Acute Cellular Xenograft Rejection Research Articles

Cardiac xenotransplantation: a promising way to treat advanced heart failure.

Cardiac xenotransplantation (CXTx) might be a promising approach to bridge the gap between the supply and demand of a donor heart. The survival of cardiac xenograft has been significantly extended in pig-to-nonhuman primate (NHP) CXTx, with records of 195days and 945days for orthotropic and heterotopic CXTx, respectively. To present the history of CXTx, we list the reported clinical CXTx, compare pigs and NHPs as sources of hearts, and compare three different kinds of preclinical CXTx models. The application of genetically modified pigs and novel immunosuppressive drugs accelerates the development of CXTx, and we summarize the reported pig-to-NHP CXTx with detailed information. Besides, we discuss the underlining mechanisms and potential preventive strategies of immunological barriers, including hyperacute rejection, acute humoral xenograft rejection, acute cellular xenograft rejection, chronic rejection, coagulation dysfunction, and systemic inflammation. Though intense cellular infiltration in cardiac xenograft has only been documented in a small number of studies, we especially stress the importance of cellular rejection in CXTx, because we believe it is often masked by the rapid and strong humoral response and it may eventually become a more important and common type of xenograft rejection. In addition, we conclude other obstacles as well as possible solutions in CXTx, such as perioperative cardiac xenograft dysfunction, detrimental xenograft overgrowth, and porcine endogenous retroviruses. Finally, we briefly introduce several other approaches that have been proposed to deal with the organ heart shortage crisis, and we firmly believe that CXTx provides the best near-term solution.

Pathologic Characteristics of Transplanted Kidney Xenografts

For xenotransplantation to become a clinical reality, we need to better understand the mechanisms of graft rejection or acceptance. We examined pathologic changes in α1,3-galactosyltransferase gene-knockout pig kidneys transplanted into baboons that were treated with a protocol designed to induce immunotolerance through thymic transplantation (n=4) or were treated with long-term immunosuppressants (n=3). Hyperacute rejection did not occur in α1,3-galactosyltransferase gene-knockout kidney xenografts. By 34 days, acute humoral rejection led to xenograft loss in all three xenografts in the long-term immunosuppression group. The failing grafts exhibited thrombotic microangiopathic glomerulopathy with multiple platelet-fibrin microthrombi, focal interstitial hemorrhage, and acute cellular xenograft rejection. Damaged glomeruli showed IgM, IgG, C4d, and C5b-9 deposition. They also demonstrated endothelial cell death, diffuse endothelial procoagulant activation with high expression of tissue factor and vWF, and low expression of the ectonucleotidase CD39. In contrast, in the immunotolerance group, two of four grafts had normal graft function and no pathologic findings of acute or chronic rejection at 56 and 83 days. One of the remaining kidneys had mild but transient graft dysfunction with reversible, mild microangiopathic glomerulopathy, probably associated with preformed antibodies. The other kidney in the immunotolerance group developed unstable graft function at 81 days and developed chronic xenograft glomerulopathy. In summary, the success of pig-to-primate xenotransplantation may necessitate immune tolerance to inhibit acute humoral and cellular xenograft rejection.

Rejection of Cardiac Xenografts Transplanted from α1,3-Galactosyltransferase Gene-Knockout (GalT-KO) Pigs to Baboons

