Heterotopic Tracheal Transplantation Research Articles

Attenuation of early airway obstruction by mesenchymal stem cells in a murine model of heterotopic tracheal transplantation

Long-term success in lung transplantation is limited by obliterative bronchiolitis (OB). Presently, complete understanding of the mechanisms of OB has been elusive. Bone marrow-derived mesenchymal stem cells (MSC) have been shown to modulate repair of the injured lung in multiple disease models. We hypothesized that the injection of MSC would prevent development of early airway obstruction (AO) in the heterotopic tracheal transplant model. Forty-four tracheas from BALB/c and C57BL/6 donors were transplanted into 22 C57BL/6 recipients. At the time of transplant, 13 of the allogeneic recipient mice were injected with 5 × 10(5) MSC from various murine sources. To confirm the role of the immune response in the generation of AO we used a permeable inhibitor of nuclear factor-kappaB (NF-κB) in 11 recipients after transplantation with 22 BALB/c tracheas. After transplantation, administration of MSC inhibited intraluminal obstruction by collagen in 98% of the mice and transforming factor-beta (TGF-β) expression decreased to levels similar to those observed in isograft controls. These effects were associated with a significant (p < 0.05) increase in expression of the anti-inflammatory cytokine interleukin-10 (IL-10). NF-κB inhibitor showed decreased expression of transforming growth factor-beta (TGF-β) in the Day 7 and Day 14 groups, resulting in a 60% reduction of luminal obstruction as well as a decrease in inflammatory cells to the airway. Our observations suggest that administration of MSC prevents development of airway occlusion in a mouse model, probably through the modulated immune response altering TGF-β expression.

Low-molecular-weight heparins do not modify obliterative airway disease in rat tracheal allografts

ABSTRACTImmunosuppressive and antiproliferative effects of heparin may be beneficial in the field of solid organ transplantation. The aim of this study was to examine the effect of low-molecular-weight heparin (LMWH) compounds on the development of obliterative airway disease (OAD) in the rat tracheal transplant model. Allogenic heterotopic tracheal transplantations were performed from Brown-Norway into Lewis rats. Recipients were treated either with nadroparin, enoxaparin, parnaparin, or vehicle from day 0 until harvesting at day 7 or 21. Graft rejection was morphometrically assessed to determine the extent of luminal obliteration end epithelial necrosis. All tracheal grafts harvested at day 7 demonstrated nearly equivalent degree of luminal obstruction regardless of treatment regimen. Likewise, at day 21 the extent of airway narrowing and the degree of inflammatory cell infiltration were similar among the groups. Moreover, loss of airway epithelium was not prevented by LMWH treatments. Finally, intragraft mRNA expression for transforming growth factor-β1 and platelet-derived growth factor-A, interleukin-2, interferon-γ, and monocyte chemoattractant protein-1 did not differ between the groups. In contrast with findings in other animal models, treatment with LMWH preparations did not modify the development of OAD in rat tracheal allografts.

580: Poly I:C Abrogates Murine Airway Allograft Tolerance Induced by CD154 Costimulation Blockade and Enhances Allospecific CD8+ T Cell Effector Responses

RNA viruses are a known risk factor for obliterative bronchiolitis (OB), the major cause of mortality in lung transplantation. Using a murine heterotopic tracheal transplant model (HTT) of obliterative airway disease (OAD), we studied the role of polyinosinic acid: polycytosinic acid (polyI:C), a synthetic RNA polymer in the induction of OAD and abrogation of allograft tolerance induced by CD154 costimulation blockade

391: T-Bet Deficiency Alters Murine Obliterative Airway Disease Immunopathology and Is Associated with Both Type 1 and Type 17 Effector T Cell Responses

T-bet is a transcription factor known to direct Type 1 lineage commitment in CD4+ and CD8+ T cells, though its role in allograft rejection remains incompletely understood. Using the murine heterotopic tracheal transplant model (HTT) for obliterative airway disease (OAD), we studied the effect of T-bet deficiency on OAD pathology and the character of T cell effector responses to airway allografts.

