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.     

Relative antigenicity of components of a vascularized limb allograft

At present, the transplantation of vascularized limb-tissue allografts can be achieved only with generalized host immunosuppression, which results in significant systemic toxicity, thereby precluding their clinical use. A better understanding of the immunogenic mechanisms of these allografts may permit less toxic and thus clinically applicable means of host immunosuppression. In this study, individual vascularized limb tissues (skin, subcutaneous tissue, muscle, bone, and blood vessels) and a whole limb were transplanted microsurgically across a strong histocompatibility barrier in rats. The respective cell-mediated and humoral immune responses generated in the hosts were determined by means of mixed lymphocyte cultures by radioactive 51Cr release assays and compared. No single tissue predominated in the elicited immune response. Rather, the various tissue components interacted with the host immune system in a complex but predictable pattern with differing timing and intensity. Surprisingly, the whole-limb allograft elicited less immune response than did allografts of its individual components. The data presented here also serve as a foundation for further elucidation of the immunogenic mechanisms of vascularized limb-tissue allografts.     

Donor modification leads to prolonged survival of limb allografts

Chronic immunosuppression is essential for maintaining human hand transplant survival because composite tissue allografts are as susceptible to rejection as visceral organ allografts. Limb allografts comprise different types of tissues with varying antigenicities, and the immunosuppressive doses required for these allografts are as high or higher than those required for solid organ allotransplantation. In particular, bone marrow is an early target of the host immune response. This study reports on donor limb modification of the marrow compartment leading to prolonged survival of limb allografts.Chimeric limb allografts comprising a Lewis rat vascularized allograft and Brown Norway rat bone marrow were created. These chimeric limbs were transplanted into three recipients: (1) Buffalo rats (n = 12), where the chimeric limb was allogeneic for both vascular graft and bone marrow; (2) Lewis rats (n = 12), where the limb was allogeneic for marrow alone; and (3) Brown Norway rats (n = 12), where the limb was allogeneic for graft alone.This study found that Brown Norway recipients elicited significantly reduced cell-mediated and humoral immune responses in comparison with the Buffalo and Lewis recipients (p < 0.001 and p < 0.01, respectively). The Buffalo and Lewis recipients both elicited high cell-mediated and humoral responses. The Brown Norway recipients also had prolonged survival of limb tissue allograft in comparison with the other experimental groups.     

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.     

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.     

Vascularized muscle allografts and the role of cyclosporine

This study examined the fate of vascularized muscle allografts using a genetically defined rat model. Its purposes were (1) to analyze the histologic/immunologic responses, (2) to study the effect of cyclosporine on graft survival, and (3) to examine the possibility of inducing tolerance. In rats differing at a major histocompatibility locus, vascularized gastrocnemius muscle transplants were performed based on the sural branches of the femoral artery and vein. Forty-two animals studied were divided into three groups: Group 1, allografts, was treated without cyclosporine; Group 2, allografts, was administered continuous cyclosporine; and Group 3, allografts, was administered cyclosporine for 6 weeks only. Evaluation consisted of gross examination, H&E histology, and immunologic studies (MLC, CML, and complement-dependent 51Cr lysis assay). Lytic units (LU) were derived from the assays and served as the indicator of immune response. Group 1 animals had uniform rejection with intense cell-mediated response (LU 23 to 47) and low humoral response. Group 2 animals had viable allografts throughout with suppressed lytic unit values of 0 to 9 initially, which rose to 14 to 29 at 6 weeks despite continuous cyclosporine treatment. Group 3 animals showed rejection similar to the untreated animals. Autografts were performed as controls and survived indefinitely. Analysis of variance was significant at p less than 0.05. Using a reliable rat model for vascularized muscle allografts, we found that in transplantation across a major histocompatibility barrier, the initial immune response was primarily cell-mediated. Cyclosporine suppressed rejection only when given continuously, and short-term cyclosporine treatment did not induce a tolerant state. These data should be useful for future studies of vascularized muscle allografts.     

