Novel factors potentially initiating acute antibody-mediated rejection in pig kidney xenografts despite an efficient immunosuppressive regimen.

Antibody-mediated rejection (AMR) is a common cause of graft failure after pig-to-nonhuman primate organ transplantation, even when the graft is from a pig with multiple genetic modifications. The specific factors that initiate AMR are often uncertain. We report two cases of pig kidney transplantation into immunosuppressed baboons in which we identify novel factors associated with the initiation of AMR. In the first, membranous nephropathy was the initiating factor that was then associated with the apparent loss of the therapeutic anti-CD154 monoclonal antibody in the urine when severe proteinuria was present. This observation suggests that proteinuria may be associated with the loss of any therapeutic monoclonal antibody, for example, anti-CD154 or eculizumab, in the urine, resulting in xenograft rejection. In the second case, the sequence of events and histopathology tentatively suggested that pyelonephritis may have initiated acute-onset AMR. The association of a urinary infection with graft rejection has been well-documented in ABO-incompatible kidney allotransplantation based on the expression of an antigen on the invading microorganism shared with the kidney graft, generating an immune response to the graft. To our knowledge, these potential initiating factors of AMR in pig xenografts have not been highlighted previously.

Research opportunities and ethical considerations for heart and lung xenotransplantation research: A report from the National Heart, Lung, and Blood Institute workshop.

Xenotransplantation offers the potential to meet the critical need for heart and lung transplantation presently constrained by the current human donor organ supply. Much was learned over the past decades regarding gene editing to prevent the immune activation and inflammation that cause early organ injury, and strategies for maintenance of immunosuppression to promote longer-term xenograft survival. However, many scientific questions remain regarding further requirements for genetic modification of donor organs, appropriate contexts for xenotransplantation research (including nonhuman primates, recently deceased humans, and living human recipients), and risk of xenozoonotic disease transmission. Related ethical questions include the appropriate selection of clinical trial participants, challenges with obtaining informed consent, animal rights and welfare considerations, and cost. Research involving recently deceased humans has also emerged as a potentially novel way to understand how xeno-organs will impact the human body. Clinical xenotransplantation and research involving decedents also raise ethical questions and will require consensus regarding regulatory oversight and protocol review. These considerations and the related opportunities for xenotransplantation research were discussed in a workshop sponsored by the National Heart, Lung, and Blood Institute, and are summarized in this meeting report.

Pediatric Cardiac Xenotransplantation: Recommendations for the Ethical Design of Clinical Trials.

For children with complex congenital heart problems, cardiac allotransplantation is sometimes the best therapeutic option. However, availability of hearts for pediatric patients is limited, resulting in a long and growing waitlist, and a high mortality rate while waiting. Cardiac xenotransplantation has been proposed as one therapeutic alternative for neonates and infants, either in lieu of allotransplantation or as a bridge until an allograft becomes available. Scientific and clinical developments in xenotransplantation appear likely to permit cardiac xenotransplantation clinical trials in adults in the coming years. The ethical issues around xenotransplantation of the heart and other organs and tissues have recently been examined, but to date, only limited literature is available on the ethical issues that are attendant with pediatric heart xenotransplantation. Here, we summarize the ethical issues, focusing on (1) whether cardiac xenotransplantation should proceed in adults or children first, (2) pediatric recipient selection for initial xenotransplantation trials, (3) special problems regarding informed consent in this context, and (4) related psychosocial and public perception considerations. We conclude with specific recommendations regarding ethically informed design of pediatric heart xenotransplantation trials.

Antibody-mediated rejection in xenotransplantation: Can it be prevented or reversed?

