Generation of tolerogenic dendritic cells by treatment with mitomycin C: inhibition of allogeneic T-cell response is mediated by downregulation of ICAM-1, CD80, and CD86.

Deliberately generated tolerogenic dendritic cells (DC) might be a useful tool for induction of donor-specific tolerance in transplantation. In this article, the authors study the effect of mitomycin C (MMC)-treated DC on rat T cells and delineate the mechanism of their conversion into tolerogenic cells. The influence of MMC treatment on the capacity of DC to activate allogeneic T cells was tested in vitro, and the expression of cell surface molecules was studied by flow cytometry. MMC-treated DC lose their allostimulatory capacity, and this cannot be attributed to cell death or release of MMC. Interestingly, suppressed T cells cannot be restimulated, indicating that MMC-treated DC induce tolerance. MMC treatment selectively decreases adhesion (intercellular adhesion molecule [ICAM]-1) and co-stimulatory (CD80, CD86) molecules. Functional blocking of these molecules with specific antibodies confers to DC the same T-cell-suppressive properties as treatment with MMC. MMC treatment converts rat DC into tolerogenic cells. This mechanism is mediated by decrease of ICAM-1, CD80, and CD86.

Generation of anergic and regulatory T cells following prolonged exposure to a harmless antigen.

Regulatory CD4(+) T cells are known to develop during the induction of donor-specific peripheral tolerance to transplanted tissues; it is proposed that such tolerance is a consequence of persistent, danger-free stimulation by Ag. To test this hypothesis, male RAG-1(-/-) mice were recolonized with small numbers of monospecific CD4(+) T cells specific for the male H-2E(k)-restricted Ag Dby. After 6 wk in the male environment, the monospecific CD4(+) T cells, having recolonized the host, had become anergic to stimulation in vitro and had acquired a regulatory capacity. CD4(+) T cells in these mice expressed higher levels of CTLA-4 and glucocorticoid-induced TNF-related receptor than naive CD4(+) T cells, but only 3% of the recolonizing cells were CD25(+) and did not express significant foxP3 mRNA. In vivo, these tolerant T cells could censor accumulation of, and IFN-gamma production by, naive T cells, with only a slight inhibition of proliferation. This suppressive effect was not reversed by the addition of fresh bone marrow-derived male dendritic cells. These results suggest that persistent exposure to Ag in conditions that fail to evoke proinflammatory stimuli leads to the development of T cells that are both anergic and regulatory.

Monoclonal antibodies as tools to induce immune tolerance

The induction of donor specific transplantation tolerance remains the ultimate goal for clinical organ grafting, as it would avoid the problems of toxicity, susceptibility to infections and tumors that arise from the current need to give long-term, nonspecific, immunosuppressive drugs. It is now more than 50 years since it was first demonstrated that such tolerance could be induced in neonatal rodents, or lethally irradiated adults, by inducing sufficient donor chimerism to maintain tolerance by clonal deletion of alloreactive T cells in the thymus. Many different attempts to extend such approaches to the clinic have generally not proven to be viable. More recently, it was found that monoclonal antibodies against various T-cell antigens, particularly combinations of CD4, CD40L, and CD8, were able to block rejection of allografts in adult rodents [1]. More important, such recipients eventually became specifically tolerant of the donor alloantigens, able to accept fresh donor-type grafts, but reject those from a third party. This induction of tolerance did not require any depletion or overt clonal deletion of donor-reactive T cells, and was independent of the thymus. Tolerance could be induced directly to skin [1] or vascularized heart grafts [2] across a full major histocompatibility complex (MHC) plus minor antigens mismatch. In vitro there were no differences in T-cell responses from tolerant or rejecting mice, but tolerance in vivo was found to be dependent on CD4 + regulatory T cells [3].

