The review introduces the challenges and potential solutions in xenotransplantation, focusing on pig-to-human organ transplant. Xenotransplantation, mainly with the use of pig organs, is a promising solution because of the reproductive capacity, size, and physiological resemblance of pigs to humans. However, immunological barriers, especially humoral and cellular immune responses, pose substantial challenges. The humoral immune response, involving antibodies targeting xenoantigens, is a substantial barrier. Anti-α-galactose antibodies, targeting α-Gal epitopes, are crucial in hyperacute rejection and acute humoral xenograft rejection. Genetic modifications, including CRISPR/Cas9 technology, aim to eliminate xenoantigens like α-Gal, potentially overcoming these challenges. This review discusses the use of genetically modified pigs for xenotransplantation, emphasizing the removal of xenoantigens, expression of human complement regulatory proteins, and transgenic expres-sion of human regulatory factors. Recent advancements, such as the world’s first porcine-to-human heart transplant, highlight the potential of genetic manipu-lation in overcoming immune rejection barriers.

Key words : Adaptive immunity, Genetic modification, Humoral immunity, Immunogenetic, Rejection

Introduction

Hemodialysis is crucial for sustaining the lives of thousands of patients with end-stage renal disease. However, hemodialysis is not the optimal, preferred, or a gratifying treatment for patients with end-stage renal disease because several drawbacks, including low quality of life, susceptibility to infection, high morbidity, and elevated mortality rates. Each addi-tional year of dialysis treatment is associated with an approximately 6% increase in the risk of death.¹ Shockingly, 40% of patients on waiting lists may succumb to various side effects within 5 years of treatment.²

Heart transplantation stands as a life-saving medical intervention for individuals with end-stage heart failure. Nevertheless, the success and availa-bility of this procedure hinge on a critical factor: the availability of suitable donor hearts. The shortage of heart donors has become a pressing issue, posing substantial challenges to patients awaiting heart transplants worldwide.² The shortage of donor hearts contributes to high mortality rates among indivi-duals who are waiting for heart transplants. The scarcity of donor hearts prolongs the time patients spend on transplant waiting lists. Extended wait times exacerbate the health conditions of those awaiting transplant and increase the risk of complications or death before a suitable donor becomes available.

The substantial gap between the supply and demand of organs for transplant creates a bottleneck. Bridging this gap necessitates considering all brain dead individuals as default organ donors, although implementing such changes involves navigating numerous legal regulations. Consequently, achieving a stable and sufficient organ supply for transplant remains a distant goal. As an alternative, ongoing developments in tissue engineering, stem cell technologies, and blastocyst completion offer highly desirable options to overcome the limitations of donated human organs. On the horizon of medical innovation, a promising solution emerges: xenotransp-lantation. This groundbreaking approach involves the use of organs from animals, offering a potential lifeline for those in need.^3-5

Xenotransplantation holds immense promise as a revolutionary solution to the persistent organ shortage crisis. As researchers continue to make strides in overcoming technical challenges and ethical concerns, the potential benefits of xenotransplant are becoming increasingly tangible. The collaboration between scientists, ethicists, policymakers, and the public is essential to navigate the complexities of this innovative approach and ensure its responsible integration into the field of organ transplantation. If successful, xenotransplantation could redefine the landscape of organ availability, offering hope to countless individuals who are waiting for life-saving transplants.^3-5 Although the idea of using animal organs for transplant is not new, recent advancements in genetic engineering and immunosuppressive therapies have rekindled interest in making xeno-transplantation a viable and safe option.

Nevertheless, xenotransplantation can have substantial challenges, including the risk of cross-species infections (xenozoonoses) and the potential for immune rejection. Researchers are actively addressing these concerns through genetic modifi-cations to create pigs with organs less likely to provoke immune responses and to develop advan-ced immunosuppressive regimens tailored for recipients of xenotransplant.^5-7

Although nonhuman primates are phylogene-tically closer to humans, they are not favored for xenotransplantation because of concerns about organ size disparities, ethical considerations, and a high risk of cross-species infections.⁴ On the other hand, pigs have emerged as the preferred source for xenotransplantation because of their reproductive capacity, size, physiological resemblance to humans, and a lower risk of xenozoonosis.^5-7 However, the genetic incompatibility between pigs and humans poses significant immunological barriers.⁷ The severity of rejection in xenotransplants surpasses allotransplants, primarily because of substantial genetic variation.^4-7 This genetic disparity highlights the need for advanced genetic modifications to enhance compatibility and to reduce the risk of rejection in xenotransplantation procedures. Over-coming immunological barriers through precise genetic modifications brings us closer to a future where xenotransplantation plays a major role in meeting the critical need for life-saving organ transplants.

The immunopathological response after xenograft of porcine organs is complex, involving inflammatory, coagulation, complement, antibody, and cellular responses.^6,7 The immediate and aggressive response, known as hyperacute rejection (HAR), is primarily mediated by preexisting antibodies against porcine antigens. Genetic modifications, such as the galactose-α-1,3-galactose (Gal) knockout, have shown promise in mitigating HAR by reducing the recognition of pig antigens.^5-7 Prolonged xenograft survival faces hurdles in the form of chronic rejection, characterized by fibrosis, vasculopathy, and persistent immune responses. Genetic modifications with CRISPR/Cas9 technology aim to create pigs with organs that are less prone to chronic rejection. However, the complex and multifaceted nature of chronic rejection demands comprehensive approaches to extend graft survival.