Immunologic and ultrastructural changes during early rejection of vascularized bone allografts

This investigation evaluated ultrastructural changes during the earliest phase of immunologic rejection of vascularized bone allografts in a genetically defined rat model. These results were correlated with the cell-mediated and humoral immunologic responses during this time period. Employing a model for heterotopic allograft transplantation, 33 rats divided into four categories were evaluated. Group I consisted of ungrafted (naive) Lewis and Brown Norway rats; group II consisted of Lewis-to-Lewis vascularized bone isografts; group III consisted of Lewis-to-Brown Norway vascularized bone allografts; and group IV consisted of Lewis-to-Brown Norway vascularized bone allografts in rats receiving cyclosporine (10 mg/kg/day). Experimental animals were sacrificed at 3, 5, and 7 days. Immunologic analysis was performed using a cell-mediated lymphocytotoxicity assay and a complement-dependent cytotoxic antibody assay. The results of this study show that rejection of vascularized bone allografts appears as early as 3 days postoperatively, with osteocytes and vascular endothelium being the first elements affected. This early rejection is probably a manifestation of the humoral response. All changes secondary to rejection were arrested by cyclosporine.     

Long-term remodeling of vascularized and nonvascularized onlay bone grafts: a macroscopic and microscopic analysis

The present study was performed to compare vascularized and nonvascularized onlay bone grafts to investigate the potential effect of graft-to-recipient bed orientation on long-term bone remodeling and changes in thickness and microarchitectural patterns of remodeling within the bone grafts. In two groups of 10 rabbits each, bone grafts were raised bilaterally from the supraorbital processes and placed subperiosteally on the zygomatic arch. The bone grafts were oriented parallel to the zygomatic arch on one side and perpendicular to the arch on the contralateral side. In the first group, vascularized bone grafts were transferred based on the auricularis anterior muscle, and in the second group nonvascularized bone grafts were transferred. Fluorochrome markers were injected during the last 3 months of animal survival, and animals were killed either 6 or 12 months postoperatively. The nonvascularized augmented zygoma showed no significant change in thickness 6 months after bone graft placement and a significant decrease in thickness 1 year after graft placement (p < 0.01). The vascularized augmented zygoma showed a slight but statistically significant decrease in thickness 6 months after graft placement (p < 0.003), with no significant difference relative to its initial thickness 1 year after graft placement. In animals killed 6 months after bone graft placement, both the rate of remodeling and the bone deposition rate measured during the last 3 months of survival were significantly higher in the vascularized bone grafts compared with their nonvascularized counterparts (p < 0.02). By 1 year postoperatively, there were no significant differences in thickness, mineral apposition rate, or osteon density between bone grafts oriented perpendicular and parallel to the zygomatic arch. These findings indicate that the vascularity of a bone graft has a significant effect on long-term thickness and histomorphometric parameters of bone remodeling, whereas the direction of placement of a subperiosteal graft relative to the recipient bed has minimal effect on these parameters. In vascularized bone grafts, both bone remodeling and deposition are accelerated during the initial period following graft placement. Continued bone deposition renders vascularized grafts better suited for the long-term maintenance of thickness and contour relative to nonvascularized grafts.     

Bone grafts: a radiologic, histologic, and biomechanical model comparing autografts, allografts, and free vascularized bone grafts

We developed an experimental model to compare the efficacy of free vascularized bone grafts, conventional segmental autografts, matchstick autografts, and fresh segmental allografts in terms of their ability to reconstruct a 7-cm segmental diaphyseal defect created in the canine femur. Forty-five adult mongrel dogs were studied and followed for 6 to 12 months prior to sacrifice. Evaluation included radiologic assessment of graft incorporation and hypertrophy, histology, and biomechanical testing. These studies indicated that microsurgically revascularized autografts were superior to all other groups in terms of early incorporation, hypertrophy, and the highest mechanical strength to failure. Union of the bone graft to the recipient femur was achieved by 6 months in 25 of 26 autografts, and no difference in union rate was seen within the autograft group. However, only two of five allografts achieved bony union during this time interval. Arteriography, microangiography, fluorochrome, and histologic studies all supported the concept that microsurgically revascularized grafts, when successful, maintain their viability. However, the premise that all osteocytes survive in a successfully revascularized bone graft is open to question. While decalcified sections showed that all microsurgically revascularized grafts maintained normal viability in the central marrow and cancellous portions compared with the other three groups, the viability of cortical bone in the vascularized autografts was less clear. Undecalcified fluorochrome sections suggested that circulation was not preserved in all portions of the cortex. Revascularization of the nonvascularized autografts was complete at 3 months, while, in the avascular allografts, the process was not complete at 6 months.     

