Neonatal induction of tolerance to skeletal tissue allografts without immunosuppression

Vascularized allogeneic skeletal tissue transplantation without the need for host immunosuppression would increase reconstructive options for treating congenital and acquired defects. Because the immune system of a fetus or neonate is immature, it may be possible to induce tolerance to allogeneic skeletal tissues by alloantigen injection during this permissive period. Within 12 hours after birth, 17 neonatal Lewis rats were injected through the superficial temporal vein with 3.5 to 5 million Brown Norway bone marrow cells in 0.1 ml normal saline. Ten weeks after the injection, peripheral blood from the Lewis rats was analyzed for the presence of Brown Norway cells to determine hemopoietic chimerism. The Lewis rats then received a heterotopic, vascularized limb tissue transplant (consisting of the knee, the distal femur, the proximal tibia, and the surrounding muscle on a femoral vascular pedicle) from Brown Norway rat donors to determine their tolerance to the allogeneic tissue. A positive control group (n = 6) consisted of syngeneic transplants from Lewis rats into naive Lewis rats to demonstrate survival of transplants. A negative control group (n = 6) consisted of Brown Norway transplants into naive Lewis rats not receiving bone marrow or other immunosuppressive treatment. The animals were assessed for transplant viability 30 days after transplantation using histologic and bone fluorochrome analysis. All the syngeneic controls (Lewis to Lewis) remained viable throughout the experiment, whereas all the Brown Norway to Lewis controls had rejected. Ten of the 17 allografts transplanted into bone marrow recipients were viable at 30 days, with profuse bleeding from the ends of the bone graft and the surrounding graft muscle. The percent of chimerism correlated with survival, with 3.31 percent (SD = 1.9) of peripheral blood, Brown Norway chimerism present in the prolonged survival groups and 0.75 percent (SD = 0.5) of Brown Norway chimerism in the rejected graft group. This study demonstrated prolonged survival of allogeneic skeletal tissue without immunosuppression after early neonatal injection of allogeneic bone marrow in a rat model.     

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.     

Immune Reconstitution Kinetics following Intentionally Induced Mixed Chimerism by Nonmyeloablative Transplantation

Establishing mixed chimerism is a promising approach for inducing donor-specific transplant tolerance. The establishment and maintenance of mixed chimerism may enable long-term engraftment of organ transplants while minimizing the use of immunosuppressants. Several protocols for inducing mixed chimerism have been reported; however, the exact mechanism underlying the development of immune tolerance remains to be elucidated. Therefore, understanding the kinetics of engraftment during early post-transplant period may provide insight into establishing long-term mixed chimerism and permanent transplant tolerance. In this study, we intentionally induced allogeneic mixed chimerism using a nonmyeloablative regimen by host natural killer (NK) cell depletion and T cell-depleted bone marrow (BM) grafts in a major histocompatibility complex (MHC)-mismatched murine model and analyzed the kinetics of donor (C57BL/6) and recipient (BALB/c) engraftment in the weeks following transplantation. Donor BM cells were well engrafted and stabilized without graft-versus-host disease (GVHD) as early as one week post-bone marrow transplantation (BMT). Donor-derived thymic T cells were reconstituted four weeks after BMT; however, the emergence of newly developed T cells was more obvious at the periphery as early as two weeks after BMT. Also, the emergence and changes in ratio of recipient- and donor-derived NKT cells and antigen presenting cells (APCs) including dendritic cells (DCs) and B cells were noted after BMT. Here, we report a longitudinal analysis of the development of donor- and recipient-originated hematopoietic cells in various lymphatic tissues of intentionally induced mixed chimerism mouse model during early post-transplant period. Through the understanding of immune reconstitution at early time points after nonmyeloablative BMT, we suggest guidelines on intentionally inducing durable mixed chimerism.     

Differential patterns of incorporation and remodeling among various types of bone grafts

The early (3 months) and later (6 months) patterns of incorporation and bone formation have been evaluated histomorphometrically for different types of bone grafts; that is, vascularized and nonvascularized autografts with and without ciclosporin, and vascularized and nonvascularized dog leukocyte antigen (DLA)-mismatched allografts with and without ciclosporin. The vascularized bones were superior to the nonvascularized ones in healing and remodeling their grafted segments. In the autograft bones, ciclosporin did not alter the incorporation process 3 months after transplantation but delayed and increased the remodeling activities in the long run (6 months). Nonvascularized allografts underwent vigorous resorption, and were markedly porotic. Ciclosporin administration significantly reduced resorption and enhanced remodeling in nonvascularized allografts. The remodeling of allografts was similar to that of autografts in the presence of ciclosporin, but stopped soon after the administration of ciclosporin ceased.     