As researchers unravel the intricacies of immuno-pathological responses, the potential for xenotransp-lantation to revolutionize organ transplant and save countless lives remains on the horizon. This review will discuss immune responses that target xeno-organs after transplant and the promising immunogenetic modifications to ameliorate destructive xenogeneic immune responses causing organ loss.

Immunological Barriers to Xenotransplantation

Xenotransplantation used in clinical practice requires xenogeneic extracellular matrices for tissue or organ repair or replacement in regenerative medicine. Porcine and bovine matrices are already in clinical use for reconstructive applications, particularly in cardiac, ophthalmologic, and orthopedic surgery.⁸ Xenogeneic extracellular matrices have demonstrated promising applications as biological materials for surgical tissue repair and whole bioartificial organs, such as bioprosthetic heart valves.⁸ Decellularized extracellular matrix products, like cornea and cardiac valves, devoid of pig and bovine cells, escape the recipient’s immune responses because of limited epithelization and vascularization. These xenografts are repopulated with human recipient cells.^3,8 In contrast, xenotransplant of vascularized tissues and organs elicits severe immune responses involving innate and acquired immunity, natural antibodies, complement, natural killer (NK) cells, and macrophages.^3,4,6-8 Consequently, 3 main types of rejection can occur sequentially: hyperacute xenograft rejection, acute humoral xenograft rejection (AHXR), and acute cellular rejection.^3,6 In addition, coagulation and inflammatory response dysregulation contribute to xenograft failure, as evidenced by coagulation disorders observed in pig-to-primate transplant,⁶ closely linked to the xenograft’s immune response and subsequent inflammation.

Humoral Immunity

The humoral component of the immune response stands as the most formidable barrier to both short- and long-term xenograft survival. Antibodies directed against xenoantigens after xenotransplantation play a pivotal role in solid-organ xenograft rejection. Moreover, through antibody production, humoral immunity contributes greatly to the rejection and destruction of xenografts.^6-9 A primary obstacle to successful xenotransplant is the presence of preformed natural antibodies, typically classified as components of innate immunity. The presence of natural antibodies directly influences the severity of coagulation dysregulation, making them a critical focus in xenotransplant.^6-10

The most crucial natural antibody is anti-α-galactose-1,3-galactose (α-Gal), which recognizes the Gala(1,3)Galβ4Glc-Nac-R epitope.¹¹ The α-Gal antigen, expressed by α-1,3 galactosyltransferase (α-1,3GalT; also known as GGTA1), is a carbohydrate expressed in pigs and other mammalian species on various cell surfaces.¹¹ In contrast, humans or Old World monkeys do not express α-Gal because of a frameshift mutation in the α-1,3-galactosyltransferase (GT) gene.^11,12 Approximately 1% of all circulating antibodies are shown to be directed against α-Gal epitopes in human blood.^11,12 These natural anti-α-Gal antibodies are universally induced during neonatal life by gut bacteria that express GGTA1.¹³

Experiments involving pig-to-nonhuman primate transplants have shown that antibodies specific to α-Gal epitopes can lead to HAR and AHXR.¹⁴ Kidneys from GGTA1 knockout pigs transplanted into nonhuman primates were rejected within days through antibody-mediated rejection.¹⁵ This finding suggested that non-Gal antigens pose an additional hurdle to organ transplant from GGTA1 knockout pigs to humans.³ In humans, approximately 1% to 4% of circulating B lymphocytes oversee the production of anti-α-Gal antibodies, encompassing both immunoglobulin M (IgM) and IgG.¹⁴ CD20+ CD138+ Ig+ B cells produce IgM-type natural anti-α-Gal antibodies, predominantly located in lymph nodes and the spleen. Immunoglobulin G-secreting anti-α-Gal cells are immature CD138+Ig+DR+ or mature CD138+Ig−DR− plasma cells in humans and nonhuman primates.⁷ After sensitization with porcine antigens, the number of anti-α-Gal Ig-producing cells increases, with storage and identification occurring in the spleen, lymph nodes, and bone marrow for up to 6 months.⁷

The formation of anti-α-Gal antibodies is primarily a T-cell-dependent process, proven by inhibiting the humoral response by blocking the CD40-CD154 interaction between B cells and T cells.^6,7 Natural antibodies against α-Gal present a critical impediment to successful xenotransplant, inducing the development of HAR within minutes to hours.^6-9,16 Persistent elimination of the α-Gal epitope from pigs using CRISPR/Cas9 technology is the sole solution to overcome α-Gal rejection.^17,18 However, delayed xenograft rejection is still observed, deve-loping over days to weeks rather than minutes to hours. This delayed-type xenograft rejection is attributed to less evident pig cellular and molecular characteristics, such as the expression of β-1,4 N-acetylgalactosaminyl transferase 2 (β4GalNT2) or N-glycolylneuraminic acid.¹⁹