Stem cell–mediated tolerance inducing strategies in organ transplantation

The scope of possible tools to modulate the recipients immune response towards tolerance induction basically includes deletional and non deletional mechanisms, which are currently targeted by various strategies including monoclonal antibodies, cytokine deviation, chimerism induction and the support of regulating T-cells. Here we summarize the main findings in the field derived from experimental animal studies and currently performed clinical studies. This review focuses to give a clinically relevant overview over relevant tolerance inducing concepts, taking into consideration risk profiles and clinical efficacy associated with specific immunosuppressive regiments currently applied in the clinical setting of transplantation.

Production of donor T cells is critical for induction of donor-specific tolerance and maintenance of chimerism.

Nonmyeloablative conditioning has significantly reduced the morbidity associated with bone marrow transplantation. The donor hemopoietic cell lineage(s) responsible for the induction and maintenance of tolerance in nonmyeloablatively conditioned recipients is not defined. In the present studies we evaluated which hemopoietic stem cell-derived components are critical to the induction of tolerance in a total body irradiation-based model. Recipient B10 mice were pretreated with mAbs and transplanted with allogeneic B10.BR bone marrow after conditioning with 100-300 cGy total body irradiation. The proportion of recipients engrafting increased in a dose-dependent fashion. All chimeric recipients exhibited multilineage donor cell production. However, induction of tolerance correlated strictly with early production of donor T cells. The chimeras without donor T cells rejected donor skin grafts and demonstrated strong antidonor reactivity in vitro, while possessing high levels of donor chimerism. These animals lost chimerism within 8 mo. Differentiation into T cells was aborted at a prethymic stage in recipients that did not produce donor T cells. Moreover, donor Ag-driven clonal deletion of recipient T cells occurred only in chimeras with donor T cells. These results demonstrate that donor T cell production is critical in the induction of transplantation tolerance and the maintenance of durable chimerism. In addition, donor T cell production directly correlates with the deletion of potentially alloreactive cells.

Anti-ICAM-1 antibody and CTLA-4Ig synergistically enhance immature dendritic cells to induce donor-specific immune tolerance in vivo

Immature dendritic cells (DC) have been demonstrated to induce T-cell hyporesponsiveness in vitro and immune tolerance in vivo. However, immature DC (iDC) may become mature once infused in vivo, thus limiting the prolongation of the allograft survival. Considering that mature DC express high level of B7, intercellular adhesion molecule-1 (ICAM-1), and T-cell activation needs costimulation signals provided by DC, we selected anti-ICAM-1 mAb and cytotoxic T lymphocyte antigen-4Ig fusion protein (CTLA-4Ig) for in vivo administration to block costimulation pathways in order to further improve the efficacy of iDC to induce immune tolerance. Seven days before allogeneic cardiac transplantations, the recipients were intravenously (i.v.) pretreated of donor-derived iDC with or without simultaneous injections of anti-ICAM-1 mAb and CTLA-4Ig. CTLA-4Ig or anti-ICAM-1 mAb administration alone resulted in significant prolongation of cardiac allograft survival induced by iDC. When used simultaneously, CTLA-4Ig and anti-ICAM-1 mAb induced permanent allografts acceptance even in 90% recipients. The recipients could keep the skin alive for a longer time in the donor-specific second transplantation, but no effect was observed on the skin from C3H third-party mice. The efficient induction of donor-specific tolerance observed above may be related to the more potent inhibition of donor-specific T-cell responses including cytotoxicity activity, Th1 cytokines production, and alloantibody production by the combined use of anti-ICAM-1 mAb and CTLA-4Ig. Our data suggest that anti-ICAM-1 antibody and CTLA-4Ig can synergistically enhance iDC to induce donor-specific immune tolerance in vivo.