Antibody-mediated rejection (AMR) is the commonest cause of failure of a pig graft after transplantation into an immunosuppressed nonhuman primate (NHP). The incidence of AMR compared to acute cellular rejection is much higher in xenotransplantation (46% vs. 7%) than in allotransplantation (3% vs. 63%) in NHPs. Although AMR in an allograft can often be reversed, to our knowledge there is no report of its successful reversal in a pig xenograft. As there is less experience in preventing or reversing AMR in models of xenotransplantation, the results of studies in patients with allografts provide more information. These include (i) depletion or neutralization of serum anti-donor antibodies, (ii) inhibition of complement activation, (iii) therapies targeting B or plasma cells, and (iv) anti-inflammatory therapy. Depletion or neutralization of anti-pig antibody, for example, by plasmapheresis, is effective in depleting antibodies, but they recover within days. IgG-degrading enzymes do not deplete IgM. Despite the expression of human complement-regulatory proteins on the pig graft, inhibition of systemic complement activation may be necessary, particularly if AMR is to be reversed. Potential therapies include (i) inhibition of complement activation (e.g., by IVIg, C1 INH, or an anti-C5 antibody), but some complement inhibitors are not effective in NHPs, for example, eculizumab. Possible B cell-targeted therapies include (i) B cell depletion, (ii) plasma cell depletion, (iii) modulation of B cell activation, and (iv) enhancing the generation of regulatory B and/or T cells. Among anti-inflammatory agents, anti-IL6R mAb and TNF blockers are increasingly being tested in xenotransplantation models, but with no definitive evidence that they reverse AMR. Increasing attention should be directed toward testing combinations of the above therapies. We suggest that treatment with a systemic complement inhibitor is likely to be most effective, possibly combined with anti-inflammatory agents (if these are not already being administered). Ultimately, it may require further genetic engineering of the organ-source pig to resolve the problem entirely, for example, knockout or knockdown of SLA, and/or expression of PD-L1, HLA E, and/or HLA-G.

Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex-vivo perfusion of GTKO pig lungs with human blood.

INTRODUCTION: Expression of human complement pathway regulatory proteins (hCPRP's) such as CD46 or CD55 has been associated with improved survival of pig organ xenografts in multiple different models. Here we evaluate the hypothesis that an increased human CD46 gene dose, through homozygosity or additional expression of a second hCPRP, is associated with increased protein expression and with improved protection from injury when GTKO lung xenografts are perfused with human blood. METHODS: Twenty three GTKO lungs heterozygous for human CD46 (GTKO.heteroCD46), 10 lungs homozygous for hCD46 (GTKO.homoCD46), and six GTKO.homoCD46 lungs also heterozygous for hCD55 (GTKO.homoCD46.hCD55) were perfused with human blood for up to 4 h in an ex vivo circuit. RESULTS: Relative to GTKO.heteroCD46 (152 min, range 5-240; 6/23 surviving at 4 h), survival was significantly improved for GTKO.homoCD46 (>240 min, range 45-240, p = .034; 7/10 surviving at 4 h) or GTKO.homoCD46.hCD55 lungs (>240 min, p = .001; 6/6 surviving at 4 h). Homozygosity was associated with increased capillary expression of hCD46 (p < .0001). Increased hCD46 expression was associated with significantly prolonged lung survival (p = .048),) but surprisingly not with reduction in measured complement factor C3a. Hematocrit, monocyte count, and pulmonary vascular resistance were not significantly altered in association with increased hCD46 gene dose or protein expression. CONCLUSION: Genetic engineering approaches designed to augment hCPRP activity - increasing the expression of hCD46 through homozygosity or co-expressing hCD55 with hCD46 - were associated with prolonged GTKO lung xenograft survival. Increased expression of hCD46 was associated with reduced coagulation cascade activation, but did not further reduce complement activation relative to lungs with relatively low CD46 expression. We conclude that coagulation pathway dysregulation contributes to injury in GTKO pig lung xenografts perfused with human blood, and that the survival advantage for lungs with increased hCPRP expression is likely attributable to improved endothelial thromboregulation.

Recruited macrophages elicit atrial fibrillation.