Adaptive Immunity

Compared with natural immune barriers, the adaptive immunity orchestrated by B and T cells often plays a crucial role in late xenograft rejection. The magnitude of the adaptive immune response is intricately regulated by innate immune cells, which, in turn, modulate the innate immune response through cytokine secretion and interactions with antibody-Fc receptors.^9,16,19,20 B cells contribute greatly to the humoral response by secreting specific antibodies and inducing cellular rejection through antigen presentation to T cells and cytokine secretion.19 These B-cell-mediated immune responses in xenograft rejection can be mitigated by appropriate T-cell immunosuppression or tolerance induction.²⁰ However, some B-cell immune responses occur independently of T cells.^9,21 The primary functions of cellular response include T-cell recruitment, activation of innate cytotoxic cells, cytotoxicity, and cytokine production. Furthermore, T-cell activation is associated with complement activation, inflammatory responses, and coagulation.^7,10,20,21

The use of genetically modified pigs is a potential solution to prevent T-cell-mediated rejection after xenotransplant. High-level expression of specific genes, such as Fas ligand, CTLA4-Ig, PD-L1, and anti-CD2 monoclonal antibodies, in donor pig tissues promotes a decrease in cellular immune responses, leading to a reduction in the need for immunosup-pressive therapy.^5,14,22,23 Combining various options targeting specific molecules could effectively control cell-mediated rejection of xenotransplants, bringing us 1 step closer to meeting global organ demands.

Hyperacute Rejection and Acute Humoral Xenograft Rejection

Antibodies directed against donor antigens can initiate 2 distinct rejection processes: HAR and AHXR.^6-9,14,16 Hyperacute rejection occurs rapidly, within minutes to hours after transplant, due to preformed antidonor antibodies in the recipient’s circulation, leading to the destruction of the xenograft.^3,4,14 Preexisting antibodies bind to the endothelium of the graft, triggering complement activation, widespread vascular deposition, destruc-tion of vasculature, and graft failure.^3,11,24,25 Leukocyte accumulation, edema, hemorrhage, fibrin-platelet-rich thrombotic occlusion, and fibrinoid necrosis further characterize this process.²⁴

Acute humoral xenograft rejection, akin to vas-cular rejection in allotransplant, occurs within days and results from an antibody response to the xenograft. Neutrophils release proinflammatory cytokines and oxygen-reactive species during AHXR, and xenoantibodies recognize crucial molecules, leading to antibody-dependent cell-mediated cyto-toxicity by NK cells.^25,26 Furthermore, xenoantibodies can identify critical molecules such as MHC class I, NKG2D/UL16 binding protein-1, CD28/CD83, and NKp44, leading to an antibody-dependent cell-mediated cytotoxicity response by NK cells.^25,26 CD4+ T cells also have a crucial role through the Fas-Fas ligand lytic pathway, leading to direct cytotoxic effects and releasing interferon-gamma, which will further activate macrophages and NK cells.^26,27

Histopathological findings of AHXR are influenced by antixenograft antibody specificity, donor organ source, and therapeutic interventions. The deposition of antibodies, complement products such as C4d, capillary leukocyte accumulation, loss of capillary integrity, endothelial cell death, and extensive fibrin deposition within capillaries are cardinal features of AHXR. Although preexisting IgM is primarily responsible for HAR, newly formed IgM and IgG antibodies drive AHXR. Complement activation is crucial for antibody-induced damage by both IgM and IgG antibodies.^3,24 The humoral response to endothelial cells causes injury and cell death through endothelial activation, inflammation, and antibody-dependent cell-mediated cytotoxicity. This response induces a shift from an anticoagulant to a pro-coagulant phenotype, activating coagulation and thrombosis.^3,24

Conventional immunosuppressive therapy is ineffective in preventing AHXR in solid-organ xeno-transplants. However, blockade of the CD40:CD154 costimulation pathway by antihuman CD154 monoc-lonal antibody efficiently prevents T-cell activation in the xenotransplant model.²⁷ Characterization of B-cell populations producing xeno-directed antibodies is critical for the development of therapeutic strategies selectively targeting the xenogeneic antibody response. Novel B-cell-targeting therapeutic agents, such as anti-CD20 and anti-CD19 monoclonal antibodies, have recently been developed. Anti-CD20 monoclonal antibodies more efficiently deplete B cells, whereas anti-CD19 monoclonal antibodies deplete memory B cells and short-lived plasma cells.²⁸

Cellular Xenograft Rejection

Cellular xenograft rejection (CXR) typically manifests days to weeks after transplant and involves innate and adaptive immune responses. The recipient’s immune cells, comprising NK cells, monocytes/macrophages, neutrophils, dendritic cells, and T and B lymphocytes, contribute to CXR.^7,24

Natural killer cells mediate xenograft rejection through direct NK cytotoxicity and antibody-dependent cellular cytotoxicity pathways. In the direct NK cytotoxicity pathway, NK cells release lytic granules, causing donor endothelial cell lysis through the interaction of activating receptors and ligands. In the antibody-dependent cellular cytotoxicity pathway, Fc-fraction on NK cells recognize antibodies deposited on the graft endothelium, releasing cytotoxic granules and triggering apoptosis in target cells.^26,29 Whereas removal of α-Gal epitopes protects porcine endothelial cells from complement-induced lysis, it does not prevent NK cell adhesion and direct cytotoxicity. Further studies are needed to fully understand the role of NK cells in porcine xenograft rejection.³