The use of alpha1,3-galactosyltransferase gene-knockout (GalT-KO) swine donors in discordant xenotransplantation has extended the survival of cardiac xenografts in baboons following transplantation. Eight baboons received heterotopic cardiac xenografts from GalT-KO swine and were treated with a chronic immunosuppressive regimen. The pathologic features of acute humoral xenograft rejection (AHXR), acute cellular xenograft rejection (ACXR) and chronic rejection were assessed in the grafts. No hyperacute rejection developed and one graft survived up to 6 months after transplantation. However, all GalT-KO heart grafts underwent graft failure with AHXR, ACXR and/or chronic rejection. AHXR was characterized by interstitial hemorrhage and multiple thrombi in vessels of various sizes. ACXR was characterized by TUNEL(+) graft cell injury with the infiltration of T cells (including CD3 and TIA-1(+) cytotoxic T cells), CD4(+) cells, CD8(+) cells, macrophages and a small number of B and NK cells. Chronic xenograft vasculopathy, a manifestation of chronic rejection, was characterized by arterial intimal thickening with TUNEL(+) dead cells, antibody and complement deposition, and/or cytotoxic T-cell infiltration. In conclusion, despite the absence of the Gal epitope, acute and chronic antibody and cell-mediated rejection developed in grafts, maintained by chronic immunosupression, presumably due to de novo responses to non-Gal antigens.

Pathology of renal xenograft rejection in pig to non‐human primate transplantation

Xenotransplantation has the potential to alleviate the critical shortage of organs for transplantation in humans. Miniature swine are a promising donor species for xenotransplantation. However, when swine organs are transplanted into primates, hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), acute cellular xenograft rejection (ACXR), and chronic xenograft rejection prevent successful engraftment. Developing a suitable regimen for preventing xenograft rejection requires the ability to accurately diagnosis the severity and type of rejection in the graft. For this purpose, histopathology remains the most definitive and reliable tool. We discuss here the characteristic features of xenograft rejection in a preclinical pig-to-non-human primate transplantation model. In miniature swine to baboon xenotransplantation, marked interstitial hemorrhage develops in HAR, and renal microvascular injury develops with multiple platelet-fibrin microthrombi in both HAR and AHXR. T-cell-mediated cellular immunity plays an important role in ACXR. Chronic humoral and cellular rejection may induce chronic xenograft rejection, and will be a major cause of graft loss in discordant xenotransplantation.

HISTOLOGICAL FEATURES OF XENOTRANSPLANTED URETERS IN HDAF PIG-TO-PRIMATE RENAL XENOTRANSPLANTATION

P1088 Aims: The histological features of xenotransplanted ureters in pig-to-primate renal transplantation have not been described in detail, apart from a few particular cases. The aim of this study was to illustrate the ureteral findings and their clinical implications in 31 cynomolgus monkey recipients of porcine renal xenografts. Methods: Thirty-one consecutive bi-nephrectomized cynomolgus monkeys received a kidney from hDAF transgenic pigs with or without a ureteral stent. The transplanted animals received 5 different immunosuppressive regimens based on induction with cyclophosphamide/methotrexate and maintenance with cyclosporine A, mycophenolate sodium/methotrexate and steroids. Results: Four monkeys were euthanized prematurely after transplantation. In the remaining 27 cases, the mean survival was 24±18 days. Except for one case, where late venous thrombosis was the cause of a diffuse fibrosis with ischemic necrosis, histological examination showed 4 different situations: no rejection of any type (3 cases), acute cellular xenograft rejection (ACXR) alone (1 case), focal acute humoral xenograft rejection (AHXR, 6 cases), diffuse AHXR (the remaining 17 cases). Diffuse AHXR was represented in 2 different patterns. The first (8 cases) consisted in diffuse AHXR associated with wall thickening and a severe reduction of the lumen, particularly in the medial part. There were signs of diffuse mucosal erosion, hemorrhage and blood vessel thrombosis. Immunohistochemical examination revealed the presence of intravascular C5b9, C3, IgG, IgM and fibrin deposits on the wall of many small vessels of all stenotic ureters. Three of these cases developed a ureteral stenosis, while positioning a stent at surgery prevented hydroureteronephrosis in the other 4 cases. The second pattern (9 cases) was characterized by histological and immunohistochemical features of diffuse AHXR associated with homogeneous ureteral wall thickening due to considerable smooth muscle hypertrophy of the external layer of the ureter. In all these 9 cases, a ureteral stent was positioned at the time of transplantation or at resurgery for ureteral stenosis. Apart from one case, there was a close relationship between ureter and kidney in terms of type and severity of rejection. Conclusions: AHXR is a common ureteral complication following xenotransplantation of hDAF pig organs into non-human primates. While renal AHXR leads to a slow and progressive decline in renal function, ureteral AHXR causes the rapid onset of renal failure due to ureteral stenosis and hydroureteronephrosis. In the light of our experience in this delicate and ethically demanding model, we recommend the routine use of suitable surgical tools, e.g. ureteral stent, when renal xenografts are transplanted into small primates.

