Transplantation of a whole ear allograft by immunological induction of donor-specific tolerance by bone marrow transplantation: an experimental study in rabbits.

Previous studies in our laboratory have shown that giving bone marrow cells through the portal vein or intraosseous route is likely to be beneficial to tolerance of induction of allografts in rabbits. Using this model, we tested whether a less severe regimen for conditioning of the host can prevent rejection of allografts. Rabbits were given a single intraosseous injection of donor bone marrow cells and total body irradiation (7 Gy) and transplantation of skin and ear allografts. Mean skin allograft survival for this treatment was 88 days, which was similar to the results of our earlier study. A donor ear was accepted for more than a year with no signs of rejection. These results suggest that a single intraosseous injection of donor bone marrow cells is sufficient for induction of donor-specific tolerance in rabbits and that immunosuppressive agents may not be needed.     

The use of skin allograft with donor-specific tolerance in a rabbit model of full-thickness burn

Previous studies in our laboratory demonstrated that portal vein or intraosseous administration of donor bone marrow cells for modulation of the central immune system is likely to be beneficial for allograft tolerance induction in rabbits. We recently reported that the severity of host conditioning for donor-specific tolerance induction was reduced without loss of efficacy by using a single intraosseous injection of donor bone marrow cells without immunosuppressive agent administration or T cell depletion. We now further investigated the feasibility and effectiveness of skin allografting using donor-specific tolerance to treat full-thickness burns. MATERIALS AND METHODS: Dutch rabbits were used as recipients, and Japanese white rabbits were used as donors. Three experimental groups of burned rabbits were studied. Group I, allograft control; group II, allograft plus total-body irradiation; group III, allograft plus total-body irradiation and bone marrow infusion. Allograft survival times were determined by macroscopic and histopathologic examinations. RESULTS: Mean (S.D.) skin allograft survival was as follows: group I, 13.2 (4.1) days; group II, 15.2 (3.7) days; group III, 108.4 (14.3) days. CONCLUSION: We have shown the potential to perform long-term skin allografts by the induction of donor-specific tolerance in a rabbit model of burn.     

Epidermal Langerhans Cells Promote Skin Allograft Rejection in Mice With NF-κB-impaired T Cells

T cells play a major role in the acute rejection of transplanted organs. Using mice transgenic for a T-cell-restricted NF-κB super-repressor (IκBαΔN-Tg mice), we have previously shown that T-cell-NF-κB is essential for the acute rejection of cardiac but not skin allografts. In this study, we investigated the mechanism by which skin grafts activate IκBαΔN-Tg T cells. Rejection was not due to residual T-cell-NF-κB activity as mice with p50/p52^−/− T cells successfully rejected skin grafts. Rather, skin but not cardiac allografts effectively induced proliferation of graft-specific IκBαΔN-Tg T cells. Rejection of skin grafts by IκBαΔN-Tg mice was in part dependent on the presence of donor Langerhans cells (LC), a type of epidermal dendritic cells (DC), as lack of LC in donor skin grafts resulted in prolongation of skin allograft survival and injection of LC at the time of cardiac transplantation was sufficient to promote cardiac allograft rejection by IκBαΔN-Tg mice. Our results suggest that LC allow NF-κB-impaired T cells to reach an activation threshold sufficient for transplant rejection. The combined blockade of T-cell-NF-κB with that of alternative pathways allowing activation of NF-κB-impaired T cells may be an effective strategy for tolerance induction to highly immunogenic organs.     

Allopeptide-pulsed dendritic cells and composite tissue allograft survival

Composite tissue allografts (CTAs) contain their own reservoir of vascularized bone marrow, offering novel aspects for the induction of donor-specific tolerance. Additionally, the manipulation of recipient dendritic cells, pulsed with donor allopeptide, has been shown to engender solid organ allograft survival. To exploit these modalities, we have developed a protocol utilizing injection of recipient bone marrow-derived dendritic cells (BMDCs) pulsed with a donor-derived peptide for use in CTA transplantation. Six days prior to orthotopic hind-limb transplantation, Lewis rats received IV injection of donor allopeptide-pulsed, recipient BMDCs, in conjunction with a single dose of anti-lymphocyte serum. Control groups displayed signs of allograft rejection within 5 days postoperatively. Animals within the primary experimental cohort demonstrated prolongation of graft survival to an average of 8 days, and exhibited low numbers of donor T cells. The use of BMDCs in conjunction with transient immunosuppression has potential therapeutic application for induction of donor-antigen-specific tolerance to hind limb allografts.     