Lack of Role for CsA‐Sensitive or Fas Pathways in the Tolerization of CD4 T Cells Via BMT and Anti‐CD40L

Anti-CD40L mAb plus bone marrow transplantation (BMT) and recipient CD8 T-cell depletion permits long-term mixed hematopoietic chimerism and systemic donor-specific tolerance to be achieved across full MHC barriers. Initial tolerance is characterized by peripheral deletion of donor-reactive CD4 cells. In regimens using costimulatory blockade without BMT to achieve allograft survival, cyclosporine inhibited graft survival, suggesting that the combination may not be clinically applicable. We assessed the role of cyclosporine-sensitive mechanisms and the mechanisms of T-cell apoptosis involved in the induction of early peripheral CD4+ T-cell tolerance by BMT with anti-CD40L. Neither a short course of cyclosporine (14 days) nor the absence of FAS-mediated activation-induced cell death (AICD) blocked the induction or maintenance of donor-specific tolerance. IL-2 production was not associated with tolerance induction, consistent with the lack of a role for Fas-mediated AICD. Mice in which passive T-cell death was impaired because of constitutive expression of a Bcl-xL transgene did not develop tolerance with this protocol. These data confirm that deletion of donor-reactive T cells is critical for the induction of mixed chimerism and tolerance. However, the mechanisms involved may differ from those involved in costimulatory blockade regimens that do not include BMT.

Towards a myeloablative regimen with clinical potential: I. Treosulfan conditioning and bone marrow transplantation allow induction of donor-specific tolerance for skin grafts across full MHC barriers.

To investigate whether we could create a radiation-free conditioning regimen to induce permanent mixed and multilineage chimerism and donor-specific tolerance, we treated recipient mice with anti-T-cell antibodies, varying and fractionated doses of Treosulfan and fully MHC disparate bone marrow cells. Treosulfan is mainly used in the treatment of ovarian cancer. It is a structural analog of busulfan, but it does not induce severe hepatotoxicity or veno-occlusive disease at or above the maximum tolerated dose, lacks significant nonhematological toxicity and has limited organ toxicity. We report here the successful induction of permanent mixed multilineage chimerism and donor-specific tolerance as was proven by skin transplantation and IFN-gamma ELISPOT. In conclusion, because of its lower nonhematological toxicity, compared with other myeloablative regimens (eg irradiation or busulfan admin- istration), Treosulfan could be a better candidate for conditioning to induce donor-specific allograft tolerance.

Towards clinical transplantation tolerance

Progress in immunosuppressive therapy has reduced the incidence of acute graft rejection and gives excellent short-term results in clinical organ transplantation. There remains, however, the problem of chronic graft-rejection and, because immunosuppressive drugs lack specificity, long-term therapy is associated with severe side-effects such as a high incidence of cancer and opportunistic infections. The obvious solution for these problems is the induction of donor-specific transplantation tolerance.

Gene therapy progress and prospects: gene therapy in organ transplantation.

One major complication facing organ transplant recipients is the requirement for life-long systemic immunosuppression to prevent rejection, which is associated with an increased incidence of malignancy and susceptibility to opportunistic infections. Gene therapy has the potential to eliminate problems associated with immunosuppression by allowing the production of immunomodulatory proteins in the donor grafts resulting in local rather than systemic immunosuppression. Alternatively, gene therapy approaches could eliminate the requirement for general immunosuppression by allowing the induction of donor-specific tolerance. Gene therapy interventions may also be able to prevent graft damage owing to nonimmune-mediated graft loss or injury and prevent chronic rejection. This review will focus on recent progress in preventing transplant rejection by gene therapy.

Primary and secondary immunocompetence in mixed allogeneic chimeras.

Targeted disruption of T cell costimulatory pathways, particularly CD28 and CD40, has allowed for the development of minimally myeloablative strategies for the induction of mixed allogeneic chimerism and donor-specific tolerance across full MHC barriers. In this study we analyze in depth the ability of mixed allogeneic chimeras in two strain combinations to mount effective host-restricted and donor-restricted antiviral CD4 and CD8 responses, as well as the impact of development of mixed chimerism on the maintenance of pre-existing memory populations. While antiviral CD8 responses in mixed chimeras following acute viral infection with lymphocytic choriomeningitis virus Armstrong or vaccinia virus are largely host-restricted, donor-restricted CD8 responses as well as host- and donor-restricted CD4 responses are also readily detected, and virus is promptly cleared. We further demonstrate that selection of donor-restricted T cells in mixed chimeras is principally mediated by bone marrow-derived cells in the thymus. Conversely, we find that mixed chimeras exhibit a deficit in their ability to deal with a chronic lymphocytic choriomeningitis virus clone 13 infection. Encouragingly, pre-existing memory populations are largely unaffected by the development of high level mixed chimerism and maintain the ability to control viral rechallenge. Our results suggest that while pre-existing T cell memory and primary immunocompetence to acute infection are preserved in mixed allogeneic chimeras, MHC class I and/or class II tissue matching may be required to fully preserve immunocompetence in dealing with chronic viral infections.