MyD88-dependent toll-like receptor signalling is not a requirement for fetal islet xenograft rejection in mice.

Rejection of pancreatic islet xenografts in mice shares immunopathological features with a Th1-associated delayed-type hypersensitivity (DTH) reaction. The aim of the present study was to investigate the mechanism of acute cellular xenograft rejection in a strain of mice with a targeted gene disruption of the toll-like receptor (TLR) signal adaptor protein MyD88. These mice have been shown to have markedly impaired Th1 immunity. The MyD88-/- and normal mice were transplanted with 2 microl of fetal porcine islet-like cell clusters (ICC) under the left kidney capsule. On days 3, 6 or 12 after transplantation the mice were killed and the grafts either prepared for immunohistochemistry or real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The number of remaining ICC and infiltrating cells with different phenotypic characteristics was assessed semi-quantitatively. Grafts used for quantitative RT-PCR were analysed for content of murine mRNA of interferon (IFN)-gamma, interleukin (IL)-12p40, IL-4 and IL-10. On day 3, the rejection process was initiated in both MyD88-/- and normal mice as characterized by a moderate infiltration of F4/80+ and MAC-1+ macrophages and occasional CD3+ and CD4+ cells. Expression of IFN-gamma and IL-12p40 was lower but still detectable in the MyD88-/- mice, when compared with control animals. By day 6, rejection was almost completed in all animals with only few ICC remaining. 12 days after transplantation all grafts were completely destroyed and heavily infiltrated by macrophages. Moderate numbers of CD3+ and CD4+ and occasional CD8+ cells were also present. Islet xenograft rejection was found to persist in MyD88-/- mice. Despite a relatively lower expression of the Th1-associated cytokines IFN-gamma and IL12-p40 within the xenograft area, both the time course and morphological pattern of the rejection were essentially similar to that found in normal animals. Hence, MyD88-dependent TLR signalling does not appear to be a crucial component of acute cellular xenograft rejection.

Maintenance triple immunosuppression with cyclosporin A, mycophenolate sodium and steroids allows prolonged survival of primate recipients of hDAF porcine renal xenografts.

To date, the best results in life-supporting pig-to-primate renal xenotransplantation have been obtained in recipients exposed to long-term immunosuppression with cyclophosphamide. As this agent is frequently associated with side-effects, we have explored the potential of a mycophenolate sodium-based maintenance immunosuppression in this model. Human decay-accelerating factor (hDAF) transgenic kidneys were transplanted into splenectomized and bilaterally nephrectomized cynomolgus monkeys immunosuppressed with mycophenolate sodium, cyclosporin A and steroids, and exposed to a brief induction course with cyclophosphamide (up to four doses). After transplantation, the primates were monitored daily for biochemical and haematological evaluations and for the measurements of haemolytic anti-pig antibodies (APA). A detailed histological analysis of each explanted graft was also performed. All the animals showed very poor initial graft function but survived for up to 51 days. In contrast to our previous studies in xenograft recipients on long-term immunosuppression with cyclophosphamide, minimal or no circulating xeno-directed antibodies, as measured by the evaluation of APA titres, were detected in this series although some degree of acute humoral rejection was observed in all the explanted grafts and was the primary cause of graft failure. Furthermore, in addition to areas of humorally mediated graft damage, we have observed for the first time areas with exclusive and prominent infiltration by CD2+ and CD8+ mononuclear cells presenting patterns compatible with tubulitis, glomerulitis and arteritis, which we have called acute cellular xenograft rejection (ACXR). In addition, CD68+ infiltrating macrophages and CD20+ B-cells were also present. This study demonstrates that a triple maintenance immunosuppression with mycophenolate sodium, cyclosporin A and steroids is a viable alternative to a cyclophosphamide-based immunosuppression to obtain prolonged survival of porcine organs transplanted into primates. However, a more stringent control of antibody forming cells remains essential to further extend the survival of xenografts in this model. In addition, the use of the immunosuppressive regimen reported here in the primate is associated with the occurrence of a new category of cell-mediated xenograft injury (ACXR) whose significance has yet to be clarified.

