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Study of lung xenografts has proven useful to understand the remaining barriers to successful transplantation of other organ xenografts. In this chapter, the history and current status of lung xenotransplantation will be briefly reviewed, and two different experimental models, the ex vivo porcine-to-human lung perfusion and the in vivo xenogeneic lung transplantation, will be presented. We will focus on the technical details of these lung xenograft models in sufficient detail, list the needed materials, and mention analysis techniques to allow others to adopt them with minimal learning curve.

Purpose of Review: Recent progress in genetic engineering has facilitated development of transgenic donor animals designed to overcome the known barriers to discordant xenotransplantation, and greatly accelerated progress in the field of xenotransplantation. Here we review and summarize recent progress in lung xenotransplantation, and discuss possible additional genetic modifications and other interventions that may further advance the use of pulmonary xenografts towards clinical applications based on known mechanisms of xeno lung injury., Recent Findings: Ex-vivo lung perfusion experiments have shown that the addition of human complement (hCD46, hCD55), coagulation (hEPCR, hVWF, hTBM, hTFPI, hCD39), or anti-inflammatory pathway regulatory genes (HO-1, HLA-E), and the knockout (KO) of major porcine carbohydrates (GalT, Neu5Gc, B4Gal) have each protective effects on lung survival and function. The use of these transgenes in multitransgenic donor organs, targeting several known xenogeneic rejection mechanisms, combined with drug treatments addressing remaining known rejection pathways, have led to prolonged recipient survival of up to 31 days with in some cases preserved live-supporting organ function of the transplanted graft for several days. Pulmonary vascular resistance elevation, which has been found to be associated with high thromboxane levels and has been the major failure reason of xenogeneic lung grafts in the past years, has been successfully attenuated by the addition of a thromboxane synthase inhibitor (1-Benzylimidazole). Currently, the predominant failure mechanism of xenogeneic lung grafts is an inflammatory process, leading to vascular barrier function injury with interstitial and trachea edema. Work with other pig organs in primate models show that regimens based on costimulatory pathway blocking antibodies prolong xenograft function for months to years, suggesting that once initial lung inflammation mechanisms are fully controlled, clinically useful application of pig lung xenografts may be feasible., Summary: The use of multitransgenic donor pigs coupled with drugs targeting complement activation, coagulation, and inflammation have significantly improved the survival of xenogeneic pig lungs both during ex vivo human blood perfusion and in life-supporting in vivo models, and for the first time allowed consistent life-supporting function of lung xenografts. Overcoming delayed loss of vascular barrier function injury appears to be within reach, and will be essential to make lung xenografts a clinically relevant treatment option.

In addition to immune barriers, molecular incompatibilities between species are predicted to limit pig liver survival in primate xenotransplantation models. Assessment and measurement of synthetic function of genetically modified porcine livers after ex vivo perfusion with human blood have not previously been described. Eight porcine livers from α1,3-galactosyl transferase knockout and human membrane cofactor (GalTKO.hCD46), six livers from GalTKO.hCD46 and N-glycolylneuraminic acid knockout (GalTKO.hCD46.Neu5GcKO), and six livers from GalTKO.hCD46 with humanized decay-accelerating factor (hCD55), endothelial protein C receptor (hEPCR), tissue factor pathway inhibitor (hTFPI), and integrin-associated protein (hCD47) (GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47) pigs were perfused with human blood under physiologic conditions. Timed blood samples were tested for liver enzymes and for pig-specific albumin production via Western blot. Porcine albumin levels increased with time in all experiments. By densitometry, GalTKO.hCD46.Neu5GcKO livers had the highest albumin levels, measured both as total produced, and when controlled for perfusion duration, compared to GalTKO.hCD46 (P = .068) and GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47 livers (P = .04). Porcine livers perfused with human blood demonstrated the synthetic ability to produce albumin in all cases. GalTKO.hCD46.Neu5GcKO pig livers demonstrated the most robust albumin production. This suggests that the Neu5GcKO phenotype provides a protective effect on the graft due to decreased human antibody recognition and graft injury., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Human organ transplantation has improved duration and quality of life for many people, but its full potential is critically limited by short supply of available organs. One solution is xenotransplantation, although this comes with its own set of challenges. Lungs in particular are highly sensitive to injury, during the transplantation process generally, and to multiple immune rejection mechanisms. Using pig lung donors, our lab has been working on lung transplants into baboons as a surrogate for a human recipient. Several ex vivo human blood perfusion models have also proven useful. The combination of these experiments allows us to test large animal models as well as whole organ or isolated endothelial reactions to perfusion with human blood. We have found that a multi-modality therapeutic approach to prevent various pathogenic cascades - such as antibody-driven complement activation, other immune pathway activation, thrombosis, and tissue ischemia-reperfusion injury - has met with progressively greater success to protect the xeno lung from injury. Pig gene knockout and human gene transfer has been perhaps the greatest contributor. This review will discuss mechanisms of xeno lung injury, relevant experimental models, as well as recent results and future targets for research., (Copyright © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.)