Atrial fibrillation disrupts contraction of the atria, leading to stroke and heart failure. We deciphered how immune and stromal cells contribute to atrial fibrillation. Single-cell transcriptomes from human atria documented inflammatory monocyte and SPP1+ macrophage expansion in atrial fibrillation. Combining hypertension, obesity, and mitral valve regurgitation (HOMER) in mice elicited enlarged, fibrosed, and fibrillation-prone atria. Single-cell transcriptomes from HOMER mouse atria recapitulated cell composition and transcriptome changes observed in patients. Inhibiting monocyte migration reduced arrhythmia in Ccr2-∕- HOMER mice. Cell-cell interaction analysis identified SPP1 as a pleiotropic signal that promotes atrial fibrillation through cross-talk with local immune and stromal cells. Deleting Spp1 reduced atrial fibrillation in HOMER mice. These results identify SPP1+ macrophages as targets for immunotherapy in atrial fibrillation.

TNX-1500, a crystallizable fragment-modified anti-CD154 antibody, prolongs nonhuman primate cardiac allograft survival.

Blockade of the CD40/CD154 T cell costimulation pathway is a promising approach to supplement or replace current clinical immunosuppression in solid organ transplantation. We evaluated the tolerability and activity of a novel humanized anti-CD154 monoclonal antibody, TNX-1500 (TNX), in a nonhuman primate heterotopic cardiac allogeneic (allo) transplant model. TNX-1500 contains a rupluzimab fragment antigen-binding region and an immunoglobin G4 crystallizable fragment region engineered to reduce binding to the crystallizable fragment gamma receptor IIa and associated risks of thrombosis. Recipients were treated for 6 months with standard-dose TNX (sTNX) monotherapy, low-dose TNX monotherapy (loTNX), or loTNX with mycophenolate mofetil (MMF) (loTNX + MMF). Results were compared with historical data using chimeric humanized 5c8 monotherapy dosed as for loTNX but discontinued at 3 months. Median survival time was similar for humanized 5c8 and both loTNX groups, but significantly longer with sTNX (>265 days) than with loTNX (99 days) or loTNX + MMF (88 days) (P < 0.05 for both comparisons against sTNX). Standard-dose TNX prevented antidonor alloantibody elaboration, inhibited chronic rejection, and was associated with a significantly reduced effector T cells/regulatory T cells ratio relative to loTNX with MMF. No thrombotic complications were observed. This study demonstrated that TNX was well tolerated, prolongs allograft survival, and prevents alloantibody production and cardiac allograft vasculopathy in a stringent preclinical nonhuman primate heart allotransplant model.

TNX-1500, a crystallizable fragment-modified anti-CD154 antibody, prolongs nonhuman primate renal allograft survival.

The blockade of the CD154-CD40 pathway with anti-CD154 monoclonal antibody has been a promising immunomodulatory approach to prevent allograft rejection. However, clinical trials of immunoglobulin G1 antibodies targeting this pathway revealed thrombogenic properties, which were subsequently shown to be mediated by crystallizable fragment (Fc)-gamma receptor IIa-dependent platelet activation. To prevent thromboembolic complications, an immunoglobulin G4 anti-CD154 monoclonal antibody, TNX-1500, which retains the fragment antigen binding region of ruplizumab (humanized 5c8, BG9588), was modified by protein engineering to decrease Fc binding to Fc-gamma receptor IIa while retaining certain other effector functions and pharmacokinetics comparable with natural antibodies. Here, we report that TNX-1500 treatment is not associated with platelet activation in vitro and consistently inhibits kidney allograft rejection in vivo without clinical or histologic evidence of prothrombotic phenomena. We conclude that TNX-1500 retains efficacy similar to that of 5c8 to prevent kidney allograft rejection while avoiding previously identified pathway-associated thromboembolic complications.

Guidelines and principles for the care of the cardiothoracic transplant patient in the intensive care unit.

Heart and lung transplant recipients require care provided by clinicians from multiple different specialties, each contributing unique expertise and perspective. The period the patient spends in the intensive care unit is one of the most critical times in the perioperative trajectory. Various organizational models of intensive care exist, including those led by intensivists, surgeons, transplant cardiologists, and pulmonologists. Coordinating timely efficient intensive care is an essential and logistically difficult goal. The present work product of the American Society of Transplantation's Thoracic and Critical Care Community of Practice, Critical Care Task Force outlines operational guidelines and principles that may be applied in different organizational models to optimize the delivery of intensive care for the cardiothoracic organ recipient.