Induction of donor-specific tolerance to rat cardiac allografts by intrathymic inoculation of bone marrow.

BACKGROUND. Induction of donor-specific tolerance to tissue or organ allografts can readily be achieved by administration of allogeneic bone marrow to neonatal rodents; however, in adult recipients induction of transplantation tolerance by this strategy generally requires intensive cytoablative conditioning. Described here is a novel method of promoting transplantation tolerance that involves inoculation of donor bone marrow into the thymus of transiently immunosuppressed adult recipients. METHODS. Prospective Wistar-Furth recipients were inoculated with allogeneic Lewis bone marrow cells (BMCs) either intrathymically or intravenously in conjunction with a single dose of antilymphocyte serum 2 to 3 weeks before receiving donor-strain cardiac allografts. Recipients were monitored for graft survival and examined for presence of hematopoietic chimerism. RESULTS. Intrathymic but not intravenous inoculation of donor BMCs led to permanent survival of donor-strain cardiac allografts, whereas third-party Dark agouti cardiac allografts were rejected promptly. Persistence of donor chimerism was demonstrated in the thymus of Wistar-Furth recipients of intrathymic Lewis BMCs for as long as 3 weeks after BMC inoculation. CONCLUSIONS. Intrathymic inoculation of BMCs concurrently with a single dose of antilymphocyte serum induces donor-specific unresponsiveness to rat cardiac allografts. The unresponsiveness may be the result of deletion or functional inactivation of alloreactive clones maturing in a thymus bearing donor alloantigen. Intrathymic inoculation of BMCs deserves further evaluation as a possible clinical strategy for the induction of transplantation tolerance.     

Skin allograft survival following intrathymic injection of donor bone marrow.

BACKGROUND: Success has been reported using intrathymic injection in the preconditioning regimen to induce allograft tolerance. Although long-term stable tolerance has been achieved in numerous rodent vascularized solid organ allograft models, tolerance to skin transplants has only been achieved across minor antigenic or concordant species disparities. This study sought to induce tolerance across an allogeneic barrier in a rat model with a major genetic disparity. MATERIALS AND METHODS: Lewis rats were injected intrathymically with 1 x 10(8) Brown-Norway (BN) bone marrow cells and intraperitoneally with 1.0 cc of rabbit anti-rat anti-lymphocyte serum (ALS). Twenty-one days later, BN skin grafts were placed on the injected animals. Control groups were included to isolate the effect of technique, thymic manipulation, strain specificity, and ALS. RESULTS: Animals receiving both intrathymic bone marrow cells and ALS had a skin graft median survival time of 24 days versus 8 days for the control group (P = 0.003). Groups receiving anti-lymphocyte serum alone or intrathymic bone marrow cell injection alone exhibited no skin graft survival prolongation. Mixed lymphocyte reactions revealed normal responsiveness of tolerant animal lymphocytes to donor strain lymphocytes. CONCLUSION: This protocol utilizing the intrathymic injection of donor bone marrow cells along with short-term immunosuppression with anti-lymphocyte serum produced markedly prolonged survival of skin allografts transplanted across a major histocompatibility barrier. Although tolerance was incomplete, significant prolongation has not previously been reported in genetic disparities of this degree. These results suggest that the application of this technique for central immune modulation may be beneficial for allograft tolerance induction and deserves further study in large animals models.     

Induction of Tolerance by Exosomes and Short-Term Immunosuppression in a Fully MHC-Mismatched Rat Cardiac Allograft Model