Background: Genetically modified pigs are a promising potential source of lung xenografts. Ex vivo xenoperfusion is an effective platform for testing the effect of new modifications, but typical experiments are limited by testing of a single genetic intervention and small sample sizes. The purpose of this study was to analyze the individual and aggregate effects of donor genetic modifications on porcine lung xenograft survival and injury in an extensive pig lung xenoperfusion series., Methods: Data from 157 porcine lung xenoperfusion experiments using otherwise unmodified heparinized human blood were aggregated as either continuous or dichotomous variables. Lungs were wild type in 17 perfusions (11% of the study group), while 31 lungs (20% of the study group) had one genetic modification, 40 lungs (39%) had 2, and 47 lungs (30%) had 3 or more modifications. The primary endpoint was functional lung survival to 4 h of perfusion. Secondary analyses evaluated previously identified markers associated with known lung xenograft injury mechanisms. In addition to comparison among all xenografts grouped by survival status, a subgroup analysis was performed of lungs incorporating the GalTKO.hCD46 genotype., Results: Each increase in the number of genetic modifications was associated with additional prolongation of lung xenograft survival. Lungs that exhibited survival to 4 h generally had reduced platelet activation and thrombin generation. GalTKO and the expression of hCD46, HO-1, hCD55, or hEPCR were associated with improved survival. hTBM, HLA-E, and hCD39 were associated with no significant effect on the primary outcome., Conclusion: This meta-analysis of an extensive lung xenotransplantation series demonstrates that increasing the number of genetic modifications targeting known xenogeneic lung injury mechanisms is associated with incremental improvements in lung survival. While more detailed mechanistic studies are needed to explore the relationship between gene expression and pathway-specific injury and explore why some genes apparently exhibit neutral (hTBM, HLA-E) or inconclusive (CD39) effects, GalTKO, hCD46, HO-1, hCD55, and hEPCR modifications were associated with significant lung xenograft protection. This analysis supports the hypothesis that multiple genetic modifications targeting different known mechanisms of xenograft injury will be required to optimize lung xenograft survival., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Xenotransplantation has undergone important progress in controlling initial hyperacute rejection in many preclinical models, with some cell, tissue, and organ xenografts advancing toward clinical trials. However, acute injury, driven primarily by innate immune and inflammatory responses, continues to limit results in lung xenograft models. The purpose of this article is to review the current status of lung xenotransplantation--including the seemingly unique challenges posed by this organ-and summarize proven and emerging means of overcoming acute lung xenograft injury., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose-α(1,3)-galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n = 16), and WT pig lungs (n = 16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 min compared to 120 for GalTKO (p = 0.27) and 10 for WT lungs (p < 0.001). Complement activation, platelet activation and histamine elaboration were significantly reduced during the first 2 h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120' 812 ± 230 vs. 1412 ± 1047, p = 0.02; ΔCD62P at 120' 9.8 ± 7.2 vs. 25.4 ± 18.2, p < 0.01; Δhistamine at 60' 97 ± 62 vs. 189 ± 194, p = 0.03). We conclude that, in addition to significant down-modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Barriers to successful lung xenotransplantation appear to be even greater than for other organs. This difficulty may be related to several macro anatomic factors, such as the uniquely fragile lung parenchyma and associated blood supply that results in heightened vulnerability of graft function to segmental or lobar airway flooding caused by loss of vascular integrity (also applicable to allotransplants). There are also micro-anatomic considerations, such as the presence of large numbers of resident inflammatory cells, such as pulmonary intravascular macrophages and natural killer (NK) T cells, and the high levels of von Willebrand factor (vWF) associated with the microvasculature. We have considered what developments would be necessary to allow successful clinical lung xenotransplantation. We suggest this will only be achieved by multiple genetic modifications of the organ-source pig, in particular to render the vasculature resistant to thrombosis. The major problems that require to be overcome are multiple and include (i) the innate immune response (antibody, complement, donor pulmonary and recipient macrophages, monocytes, neutrophils, and NK cells), (ii) the adaptive immune response (T and B cells), (iii) coagulation dysregulation, and (iv) an inflammatory response (e.g., TNF-α, IL-6, HMGB1, C-reactive protein). We propose that the genetic manipulation required to provide normal thromboregulation alone may include the introduction of genes for human thrombomodulin/endothelial protein C-receptor, and/or tissue factor pathway inhibitor, and/or CD39/CD73; the problem of pig vWF may also need to be addressed. It would appear that exploration of every available therapeutic path will be required if lung xenotransplantation is to be successful. To initiate a clinical trial of lung xenotransplantation, even as a bridge to allotransplantation (with a realistic possibility of survival long enough for a human lung allograft to be obtained), significant advances and much experimental work will be required. Nevertheless, with the steadily increasing developments in techniques of genetic engineering of pigs, we are optimistic that the goal of successful clinical lung xenotransplantation can be achieved within the foreseeable future. The optimistic view would be that if experimental pig lung xenotransplantation could be successfully managed, it is likely that clinical application of this and all other forms of xenotransplantation would become more feasible., (© 2012 John Wiley & Sons A/S.)

