The replacement of graft endothelium by recipient-type cells conditions allograft rejection mediated by indirect pathway CD4+ T cells

BACKGROUND: Whereas the participation of alloreactive T cells sensitized by indirect allorecognition in graft rejection is well documented, the nature of recipient antigen presenting cells recognized by indirect pathway CD4+ T cells within the graft has yet to be identified. The purpose of this study was to determine the role played by graft endothelium replacement in the immune recognition of cardiac allografts rejected by indirect pathway CD4+ T cells. METHODS: Transgenic RAG2-/- mice expressing I-Ab-restricted male antigen H-Y-specific TcR were studied for their capacity to reject H-2k male cardiac allografts. Chronic vascular rejection in this model was due to the indirect recognition of H-Y antigen shed from H-2k male allograft and presented by the recipient's own I-Ab APC to transgenic T cells. RESULTS: Immunohistochemical analysis of rejected grafts revealed the presence of numerous microvascular endothelial cells (EC) that expressed recipient's I-Ab MHC class II molecules. This observation suggested that graft endothelium replacement by I-Ab-positive cells of recipient origin could stimulate the rejection of male H-2k graft by I-Ab-restricted H-Y-specific T cells. To investigate further this possibility, hearts from H-2b-into-H-2k irradiation bone marrow (BM) chimera were transplanted in transgenic recipients. A direct correlation was observed between the presence of I-Ab-positive EC within myocardial microvessels and the induction of acute rejection of chimeric H-2k male cardiac allografts transplanted in transgenic recipients. CONCLUSIONS: We conclude that graft endothelium replacement by recipient-type cells is required for the rejection of cardiac allograft mediated by indirect pathway alloreactive CD4+ T cells.     

The replacement of graft endothelium by recipient-type cells conditions allograft rejection mediated by indirect pathway CD4(+) T cells

BACKGROUND: Whereas the participation of alloreactive T cells sensitized by indirect allorecognition in graft rejection is well documented, the nature of recipient antigen presenting cells recognized by indirect pathway CD4(+) T cells within the graft has yet to be identified. The purpose of this study was to determine the role played by graft endothelium replacement in the immune recognition of cardiac allografts rejected by indirect pathway CD4(+) T cells. METHODS: Transgenic RAG2(-/-) mice expressing I-A(b)-restricted male antigen H-Y-specific TcR were studied for their capacity to reject H-2(k) male cardiac allografts. Chronic vascular rejection in this model was due to the indirect recognition of H-Y antigen shed from H-2(k) male allograft and presented by the recipient's own I-A(b) APC to transgenic T cells. RESULTS: Immunohistochemical analysis of rejected grafts revealed the presence of numerous microvascular endothelial cells (EC) that expressed recipient's I-A MHC class II molecules. This observation suggested that graft endothelium replacement by I-A(b)-positive cells of recipient origin could stimulate the rejection of male H-2(k) graft by I-A(b)--restricted H-Y--specific T cells. To investigate further this possibility, hearts from H-2(b)--into--H-2(k) irradiation bone marrow (BM) chimera were transplanted in transgenic recipients. A direct correlation was observed between the presence of I-A(b)-positive EC within myocardial microvessels and the induction of acute rejection of chimeric H-2(k) male cardiac allografts transplanted in transgenic recipients. CONCLUSIONS: We conclude that graft endothelium replacement by recipient-type cells is required for the rejection of cardiac allograft mediated by indirect pathway alloreactive CD4(+) T cells.     