Macrophages are pivotal in xenograft rejection, contributing to adaptive immunity and direct cytotoxicity.^24,30,31 Recruited and activated by T cells, macrophages cause diffuse infiltration and dest-ruction within the graft. Increased macrophage infiltration amplifies the T-cell response, whereas direct cytotoxic effects involve the release of proinflammatory cytokines such as interleukin-1(IL-1), IL-6, and tumor necrosis factor-alpha (TNF-α). Regulation of macrophage activation and recruitment is crucial for enhancing xenograft survival.³⁰

T lymphocytes are central to acute CXR, activated through direct and indirect pathways similar to allotransplantation.^3,32 Porcine antigen-presenting cells directly activate primate T cells in direct T-cell activation, whereas indirect activation occurs through donor-derived peptides presented by recipient antigen-presenting cells. MHC class II of the recipient recognizes porcine xenoantigens, leading to CD4-positive T-cell activation, B-cell stimulation, antibody production, and AHXR.³² Activated T cells release cytokines that augment the cytotoxicity of NK cells and macrophages.²⁶

Despite their shared similar immunological pathways, xenotransplant elicits a more robust indirect T-cell response against porcine antigensthan alloantigens. Acute cellular rejection is rarely documented after pig-to-nonhuman primate xenotransplant, possibly because of the over-whelming strength of AHXR or the adequacy of current immunosuppression treatments to control T-cell-mediated rejection.

Genetically Modified Pigs For Xenotransplantation

The primary challenge hindering the success of xenotransplantation lies in the extent of immune incompatibility between the donor and recipient. Immunological barriers such as HAR, AHXR, CXR, coagulation dysregulation, and inflammatory res-ponses pose major hurdles, reducing the efficacy of xenotransplantation. CRISPR/Cas9 and advanced genetic editing techniques have created a diverse range of genetically modified pigs tailored for xenotransplantation, aiming to bridge molecular incompatibilities across species and surmount these obstacles.³³

Removal of xenoantigens

Plasmapheresis, immunoabsorption, and comple-ment activation inhibition have emerged as primary treatment avenues to quell the humoral response in xenotransplant cases, mirroring approaches used for allotransplant recipients. Plasmapheresis and immunoadsorption have been used to eliminate preformed anti-Gal antibodies from the recipient’s plasma.³⁴ However, these methods yielded limited success as antibodies could swiftly return, preci-pitating AHXR.^3,34 A transformative strategy in xenotransplantation involves the knockout of the GGTA1 gene (GGTA1KO) in donor pigs.^18,35

The use of GGTA1KO pigs as donors was shown to substantially prolong renal and cardiac graft survival in baboons. GGTA1KO eliminated Gal-mediated HAR, forming the foundation for sub-sequent genetic modifications.^36,37 Beyond Gal antigens, various non-Gal antigens on pig cell surfaces, such as Sda and N-glycolylneuraminic acid, produced by β4GalNT2 and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), respectively, can combine with Gal-specific antibodies, triggering AHXR.^24,25,38 Studies demonstrated that simultaneous inactivation of GGTA1/CMAH/β4GalNT2 genes attenuated human antibody binding to pig renal tissues.³⁹ Porcine bioprosthetic heart valves showed reduced im-munoreactivity after concurrently deleting 3 genes (GGTA1, CMAH, and β4GalNT2). Pigs with GGTA1/CMAH/β4GalNT2 inactivation exhibited a notable decrease in human antibody binding to porcine blood mononuclear cells compared with those lacking GGTA1 and CMAH genes.^39,40 Although GGTA1/CMAH/β4GalNT2 triple knoc-kout gene modification has offered enhanced protection against immunoreactivity and rejection, its ability to prolong graft survival has remained contentious. Further extensive studies are warranted to elucidate this matter comprehensively.

Expression of human complement regulatory proteins

Although the removal of Gal and non-Gal antigens can effectively prevent both HAR and AHXR,^25,39,40 immune rejection and xenograft failure still involve complement cascade activation. Although pigs possess complement regulation proteins (CRPs) akin to humans, they fall short in shielding xenografts from human complement-mediated injury. Consequently, incorporation of human CRPs into donor pigs becomes imperative to overcome this obstacle.³

Porcine expression of human CRP is achieved through the microinjection of DNA into fertilized eggs.⁴¹ Advances in gene editing have enabled various genetic modifications in pigs for xenot-ransplantation. Notably, numerous transgenic pigs expressing human complement regulators like CD55, CD46, and CD59 have demonstrated pro-tection against complement-mediated xenograft injury, leading to prolonged xenograft survival.^3,42,43 However, this approach does not consistently succeed in cases of AHXR, where the complement system is activated through both classical and alternative pathways.

The insertion of 2 or 3 human complement regulators has proven more effective in protecting against complement activation than a single regulator’s transgenic expression. Combining GGTA1KO porcine with transgenic expression of human CD46 and CD55 significantly reduced early kidney and heart graft failure in baboons.^37,44 These results strongly advocate using GGTA1KO porcine with 1 or 2 human complement regulators as more favorable donors for kidney and other solid-organ xenotransplants.