Study of lung xenografts has proven useful to understand the remaining barriers to successful transplantation of other organ xenografts. In this chapter, the history and current status of lung xenotransplantation are briefly reviewed and two different experimental models, the ex vivo porcine-to-human lung perfusion and the in vivo xenogeneic lung transplantation, are presented. We focus on the technical details of these lung xenograft models in sufficient detail, list the needed materials, and mention analysis techniques to allow others to adopt them with minimal learning curve.

Background: Galactosyl transferase gene knock-out (GalTKO) swine offer a unique tool to evaluate the role of the Gal antigen in xenogenic lung hyperacute rejection., Methods: We perfused GalTKO miniature swine lungs with human blood. Results were compared with those from previous studies using wild-type and human decay-accelerating factor-transgenic (hDAF(+/+) ) pig lungs., Results: GalTKO lungs survived 132 ± 52 min compared to 10 ± 9 min for wild-type lungs (P = 0.001) and 45 ± 60 min for hDAF(+/+) lungs (P = 0.18). GalTKO lungs displayed stable physiologic flow and pulmonary vascular resistance (PVR) until shortly before graft demise, similar to autologous perfusion, and unlike wild-type or hDAF(+/+) lungs. Early (15 and 60 min) complement (C3a) and platelet activation and intrapulmonary platelet deposition were significantly diminished in GalTKO lungs relative to wild-type or hDAF(+/+) lungs. However, GalTKO lungs adsorbed cytotoxic anti-non-Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points., Conclusions: In summary, GalTKO lungs are substantially protected from injury but, in addition to anti-non-Gal antibody and complement, platelet adhesion and non-physiologic intravascular coagulation contribute to Gal-independent lung injury mechanisms., (© 2011 John Wiley & Sons A/S.)

Objective: During ex vivo perfusion with human blood, homozygous galactosyl transferase knockout swine lungs exhibit prolonged survival (approximately 2 hours) relative to wild-type (<15 minutes) and swine lungs expressing human decay accelerating factor (<1 hour). In this study, the in vivo behavior of galactosyl transferase knockout lungs was evaluated., Methods: Three galactosyl transferase knockout swine left lungs were transplanted into baboons in a life-supporting model. One baboon lung allograft and two swine lung xenografts transgenic for human membrane cofactor protein (CD46) served as controls., Results: Whereas two membrane cofactor protein lungs exhibited high pulmonary vascular resistance (>500 mm Hg x min/L) and failed to support life within 21 minutes, two of three galactosyl transferase knockout lungs supported life, for 90 and 215 minutes, and displayed low peripheral vascular resistance (48 +/- 12 mm Hg x min/L at 60 minutes), similar to the allogeneic control. Complement activation (delta C3a < 250 ng/mL through 60 minutes) and C5b-9 deposition were minimal in both galactosyl transferase knockout and membrane cofactor protein lungs. Neutrophils, monocytes, and platelets were rapidly sequestered in galactosyl transferase knockout and human membrane cofactor protein lung recipients, unlike the allogeneic control (<20%); and thrombin formation (delta plasma fraction 1+2 > 0.5 nmol/L) was seen in the galactosyl transferase knockout recipients. Platelet activation (beta-thromboglobulin rise > 200) and appearance of capillary congestion and vessel thrombosis confirmed coagulation activation associated with galactosyl transferase knockout lung failure., Conclusions: Galactosyl transferase knockout swine lungs are significantly protected in vivo from the physiologic consequences (increased pulmonary vascular resistance, capillary leak) associated with hyperacute lung rejection. As during ex vivo perfusion, dysregulated coagulation-thrombin elaboration, platelet activation, and intravascular thrombosis-mediates galactosyl transferase knockout lung xenograft injury.