Recipient bone marrow engraftment in donor tissue after long-term tolerance to a composite tissue allograft

BACKGROUND: An important component of a composite tissue limb allograft (CTA) is the vascularized bone marrow and bone marrow stroma, which when transplanted could create immediate marrow space and engraftment. We have previously demonstrated that tolerance to musculoskeletal allografts can be achieved with a 12-day course of cyclosporine without the presence of long-term peripheral donor cell chimerism. The objective of this study was to determine the fate of the donor bone marrow after transplantation of a limb allograft in a miniature swine model. METHODS: CTAs from donor swine were heterotopically transplanted into six MHC-matched, minor-antigen-mismatched recipients, and a 12-day course of cyclosporine was given. Previous animals transplanted without cyclosporine rejected their grafts in less than 42 days. A non-MHC-linked marker, pig allelic antigen (PAA), was used to distinguish host and donor cells. Three PAA- animals received PAA+ CTAs, and three PAA+ animals received PAA- CTAs. Bone marrow was harvested from the donor limb grafts and the recipient and analyzed by flow cytometry and histology. Thymus, spleen, and mesenteric lymph nodes were also harvested from the recipient swine and evaluated for the presence of donor cells by flow cytometry. RESULTS: All animals receiving cyclosporine demonstrated permanent tolerance to their allografts. Donor bone marrow cells were present in all grafts at the time of transplantation and during the immediate postoperative period. By 48 weeks, donor cells were no longer detectable within the marrow space of the allograft. In long-term animals host bone marrow cells replaced donor cells in the graft marrow space. No evidence of donor cell engraftment was found in recipient animals. CONCLUSION: This study demonstrates that in long-term tolerant recipients of musculoskeletal allografts there is no evidence of persistent donor bone marrow cells in the hematopoietic tissues of the graft or the host. Rather, the recipient's bone marrow cells and lymphocytes repopulate the donor marrow space of the graft.     

Autologous Osteoblast Transplantation,an Innovative Method of Bone Defect Treatment: Role of a Tissue and Cell Bank in the Process

Background

The idea of cell treatment of various diseases and medical conditions has become very popular. Some procedures are well established, as is autologous chondrocyte implantation, whereas others are still in the process of early development, laboratory experiments, and some clinical trials.

Methods

This report is devoted to an example of an emerging cell treatment: bone augmentation with the use of autologous cells and its legal and technical background. Various requirements set by law must be met by tissue banks performing cell seeding of grafts. In Europe, the requirements are described in directives 2004/23/EC, 2006/17/EC, 2006/86/EC, and in the regulation 2007/1394/EC.

Results

Revitalization of biostatic allografts gives new, promising tools for creation of functional parts of organs; brings the methodology used in tissue banks closer to tissue engineering; places the enterprise in the mainstream of advanced biotechnology; allows the full potential of tissue allografts; and opens a new, large area for clinical and laboratory research. Cell and tissue processing also have a financial impact on the treatment: it produces additional expenditures.

Conclusions

Clinical effectiveness will be the most decisive factor of whether this innovative treatment will be applied in a particular type of medical condition. From a tissue establishment perspective, the most important issue is to develop a procedure that ensures safety for the patient in graft quality terms.     

Induction of transplantation tolerance in rats by spleen allografts. III. The role of T suppressor cells in the induction of specific unresponsiveness

Heterotopic (WAG x AGUS)F1 spleen allografts survive indefinitely when transplanted to normal AGUS recipients and induce long-term donor-specific unresponsiveness. In this report, we have examined the immune reactivity of spleen graft recipients soon after transplantation, in an attempt to define the immunological mechanisms responsible for the induction of donor-specific unresponsiveness. Unresponsiveness develops as early as one week after splenic transplantation. T cells obtained from the recipient lymph node and spleen exhibit reduced mixed lymphocyte reaction responses to donor (WAG) but respond normally to third-party (PVG) stimulators. In contrast, T cells obtained from the spleen graft are unresponsive to both donor and third-party stimulators. Donor specific T suppressor cells (Ts) appear in the recipient's lymph node and spleen by one week posttransplantation--however, at this time antigen nonspecific suppressor cells predominate in the spleen graft. Only minimal cytotoxic T cell activity could be detected in the spleen graft, with the host spleen and lymph nodes being devoid of cytotoxic T lymphocytes. Sera obtained one or two weeks following splenic transplantation did not contain cytotoxic alloantibodies, and only a very weak response could be detected at one month. These data demonstrate that the unresponsiveness associated with the spontaneous acceptance of spleen allografts is correlated with the early induction of antigen specific Ts in recipient lymphoid tissue and the presence of nonspecific suppressor cells at the graft site.     