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7). To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations. For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model. In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7). This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

CD154 Deficiency Uncouples Allograft CD8+ T-Cell Effector Function from Proliferation and Inhibits Murine Airway Obliteration

Obliterative bronchiolitis (OB) limits the long-term success of lung transplantation, while T-cell effector mechanisms in this process remain incompletely understood. Using the murine heterotopic tracheal transplant model of obliterative airway disease (OAD) to characterize airway allograft rejection, we previously reported an important role for CD8(+) T cells in OAD. Herein, we studied the role of CD154/CD40 costimulation in the regulation of allospecific CD8(+) T cells, as airway rejection has been reported to be CD154-dependent. Airway allografts from CD154(-/-) recipients had significantly lower day 28 OAD scores compared to wild-type (WT) recipients, and adoptive transfer of CD8(+) T cells from WT recipients, but not CD154(-/-) recipients, were capable of airway rejection in fresh CD154(-/-) allograft recipients. Intragraft CD8(+) T cells from CD154(-/-) mice showed similar expression of the surface markers CD69, CD62L(low) CD44(high) and PD-1, but markedly impaired IFN-gamma and TNF-alpha secretion and granzyme B expression versus WT controls. Unexpectedly, intragraft and systemic CD8(+) T cells from CD154(-/-) recipients demonstrated robust in vivo expansion similar to WT recipients, consistent with an uncoupling of proliferation from effector function. Together, these data suggest that a lack of CD154/CD40 costimulation results in ineffective allospecific priming of CD8(+) T cells required for murine OAD.

Suppression of the Obliteration Process by Ventilation in a Mouse Orthotopic Tracheal Transplantation Model

Obliterative airway disease (OAD) has been a major obstacle to long-term survival after lung or tracheal transplantations, but the role of airflow has not been examined in the orthotopic or the heterotopic tracheal transplantation models. Sixty mice were assigned to two experimental groups. Two C57BL/6 tracheal segments were surgically prepared and then orthotopically transplanted into allogeneic BALB/c recipients. In group A mice, both segments were left patent, whereas in group B mice, one of the donor tracheas was occluded with a silk knot to obstruct airflow. Histology, quantitative OAD measurements, electron microscopy, immunohistochemical staining, and apoptosis measurement of the epithelium were performed. Gross examination at harvest showed patent lumens of all tracheal segments. Group A allografts (ventilating tracheas) showed a markedly higher proportion of ciliated epitheliums and less lymphocyte infiltration in the lamina propria, whereas the epithelium appeared metaplastic in group B, with a higher proportion of flattened attenuated epithelium and loss of the normal ciliate architecture. Quantitative morphometric measurements suggested more prominent OAD manifestations in the nonventilating allografts of group B than were present in group A, although recipient-derived epithelium was observed in all allografts under immunohistochemical staining. The apoptotic indexes of the epithelium were 12.1% in allografts with adequate ventilation (group A) and 66.2% in ventilation-occluded allotracheas (group B). OAD severity and the epithelial repopulation process are closely related to the physiologic environment of airflow. Further research is warranted to explore the underlying mechanisms of this phenomenon.

Gene expression profile in chronic rejected transplant lungs detected by microarray

Objective To investigate the pathogenesis of obliterative brenchiolitis (OB) follow-ing lung transplantation.Methods Heterotopic tracheal transplantation model was created.Tracheal grafts were harvested on the day 5,15, and 15, and pathological changes were observed in one half over time, and the other half were subjected to mierearray analyses to examine changes in gene expression patterns on the day 15.Results Compared with the isngraft control, expression levels of 422 genes were up-regulated in the allograft.A large proportion of the up-regulated genes in this transplant-associated profile were func-tionally linked with cell survival and differentiation.Conclusion OB presented as a multistage, ongoing and nonequilibrium process.The analyses with much more genes associated with cell differentiation and less with immunological cells suggest the development of new therapeutic strategies. Key words: Lung transplantation; Chronic rejection; Obliterative bronchiolitis; Gene expression