The use of animal organs for transplantation in humans is seen as a potential solution to the short supply of human donor organs available for clinical transplantation. However, to develop this therapeutic option as clinical reality will require surmounting formidable obstacles. The primary immunologic barrier to pig-to-human xenotransplantation is hyperacute rejection (HAR), a phenomenon previously characterized as resulting from antibody binding and complement activation. This article will first review recent progress in the development of specific strategies to overcome hyperacute lung rejection (HALR), through production of genetically engineered pig organs, modification of the host innate immunity and control of antibody and complement. Additional therapeutic targets identified in HALR are reviewed, with particular emphasis on recent studies describing a critical role for the coagulation cascade in HAR.

Background: We assessed whether the combination of complement regulation and depletion of xenoreactive antibodies improves the outcome of pulmonary xenografts compared with either strategy alone., Methods: Lungs from pigs heterozygous (hDAF(+/-)) or homozygous (hDAF(+/+)) for the human decay accelerating factor transgene (hDAF) or their nontransgenic litter mates (hDAF(-/-)) were perfused with heparinized whole human blood. In additional groups, xenoreactive natural antibodies (XNA) were depleted by pig lung perfusion (hDAF(-/-)/AbAbs, hDAF(+/-)/AbAbs) before the experiment. This combined approach was augmented by adding soluble complement receptor 1 (sCR1) to the perfusate in one further group (hDAF(+/-)/AbAbs/sCR1)., Results: HDAF(-/-) lungs perfused with unmodified human blood were rejected after 32.5 min (interquartile range, IQR 5 to 210). HDAF(+/-) lungs survived for 90 min (IQR 10 to 161, P = 0.54). Both groups showed a rapid rise in pulmonary vascular resistance (PVR), which is a characteristic feature of hyperacute rejection (HAR). This phenomenon was blunted in the hDAF(+/+) group, although survival (48 min, IQR 14 to 111) was not further prolonged. Antibody depletion (AbAbs) led to a significant increase in survival time (hDAF(-/-)/AbAbs: 315 min, IQR 230 to 427; hDAF(+/-)/AbAbs: 375 min, IQR 154 to 575), reduced PVR and less complement production. Addition of sCR1 reduced complement elaboration but did not further improve survival (200 min, IQR 128 to 580) and surprisingly tended to increase PVR., Conclusions: Depletion of xenoreactive antibodies is more effective than membrane-bound complement regulation to blunt hyperacute rejection of pulmonary xenografts, but even the combined approach including soluble-phase complement inhibition is not sufficient to reliably prevent organ failure within hours. It therefore seems likely that other factors independent of antibody and complement contribute to HAR in this model.

Background: Hyperacute rejection (HAR) is one of the principal obstacles to successful xenotransplantation. Homozygous alpha-1,3-galactosyltransferase knockout (GalT-KO) miniature swine now offer the prospect of overcoming this barrier to xenotransplantation. In this study, the short-term function of GalT-KO swine lungs was evaluated in a well-established ex vivo model of swine-to-human lung xenotransplantation., Methods: Lungs from homozygous GalT-KO swine (n = 3) and control lungs from pigs of the background strain used to create the GalT-KO pig line (n = 2) were perfused ex vivo with freshly collected heparinized human blood. Graft function was assessed by various physiologic measurements, serial histologic and immunohistochemical evaluation, and assays of complement and platelet activation., Results: Xenoperfused control swine lungs exhibited HAR with graft survival times <5 minutes. In contrast, GalT-KO swine lungs retained their function for approximately 2 hours, on average. GalT-KO swine lungs showed decreased complement and platelet activation compared with controls. Nonetheless, activation of complement and coagulation cascades was not completely eliminated in the GalT-KO swine lungs., Conclusions: The survival of xenoperfused GalT-KO swine lungs was significantly prolonged, as compared with control lungs expressing Gal. This appears to have been due largely to substantially reduced complement activation. Nonetheless, the xenoperfused GalT-KO lungs still showed some evidence of complement fixation and intravascular coagulopathy by the time of graft demise.