Prevention of chronic rejection in murine cardiac allografts: a comparison of chimerism- and nonchimerism-inducing costimulation blockade-based tolerance induction regimens

We have previously described a nonirradiation-based regimen combining costimulation blockade, busulfan, and donor bone marrow cells that promotes stable, high level chimerism, deletion of donor-reactive T cells, and indefinite survival of skin allografts in mice. The purpose of the current study is to determine the efficacy of this tolerance regimen in preventing acute and chronic rejection in a vascularized heart graft model and to compare this regimen with other putative tolerance protocols. Mice receiving costimulation blockade (CTLA4-Ig and anti-CD40 ligand) alone or in combination with donor cells enjoyed markedly prolonged heart graft survival and initially preserved histological structure. However, tolerance was not achieved, as evidenced by the eventual onset of chronic rejection characterized by obliterative vasculopathy and the rejection of secondary skin grafts. In contrast, following treatment with costimulation blockade, busulfan, and bone marrow, heart grafts survived indefinitely without detectable signs of chronic rejection or structural damage, even 100 days after placement of a secondary donor skin graft. We detected multilineage chimerism in peripheral blood, spleen, lymph nodes, and thymus, and peripheral deletion of donor-reactive cells was complete by day 90. These findings indicate that only the CD40/CD28 blockade chimerism induction regimen prevents both acute and chronic rejection of vascularized organ transplants. Further testing of these strategies in a preclinical large animal model is warranted.     

The abrogation of allosensitization following the induction of mixed allogeneic chimerism

The association of preformed anti-donor Abs with the hyperacute rejection of bone marrow and solid organ allografts and the persistence of the anti-donor immune response secondary to immunologic memory make allosensitization an absolute contraindication to transplantation. Mixed allogeneic (A + B-->A) bone marrow chimerism has been demonstrated to confer donor-specific tolerance in nonsensitized recipients, but has not been evaluated in the setting of allosensitization. The current study documents that despite significant anti-donor sensitization, mixed allogeneic engraftment is possible and provides a marked advantage over fully allogeneic (B-->A) models. Moreover, the acceptance of donor skin grafts and loss of circulating anti-donor Abs suggest that allosensitization can be abrogated with the induction of stable mixed allogeneic chimerism.     

Nonmyeloablative bone marrow transplantation of BXSB lupus mice using fully matched allogeneic donor cells from green fluorescent protein transgenic mice

Male BXSB mice, a mouse model of systemic lupus erythematosus, were given bone marrow transplants (BMT) at 20 wk of age using MHC-matched donor cells and nonmyeloablative conditioning (550 cGy irradiation). Transplanted mice and irradiation controls were followed for a period of 20 wk. Mice transgenic for green fluorescent protein were used as donors to allow tracking of donor cells and a determination of chimerism. Radiation controls had reduced renal pathology at 10 wk posttransplant, but not at 20 wk compared with untreated mice, while nonmyeloablative BMT mice had significantly reduced pathology at both time intervals. The monocytosis characteristic of older BXSB mice was also reduced by BMT, but the treatment did not prevent production of Ab to dsDNA. A stable chimerism of 24-40% donor CD45-positive cells was achieved in spleen and bone marrow, and there was no evidence of clinical graft vs host disease. Donor cells were detected in most recipient organs, notably the thymus and renal glomeruli. The results suggest that complete depletion of mature lymphocytes or of progenitor stem cells is not required to control lupus nephritis in BXSB mice.     

Relationship between chimerism and tolerance in a kidney transplantation model

The persistence of donor leukocytes in recipients of organ allografts has been associated with long-term graft acceptance. However, it remains unclear whether this peripheral donor cell microchimerism plays an active role in graft acceptance or is simply a consequence of the maintenance of sufficient immunosuppression to avoid rejection. A model of kidney transplantation between swine leukocyte Ag (SLA)-matched miniature swine, in which tolerance can be established with or without immunosuppressive treatment, has been used to study the correlation between donor leukocyte chimerism and kidney graft acceptance. SLA-identical kidney transplants were performed from animals positive for an allelic pig leukocyte Ag to animals negative for this marker. SLA-identical kidney transplant recipients given a 12-day course of cyclosporine (CyA) (n = 3) became tolerant, showing stable serum creatinine levels (1-2 mg/dl) after cessation of CyA treatment. Donor cell chimerism (0.2-0.7%) was present by FACS in all three animals with peak levels detected at 3 wk. Two control animals receiving SLA-identical kidney grafts without CyA also showed stable serum creatinine levels and became tolerant. However, in neither of these animals could donor leukocytes be detected in the peripheral blood beyond 1 wk following transplantation. In one additional control animal, ureteral obstruction occurred at day 10, and was associated with additional peripheral chimerism, presumably related to inflammation rather than to immune status. These results indicate that the persistence of donor cell chimerism is not a requirement for the maintenance of tolerance to organ allografts in this model.     