Alcohol Primes the Airway for Increased Interleukin‐13 Signaling

Using an experimental model of airway fibrosis following lung transplantation, we recently showed that chronic alcohol ingestion by donor rats amplifies airway fibrosis in the recipient. Associated with alcohol-mediated amplification of airway fibrosis is increased transforming growth factor beta-1(TGFbeta(1)) and alpha-smooth muscle actin expression. Other studies have shown that interleukin-13 (IL-13) modulates TGFbeta(1) signaling during experimentally-induced airway fibrosis. Therefore, we hypothesized that IL-13 is a component of alcohol-mediated amplification of pro-fibrotic mediators in the alcoholic lung. To test this hypothesis, we analyzed tracheal epithelial cells and type II alveolar cells from control- or alcohol-fed rats, alcohol-treated mouse lung fibroblasts, and human bronchial epithelial cells in vitro for expression of various components of the IL-13 signaling pathway. Signaling via the IL-13 pathway was assessed by measuring levels of phosphorylated signal transducers and activators of transcription-6 (STAT6). In addition, we performed heterotopic tracheal transplantation using control-fed and alcohol-fed donor rats and analyzed tracheal allografts for expression of components of the IL-13 signaling pathway by RT-PCR and immunocytochemical analyses. Interleukin-13 expression was detected in type II alveolar epithelial cells and human bronchial epithelial cells, but not in lung fibroblasts. IL-13 expression was decreased in whole lung and type II cells in response to alcohol exposure. In all cell types analyzed, expression of IL-13 signaling receptor (IL-13R alpha(1)) mRNA was markedly increased. In contrast, mRNA and protein expression of the IL-13 decoy receptor (IL-13R alpha(2)) were decreased in all cells analyzed. Exposure to alcohol also increased STAT6 phosphorylation in response to IL-13 and lipopolysaccharide. Data from multiple cell types in the pulmonary system suggest that IL-13 and its receptors play a role in alcohol-mediated activation of pro-fibrotic pathways. Taken together, these data suggest that alcohol primes the airway for increased IL-13 signaling and subsequent tissue remodeling upon injury such as transplantation.

Kinetics of systemic host immune response after experimental heterotopic and orthotopic tracheal transplantations

Kinetics of systemic host immune response after experimental heterotopic and orthotopic tracheal transplantations

Attenuation of Obliterative Bronchiolitis by a CXCR4 Antagonist in the Murine Heterotopic Tracheal Transplant Model

Long-term success in lung transplantation is limited by obliterative bronchiolitis (OB), yet the mechanism for this disease is not well understood. Chemokine SDF-1 and its receptor, CXCR4, have been reported to be involved in several fibrogenic processes by recruiting inflammatory and fibroblast progenitor cells into injured tissues. We hypothesized that the SDF-1/CXCR4 axis also plays a role in the pathogenesis of OB. Using the mouse heterotopic allogeneic airway transplant model, we transplanted mouse tracheas from BALB/c donors into C57BL/6 recipients. At Day 10 after transplant, we found high expression of SDF-1 in cells in the sub-epithelial layers of the allograft. Approximately 26% of cells infiltrating the allograft were CD45(+)CXCR4(+), as determined by flow cytometry analysis. Treatment of the recipients with a CXCR4 antagonist, TN14003, decreased cell infiltration into the grafts at Day 10 post-implantation. At Day 42, a significant reduction in luminal occlusion was found in the TN14003-treated animals compared with controls (57.40% vs 98.21%, p < 0.01). To demonstrate the relevance of the SDF-1/CXCR4 axis in OB, sections of lung tissue obtained from lung transplant patients with OB were examined for SDF-1 and CXCR4 expression. We found a higher number of CXCR4- and SDF-1-positive cells in samples from patients with OB as compared with normal lungs. These findings provide new insights into the mechanisms of lung chronic rejection and may lead to new intervention tools for the treatment of OB.