Exosomes are MHC‐bearing vesicles secreted by a wide array of cells. We have previously shown that donor‐haplotype exosomes from bone marrow dendritic cells (DCs) injected before transplantation significantly prolong heart allograft survival in congenic and fully MHC‐mismatched Lewis rats. Here we show that donor exosomes administered after transplantation are similarly able to prolong allograft survival, however, without inducing tolerance. We therefore tested the effect of exosomes combined with short‐term LF 15‐0195 (LF) treatment, which blocks the maturation of DCs, so that donor‐MHC antigens from exosomes could be presented in a more tolerogenic environment. LF treatment does not preclude the development of a strong antidonor cellular response, and while LF, but not exosome, treatment inhibits the antidonor humoral response and decreases leukocyte graft infiltration, allografts from LF‐treated recipients were either acutely or strongly chronically rejected. Interestingly, when combined with LF treatment, exosomes induced a donor‐specific allograft tolerance characterized by a strong inhibition of the antidonor proliferative response. This donor‐specific tolerance was transferable to naïve allograft recipients. Moreover, exosomes/LF treatment prevented or considerably delayed the appearance of chronic rejection. These results suggest that under LF treatment, presentation of donor‐MHC antigens (from exosomes) can induce regulatory responses that are able to modulate allograft rejection and to induce donor‐specific allograft tolerance.     

Indirect allorecognition in acquired thymic tolerance: induction of donor-specific tolerance to rat cardiac allografts by allopeptide-pulsed host dendritic cells

BACKGROUND: Presentation of peptides either by recipient or donor MHC molecules displayed on the surface of antigen-presenting cells is an essential element in the induction of T cell responses to transplant antigens. The finding that intrathymic (IT) injection of an immunodominant peptide induces acquired thymic tolerance suggests an indirect pathway of allorecognition in the thymus. To address this theory, we studied the effects of IT injection of host bone marrow (BM)-derived dendritic cells (DC)-pulsed with the immunodominant Wistar Furth (WF) MHC class I (RT1.Au) peptide 5 (93-109) on cardiac allograft survival in the WF-to-ACI rat combination. METHODS: DC were propagated from cultures of ACI (recipient) bone marrow (BM) maintained in a medium supplemented with granulocyte-macrophage colony-stimulating factor and IL-4. The BM-derived DC after 8 days of culture were pulsed in vitro with a single WF MHC class I peptide (Residue 93-109) with the dominant epitope, washed, and injected into the thymus of ACI rats. The ACI recipients received donor-type (WF) or 3rd party (Lewis) cardiac allografts 7 days after IT immunization with peptide-pulsed DC. RESULTS: BM-derived DC cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 for 8 days have a strong allostimulatory ability and present peptide 5 to naive syngeneic T cells in mixed lymphocyte reaction. IT inoculation of 300 microg RT1.Au peptide 5 combined with transient antilymphocyte serum immunosuppressive therapy induced donor-specific tolerance to cardiac allografts. Extension of this finding to peptide-pulsed self DC showed that IT injection of peptide 5-pulsed host DC consistently led to permanent acceptance (>150 days) of donor-type (WF) cardiac allografts, whereas third-party (Lewis) grafts were acutely rejected. The long-term unresponsive recipients challenged with second-set grafts accepted permanently (>100 days) donor-type(WF) grafts while rejecting third-party (Lewis) grafts without the rejection of the primary WF grafts. CONCLUSION: This novel finding that allopeptide-pulsed host DC induces tolerance to cardiac allografts suggests that the induction of acquired tolerance is dependent on the indirect allorecognition pathway. The results further suggest that genetically engineered DC expressing donor MHC class I or II molecules or a peptide analogue might have therapeutic potential in the induction of transplant tolerance and in the treatment of autoimmune diseases.     

Intrathymic inoculation of donor bone marrow induces long-term acceptance of lung allografts

BACKGROUND: We investigated whether intrathymic inoculation of donor bone marrow at the time of transplantation induced long-term acceptance of lung allografts. METHODS: Four- to-six-week-old August Copenhagen Irish (ACI) and Wistar Furth (WF) rats were used as donors and recipients, respectively. After being inoculated intrathymically with either donor-specific (ACI) or third-party (F344) bone marrow (2.0 x 10(7) cells/lobe), the recipient (WF) animal received a left lung transplant from an ACI donor. A short course of tacrolimus (1 mg/kg per day for 5 days) was administered. Animals were sacrificed at timed intervals after transplantation, and rejection was graded on a scale of 0 (none) to 4 (severe). RESULTS: At 28 days, animals receiving donor-specific bone marrow have lower (p < 0.01) median rejection grade (MRG = 0.25; n = 6) than those receiving third-party bone marrow (MRG = 3; n = 6) and controls (no bone marrow; MRG = 2.5; n = 6). Animals receiving intrathymic donor bone marrow accepted lung allografts up to 380 days with minimal rejection (MRG = 2; n = 6). Long-term lung recipients also accepted a challenging donor-specific heart graft (n = 4) for more than 150 days. In mixed lymphocyte reaction assays, T lymphocytes of WF recipients that had received intrathymic bone marrow (from ACI donor) exhibited low response (similar to self antigens) to donor (ACI) cells, but reacted strongly (five times higher) to third-party (F344) cells. CONCLUSIONS: Intrathymic inoculation of donor bone marrow at the time of transplantation along with a short course of tacrolimus induces long-term acceptance of lung allografts in rats. This simple approach of tolerance induction may have clinical application.     