Primary vascularization of the graft determines the immunodominance of murine minor H antigens during organ transplantation

Grafts can be rejected even when matched for MHC because of differences in the minor histocompatibility Ags (mH-Ags). H4- and H60-derived epitopes are known as immunodominant mH-Ags in H2(b)-compatible BALB.B to C57BL/6 transplantation settings. Although multiple explanations have been provided to explain immunodominance of Ags, the role of vascularization of the graft is yet to be determined. In this study, we used heart (vascularized) and skin (nonvascularized) transplantations to determine the role of primary vascularization of the graft. A higher IFN-γ response toward H60 peptide occurs in heart recipients. In contrast, a higher IFN-γ response was generated against H4 peptide in skin transplant recipients. Peptide-loaded tetramer staining revealed a distinct antigenic hierarchy between heart and skin transplantation: H60-specific CD8(+) T cells were the most abundant after heart transplantation, whereas H4-specific CD8(+) T cells were more abundant after skin graft. Neither the tissue-specific distribution of mH-Ags nor the draining lymph node-derived dendritic cells correlated with the observed immunodominance. Interestingly, non-primarily vascularized cardiac allografts mimicked skin grafts in the observed immunodominance, and H60 immunodominance was observed in primarily vascularized skin grafts. However, T cell depletion from the BALB.B donor prior to cardiac allograft induces H4 immunodominance in vascularized cardiac allograft. Collectively, our data suggest that immediate transmigration of donor T cells via primary vascularization is responsible for the immunodominance of H60 mH-Ag in organ and tissue transplantation.     

A biomechanical comparison of vascularized and conventional autogenous bone grafts

Vascularized and conventional autogenous rib grafts were used to reconstruct 6-cm ulnar defects in the forelegs of the nine dogs. Each dog served as its own control. Biomechanical torsional testing of the grafted ulnas showed that vascularized grafts were 234 percent stronger than the conventional grafts. Bone toughness (energy absorbed) was 483 percent greater in the vascularized grafts, and elastic modulus and proportional limits were 263 and 246 percent greater, respectively. We conclude that vascularized bone grafts are significantly stronger than conventional autogenous bone grafts after 3 months of healing in the dog ulna model.     

Growth potential in onlay bone grafts: a comparison of vascularized and free calvarial bone and suture bone grafts

Vascularized bone grafts are characterized by a viable cell population with osteogenic potential. These features suggest that continued growth can be anticipated following vascularized membranous bone transfer in a growing craniofacial skeleton. The present paper compares the potential for appositional bone growth in vascularized and free calvarial onlay bone grafts. In seven 8-week-old beagles, growth was assessed by direct caliper measurements of graft dimensions intraoperatively and 16 weeks postoperatively. Vascularized grafts demonstrated a 50 to 60 percent increase in size in all dimensions compared to 10 to 20 percent growth in free grafts (p less than 0.01). Microradiography revealed preservation of calvarial bony architecture and minimal resorption in vascularized grafts, while triple-fluorochrome labeling confirmed subperiosteal appositional bone formation. Free grafts were characterized by significant resorption and a delay in subperiosteal bone formation.     

The effects of vascularity and cyclosporin A on the mechanical properties of canine fibular autografts.

We studied the biomechanical behavior of orthotopic canine autografts as influenced by vascularized supply and the administration of cyclosporin A at three months and six months post-surgery. The model was the proximal 8 cm of the fibula in young adult dogs. In vascularized grafts, blood supply was re-established by microvascular re-anastomosis. Experimental controls were sham-operated and unoperated bones. Mid-graft test sections were subjected to loading-to-failure in torsion to determine the strength and stiffness. In both three- and six-month groups, vascularized grafts were significantly stronger and stiffer than contralateral nonvascularized grafts. Vascularized grafts were not significantly different from sham-operated bones. A 30-day regimen of cyclosporin A was found to have no measurable effect on mechanical properties for any individual treatment group. The results indicate that re-established blood supply can be a major factor in maintaining the mechanical integrity in large-segment cortical autografts.     