Attenuation of Airway Obliteration by Ciprofloxacin in Experimental Posttransplant Bronchiolitis Obliterans

Ciprofloxacin is widely used to treat respiratory tract infections. Like other fluoroquinolones, ciprofloxacin has immunomodulatory effects; however, it is unknown whether these effects are beneficial in the setting of lung transplantation. We investigated potential immunomodulatory effects of ciprofloxacin in a model of posttransplant bronchiolitis obliterans. The heterotopic tracheal transplantation model in rats was used. Three groups received ciprofloxacin and underwent different immunosuppressive regimens of cyclosporine A, that is, no immunosuppression, insufficient immunosuppression, or low-dose immunosuppression. Three groups underwent the same immunosuppressive regimen but had no ciprofloxacin treatment. Tracheas were harvested after 21 days and examined with respect to histology and expression of selected cytokines. The allografts of animals treated with ciprofloxacin showed less airway obliteration compared with allografts of untreated animals. When combined with low-dose immunosuppression ciprofloxacin showed beneficial effects in preventing airway obliteration and rejection of the respiratory epithelium. Cytokine gene expression of the allografts treated with ciprofloxacin was higher with respect to transforming growth factor-beta and equal with respect to tumor necrosis factor-alpha and interferon-gamma compared with controls. When applied in combination with cyclosporine A, ciprofloxacin lowered the expression of transforming growth factor-beta and tumor necrosis factor-alpha and increased interferon-gamma expression. Ciprofloxacin attenuates airway rejection after tracheal transplantation. Genetic expression of mediators that are known to play an important role in mediating rejection in this model supports an immunomodulatory and antifibrotic role of ciprofloxacin. These findings suggest that further clinical studies are needed to investigate whether ciprofloxacin in addition to its bactericidal effect might be beneficial in the treatment of human posttransplant bronchiolitis obliterans.

424: BOS-Development: Comparing the Kinetics of Systemic Host Immune Response after Experimental Heterotopic and Orthotopic Tracheal Transplantations

Different animal models have been developed to study the pathogenesis and treatment of obliterative airway disease (= chronic allograft rejection after airway transplantation; OAD). Here we describe the techniques of heterotopic and orthotopic tracheal transplantations in the rat, comparing the kinetics of systemic host immune response and of histopathologic OAD development.

Pirfenidone: A Novel Potential Therapeutic Agent in the Management of Chronic Allograft Rejection

Chronic allograft dysfunction is a leading cause of allograft failure, morbidity, and mortality after solid organ transplantation. The pathogenesis of chronic allograft failure has a final common pathway leading to organ fibrosis. Pirfenidone is an effective and novel antifibrotic agent with anti-inflammatory properties. Clinical use of the agent has been tested in a number of nontransplant recipients and has a favorable safety profile based on available clinical data. Building on these observations and findings, and considering the role of fibrosis in chronic allograft rejection, pirfenidone was initially investigated as adjunct therapy in a rat heterotopic tracheal transplantation model. This led to several studies confirming that pirfenidone may well be worth considering for further investigation. This paper reviews the possibility of using pirfenidone in clinical transplantation management.

Maintenance of Airway Epithelium in Acutely Rejected Orthotopic Vascularized Mouse Lung Transplants

Lung transplantation remains the only therapeutic option for many patients suffering from end-stage pulmonary disease. Long-term success after lung transplantation is severely limited by the development of bronchiolitis obliterans. The murine heterotopic tracheal transplantation model has been widely used for studies investigating pathogenesis of obliterative airway disease and immunosuppressive strategies to prevent its development. Despite its utility, this model employs proximal airway that lacks airflow and is not vascularized. We have developed a novel model of orthotopic vascularized lung transplantation in the mouse, which leads to severe vascular rejection in allogeneic strain combinations. Here we characterize differences in the fate of airway epithelial cells in nonimmunosuppressed heterotopic tracheal and vascularized lung allograft models over 28 days. Up-regulation of growth factors that are thought to be critical for the development of airway fibrosis and interstitial collagen deposition were similar in both models. However, while loss of airway epithelial cells occurred in the tracheal model, airway epithelium remained intact and fully differentiated in lung allografts, despite profound vascular rejection. Moreover, we demonstrate expression of the anti-apoptotic protein Bcl-2 in airway epithelial cells of acutely rejected lung allografts. These findings suggest that in addition to alloimmune responses, other stimuli may be required for the destruction of airway epithelial cells. Thus, the model of vascularized mouse lung transplantation may provide a new and more physiologic experimental tool to study the interaction between immune and nonimmune mechanisms affecting airway pathology in lung allografts.