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.     

Graft volume as the surrogate marker for nephron number affects the outcomes of living-donor kidney transplantation

Post-transplant outcome of kidney allografts depends on various factors, one of which may be the compatibility in volume between graft and recipient. However, previous studies adjusted the graft volume only for recipient's size. As the adjusted graft volume for donor's size would be substituted of nephron number more accurately, we adjusted the graft volume for both recipient's and donor's sizes. In 351 cases of living-donor kidney transplantation, we found that the adjusted graft volume for both recipient's and donor's body surface areas (BSAs) yielded larger area under the curves for the transplant outcomes than looking only at the adjusted volume for the recipient's BSA. The recipients were separated into two groups according to the low and high adjusted graft volumes. During the follow-up period (mean 55.6 months), the low-graft-volume group conferred greater risk of rejection, chronic change, glomerulonephritis, and graft loss than the high-graft-volume group (all p's < 0.05). However, the frequency of T-cell infiltration, as evaluated in protocol biopsy, was not different between the two adjusted graft volume groups. In conclusion, the graft volume as the surrogate marker for nephron number should be considered in kidney transplantation, especially in otherwise similar donor conditions.     

Recipient immature dendritic cells do not prolong allograft survival

Experimental studies on allogeneic transplantation have shown that recipient dendritic cells (DC) play a role in peripheral tolerance as well as in rejection of allografts. It is not known whether DCs exert their tolerogenic function in the graft or in recipient lymphoid tissue. To answer this question we created a chimeric heart model deprived of its own DCs and repopulated with recipient DCs. The rationale for this model was to observe whether recipient mature and immature DCs located in the graft attenuate recruitment and stimulation of recipient lymphocytes, subsequently prolonging graft survival. Vascularized bone marrow transplants from the prospective recipient to the lethally irradiated heart donor, which function for a period of 14 days, were used to replace donor DCs with prospective recipient either mature or immature DCs. Replacement of the donor heart with either of these cells did not prolong graft survival. The intragraft microchimerism did not mitigate the allogeneic rejection reaction.     

Effects of low intensity pulsed ultrasound with and without increased cortical porosity on structural bone allograft incorporation

Background  

Though used for over a century, structural bone allografts suffer from a high rate of mechanical failure due to limited graft revitalization even after extended periods in vivo. Novel strategies that aim to improve graft incorporation are lacking but necessary to improve the long-term clinical outcome of patients receiving bone allografts. The current study evaluated the effect of low-intensity pulsed ultrasound (LIPUS), a potent exogenous biophysical stimulus used clinically to accelerate the course of fresh fracture healing, and longitudinal allograft perforations (LAP) as non-invasive therapies to improve revitalization of intercalary allografts in a sheep model.     

Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.

A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that approximately 70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP-2-producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene-enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair. INTRODUCTION: The loss of cellular activity in a structural bone allograft markedly reduces its healing potential compared with a live autograft. To further understand the cellular mechanisms for structural bone graft healing and repair and to devise a therapeutic strategy aimed at enhancing the performance of allograft, we established a segmental femoral structural bone graft model in mice that permits qualitative and quantitative analyses of graft healing and neovascularization. MATERIALS AND METHODS: Using this segmental femoral bone graft model, we transplanted live isografts harvested from Rosa 26A mice that constitutively express beta-galactosidase into their wildtype control mice. In an attempt to emulate the osteogenic and angiogenic properties of periosteum, we applied a cell-based, adenovirus-mediated gene therapy approach to engraft BMP-2-producing bone marrow stromal cells onto devitalized allografts. RESULTS: X-gal staining for donor cells allowed monitoring the progression of periosteal progenitor cell fate and showed that 70% of osteogenesis was attributed to cellular proliferation and differentiation of donor progenitor cells on the surface of the live bone graft. Quantitative muCT analyses showed a 3-fold increase in new bone callus formation and a 6.8-fold increase in neovascularization for BMP-2/stromal cell-treated allograft compared with control acellular allografts. Histologic analyses showed the key features of autograft healing in the BMP-2/stromal cell-treated allografts, including the formation of a mineralized bone callus completely bridging the segmental defects, abundant neovascularization, and extensive resorption of bone graft. CONCLUSIONS: The marked improvement of healing in these cellularized allografts suggests a clinical strategy for engineering a functional periosteum to improve the osteogenic and angiogenic properties of processed allografts.     