Hematopoietic chimerism in sheep and nonhuman primates by in utero transplantation of fetal hematopoietic stem cells.

Bone marrow transplantation to reconstitute defective hematopoietic cell lines in children with congenital defects is limited by donor availability, graft rejection, and graft-versus-host disease (GVHD). These problems can be eliminated by transplanting normal preimmune fetal hematopoietic stem cells (HSC) into an unrelated preimmune fetal recipient. We show here that injections of allogeneic fetal stem cells into preimmune fetal lambs and monkeys result in long-term stable hematopoietic chimerism. HSCs harvested from the livers of preimmune fetal sheep and monkeys when injected into the peritoneal cavity of young unrelated fetal sheep and monkey recipients results in stable, long-term postnatal hematopoietic chimerism involving lymphoid, erythroid, and myeloid cells of donor origin. Donor cell engraftment was achieved without the use of cytoablative procedures and without the development of GVHD.     

Vascular knee allograft transplantation in a rabbit model

Using a rabbit model in which vascularized knee autograft transplantation was successful, vascularized knee allograft transplants survived an average of 9 days, as determined by serial bone scan. The longest surviving allograft was one of 3 months. Immunosuppression and irradiation did not significantly increase survival. Both vascularized and nonvascularized allografts elicited a second-set skin graft response but no histologic evidence of rejection. This suggests that joint allografts are clearly immunogenic but do not undergo the same destructive rejection process with a clear end point seen with soft-tissue grafts. Donor vessels did show a classic rejection picture with severe intimal damage presumably predisposing to vessel thrombosis and graft loss. Vascular rejection, therefore, limited joint allograft survival. Immediate vascularization of the allograft with subsequent limited survival does not enhance host revascularization and creeping substitution at 1, 3, or 6 months. These findings do not suggest clinical applicability for vascularized joint allograft transplantation at this time. Future experimental studies should employ genetically defined models.     

Vascularized bone allograft transplantation in a genetically defined rat model

A heterotopic subcutaneous model for experimental vascularized bone allograft transplantation has been presented. This model uses genetically defined rats and allows serial assessment of graft viability. The reliability of this model has been proven by successful isograft transplantation. This model was used to study the effect of matching at the major histocompatibility complex on vascularized bone allograft survival. Whereas grafts transplanted across a minor histocompatibility barrier survived until sacrifice, grafts transplanted across a major histocompatibility barrier were victims of an acute rejection process. This study, therefore, showed genetic disparity to be a critical determinant of vascularized bone allograft survival. It indicates that primary vascularized bone allografts are as susceptible to rejection as heart and kidney allografts. For these reasons, it can be anticipated that genetic matching will be important in clinical vascularized bone allograft transplantation. The model used in this study should be useful for obtaining further fundamental immunologic information concerning vascularized bone allograft transplantation.     

One-stage reconstruction of composite bone and soft-tissue defects in traumatic lower extremities

Management of bone loss that occurs after severe trauma of open lower extremity fractures continues to challenge reconstructive surgeons. Sixty-one patients who had 62 traumatic open lower extremity fractures and combined bone and composite soft-tissue defects were treated with the following protocol: extensive debridement of necrotic tissues, eradication of infection, and vascularization of osteocutaneous tissue for one-stage bone and soft-tissue coverage reconstruction. The mechanism of injury included 49 motorcycle accidents (80.3 percent), five falls (8.2 percent), three crush injuries (4.9 percent), two pedestrian-automobile accidents (3.3 percent), and two motor vehicle accidents (3.3 percent). The bone defects were located in the tibia in 49 patients (79 percent; one patient had bilateral open tibial fractures), in the femur in seven patients (11.3 percent), in the calcaneus bone in four patients (6.5 percent), and in the metatarsal bones in two patients (3.2 percent). The size of soft-tissue defects ranged from 5 x 9 cm to 30 x 17 cm. The average length of the preoperative bony defect was 11.7 cm. The average duration from injury to one-stage reconstruction was 27.1 days, and the average number of previous extensive debridement procedures was 3.4. Fifty patients had vascularized fibula osteoseptocutaneous flaps, six had vascularized iliac osteocutaneous flaps, and five patients had seven combined vascularized rib transfers with serratus anterior muscle and/or latissimus dorsi muscle transfers. One patient received a second combined rib flap because the first combined rib flap failed. The rate of complete flap survival was 88.9 percent (56 of 63 flaps). Two combined vascularized rib transfers with serratus anterior muscle and latissimus dorsi muscle flaps were lost totally (3.2 percent) because of arterial thrombosis and deep infection, respectively. Partial skin flap losses were encountered in the five fibula osteoseptocutaneous flaps (7.9 percent). Postoperative infection for this one-stage reconstruction was 7.9 percent. Excluding the failed flap and the infected/amputated limb, the primary bony union rate after successful free vascularized bone grafting was 88.5 percent (54 of 61 transfers). The average primary union time was 6.9 months. The overall union rate was 96.7 percent (59 of 61 transfers). The average time to overall union was 8.5 months after surgery. Seven transferred vascularized bones had stress fractures, for a rate of 11.5 percent. Donor-site problems were noted in six fibular flaps, in two iliac flaps, and in one rib flap. The fibular donor-site problems were foot drop in one patient, superficial peroneal nerve palsy in one patient, contracture of the flexor hallucis longus muscle in two patients, and skin necrosis after split-thickness skin grafting in two patients. The iliac flap donor-site problems were temporary flank pain in one patient and lateral thigh numbness in the other. One rib flap transfer patient had pleural fibrosis. Transfer of the appropriate combination of vascularized bone and soft-tissue flap with a one-stage procedure provides complex lower extremity defects with successful functional results that are almost equal to the previously reported microsurgical staged procedures and conventional techniques.     