The mechanism of transforming growth factor beta1 in myofibroblast differentiation

To investigate the mechanism underlying myofibroblast differentiation induced by transforming growth factor (TGF) beta1 in obliterative bronchiolitis following lung transplantation. Heterotopic tracheal transplantation was performed in Smad3 wild-type and knock-out mice to simulate the lung transplantation in human. Murine tracheal fibroblasts cultivated in primary culture were used for in vitro study. Immunohistochemistry, immunocytochemistry, Western Blotting, RT-PCR and DNA electrophoresis mobility gel shift assay were conducted to detect the expression of alpha-smooth muscle actin (alphaSMA), the marker of fibroblast-myofibroblast differentiation, and the activation of Smad3, p38 and ERK1/2. In affected airways of experimental obliterative bronchiolitis, abundant expression of alphaSMA were found. In vitro study for tracheal fibroblasts, the activation of Smad3 by TGF-beta1 presents as three major forms, phosphorylation, nuclear translocation and DNA binding. In Smad3 wild-type fibroblasts, TGF-beta1 induces the increase of the myofibroblasts transformation, characterized by the elevation of alphaSMA, both at transcription and protein level. While in Smad3 knock-out fibroblasts, the transformation of myofibroblasts induced by TGF-beta1 is significantly decreased (t = 2.080, P = 0.027; t = 1.982, P = 0.032), but not completely abolished. Further study in Smad3-deficient fibroblasts demonstrates that p38 and ERK1/2 could be activated by TGF-beta1 and result in fibroblast differentiation. TGF-beta1 could promote the transformation of fibroblasts into myofibroblasts in Smad3 dependent and independent signal pathways, especially the Smad3 dependent path, and result in the development of obliterative bronchiolitis.

Keratinocyte Growth Factor Improves Repair in the Injured Tracheal Epithelium

Keratinocyte growth factor (KGF) is a critical growth factor in lung development and is a protective agent after lung injury, although the exact mechanisms of this protective effect have not yet been elucidated. Our laboratory has shown that circulating epithelial progenitor cells can traffic to the airway and that they appear to be derived from the bone marrow. On this basis, we hypothesized that KGF and its putative receptor (KGFR) would be important to these cells. We showed that the KGFR, which is found almost exclusively on epithelial cells, was present on cells in the bone marrow and circulation of mice that identified a subpopulation of cytokeratin 5+ circulating epithelial progenitor cells (CEPC). In addition, the KGFR co-localized with a population of cytokeratin 5+ basal cells in the repairing proximal airway. Systemic administration of KGF resulted in a significant increase in mobilization of cytokeratin 5+ CEPC at 6 h after injection. Administration of KGF to mouse recipients of heterotopic syngeneic tracheal transplants resulted in protection and more rapid repair of the tracheal epithelium, with an increase in the number of CEPC in the epithelium of the airway, and this effect was abrogated by blocking CEPC with anti-CXCL12 antibodies. KGF therefore appears to be an important growth factor for local resident progenitor epithelial cell repair and for mobilization and enhanced engraftment of CEPC to the injured proximal airway epithelium.

Techniques for experimental heterotopic and orthotopic tracheal transplantations — When to use which model?