Pathology of xenograft rejection: a commentary

Trends in solid organ xenograft pathology are presented, with the focus on pig-to-nonhuman primate models. A simplified classification of rejection is followed, including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), and acute cellular xenograft rejection (ACXR). The main components in HAR are natural xenoreactive antibodies in combination with complement activation. This is evident from the prevention of HAR in recipients in whom either antibodies or complement activation is depleted or inhibited. However, these strategies generally fail to prevent AHXR, which occurs later. AHXR is a multifactorial process in which natural and elicited antibodies may play roles, possibly in conjunction with complement, coagulation factors, and white blood cells. A main target appears to be the microvasculature which, in kidney grafts, is associated with a glomerular thrombotic microangiopathy. It is not clear to what extent species-specific physiologic disparities in complement and coagulation processes may play a role, separate from antibody-initiated processes. As rejection of solid organ xenografts is currently from AHXR, ACXR has not yet received close attention. In addition to intragraft rejection events, systemic complications following host-graft interactions have emerged, including (often fatal) consumptive coagulopathy and immune complex disease. It is anticipated that rejection processes will change when pigs with new genetic modifications become available. For instance, the precise role of natural antibodies to Galalpha1,3Gal will be able to be distinguished from other factors when pigs that lack the target antigen are available, and their organs can be evaluated in large animal xenotransplantation models.

A distinct Th1 immune response precedes the described Th2 response in islet xenograft rejection.

Previous studies using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) have demonstrated that islet xenograft rejection in mice is dominated by Th2-associated cytokines, i.e., interleukin (IL)-4 and IL-10. However, immunohistochemical stainings show that the morphological pattern in this model is more reminiscent of a delayed-type hypersensitivity (DTH) reaction, which is associated with a Th1 response. This study was designed to resolve the mechanisms of acute cellular xenograft rejection in rats transplanted with fetal porcine islet-like cell clusters (ICCs). Real-time quantitative RT-PCR was used to quantify the mRNA expression of cytokines in the grafts and lymph nodes, and the findings were related to the immunopathology of the rejecting grafts. By day 1, mRNA expression levels of IL-1 beta, IL-2, IL-12p40, interferon-gamma, and tumor necrosis factor-alpha were already induced in the lymph nodes. From days 3 to 12, an increasing amount of activated macrophages was seen in the grafts, whereas T- and NK-cells were fewer and mainly accumulated in the periphery of the grafts. Most of the ICCs were rejected by day 5. Transcripts of Th1-associated cytokines were dominant in both regional lymph nodes and in the grafts, with peak levels on days 3 and 5, respectively. The mRNA expression of IL-4 was increased on day 12, and it correlated with the infiltration of eosinophils and an increased level of xenoreactive IgG. The data presented indicate that an islet xenograft triggers a sequential activation of 1) a Th1-associated response characterized by graft destruction in a DTH-like reaction and then 2) a subsequent Th2-associated response characterized by increased levels of xenoreactive antibodies.