Induction of transplantation tolerance by intraportal injection of allogeneic bone marrow cells

Intraportal inoculation of C57BL/6 marrow cells into sublethally (400 rad) irradiated BALB/c recipients resulted in durable chimerism and the permanent acceptance of C57BL/6 skin allografts. Sublethally irradiated recipients of a similar number of marrow cells inoculated systemically did not develop chimerism or any significant prolongation of the survival of C57BL/6 skin allografts. Consequently, lethal graft-versus-host disease developed only in recipients of intraportal marrow allografts (80%). The intraportal injection of allogeneic C57BL/6 marrow cells into nonirradiated recipients resulted in significant, although not permanent, prolongation of skin allograft survival without durable chimerism, suggesting that the introduction of alloantigens intraportally may favor the induction of nonresponsiveness to alloantigens even across strong major histocompatibility barriers. The relevance of these findings is discussed regarding the intraportal inoculation of allogeneic bone marrow cells for the treatment of genetic disorders in utero through the induction of neonatal tolerance.     

Mesenchymal Stem Cells Facilitate the Induction of Mixed Hematopoietic Chimerism and Islet Allograft Tolerance without GVHD in the Rat

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.     

Prolonged improvement of total pancreatic allograft function by previous intrathymic bone marrow cell injection in rats

Donor-specific induction of tolerance was previously achieved in the diabetic rat by intrathymic injection of pancreatic islets. It allowed a secondary islet graft in any site without immunosuppression. Since total pancreatic graft in man is metabolically more proficient than islet graft, we attempted tolerance induction for total vascularized pancreas transplantation in diabetic BN recipient rats by an intrathymic bone marrow cell (BMC) injection from Lewis donor rats, associated to an antilymphocyte antibody (ALS) administration. Control groups consisted of isogenic grafts, allogenic grafts without tolerance induction and allogenic grafts with ALS alone. In all grafted groups, mean blood glucose and plasma insulin were normalised within 24 h. Graft rejection (clinically suggested by diabetes recurrence and later confirmed by histology) appeared at 18 +/- 2 postoperative days in the absence of intrathymic BMC injection and at 36 +/- 8 days in the group with BMC injection (p < 0.05). Intrathymic bone marrow graft was successful in delaying rejection in our study.     

Induction of transplantation tolerance by intraportal injection of allogeneic bone marrow cells: Possible implications for intrauterine bone marrow transplantation across major histocompatibility barriers

Abstract. Intraportal inoculation of C57BL/6 marrow cells into sublethally (400 rad) irradiated BALB/c recipients resulted in durable chimerism and the permanent acceptance of C57BL/6 skin allografts. Sublethally irradiated recipients of a similar number of marrow cells inoculated systemically did not develop chimerism or any significant prolongation of the survival of C57BL/6 skin allografts. Consequently, lethal graft-versus-host disease developed only in recipients of intraportal marrow allografts (80%). The intraportal injection of allogeneic C57BL/6 marrow cells into nonirradiated recipients resulted in significant, although not permanent, prolongation of skin allograft survival without durable chimerism, suggesting that the introduction of alloantigens intraportally may favor the induction of nonresponsiveness to alloantigens even across strong major histocompatibility barriers. The relevance of these findings is discussed regarding the intraportal inoculation of allogeneic bone marrow cells for the treatment of genetic disorders in utero through the induction of neonatal tolerance.     