Background Different animal models have been developed to study the pathogenesis and treatment of obliterative airway disease (OAD). Here we describe the techniques of heterotopic and orthotopic tracheal transplantations in the rat, comparing the kinetics of systemic host immune response and of histopathologic OAD development. Methods Heterotopic and orthotopic tracheal transplantations were performed in both allogeneic (Brown Norway-to-Lewis) and syngeneic (Lewis-to-Lewis) models. Grafts were harvested after 7, 30, and 60 days post-transplant for histologic evaluation and analysis of host cellular and humoral response. Results Syngeneic tracheal grafts did not develop luminal obliteration and were morphologically indistinguishable from native tracheas. In heterotopic allografts, airway epithelium was rapidly destroyed and OAD progressed with complete luminal occlusion by 30 days. Orthotopic allografts showed enhanced early infiltration (1298 ± 45 vs. 674 ± 75 cells/high power field, p < 0.001) with concomitant greater day 7 luminal narrowing (45 ± 6% vs. 14 ± 3%, p < 0.001). In this model, donor-type BN epithelium (62 ± 17%, 21 ± 19%, and 1 ± 1% on days 7, 30, and 60) was gradually replaced by recipient-type epithelial cells (2 ± 4%, 70 ± 22%, and 98 ± 2%). OAD developed with circular orientation of cells and connective tissue fibers to 45 ± 6% obliteration by day 60. Cellular host response, as determined by IFN-γ-ELISPOT assay (548 ± 132 vs. 402 ± 197 spots, p = 0.046) and anti-donor alloreactive IgM antibody production (2827 ± 148 vs. 1565 ± 393 mean channel fluorescence, p < 0.001) were significantly stronger in rats bearing orthotopic vs. heterotopic allografts. Conclusions The orthotopic tracheal transplantation model may be more representative of OAD found in human lung transplant recipients and we therefore encourage the wider use of this model.

Presensitization accelerates chronic allograft rejection in a heterotopic rat tracheal allograft model with immunosuppression

Background In the field of organ transplantation, the effect of pretransplant humoral allosensitization on the organ transplant outcome has been highlighted. To clarify the correlation of presensitization with chronic rejection that can lead to a poor prognosis in transplant recipients, we examined humoral alloreactivity and allograft rejection in sensitized recipients by using a rat heterotopic tracheal transplant model. Methods An MHC fully incompatible combination strain was used in this study. Lewis (LEW) rats sensitized by transplantation with Brown Norway (BN) skin grafts received tracheal segments from BN rats in the dorsal subcutaneous pouch. Four allogenic groups ( n = 5) were investigated. Group1, non-sensitized recipients without cyclosporine A (CsA) administration; group 2, non-sensitized recipients with CsA administration; group 3, sensitized recipients without CsA administration; and group 4, sensitized recipients with CsA administration. In the immunosuppressant groups (groups 2 and 4), the recipients were administered a subcutaneous injection of CsA (25 mg·kg − 1 ·day − 1 ) for 3 days from the day of operation. All recipients were sacrificed 21 days after transplantation. Tracheal segments were extracted from the recipients and histologically evaluated with regard to the following parameters: (1) airway lining epithelial loss, (2) lymphocyte/plasma cell infiltration, and (3) luminal obliteration due to granulation tissue formation and/or fibrosis. In order to analyze alloantibody (allo-Ab) responses, sera samples were tested by the flow cytometric cross-match (FCXM) technique. Results Histological findings revealed that the chronic rejection score in sensitized recipients treated with CsA was significantly higher than that in non-sensitized recipients treated with CsA (9.0 ± 1.2 vs. 3.0 ± 0.54, p < 0.05). In other words, CsA therapy reduced the rejection score in non-sensitized recipients, but not in sensitized recipients. No significant differences were observed in the level of IgM Abs among the groups. However, donor-specific IgG Abs were induced after presensitization by donor skin grafting prior to tracheal transplantation. Heterotopic tracheal implantation also induced IgG Ab production. This elevation in the Ab levels was inhibited by CsA treatment in non-sensitized recipients. Conversely, the Ab levels were significantly higher in sensitized recipients than in non-sensitized recipients, regardless of CsA administration. Conclusions Our data showed that presensitization accelerates chronic allograft rejection with a marked elevation in the level of donor specific IgG Abs. These results suggest that presensitization will be a significant risk factor for the lung transplant recipient, furthermore, the effect of immunosuppression might be insufficient in sensitized recipients.