Background: The pathophysiology of hyperacute lung rejection (HALR) is not fully understood. A mouse model of HALR by human blood would be valuable to efficiently dissect the molecular mechanisms underlying this complex process, but it has not been described., Methods: We developed a xenogenic mouse lung-perfusion model. Perfusion with heparinized autologous blood (n=3) was compared with human blood unmodified (n=7) or pretreated with C1 inhibitor (n=5) or soluble complement receptor type 1 (n=6) at unchanged flow conditions., Results: Perfusion with autologous blood was associated with stable physiologic parameters and no overt evidence of lung injury for up to 2 hr. Pulmonary artery perfusion pressure increased rapidly after introduction of unmodified human blood, plasma anti-Gal(alpha)1,3Gal antibodies declined (90% immunoglobulin [Ig]M, 80% IgG), and lungs reliably met survival endpoints within 11 min (median 10 min, confidence interval [CI]: 9-11). Human Ig and neutrophils were rapidly sequestered in the lung. Survival was significantly prolonged in the soluble complement receptor type 1 group (36 min, CI: 26-46) (P<0.01) and in the C1 inhibitor group (23 min, CI: 21-25) (P<0.05), and pulmonary vascular resistance elevation and complement activation were significantly attenuated but not prevented., Conclusions: Hyperacute rejection of mouse lung by human blood occurs with kinetics, physiology, and histology closely analogous to the pig-to-human model. In addition, as in that model, neither of two potent soluble-phase complement inhibitors prevented complement activation or HALR. We conclude that the mouse lung model is relevant to dissect the cellular and molecular mechanisms governing HALR.

Background: The influence of platelet von Willebrand factor (vWF)-glycoprotein (GP)Ib-V-IX and GPIIb-IIIa receptor interactions in the context of hyperacute rejection (HAR) of pulmonary xenografts has not previously been explored., Methods: Aurintricarboxylic acid (ATA, an inhibitor of platelet-GPIb interactions with vWF), SC52012A (SC, a synthetic GPIIb/IIIa inhibiting peptide), or both were added to heparinized whole human blood before perfusion of isolated piglet lungs. Results were compared with unmodified blood ("unmodified")., Results: Perfusion of porcine lungs with unmodified human blood resulted in an immediate rise in pulmonary vascular resistance (PVR), fluid and platelet sequestration in the lung, and, without exception, cessation of function within 15 minutes with a mean survival of 8 minutes. Addition of ATA or SC before lung perfusion significantly decreased the rise in PVR, diminished histamine release, and prolonged survival to 31+/-11 and 31+/-22 minutes, respectively. When the therapies were combined, mean survival was 156+/-77 minutes (P<0.05 vs. either monotherapy). Complement activation was synergistically attenuated only when the drugs were used together., Conclusions: Platelet protein receptor adhesive interactions play an important role in amplification of complement activation during hyperacute lung rejection. Inhibiting recruitment of platelets at the site of initial immunologic injury to endothelial cells may protect porcine organs against thrombosis and inflammation during the initial exposure to human blood.

Background: Previous work has shown that antibodies against porcine antigens are an important trigger of hyperacute lung rejection (HALR). The relative importance of Gal alpha1,3Gal epitopes and other antigens, such as those expressed on pig platelet membranes or lung itself, has not been defined. This study compares the efficiency of three anti-pig antibody depletion strategies, and their efficacy with regard to attenuation of HALR., Methods: Plasma pooled from three human donors was adsorbed against Gal alpha1,3Gal disaccharide or porcine platelet extract (PPE), or passed through pig lung vasculature. Whole blood reconstituted using adsorbed plasma was then used to perfuse piglet lung, and results were compared with unmodified human blood., Results: Depletion of lung-reactive anti-Gal alpha1-3Gal antibodies was most efficient with the alphaGal column (99% +/- 0.5% vs 87% to 93% +/- 11% for PPE and 92% to 95% +/- 8% for lung, p < 0.01 vs alphaGal column). PPE column tended to be more efficient (77% to 84% +/- 12%) in removing anti-PPE antibodies than pig lung (66% to 70% +/- 14%) or the alphaGal column (56% to 63% +/- 16%, p < 0.05). Lung survival and function with each antibody depletion strategy was improved relative to unmodified controls (mean survival > or = 146 minutes vs 8 minutes for controls). Although alphaGal and lung adsorption yielded more consistent lung protection (survival beyond 2 hours) than did PPE, no approach proved significantly superior. Complement C3a elaboration at 10 minutes was attenuated > 80% by each adsorption strategy, an effect that was most pronounced in the lung adsorption group (95%, p < 0.01). Histamine elaboration was blunted significantly by PPE adsorption but not in other groups (p < 0.05). Platelet but not leukocyte sequestration was decreased with antibody depletion compared with the nondepleted group (44% to 50% vs 82%, p < 0.01)., Conclusions: Each antibody depletion strategy tested significantly prolongs lung xenograft survival and function compared with unmodified human blood, but none was sufficient to reliably prevent HALR. Depletion of antibodies against both alphaGal and additional cell membrane antigens, or control of antibody-independent pathogenic pathways, may be necessary to consistently prevent HALR.