Tolerance induction in rats, using a combination of anti-CD154 and donor splenocytes, given once on the day of transplantation.

Donor-specific tolerance induction remains an attractive objective that generates much research in the field of transplantation. Unfortunately, most of the protocols available involve pregraft conditioning, making these treatments incompatible with clinical applications. LEW.1A rats were grafted with histoincompatible LEW.1W hearts. On the day of transplantation, recipients were treated with anti-CD40L combined with donor splenocytes. The hearts were evaluated for graft survival; cellular infiltrate and intragraft cytokines were determined using real-time reverse transcriptase-polymerase chain reaction. Tolerance induction was assessed by skin grafting and adoptive transfers. The combination of a single injection of anti-CD40L and donor splenocytes, given on the day of surgery, allowed 40% of cardiac allografts to survive long-term (mean survival time=66.3 day). The cellular composition or the extent of graft infiltrate was not modified but was associated with a massive decrease of proinflammatory cytokines expression within the graft. Long-term survivors accepted donor-matched skin grafts, and leukocytes harvested from these animals transferred tolerance into irradiated freshly grafted recipients. A combination of costimulation blockade and donor cells, given once at the time of transplantation, is sufficient to induce allograft tolerance in rats.

Induction of donor-specific tolerance to allogeneic hepatocytes by allogeneic bone marrow transplantation

Allogenic hepatocytes are rejected within a few days after transplantation without immunosuppression. We previously showed that the peripheral tolerance did not effectively prolong the survival of allogeneic hepatocytes transplanted in the spleen, because anergic T cells lysed allogeneic hepatocytes through Fas/Fas ligand system. The aim of this study was to address whether or not the central tolerance induced by allogeneic bone marrow transplantation (BMT) allows transplanted hepatocytes to survive in the spleen of the recipient. Bone marrow cells obtained from C57BL/6 (B6) donor mice were injected intravenously into lethally irradiated BALB/c recipients. Ninety percent of the recipient mice were able to survive more than 30 days, although all of the non-BMT controls died within 12 days. Full donor chimerism was observed in the recipient. Then hepatocytes from B6 mice were transplanted into the spleen of BALB/c mice at 30 days post-BMT. The transplanted donor-hepatocytes were able to survive for over 7 days, while those from third-party mice (C3H/He) were rejected within a few days. These findings demonstrate that donor-specific tolerance was induced by allogeneic BMT. Furthermore, the induction of central tolerance, but not peripheral tolerance, may be a useful strategy for prolonging the survival of allogeneic hepatocytes in the spleen.

Induction of tolerance in fully MHC disparate composite tissue allografts

Induction of donor-specific tolerance in composite tissue allografts (CTA) continues to be one of the most challenging goals. In our previous study, using combined TCR mAb + CsA treatment we induced tolerance between LBN donors (RTln/l; FI) and Lewis recipients (LEW; RTII) in CTAs model. The primary aim of this study is to induce tolerance in the fully MHC mismatched allograft recipients under combined alpha-betaTCR+CSA protocol. Thirty transplantations across strong MHC barrier between BrownNorway donors (RTln) and LEW recipients were performed. The animals from experimental groups received for 7 days either rat TCR and CsA or a combination of alpha-betaTCR+CsA, The immunosuppressive treatment efficacy and chimerism was monitored by flow cytometry. The survival > lOOdays was noted in allograft recipients treated with combination of TCXUCsA. Flow cytometry analysis demonstrated mixed chime&m in the periphery of tolerated recipients. Donor specific tolerance was confirmed by rejection of the third-party skin graft and by MLR. Tolerance can be induced in the short term using a TCRKsA protocol in the fully mismatched CTAs. Stable mixed chimerism can be achieved without myeloahlative bone marrow modifications of the recipients.