Transgenic expression of human regulatory factors involving immune responses

Transgenic pigs expressing human regulatory factors related to innate or adaptive immune responses are already accessible for xenotransplantation. CD47, an immune inhibitory receptor expressed on macrophages, undergoes species-specific changes. To counter incompatibility between human SIRP-α and pig CD47, generating porcine cells expressing human CD47 is a crucial step in immune response inhibition.³ Human CD47-expressing porcine cells can evade phagocytosis by human macrophages.45 CD47 expression in porcine endothelial cells can suppress macrophage-mediated cytotoxicity and the release of inflammatory cytokines such as IL-6 and TNF-α.⁴⁶ In addition, CD47 expression inhibits human T-cell infiltration in xenografts.⁴⁶ Transgenic porcine expression of CD47 has increased xenogeneic hematopoietic engraftment chimerism in murine models and extended the survival of porcine skin grafts.^47,48 These findings have suggested that introducing human CD47 into porcine glomerular cells may inhibit proteinuria after kidney xenotransplant. Although evidence supports the beneficial effect of human CD47 expression in xenografts, transgenic human CD47 expression alone may not completely prevent phagocytosis by human macrophages. Therefore, it may be necessary to suppress macrophages activated by xenoantigens.³

Xenografts from transgenic pigs expressing human CD200 have demonstrated inhibition of human macrophage phagocytic and cytotoxic activities against porcine endothelial cells.^46,49 Transgenic porcine expression of human CD274 has greatly decreased the capacity to induce CD4-positive T-cell proliferation. The costimulatory molecule CTLA4, which inhibits the T-cell activation pathway, may play a crucial role in xenotransplant. Transgenic porcine expression of hCTLA4-Ig has demonstrated prolonged allograft survival in nonhuman primates.⁴⁸ Although this finding suggests the potential to prevent T-cell activity in xenografts, hCTLA4-Ig alone may not completely prevent xenograft rejection.³ Recently, transgenic pigs expressing neuronal hCTLA4-Ig were shown to reduce human T-cell proliferation versus porcine cells, resulting in long-term xenograft survival in a rat skin model.⁵⁰ It was also observed that hCTLA4-Ig alone could not prevent xenograft rejection, aligning with results from blocking the costimulatory pathway against B7-CD28 only.³ Transgenic pigs expressing pCTLA4-Ig have also been produced, with pCTLA4-Ig ubiquitously expressed in these pigs.⁵¹ However, pigs ubiquitously expressing pCTLA4-Ig demonstrated susceptibility to infection because of high levels of expression in the blood. Consequently, designing transgenic pigs with pCTLA4-Ig expression limited to specific target cells may be a more suitable approach.

Genetically Modified Porcine-to-Human Cardiac Xenotransplant

Over an extended period, preclinical investigations into xenotransplant have been conducted; however, the lack of a comprehensive understanding of xenotransplant coupled with ineffective immuno-suppression strategies led to repeated failures.One notable instance of such setbacks occurred in 1983 when an infant who underwent a heart transplant from a baboon only survived for 20 days.⁵²

Recent advancements in genetic manipulation technologies have ushered in a new era, paving the way for more promising donor organs, particularly those engineered from animals. Notably, the genetically modified pig heart, a cutting-edge technology product, underwent an impressive array of 10 genetic modifications. These modifications included the knockdown of 3 immune rejection-related genes and the insertion of 6 human genes, along with 1 growth gene to regulate the heart size. Revivicor, a regenerative medicine company headquartered in Blacksburg, Virginia (USA), made this groundbreaking achievement possible. Their innovative approach to genetic manipulation has opened new possibilities for overcoming historical challenges in xenotransplantation and has shown potential to revolutionize organ transplantation.

Gene manipulation is essential in eliminating xenoantigens and mitigating the human immune rejection response. For example, the cell surface xenoantigen α-1,3-galactosidase, encoded by GGTA1, represents a critical target. Similarly, cross-species immunity response involves N-glycolylneuraminic acid, encoded by CMAH, and β4GalNT2. Knocking out these genes ensures pig health, minimizing the likelihood of evoking immune responses from human immune cells.³⁹

In a distinct approach, a group of genetically modified pig cells that retain immunoreactivity to human cells may attribute immune responses to swine leukocyte antigen class I, equivalent to human leukocyte antigen class I. Deletion of swine leukocyte antigen genes could enhance host tolerance to pig organs.⁵³ Another avenue to reduce host rejection involves the genetic engineering of complement components and pathways, a concept termed immune cloaking. This manipulation regulates the expression of cell surface molecules from host species in donor cells. Several human proteins, including CD55 (a complement decay-accelerating factor), CD59 (a membrane attack complex-inhibitory protein), CD46 (a complement regulatory protein), and the CD47 signal protein, can downregulate human complement activity, aiding transplants in evading recognition by the human immune system. With continuous advancements in genetic engi-neering technologies, the prospect of eliminating host rejections becomes increasingly promising.