Chimerism induction in vascularized bone marrow transplants augmented with bone marrow cells

Composite tissue allografts (CTAs) are currently accepted in the clinic; however, long-term immunosuppression is still needed for allograft survival. The presence of donor-specific chimerism may induce tolerance. Thirty-six vascularized bone marrow transplantation (VBMT) allotransplantation were performed across MHC barrier under short-term protocol of 7-day alphabeta-TCRmAb and Cyclosporin A therapy to determine the efficacy of VBMT alone and VBMT augmented with donor bone marrow transplantation (BMT) in chimerism induction. Flow cytometry analysis revealed that VBMT supported with donor BMT directly into the bone resulted in chimerism augmentation and maintenance compared to VBMT. In vivo and in vitro tolerance testing showed prolonged survival of donor skin graft up to 35 days and moderate reactivity in MLR assay that suggests only tolerance induction. Transplantation of vascularized bone without chronic immunosuppression provides a substantial source of bone marrow cells, leading to the development of stable donor-specific chimerism.     

Induction of stable chimerism and elimination of graft-versus-host disease by depletion of T lymphocytes from bone marrow using immunomagnetic beads

The goal of transplantation is the induction of immunologic tolerance. At present, nonspecific immunosuppression is used to prevent graft rejection and, commonly, graft-versus-host disease (GVHD). Nevertheless, nonspecific immunosuppressive therapy is frequently complicated by infection, malignant tumors, and drug toxicity. In order to examine whether hematopoietic chimerism can be used to induce specific allograft tolerance, we have reconstituted lethally irradiated Lewis rats with ACI bone marrow that has been depleted of T cells with use of immunomagnetic beads. This technique consists of binding OX-19, a mouse anti-rat pan-T lymphocyte monoclonal antibody, to magnetic polymer beads. Mixing of bone marrow or splenocytes with the bead/OX-19 complexes, followed by magnetic separation, results in significant depletion of T cells with minimal nonspecific cell loss. Immunomagnetic T-cell depletion of bone marrow, followed by reconstitution of a lethally irradiated host, allows for the development of stable, mixed hematopoietic chimerae without evidence of GVHD. These hosts are immunocompetent by clinical criteria. Recipients of untreated donor bone marrow that did or did not receive nonspecific immunosuppression demonstrated varying degrees of GVHD and reduced survival. The ability to rapidly and simply deplete T lymphocytes from bone marrow and produce stable, immunocompetent hematopoietic chimerae without GVHD may be an important method for tolerance induction to vascularized allografts.     

Infusion of Lin- bone marrow cells results in multilineage macrochimerism and skin allograft tolerance in minimally conditioned recipient mice

Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk. In the present study, we demonstrate that the infusion of low numbers of donor Lin(-) bone marrow cells (Lin(-) BMC) 7 days post allograft transplantation facilitates high level macrochimerism induction and graft tolerance. Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, +2, +5, +7 and +14 along with low dose busulfan on day +5. A low dose of highly purified Lin(-) BMC from BALB/c donor mice was infused on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). Lin(-) BMC infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (>300 days). Mixed donor-recipient macrochimerism was observed. Donor-reactive T cells were clonally deleted and a 130% increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. We conclude that the infusion of donor Lin(-) BMC without cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4(+)Foxp3(+) Tregs.     

Permanent acceptance of both cardiac and skin allografts using a mild conditioning regimen for the induction of stable mixed chimerism in mice

Patients who are receiving an organ transplant nowadays are sentenced to the life-long administration of immunosuppressive drugs, which have serious side effects. The reliable induction of donor-specific tolerance therefore remains a major goal in organ transplantation. Previously, we have developed a sublethal, non-myeloablative murine model in which permanent mixed, multilineage chimerism and donor-specific tolerance are established. Our model involves engraftment of fully allogeneic T cell depleted donor bone marrow cells in low dose irradiated and anti-CD3 treated major histocompatibility complex (MHC)-disparate recipient mice. To investigate whether vascularized organ grafts are accepted in our model, we performed heterotopic heart transplantations in our mixed chimeric mice. Chimeric mice permanently accepted hearts from the bone marrow donor (>130 days) and rapidly rejected third party-type allografts (median survival time 9 days). Untreated control recipient mice rejected both donor- and third party-type allografts. In addition, mice that accepted their cardiac grafts, donor-specific acceptance of skin grafts was observed. In conclusion, the establishment of stable mixed chimerism with this low-toxicity regimen resulted in permanent donor-specific acceptance of vascularized organ as well as skin grafts across a full MHC barrier.     