Mixed xenogeneic chimerism induces donor-specific humoral and cellular immune tolerance for cardiac xenografts

Xenotransplantation has been suggested as a potential solution to the critical shortage of donor organs. However, success has been limited by the vigorous rejection response elicited against solid organs transplanted across species barriers. Mixed xenogeneic bone marrow chimeras resulting from the transplantation of a mixture of host and donor marrow (B10 mouse + F344 rat --> B10 mouse) results in donor-specific cross-species transplantation tolerance for subsequent nonvascularized skin and islet grafts. Furthermore, compared with fully xenogeneic chimeras (rat --> mouse), mixed xenogeneic chimeras exhibit superior immunocompetence for infectious agents in vivo and in vitro, suggesting that the immune system is intact. The ability to establish long-term humoral and cellular tolerance for primarily vascularized xenografts in vivo, in the setting of both recipient and donor Ig and effector cell production, has not previously been characterized. Mixed xenogeneic chimeras exhibit donor-specific humoral tolerance as evident by the absence of anti-donor Ab and Ab-dependent donor-specific cytotoxicity in vitro and intravascular IgM deposition within donor-strain (F344) cardiac xenografts in vivo. F344 cardiac xenografts are accepted (median > or =180 days) without clinical or histologic evidence of rejection, suggesting cellular tolerance. In contrast, MHC-disparate third-party mouse (B10.BR) and rat (ACI or WF) grafts are rejected (median of 23 and 41 days, respectively) in association with extensive mononuclear cell infiltration and vascular deposits of mouse IgM. These results demonstrate that mixed xenogeneic chimerism establishes donor-specific humoral and cellular tolerance and permits the successful transplantation of even primarily vascularized xenografts in the setting of intact Ab production.     

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.     

Growth and survival of vascularized and nonvascularized membranous bone: an experimental study

Although nonvascularized membranous bone grafts to the craniofacial skeleton demonstrate improved survival over similar grafts of endochondral origin, the comparative fate of vascularized membranous grafts is unknown. It is also unknown whether onlay membranous bone grafts in immature animals have the ability to grow. To examine these questions, a model was developed in New Zealand white rabbits in which a segment of the zygomatic arch was transferred to the subjacent mandible as either a vascularized or nonvascularized transfer. At harvest 16 weeks later, residual graft volume and bone architecture were analyzed. Results demonstrate no improved survival for vascularized membranous grafts in adult animals (n = 7), while in the immature animals (n = 6), growth of the vascularized bone transfers was documented. We conclude that in the majority of instances in craniofacial reconstruction, nonvascularized onlay membranous grafts are to be preferred. Specific instances for the use of vascularized transfers will be discussed.     

The role of cyclosporin in prolonging survival in vascularized bone allografts

It is known that experimental vascularized bone allografts are subject to host rejection. To be useful clinically, this rejection response would need to be controlled. Cyclosporin is a potent immunosuppressant whose precise role in vascularized bone allograft transplantation has not been established. Using a proven reliable vascularized knee allograft model in inbred rats, cyclosporin was used postoperatively both continuously and short term (14 days after transplant) at 10 mg/kg per day as recipient treatment. Across a strong histocompatibility barrier, continuous cyclosporin was required for long-term graft survival. Short-term therapy delayed rejection for 4 to 6 weeks. However, across a weak histocompatibility barrier, short-term therapy was as effective as continuous therapy in achieving long-term graft survival. The implication is that a limited course of cyclosporin may be clinically successful in sustaining vascularized bone allograft survival, provided the genetic disparity between graft and host has been minimized by genetic matching techniques.     