Prolonged skin allograft survival through enhancement effects on donor tissue

A skin allograft retransplantation model was utilized to study the mechanism of immunological enhancement in a murine system. Enhancement was accomplished by treating allografted recipients with host antidonor serum (B6AF1 anti-B10.D2 alloantiserum). Grafts from enhanced or untreated hosts were retransplanted after seven days onto a second recipient. Enhanced retransplanted grafts had significantly prolonged survival as compared with unenhanced grafts. The survival of enhanced retransplanted grafts was as prolonged as that of primary skin grafts on antiserum-treated hosts. Splenocytes harvested from recipients of enhanced retransplanted allografts showed delayed and diminished development of T cell responses to graft alloantigens. Sensitization of the second recipient abrogated prolonged survival of enhanced retransplanted grafts. Also, enhancement prevented sensitization of allografted recipients. One interpretation of these studies suggests that a sensitization block is a sufficient mechanism of skin allograft enhancement. The site of action of antisera is within the graft itself. Decreased T cell responses in the host are indirect effects of diminished antigenicity of enhanced grafts. Further studies of immunological enhancement should be directed to the graft, not toward the graft recipient.     

Immunological characteristics of purified pancreatic islet grafts

In a DBA/2 (H-2d) pancreatic islet-to-B6AF1 (H-2b/k.d) recipient combination, the graft survival of hand-picked islets was compared with that of "crude digested" islets that were prepared simply by collagenase digestion and Ficoll gradient separation and were contaminated with lymph nodes and vascular and ductal tissue. Islet allografts were transplanted into the renal subcapsular space of streptozotocin-induced diabetic recipients. No immunosuppression was used. All the crude digested islet allografts were acutely rejected between days 7 and 18 with a median survival time (MST) of 10.2 +/- 2.5 days. In contrast, 33% (3/9) of the purified islet allografts survived more than 100 days. Simultaneous transplantation of purified islets and contaminating tissue resulted in a shorter graft survival (MST of 15.6 +/- 3.7 days). When 5 X 10(7) donor strain spleen cells were injected i.v. at the time of transplantation, all purified islet grafts were acutely rejected within 9 days. In addition, the rejection time of the purified islet allografts was inversely correlated with the number of donor spleen cells injected. These results indicate that contaminating tissues such as lymph nodes, vascular tissue, and ductal fragments present in the crude digested islet allografts are a major stimulus for induction of an immune response resulting in acute rejection of islet allografts.     

Lung allograft rejection in the rat. II. Specific immunological properties of lung grafts

The immunological mechanism of lung allograft rejection was studied in inbred rats, in order to explain the rapid progress of the rejection response against RT1-incompatible lung grafts. Histological appearances of the graft and of the recipient's spleen were studied, migration patterns of graft and recipient lymphocytes were assessed, and titers of circulating alloantibodies were determined. Histologically, we discriminated four phases of the rejection response in lung grafts: sequentially the latent, vascular, alveolar, and destruction phases. Early in the vascular phase, recipient lymphocytes primarily infiltrated the bronchus-associated lymphoid tissue (BALT) of the graft, causing a local immune response. Concurrent with these local rejection phenomena in the graft, a strong systemic immune response developed in the recipient's spleen, presumably induced by the great number of lymphocytes that migrated from the graft's BALT into the recipient's lymphoid tissues. We conclude that BALT facilitates a fast and intensive interaction between lung graft and recipient that is likely to accelerate the induction of the rejection response both locally in the graft and systemically in the recipient's lymphoid organs.     