214. Induction of the donor specific chimerism and tolerance in fully MHC mismatched limb allograft recipients

Purpose Induction of donor-specific tolerance in composite tissue allografts (CTA) remains one of the most challenging goals in transplant surgery. Recently we have developed a protocol (ab TCR mAb/ CsA) for tolerance induction in the semiallogeneic rat hind-limb transplantations!. In this report we induced tolerance in the fully MHC mismatched allograft recipients under, combined ab-TCR/CSA protocol. Methods Thirty transplantations across strong MHC barrier were performed between Brown-Norway donors (RT1n) and LEW recipients. Isograft and allograft rejection control groups received no treatment. Experimental groups received either rat abTCR or CsA or combination of abTCR and CsA at the day of transplantation and for 7 days thereafter. The efficacy of immunosuppressive treatment and chimerism were monitored by flow cytometry FC. Donor specific tolerance and immunocompetence of the limb recipients were determined in vivo by secondary skin graft from the recipient (LEW), the donor (BN) and the third party (ACI) graft. Mixed lymphocyte reaction (MLR) was performed for the assessment of donor specific tolerance in vitro. Results Only fully mismatched allograft recipients under abTCR/CsA protocol survived indefinite (over 250 days). Three color FC analysis at day 120 post-transplant demonstrated stable, multilineage, donor specific chimerism in the periphery of the tolerated recipients (CD4+PE/RT1n+APC-7.6% and 1.3% of CD8+PE/RT1n+APC positive T cell sub-populations and CD45RA+PE/RT1n+APC-16.5% B cell population). Donor specific tolerance and immunocompetence in vivo was confirmed by acceptance of the secondary skin graft from the donor and rejection of the third-party grafts. MLR revealed no reactivity to host (LEW) and donor (BN) antigens but strong reactivity to the third-party alloantigens. Conclusions Donor specific tolerance was induced under 7 days protocol of abTCR/CsA therapy in the fully mismatched limb allograft transplanrs. Stable mixed chimerism was achieved without the need for the myeloablative bone marrow modification of the recipients and without the need for chronic immunosuppression.

Long-Term Immunologic Induction of Donor-Specific Tolerance to Skin Allografts by Bone Marrow Transplant in Rabbits

The induction of donor-specific tolerance to skin allografts was investigated in rabbits using bone marrow transplantation techniques reported to be effective in mice. Various routes of bone marrow transplantation (i.e., intravenous, portal venous, or intraosseous) were also examined. In regimen A, the animals were treated with portal venous injection of bone marrow cells from the donor on day 0 and intravenous injection of bone marrow cells from the same donor on posttransplant day 5. In regimen B, the animals were treated with portal venous and intraosseous injections of donor bone marrow cells on day 0 and intravenous injection of bone marrow cells from the same donor on posttransplant day 5. In regimen C, the animals were given intraosseous injection of donor bone marrow cells on day 0 and intravenous injection of bone marrow cells from the same donor on posttransplant day 5. It was found that regimens B and C were more effective than regimen A in prolonging allograft survival. The results demonstrate that induction of allograft tolerance can be achieved by bone marrow transplantation in a rabbit model. This protocol deserves further study in other large animal models. (Plast. Reconstr. Surg. 111: 291, 2003.)

Long-Term Immunologic Induction of Donor-Specific Tolerance to Skin Allografts by Bone Marrow Transplant in Rabbits; Yi Biao Wang, M.D., Yutaka Ogawa, M.D., Hideaki Doi, M.D., Kenji Kusumoto, M.D., Tie Nan Jin, M.D., and Susumu Ikehara, M.D.

Received for publication May 22, 2002. Jean Michel Dubernard, M.D. Service de Chirurgie de la Transplantation Hopital Edouard Herriot Place d'Arsonval 69437 Lyon, France [email protected]