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.     

Growth in revascularized bone grafts in young puppies.

The results of microvascular transfers of growing ulnas in puppy forelegs have been studied. These transfers were carried out both heterotopically and orthotopically. The growth in the revascularized bone grafts has been compared to that in heterotopic, nonvascularized ulna transfers and to normal ulnar growth. The growth in the vascularized bone grafts was significantly greater than in the nonvascularized grafts, but significantly less than in normal ulnar growth. A metaphyseal contribution to the blood supply of the growing portion of long bones is suggested.     

Immunologic 'ignorance' of vascularized organ transplants in the absence of secondary lymphoid tissue

Secondary lymphoid organs (the spleen, lymph nodes and mucosal lymphoid tissues) provide the proper environment for antigen-presenting cells to interact with and activate naive T and B lymphocytes. Although it is generally accepted that secondary lymphoid organs are essential for initiating immune responses to microbial antigens and to skin allografts, the prevailing view has been that the immune response to primarily vascularized organ transplants such as hearts and kidneys does not require the presence of secondary lymphoid tissue. The assumption has been that the immune response to such organs is initiated in the graft itself when recipient lymphocytes encounter foreign histocompatibility antigens presented by the graft's endothelial cells. In contrast to this view, we show here that cardiac allografts are accepted indefinitely in recipient mice that lack secondary lymphoid tissue, indicating that the alloimmune response to a vascularized organ transplant cannot be initiated in the graft itself. Moreover, we demonstrate that the permanent acceptance of these grafts is not due to tolerance but is because of immunologic 'ignorance'.     

Prolonged survival of musculoskeletal xenografts with combined cyclosporine and 15-deoxyspergualin

This study was undertaken to evaluate the feasibility of performing vascularized musculoskeletal xenografts between mice and rats using immunosuppression. Vascularized musculoskeletal grafts were harvested from the hind limb of C57BL/6J (B6) mice, transplanted heterotopically into Lewis rats, and revascularized by microanastomoses of the graft artery and the recipient femoral artery and the graft vein to the recipient femoral vein. Recipient rats were divided into four groups. Group 1 received no immunosuppression (n = 10), group 2 was treated with cyclosporine (10 mg/kg/day; n = 10), group 3 was treated with 15-deoxyspergualin (5 mg/kg/day; n = 10), and group 4 received both cyclosporine and 15-deoxyspergualin (n = 10). Graft survival was directly examined on postoperative days 4, 7, and 14. In vitro assays were performed using mixed lymphocyte reactions and anti-donor cytotoxic antibody assays to assess the recipient's immune response. Grafts were examined by histology and immunohistochemistry. All grafts in group 1 were rejected by day 4. In groups 2 and 3, all grafts were rejected by day 7. In group 4, however, 8 of 10 recipients had viable grafts on day 14. Data from mixed lymphocyte reactions showed that cell-mediated immune responses were uniformly suppressed in groups 2, 3, and 4 compared with group 1. However, anti-donor antibody production was only partly suppressed in groups 2 and 3, suggesting that graft rejection was primarily caused by circulating cytotoxic anti-donor antibodies in groups 1, 2, and 3. Histologic observations in groups 1, 2, and 3 confirmed the important role of the humoral mechanism in xenograft rejection. Furthermore, immunohistochemical results demonstrated that the small vessels in the rejected grafts showed anti-rat immunoglobulin and complement depositions. Only a combination therapy of cyclosporine and 15-deoxyspergualin attenuated the rejection of xenografts.     