Generation of donor hematolymphoid cells after rat-limb composite grafting

BACKGROUND: Composite tissue allografts are unique because they provide the vascularized bone marrow with stroma, which is the supportive microenvironment. In this study, we investigated the beneficial effect of donor-derived bone marrow cells within the long-surviving recipient rats after limb transplantation. METHODS: Green fluorescent protein (GFP) transgenic rats developed for paramount cell marking were donors, and wild Wistar rats were recipients. Orthotopic hind-limb transplantation was performed using a microsurgical technique. Tacrolimus (1.0 mg/kg) was intramuscularly injected for 14 days postoperatively. The skin graft from GFP donor onto the GFP recipient was performed as a control. Flow cytometric analyses of recipient peripheral blood and bone marrow were carried out at 4 to 6 days, 18 to 21 days, 6 weeks, and 2, 4, 6, 9, and 12 months after transplantation. RESULTS: The rats that received tacrolimus therapy achieved prolonged composite graft acceptance more than 12 months, whereas GFP skin grafts were rejected at 47 days under the same immunosuppressive protocol. Numerous GFP lymphocytes and granulocytes were detected within the recipient bone marrow for the first 6 weeks post limb transplantation. These cells remained relatively stable for more than 12 months. CONCLUSIONS: The results showed that donor-derived hematopoietic stem cells engrafted in recipient bone marrow and differentiated to lymphocytes and granulocytes after limb transplantation. The vascularized bone marrow, transplanted as a part of the hind limb, could have contributed to mixed chimerism and worked as the bone-marrow source in the recipients.     

Selection of massive bone allografts using shape-matching 3-dimensional registration

Background and purpose Massive bone allografts are used when surgery causes large segmental defects. Shape-matching is the primary criterion for selection of an allograft. The current selection method, based on 2-dimensional template comparison, is inefficient for 3-dimensional complex bones. We have analyzed a 3-dimensional (3-D) registration method to match the anatomy of the allograft with that of the recipient.

Methods 3-D CT-based registration was performed to match the shapes of both bones. We used the registration to align the allograft volume onto the recipient's bone. Hemipelvic allograft selection was tested in 10 virtual recipients with a panel of 10 potential allografts, including one from the recipient himself (trap graft). 4 observers were asked to visually inspect the superposition of allograft over the recipient, to classify the allografts into 4 categories according to the matching of anatomic zones, and to select the 3 best matching allografts. The results obtained using the registration method were compared with those from a previous study on the template method.

Results Using the registration method, the observers systematically detected the trap graft. Selections of the 3 best matching allografts performed using registration and template methods were different. Selection of the 3 best matching allografts was improved by the registration method. Finally, reproducibility of the selection was improved when using the registration method.

Interpretation 3-D CT registration provides more useful information than the template method but the final decision lies with the surgeon, who should select the optimal allograft according to his or her own preferences and the needs of the recipient.     

The origin of cells that repopulate patellar tendons used for reconstructing anterior cruciate ligaments in man

Cryopreserved patellar tendon allografts are often recommended for reconstruction of anterior cruciate ligaments (ACLs) because living donor fibroblasts are thought to promote repair. Animal studies, however, indicate that ligaments regenerate from recipient rather than donor cells. If applicable to man, these observations suggest that allograft cell viability is unimportant. We therefore used short tandem repeat analysis with polymerase chain reaction (PCR) amplification to determine the source of cells in nine human ACLs reconstructed with cryopreserved patellar tendon allografts. PCR amplification of donor and recipient DNA obtained before operation and DNA from the graft obtained two to ten months after transplantation revealed the genotype of cells and showed only recipient cells in the graft area. Rather than preserve the viability of donor cells, a technique is required which will facilitate the introduction of recipient cells into patellar tendon allografts.     