In a groundbreaking medical milestone, the University of Maryland School of Medicine suc-cessfully conducted the world’s first porcine-to-human heart transplant. This remarkable feat involved transplant of a genetically modified pig heart into a 57-year-old man in the advanced stages of heart disease.⁵⁴ The source pig was obtained from a highly secure Revivicor facility. This genetically engineered pig was clonally derived from fibroblasts, incorporating 10 gene edits. These modifications aimed to enhance the cardiac xenograft’s com-patibility for human transplant, a process previously outlined. The gene edits addressed 3 immuno-dominant xenoantigen carbohydrates (galactose-α-1,3-galactose, Sda blood group antigen, and N-glycolylneuraminic acid). The growth hormone receptor was also knocked out to limit intrinsic xenograft growth. The pig expressed human CD46 and decay-accelerating factor to minimize antibody-dependent complement graft injury. Thromboregulatory proteins, including human thrombomodulin and endothelial cell protein C receptor, were introduced to enhance the deficiencies of porcine-derived blood factors in activating protein C. Furthermore, anti-inflammatory proteins CD47 and heme oxygenase-1 were expressed as additional human transgenic proteins.

This highly experimental surgery marked a substantial leap in medical science, as the patient could move freely without the need for cardiopulmonary bypass assistance. The historic operation effectively addressed the major challenge of hyperacute immune rejection, marking a pivotal achievement in over-coming barriers to xenotransplantation. The initial short-term results were promising, showcasing the potential of porcine hearts as a viable option for human transplant. After a successful xenotransplant, the graft exhibited robust function on echocar-diography, sustaining cardiovascular and other organ system functions until postoperative day 47. However, at this juncture, diastolic heart failure surfaced. A subsequent endomyocardial biopsy on postoperative day 50 revealed compromised capillaries marked by interstitial edema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Under electron microscopy examination, it was evident that around 50% of the capillaries exhibited severe endothelial injury characterized by endothelial cell necrosis or pronounced cytoplasmic swelling, accompanied by regions of membrane fragmentation. Concurrently, the neighboring myocytes manifested degenerative alterations, including cytoplasmic swelling, myofi-lament disarray, and focal myocyte necrosis.⁵⁵

Notably, an escalation in anti-pig xenoantibodies was shown, primarily IgG, after intravenous immunoglobulin (IVIG) administration for hypogammaglobulinemia and during the initial plasma exchange. Further insight emerged from an endomyocardial biopsy on postoperative day 56, indicating ischemic myocyte necrosis affecting 40% of the cells and fibrotic changes consistent with progressive myocardial stiffness. The microvasculature exhibited rare microthrombi, and endothelial cells were marked with C4d, IgG, and IgM immunostains. Electron microscopy further revealed that 80% to 85% of the capillaries displayed marked abnormalities characterized by endothelial nuclear enlargement, cytoplasmic swelling, and areas of denuded basal lamina. Microbial cell-free DNA testing detected increasing titers of porcine cytomegalovirus/pig retrovirus cell-free DNA.⁵⁵

However, despite the groundbreaking success, the patient’s health took a downturn, and he passed away in March 2022, just 2 months after transplant surgery.⁵⁴ Postmortem myocardial biopsy findings depicted a similar pattern to that observed in the postoperative day 56 endomyocardial biopsy. Electron microscopy provided a comprehensive view of extensive damage to the capillary network, widespread endothelial cell lysis and fragmentation, and early fibrosis in most interstitial areas. Electron microscopic examination showed no viral particles in any tissue compartment. Postmortem single-cell RNA sequencing illuminated overlapping causes.⁵⁵ Although HAR was averted, Mohiuddin and colleagues discovered potential mediators of the observed endothelial injury. They first concluded that widespread endothelial injury pointed to antibody-mediated rejection. Second, IVIG demonstrated strong binding to the donor endothelium, suggesting a potential cause for immune activation. Finally, the reactivation and replication of latent porcine cytomegalovirus/pig retrovirus in the xenograft were identified as possible triggers for a damaging inflammatory response.

The patient, who had experienced severe hypo-gammaglobulinemia, received exogenous IVIG, a common practice because of the well-documented benefits observed in allotransplant. In response to concerns about the potential contribution of IVIG to endothelial cell damage, 3 distinct lots of com-mercially available IVIG underwent testing with porcine aortic endothelial cells from the donor pig heart (A328.1). The results revealed a pronounced binding affinity between these lots of IVIG and porcine aortic endothelial cells. Although in vitro experiments did not detect complement-mediated cytotoxicity, it is plausible that noncomplement-dependent inflammatory pathways, such as antibody-dependent cell cytotoxicity, might have played a role in endothelial cell destruction.⁵⁵ Interestingly, the patient’s serum exhibited high concentrations of anti-pig IgG, suggesting an exogenous source of IgG. This finding, coupled with increased binding of anti-pig IgG and, to a lesser extent, IgM observed in immunohistochemistry, raised questions about the potential role of anti-pig antibodies in IVIG preparations. The effects of these antibodies on endothelial damage appear to be more important in xenotransplantation than allotransplantation, war-ranting further investigation. These findings underscore the importance of specific measures to enhance xenotransplant outcomes in the future.

Conclusions

The use of xenotransplant involving pig tissues and organs has emerged as a promising alternative to address chronic organ shortages. Despite immuno-logical challenges, advancements in genetic engineering offer potential solutions to overcome rejection barriers and reduce reliance on immuno-suppressive drugs. This review emphasized the complexities of xenotransplantation, mainly focusing on humoral and cellular immune responses. The presence of natural antibodies, such as anti-α-Gal, poses a critical challenge, and genetic modifications in donor pigs are a potential solution. Although adaptive immunity plays a crucial role in late xenograft rejection, strategies targeting specific genes in donor pigs offer a pathway to mitigate T-cell-mediated rejection. Further research and deve-lopments in immunogenetic modifications hold the key to improving the success of xenotransplant, bringing us closer to meeting the global demand of organs needed for organ transplantation.