Rabbit trochlear model of osteochondral allograft transplantation

Allografting and autografting of osteochondral tissues is a promising strategy to treat articular cartilage lesions in damaged joints. We developed a new model of fresh osteochondral allografting using the entire rabbit trochlea. The objective of the current study was to demonstrate that this model would achieve reproducible graft-host healing and maintain normal articular cartilage histologic, immunolocalization, and biochemical characteristics after transplantation under diverse storage and transplantation conditions. New Zealand white (n = 8) and Dutch belted (n = 8) rabbits underwent a 2-stage transplantation operation using osteochondral grafts that had been stored for 2 or 4 wk. Trochlear grafts harvested from the left knee were transplanted to the right knee as either autografts or allografts. Grafts were fixed with 22-gauge steel wire or 3-0 nylon suture. Rabbits were euthanized for evaluation at 1, 2, 4, 6, and 12 wk after transplantation. All grafts that remained in vivo for at least 4 wk demonstrated 100% interface healing by microCT. Trabecular bridging was present at the host-graft interface starting at 2 wk after transplantation, with no significant difference in cartilage histology between the various groups. The combined histology scores indicated minimal evidence of osteoarthritis. Immunostaining revealed that superficial zone protein was localized at the surface of all transplants. The rabbit trochlear model met our criteria for a successful model in regard to the ease of the procedure, low rate of surgical complications, relatively large articular cartilage surface area, and amount of host-graft bone interface available for analysis.     

Early chemokine cascades in murine cardiac grafts regulate T cell recruitment and progression of acute allograft rejection

The identification of early inflammatory events after transplant in solid tissue organ grafts that may direct T cell recruitment and promote acute allograft rejection remain largely unknown. To better understand temporal aspects of early inflammatory events in vascularized organ grafts, we tested the intragraft expression of four different chemokines in heterotopically transplanted A/J (H-2(a)) and syngeneic heart grafts in C57BL/6 (H-2(b)) recipient mice from 1.5 to 48 h after transplant. Similar temporal expression patterns and equivalent levels of chemokine expression were observed in both syngeneic and allogeneic cardiac allografts during this time period. Expression of the neutrophil chemoattractant growth-related oncogene alpha (KC) was observed first and reached peak levels by 6 h after transplant and was followed by the monocyte/macrophage chemoattractant protein-1 (JE) and then macrophage inflammatory proteins 1beta and 1alpha. Administration of rabbit KC antiserum to allograft recipients within 30 min of cardiac transplantation attenuated downstream events including intra-allograft expression of the T cell chemoattractants IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma, cellular infiltration into the allograft, and graft rejection. Similarly, depletion of recipient neutrophils at the time of transplantation significantly extended allograft survival from day 8 to 10 in control-treated recipients up to day 21 after transplant. These results indicate the induction of highly organized cascades of neutrophil and macrophage chemoattractants in cardiac grafts and support the proposal that early inflammatory events are required for optimal recruitment of T cells into allografts during the progression of acute rejection of cardiac allografts.     

Acquisition of humoral transplantation tolerance upon de novo emergence of B lymphocytes

A major obstacle to transplantation tolerance is humoral immunity. In this paper, we demonstrate that the intrinsic developmental propensity of the B lymphocyte compartment for acquisition of self-tolerance can be harnessed to induce humoral unresponsiveness to transplanted alloantigens. In the current study, when transitional B cells developed in the presence of donor lymphoid cells, the mature B lymphocyte compartment failed to mount a donor-specific alloantibody response to an organ transplant--despite unrestrained acute T cell-mediated allograft rejection. Specifically, we generated an experimental system wherein a B6 strain B cell compartment developed de novo in the presence of F1 (B6xBALB/c) lymphoid cells and in a T cell-deficient setting. Following establishment of a steady-state B cell compartment, these B6 mice were transplanted with heterotopic cardiac allografts from allogeneic BALB/c donors. The mice were then inoculated with purified syngeneic B6 T cells. As expected, all cardiac allografts were acutely rejected. However, the B lymphocyte compartment of these mice was completely inert in its capacity to form a BALB/c-specific alloantibody response. Using an alloantigen-specific Ig transgenic system, we demonstrated that this profound degree of humoral tolerance was caused by clonal deletion of alloreactive specificities from the primary B cell repertoire. Thus, de novo B cell compartment development at the time of transplantation is of critical importance in recipient repertoire "remodeling" to a humoral tolerant state.