Milestones on the path to clinical pig organ xenotransplantation.

Progress in pig organ xenotransplantation has been made largely through (1) genetic engineering of the organ-source pig to protect its tissues from the human innate immune response, and (2) development of an immunosuppressive regimen based on blockade of the CD40/CD154 costimulation pathway to prevent the adaptive immune response. In the 1980s, after transplantation into nonhuman primates (NHPs), wild-type (genetically unmodified) pig organs were rejected within minutes or hours. In the 1990s, organs from pigs expressing a human complement-regulatory protein (CD55) transplanted into NHPs receiving intensive conventional immunosuppressive therapy functioned for days or weeks. When costimulation blockade was introduced in 2000, the adaptive immune response was suppressed more readily. The identification of galactose-α1,3-galactose as the major antigen target for human and NHP anti-pig antibodies in 1991 allowed for deletion of expression of galactose-α1,3-galactose in 2003, extending pig graft survival for up to 6 months. Subsequent gene editing to overcome molecular incompatibilities between the pig and primate coagulation systems proved additionally beneficial. The identification of 2 further pig carbohydrate xenoantigens allowed the production of 'triple-knockout' pigs that are preferred for clinical organ transplantation. These combined advances enabled the first clinical pig heart transplant to be performed and opened the door to formal clinical trials.

Future of Lung Transplantation: Xenotransplantation and Bioengineering Lungs.

Xenotransplantation promises to alleviate the issue of donor organ shortages and to decrease waiting times for transplantation. Recent advances in genetic engineering have allowed for the creation of pigs with up to 16 genetic modifications. Several combinations of genetic modifications have been associated with extended graft survival and life-supporting function in experimental heart and kidney xenotransplants. Lung xenotransplantation carries specific challenges related to the large surface area of the lung vascular bed, its innate immune system's intrinsic hyperreactivity to perceived 'danger', and its anatomic vulnerability to airway flooding after even localized loss of alveolocapillary barrier function. This article discusses the current status of lung xenotransplantation, and challenges related to immunology, physiology, anatomy, and infection. Tissue engineering as a feasible alternative to develop a viable lung replacement solution is discussed.

Cardiac xenotransplantation: from concept to clinic.

For many patients with terminal/advanced cardiac failure, heart transplantation is the most effective, durable treatment option, and offers the best prospects for a high quality of life. The number of potentially life-saving donated human organs is far fewer than the population who could benefit from a new heart, resulting in increasing numbers of patients awaiting replacement of their failing heart, high waitlist mortality, and frequent reliance on interim mechanical support for many of those deemed among the best candidates but who are deteriorating as they wait. Currently, mechanical assist devices supporting left ventricular or biventricular heart function are the only alternative to heart transplant that is in clinical use. Unfortunately, the complication rate with mechanical assistance remains high despite advances in device design and patient selection and management, and the quality of life of the patients even with good outcomes is only moderately improved. Cardiac xenotransplantation from genetically multi-modified (GM) organ-source pigs is an emerging new option as demonstrated by the consistent long-term success of heterotopic (non-life-supporting) abdominal and life-supporting orthotopic porcine heart transplantation in baboons, and by a recent 'compassionate use' transplant of the heart from a GM pig with 10 modifications into a terminally ill patient who survived for 2 months. In this review, we discuss pig heart xenotransplantation as a concept, including pathobiological aspects related to immune rejection, coagulation dysregulation, and detrimental overgrowth of the heart, as well as GM strategies in pigs to prevent or minimize these problems. Additional topics discussed include relevant results of heterotopic and orthotopic heart transplantation experiments in the pig-to-baboon model, microbiological and virologic safety concepts, and efficacy requirements for initiating formal clinical trials. An adequate regulatory and ethical framework as well as stringent criteria for the selection of patients will be critical for the safe clinical development of cardiac xenotransplantation, which we expect will be clinically tested during the next few years.