References:

1. Jagdale A, Cooper DKC, Iwase H, Gaston RS. Chronic dialysis in patients with end-stage renal disease: relevance to kidney xenotransplantation. Xenotransplantation. 2019;26(2):e12471. doi:10.1111/xen.12471
   CrossRef - PubMed
2. United States Renal Data System. Annual data report; 2017. Accessed March 22, 2020. https://www.usrds.org/adr.aspx.
   CrossRef - PubMed
3. Lu T, Yang B, Wang R, Qin C. Xenotransplantation: current status in preclinical research. Front Immunol. 2020;10:3060. doi:10.3389/fimmu.2019.03060
   CrossRef - PubMed
4. Cooper DKC, Gaston R, Eckhoff D, et al. Xenotransplantation-the current status and prospects. Br Med Bull. 2018:125(1):5-14. doi:10.1093/bmb/ldx043
   CrossRef - PubMed
5. Cooper DK, Hara H, Iwase H, et al. Pig kidney xenotransplantation: progress toward clinical trials. Clin Transplant. 2021;35(1):e14139. doi:10.1111/ctr.14139
   CrossRef - PubMed
6. Carvalho-Oliveira M, Valdivia E, Blasczyk R, Figueiredo C. Immunogenetics of xenotransplantation. Int J Immunogenet. 2021;48(2):120-134. doi:10.1111/iji.12526
   CrossRef - PubMed
7. Vadori M, Cozzi E. The immunological barriers to xenotransplantation. Tissue Antigens. 2015;86(4):239-253. doi:10.1111/tan.12669
   CrossRef - PubMed
8. Peloso A, Dhal A, Zambon JP, et al. Current achievements and future perspectives in whole-organ bioengineering. Stem Cell Res Ther. 2015;6(1):107. doi:10.1186/s13287-015-0089-y
   CrossRef - PubMed
9. Fischer-Lougheed J, Gregory C, White Z, Shulkin I, Gunthart M, Kearns-Jonker M. Identification of an anti-idiotypic antibody that defines a B-cell subset(s) producing xenoantibodies in primates. Immunology. 2008;123(3):390-397. doi:10.1111/j.1365-2567.2007.02704.x
   CrossRef - PubMed
10. Cowan PJ, Robson SC, d'Apice AJ. Controlling coagulation dysregulation in xenotransplantation. Curr Opin Organ Transplant. 2011;16(2):214-221. doi:10.1097/MOT.0b013e3283446c65
    CrossRef - PubMed
11. Galili U. Interaction of the natural anti-Gal antibody with alpha-galactosyl epitopes: a major obstacle for xenotransplantation in humans. Immunol Today. 1993;14(10):480-482. doi:10.1016/0167-5699(93)90261-i
    CrossRef - PubMed
12. Galili U. The alpha-gal epitope and the anti-Gal antibody in xenotransplantation and in cancer immunotherapy. Immunol Cell Biol. 2005;83(6):674-686. doi:10.1111/j.1440-1711.2005.01366.x
    CrossRef - PubMed
13. Galili U, Mandrell RE, Hamadeh RM, Shohet SB, Griffiss JM. Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora. Infect Immun. 1988;56(7):1730-1737. doi:10.1128/iai.56.7.1730-1737.1988
    CrossRef - PubMed
14. Lin SS, Hanaway MJ, Gonzalez-Stawinski GV, et al. The role of anti-Galalpha1-3Gal antibodies in acute vascular rejection and accommodation of xenografts. Transplantation. 2000;70(12):1667-1674. doi:10.1097/00007890-200012270-00002
    CrossRef - PubMed
15. Chen G, Qian H, Starzl T, et al. Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys. Nat Med. 2005;11(12):1295-1298. doi:10.1038/nm1330
    CrossRef - PubMed
16. Ierino FL, Sandrin MS. Spectrum of the early xenograft response: from hyperacute rejection to delayed xenograft injury. Crit Rev Immunol. 2007;27(2):153-166. doi:10.1615/critrevimmunol.v27.i2.30
    CrossRef - PubMed
17. Lai L, Kolber-Simonds D, Park KW, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science. 2002;295(5557):1089-1092. doi:10.1126/science.1068228
    CrossRef - PubMed
18. Dai Y, Vaught TD, Boone J, et al. Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol. 2002;20(3):251-255. doi:10.1038/nbt0302-251
    CrossRef - PubMed
19. Byrne G, Ahmad-Villiers S, Du Z, McGregor C. B4GALNT2 and xenotransplantation: a newly appreciated xenogeneic antigen. Xenotransplantation. 2018;25(5):e12394. doi:10.1111/xen.12394
    CrossRef - PubMed
20. Griesemer A, Yamada K, Sykes M. Xenotransplantation: immunological hurdles and progress toward tolerance. Immunol Rev. 2014;258(1):241-258. doi:10.1111/imr.12152
    CrossRef - PubMed
21. Yi S, Feng X, Hawthorne WJ, Patel AT, Walters SN, O'Connell PJ. CD4+ T cells initiate pancreatic islet xenograft rejection via an interferon-gamma-dependent recruitment of macrophages and natural killer cells. Transplantation. 2002;73(3):437-446. doi:10.1097/00007890-200202150-00019