Rapid deletional peripheral CD8 T cell tolerance induced by allogeneic bone marrow: role of donor class II MHC and B cells

Mixed chimerism and donor-specific tolerance are achieved in mice receiving 3 Gy of total body irradiation and anti-CD154 mAb followed by allogeneic bone marrow (BM) transplantation. In this model, recipient CD4 cells are critically important for CD8 tolerance. To evaluate the role of CD4 cells recognizing donor MHC class II directly, we used class II-deficient donor marrow and were not able to achieve chimerism unless recipient CD8 cells were depleted, indicating that directly alloreactive CD4 cells were necessary for CD8 tolerance. To identify the MHC class II(+) donor cells promoting this tolerance, we used donor BM lacking certain cell populations or used positively selected cell populations. Neither donor CD11c(+) dendritic cells, B cells, T cells, nor donor-derived IL-10 were critical for chimerism induction. Purified donor B cells induced early chimerism and donor-specific cell-mediated lympholysis tolerance in both strain combinations tested. In contrast, positively selected CD11b(+) monocytes/myeloid cells did not induce early chimerism in either strain combination. Donor cell preparations containing B cells were able to induce early deletion of donor-reactive TCR-transgenic 2C CD8 T cells, whereas those devoid of B cells had reduced activity. Thus, induction of stable mixed chimerism depends on the expression of MHC class II on the donor marrow, but no requisite donor cell lineage was identified. Donor BM-derived B cells induced early chimerism, donor-specific cell-mediated lympholysis tolerance, and deletion of donor-reactive CD8 T cells, whereas CD11b(+) cells did not. Thus, BM-derived B cells are potent tolerogenic APCs for alloreactive CD8 cells.     

Thymic transplantation in miniature swine. II. Induction of tolerance by transplantation of composite thymokidneys to thymectomized recipients

Previous studies in our laboratory have demonstrated that the presence of the thymus is essential for rapid and stable tolerance induction in allotransplant models. We now report an attempt to induce tolerance to kidney allografts by transplanting donor thymic grafts simultaneously with the kidney in thymectomized recipients. Recipients were thymectomized 3 wk before receiving an organ and/or tissues from a class I-mismatched donor. Recipients received 1) a kidney allograft alone, 2) a composite allogeneic thymokidney (kidney with vascularized autologous thymic tissue under its capsule), or 3) separate kidney and thymic grafts from the same donor. All recipients received a 12-day course of cyclosporine. Thymectomized animals receiving a kidney allograft alone or receiving separate thymic and kidney grafts had unstable renal function due to severe rejection with the persistence of anti-donor cytotoxic T cell reactivity. In contrast, recipients of composite thymokidney grafts had stable renal function with no evidence of rejection histologically and donor-specific unresponsiveness. By postoperative day 14, the thymic tissue in the thymokidney contained recipient-type dendritic cells. By postoperative day 60, recipient-type class I positive thymocytes appeared in the thymic medulla, indicating thymopoiesis. T cells were both recipient and donor MHC-restricted. These data demonstrate that the presence of vascularized-donor thymic tissue induces rapid and stable tolerance to class I-disparate kidney allografts in thymectomized recipients. To our knowledge, this is the first evidence of functional vascularized thymic grafts permitting transplantation tolerance to be induced in a large animal model.