Reconstructive surgery using composite tissue allografts: perspectives from the first clinical cases

Tissue allotransplantation is defined by the transfer of tissues harvested from a cadaveric donor (in a brain dead condition) and revascularized to a recipient subject. This is based on the mastery of microsurgical techniques and on the induction of a therapeutic immunosuppression for the recipient. The first allografts (flexor tendon systems, knees and femoral diaphysis, hands, nerves, larynx) opened the field of composite tissue allotransplantation (C.T.A.) in reconstructive surgery. Immunosuppressive agents are now efficient enough to prevent the rejection of the allografts. The first patients operated on did not suffer from major or irreversible complications, but some potential side-effects of the immunosuppression remain an obstacle to the development of allografts in reconstructive surgery. The early functional results of the allografts are encouraging but their long term evolution remains uncertain. Some immunological (the chronic rejection) or psychological (the refusal of the graft and of its constraints) phenomenon might curtail the functional, aesthetic and psychological result. Currently considered as an extreme solution to exceptional indications, C.T.A. represents a tremendous hope for the future of reconstructive surgery.     

Perioperative anesthetic management of the kidney-pancreas transplant recipient

Patients undergoing simultaneous pancreas-kidney transplantation are at risk for a variety of serious perioperative complications. These are related to the chronic and acute problems associated with end-stage renal disease and insulin-dependent diabetes mellitus and the prolonged, vascular and ductal surgery required to implant the two allografts. A number of strategies need to be integrated and diligently implemented to minimize the physiologic perturbations and complications related to the recipient's comorbid conditions and revascularization of the allografts. A major objective of the perioperative anesthetic management of simultaneous pancreas-kidney transplantation is to maximize cardiovascular performance in a way that provides optimum graft perfusion and recovery, while avoiding myocardial ischemia. Adherence to this objective, along with very effective immunosuppressants, surgical refinements, meticulous anesthetic preparation, extensive and frequent physiologic and metabolic monitoring, and quick response to abnormal findings has resulted in remarkably low recipient morbidity and mortality, and very high graft survival rates.     

The influence of MHC-compatible and MHC-incompatible antigen-presenting cells on the survival of MHC-compatible cultured murine keratinocyte allografts

Our group has shown previously that APC-depleted cultured epidermal keratinocytes show prolonged survival when grafted onto normal MHC-incompatible adult mice. We show here that in vitro culture also improves significantly the survival of MHC-compatible keratinocyte allografts, although these nonrejected grafts are repopulated by host cells identified by their dendritic morphology and phenotype (class II+, leukocyte-common antigen+) as APCs. Reconstitution of cultured grafts, immediately prior to transplantation, with MHC-compatible dendritic cells of either donor or recipient origin, results in graft rejection--thus demonstrating that cultured cells can be rejected by the recipient animal--and suggests that a paucity of APCs in the immediate postgrafting period is responsible for the privilege afforded these grafts.     

Repopulation of donor heart by recipient bone marrow-derived dendritic cells prior to transplantation causes acute rejection by both the allogeneic and syngeneic recipient

Experimental studies on allogeneic transplantation have shown that recipient dendritic cells (DC) play a role in peripheral tolerance as well as in rejection of allografts. It is not known whether DC exert their tolerogenic function in recipient lymphoid tissue, and whether they process shed alloantigen in the graft itself. To answer this question we created a chimeric heart model deprived of its own DC and repopulated by recipient DC. The rationale for this model was to observe whether recipient DC located in the graft attenuate recruitment and stimulation of recipient lymphocytes, subsequently prolonging graft survival. Vascularized bone marrow transplants (VBMTx) from the prospective recipient to the lethally irradiated heart donor, which function for a period of 14 days, were used to replace donor DC with prospective recipient DC. Hearts from chimeric LEW rats (with BN DC) were transplanted to untreated BN rats. Also, hearts from chimeric LEW rats (with BN DC) were returned to untreated LEW rats. Replacement of the donor heart with recipient DC did not prolong graft survival. Rather, it initiated a rejection reaction that was already present in the donor. Recipient DC retained their immunogenic properties also when the graft was returned back to a donor strain animal.