Allospecific Rejection of MHC Class I‐Deficient Bone Marrow by CD8 T Cells

Avoidance of long‐term immunosuppression is a desired goal in organ transplantation. Mixed chimerism offers a promising approach to tolerance induction, and we have aimed to develop low‐toxicity, nonimmunodepleting approaches to achieve this outcome. In a mouse model achieving fully MHC‐mismatched allogeneic bone marrow engraftment with minimal conditioning (3 Gy total body irradiation followed by anti‐CD154 and T cell–depleted allogeneic bone marrow cells), CD4 T cells in the recipient are required to promote tolerance of preexisting alloreactive recipient CD8 T cells and thereby permit chimerism induction. We now demonstrate that mice devoid of CD4 T cells and NK cells reject MHC Class I‐deficient and Class I/Class II‐deficient marrow in a CD8 T cell–dependent manner. This rejection is specific for donor alloantigens, since recipient hematopoiesis is not affected by donor marrow rejection and MHC Class I‐deficient bone marrow that is syngeneic to the recipient is not rejected. Recipient CD8 T cells are activated and develop cytotoxicity against MHC Class I‐deficient donor cells in association with rejection. These data implicate a novel CD8 T cell–dependent bone marrow rejection pathway, wherein recipient CD8 T cells indirectly activated by donor alloantigens promote direct killing, in a T cell receptor–independent manner, of Class I‐deficient donor cells.     

Examination of the mechanisms responsible for tolerance induction after intrathymic inoculation of allogeneic bone marrow.

OBJECTIVE: This study examined the immunologic mechanism(s) responsible for the induction of transplantation tolerance in rats pretreated with intrathymic inoculation of donor strain bone marrow. SUMMARY BACKGROUND DATA: Induction of unresponsiveness may involve deletion and/or inactivation of donor-reactive T-cell precursors maturing in a thymus harboring donor alloantigen or generation of regulatory/suppressor cells. It was reasoned that, if unresponsiveness is caused by deletion of alloreactive clones, the presence of additional thymic tissue devoid of donor alloantigen permits normal maturation of T-cells and, thus, prevents induction of tolerance. However, if unresponsiveness were primarily mediated by regulatory/suppressor cells, the presence of noninoculated thymic tissue should not affect the induction of tolerance. METHODS: Three strategies were used to define the cellular basis of cardiac and islet allograft survival in WF recipients of intrathymic LEW donor bone marrow as follows: (1) inoculation of bone marrow either into the native thymus and/or into an ectopic thymus, (2) limiting dilution analyses of the frequency of precursor cytotoxic T-lymphocytes (CTLp), and (3) adoptive transfer to syngeneic secondary hosts. RESULTS: Inoculation of bone marrow into only one lobe of the native thymus and/or into an ectopic thymus did not promote consistent survival of subsequent LEW cardiac allografts. Tolerant hosts displayed significant reductions in CTLp frequencies against donor alloantigens. Adoptive transfer of spleen cells from tolerant WF hosts harboring long-standing cardiac allografts led to permanent survival of LEW cardiac allografts in all secondary recipients. However, transfer of spleen cells from WF animals that received intrathymic LEW bone marrow (but no cardiac allograft) did not promote survival of LEW cardiac allografts in naive secondary hosts. CONCLUSIONS: These results indicate that the unresponsive state after intrathymic inoculation of bone marrow cells is primarily mediated by deletion and/or inactivation of donor-specific T-cell precursors maturing in a chimeric thymus. The demonstration by adoptive transfer studies of putative regulatory/suppressor cells suggested an important role for the persistence of donor alloantigen (supplied by a vascularized allograft) in the maintenance of the unresponsive state.     

CD8+ T‐Cell Depletion and Rapamycin Synergize with Combined Coreceptor/Stimulation Blockade to Induce Robust Limb Allograft Tolerance in Mice

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti‐CD154 mAb, nondepleting anti‐CD4 combined to either depleting or nondepleting anti‐CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long‐term allograft survival in both combinations. Nevertheless, robust donor‐specific tolerance, defined by the acceptance of a fresh donor‐type skin allograft and simultaneous rejection of third‐party grafts, required initial CD8⁺ T‐cell depletion. Mixed donor‐recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL‐2, IFN‐γ and TNF‐α in MLC with donor antigen while significant alloreactivity persisted against third‐ party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor‐recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8⁺ T‐cell depletion and costimulation/coreceptor blockade.