Immunohistochemical study of a new experimental model of acute cellular xenograft rejection

ENOGRAFT rejection must be overcome before clinical application of xenotransplantation. However, the complete knowledge of all the humoral and cellular mechanisms involved is not well defined. We recently described an experimental model of acute cellular rejection, 1 which should contribute to better understanding of those mechanisms. Here we present the immunohistochemical study of this ex vivo perfusion model of pig-to-human kidney xenotransplantation, in which human blood was manipulated to achieve an immunomodulatory effect. MATERIALS AND METHODS Pig kidneys were connected to an ex vivo perfusion circuit and perfused with manipulated human blood as we previously described. 1 The number of experiments was five per group. The experiment took place for 180 minutes or until hyperacute rejection was evident. Specimens were taken from all hemoperfused kidneys after the end of the experiment and frozen. Immunofluorescence assays included CD2, CD20, CD68, C5b9, C3, IgG, IgM, and NK cell deposits.

Fetal porcine islet-like cell clusters transplanted to cynomolgus monkeys: an immunohistochemical study.

The mechanism(s) involved in acute cellular xenograft rejection have hitherto been generated in vitro or in different experimental models, with pig tissue being transplanted to rodents. There is an urgent need to validate these results in a clinically more relevant combination of species. Fetal porcine islet-like cell clusters (ICC) were transplanted under the kidney capsule in cynomolgus monkeys, either untreated or given immunosuppression with cyclosporine (CsA; 10 mg/kg body weight, intramuscularly) and 15-deoxyspergualin (DSG; 5 mg/kg body weight, intramuscularly). ICC xenografts were examined at 1, 3, 6, or 10-12 days after transplantation, using immunohistochemical techniques. Serum levels of xenoreactive antibodies were measured with ELISA. No deposits of IgM, IgG, Clq, or C3 were detected within the ICC xenograft in any of the monkeys. Likewise, no significant increase in the levels of xenoreactive antibodies were found after transplantation. In untreated animals, a few N-Elastase-positive cells (neutrophil granulocytes) were seen in the xenograft at day 1. A few mononuclear cells were present in the adjacent renal parenchyma, but they did not infiltrate the xenograft. At this time (day 1), early signs of necrosis were observed in the central parts of the graft. On day 3, the graft had a large, central necrotic area that contained polymorphonuclear cells; the remaining parts of the xenograft showed severe infiltration with CD8+ T cells. Occasional CD68+ cells (macrophages) were seen on days 1 and 3. On day 6, large numbers of macrophages were found infiltrating the entire graft. A few CD20+ B cells, accumulated as small clusters, were also found. Only a few natural killer cells (CD56+) were detected. The CsA/DSG-treated monkeys showed markedly fewer CD2+/CD8+ T cells on day 6 than the untreated monkeys, and the ICC graft was clearly better preserved. However, the number of CD8+ and CD68+ cells had increased considerably at 12 days after transplantation and diffusely infiltrated the whole ICC xenograft. Porcine ICC transplanted under the kidney capsule in cynomolgus monkeys were rejected by an acute cell-mediated rejection progressing during the first 6 days after transplantation. The process was not dependent on host Ig or C3 binding to the graft. Although the rejection of porcine ICC was significantly delayed in CsA/DSG-treated monkeys, the ICC xenografts were almost completely destroyed 12 days after transplantation.

Acute cellular xenograft rejection

Abstract: The acute cellular rejection process after xenotransplantation of vascularized organs, i.e. heart, kidney and liver, has so far been difficult to study since the implanted organs are lost in a hyperacute rejection before cellular rejection develops. Primarily non‐vascularized xenografts‐e.g. pancreatic islets‐escape the hyperacute rejection, but succumb to cell‐mediated rejection during the first week after transplantation in either mice, rats or cynomolugus monkeys. The present view on the mechanisms orchestrating islet xenograft rejection in these animal models will be summarized and in part generalized to give insight into the process of acute cellular xenograft rejection.