Knock-out of N-glycolylneuraminic acid attenuates antibody-mediated rejection in xenogenically perfused porcine lungs.

BACKGROUND: Antibody-mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3-galactose (α1,3Gal) epitope, N-Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre-formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene knock-out (Neu5GcKO) in a xenogeneic ex vivo perfusion model METHODS: Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6 h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1-thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n = 18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre-defined time points throughout the perfusion RESULTS: All but one Neu5GcKO organs maintained adequate blood oxygenation and "survived" until elective termination at 6 h whereas two reference lungs failed before elective termination at 4 h. Human anti-Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (∼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p = .007; IgG p < .001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p = .007, .005, and .037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p = .023), whereas complement activation (as C3a rise in plasma) was not altered. MCP-1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p = .007 and .032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p = .003) CONCLUSION: Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody-mediated inflammation and activation of the coagulation cascade. Knock-out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients.

Genetic modifications designed for xenotransplantation attenuate sialoadhesin-dependent binding of human erythrocytes to porcine macrophages.

The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec-1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild-type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and "rosettes" (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near-complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation.

The first clinical pig heart transplant: Was IVIg or pig cytomegalovirus detrimental to the outcome?

The clinical course of the first patient to receive a gene-edited pig heart transplant was recently reported by the University of Maryland team. Although the pig heart functioned well for >40 days, serum anti-pig antibodies then increased, and the patient sadly died after 60 days. Because of his debilitated pre-transplant state, the patient never thrived despite excellent graft function for several weeks, and the cause of his demise continues to be uncertain. A few days before an increase in anti-pig antibodies was observed, the patient had received intravenous human immunoglobulin (IVIg), and whether this played a role in his cardiac deterioration has been discussed. Furthermore, mcfDNA testing indicated an increase in pig cytomegalovirus (CMV), and its possible role in the development of cardiac dysfunction has also been considered. On the basis of the limited data provided in the publication and on our previous investigations into whether IVIg contains anti-TKO pig antibodies and therefore might be deleterious to TKO pig organ xenografts, we suggest that the steady rise in anti-pig antibody titer was more consistent with the failure of the immunosuppressive regimen to prevent elicited anti-TKO pig antibody production, rather than from the passive transfusion of IVIg or the presence of pig CMV in the graft. Although the outcome of the Maryland experience was disappointing, valuable lessons were learned. Our attention was drawn to the potential risks of heart transplantation in a "deconditioned" patient, the administration of IVIg, the transmission of pig CMV, and of the difficulties in interpreting myocardial biopsy findings.

Pig heart and lung xenotransplantation: Present status.

The recent pig heart transplant in a patient at the University of Maryland Medical Center has stimulated renewed interest in the xenotransplantation of organs from genetically engineered pigs. The barriers to the use of pigs as sources of organs have largely been overcome by 2 approaches - (1) the deletion of expression of the three known pig carbohydrate xenoantigens against which humans have preformed antibodies, and (2) the transgenic introduction of human 'protective' proteins, such as complement-regulatory proteins. These gene modifications, coupled with immunosuppressive therapy based on blockade of the CD40/CD154 costimulation pathway, have resulted in survival of baboons with life-supporting pig heart grafts for almost 9 months. The initial clinical success at the University of Maryland reinforces encouraging preclinical results. It suggests that pig hearts are likely to provide an effective bridge to an allotransplant, but their utility for destination therapy remains uncertain. Because of additional complex immunobiological problems, the same approach has been less successful in preclinical lung xenograft transplantation, where survival is still measured in days or weeks. The first formal clinical trials of pig heart transplantation may include patients who do not have access to an allotransplant, those with contraindications for mechanical circulatory support, those in need of retransplantation or with a high level of panel-reactive antibodies. Infants with complex congenital heart disease, should also be considered.