    CrossRef - PubMed
22. Bahr A, Kaser T, Kemter E, et al. Ubiquitous LEA29Y expression blocks T cell co-stimulation but permits sexual reproduction in genetically modified pigs. PLoS One. 2016;11(5):e0155676.doi:10.1371/journal.pone.0155676
    CrossRef - PubMed
23. Nottle MB, Salvaris EJ, Fisicaro N, et al. Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9. Sci Rep. 2017;7(1):8383. doi:10.1038/s41598-017-09030-6
    CrossRef - PubMed
24. Schuurman HJ, Cheng J, Lam T. Pathology of xenograft rejection: a commentary. Xenotransplantation. 2003;10(4):293-299. doi:10.1034/j.1399-3089.2003.02092.x
    CrossRef - PubMed
25. Cooper DKC, Ekser B, Tector AJ. Immunobiological barriers to xenotransplantation. Int J Surg. 2015;23(Pt B):211-216. doi:10.1016/j.ijsu.2015.06.068
    CrossRef - PubMed
26. Puga Yung G, Schneider MKJ, Seebach JD. The role of NK cells in pig-to-human xenotransplantation. J Immunol Res. 2017;2017:4627384. doi:10.1155/2017/4627384
    CrossRef - PubMed
27. Ekser B, Cooper DK. Overcoming the barriers to xenotransplantation: prospects for the future. Expert Rev Clin Immunol. 2010;6(2):219-230. doi:10.1586/eci.09.81
    CrossRef - PubMed
28. Mei HE, Schmidt S, Dorner T. Rationale of anti-CD19 immunotherapy: an option to target autoreactive plasma cells in autoimmunity. Arthritis Res Ther. 2012;14 Suppl 5(Suppl 5):S1. doi:10.1186/ar3909
    CrossRef - PubMed
29. Matter-Reissmann UB, Forte P, Schneider MK, Filgueira L, Groscurth P, Seebach JD. Xenogeneic human NK cytotoxicity against porcine endothelial cells is perforin/granzyme B dependent and not inhibited by Bcl-2 overexpression. Xenotransplantation. 2002;9(5):325-337. doi:10.1034/j.1399-3089.2002.01074.x
    CrossRef - PubMed
30. Cadili A, Kneteman N. The role of macrophages in xenograft rejection. Transplant Proc. 2008;40(10):3289-3293. doi:10.1016/j.transproceed.2008.08.125
    CrossRef - PubMed
31. Shimizu A, Yamada K, Robson SC, Sachs DH, Colvin RB. Pathologic characteristics of transplanted kidney xenografts. J Am Soc Nephrol. 2012;23(2):225-235. doi:10.1681/ASN.2011040429
    CrossRef - PubMed
32. Scalea J, Hanecamp I, Robson SC, Yamada K. T-cell-mediated immunological barriers to xenotransplantation. Xenotransplantation. 2012;19(1):23-30. doi:10.1111/j.1399-3089.2011.00687.x
    CrossRef - PubMed
33. Naeimi Kararoudi M, Hejazi SS, Elmas E, et al. Clustered regularly interspaced short palindromic repeats/Cas9 gene editing technique in xenotransplantation. Front Immunol. 2018;9:1711. doi:10.3389/fimmu.2018.01711
    CrossRef - PubMed
34. Taniguchi S, Neethling FA, Korchagina EY, et al. In vivo immunoadsorption of antipig antibodies in baboons using a specific Gal(alpha)1-3Gal column. Transplantation. 1996;62(10):1379-1384. doi:10.1097/00007890-199611270-00001
    CrossRef - PubMed
35. Dai Y, Vaught TD, Boone J, et al. Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol. 2002;20(3):251-255. doi:10.1038/nbt0302-251
    CrossRef - PubMed
36. Cooper DK, Satyananda V, Ekser B, et al. Progress in pig-to-non-human primate transplantation models (1998-2013): a comprehensive review of the literature. Xenotransplantation. 2014;21(5):397-419. doi:10.1111/xen.12127
    CrossRef - PubMed
37. Mohiuddin MM, Corcoran PC, Singh AK, et al. B-cell depletion extends the survival of GTKO.hCD46Tg pig heart xenografts in baboons for up to 8 months. Am J Transplant. 2012;12(3):763-771. doi:10.1111/j.1600-6143.2011.03846.x
    CrossRef - PubMed
38. Padler-Karavani V, Varki A. Potential impact of the non-human sialic acid N-glycolylneuraminic acid on transplant rejection risk. Xenotransplantation. 2011;18(1):1-5. doi:10.1111/j.1399-3089.2011.00622.x
    CrossRef - PubMed
39. Estrada JL, Martens G, Li P, et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/beta4GalNT2 genes. Xenotransplantation. 2015;22(3):194-202. doi:10.1111/xen.12161
    CrossRef - PubMed
40. Mohiuddin MM, Singh AK, Corcoran PC, et al. Role of anti-CD40 antibody-mediated costimulation blockade on non-Gal antibody production and heterotopic cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon model. Xenotransplantation. 2014;21(1):35-45. doi:10.1111/xen.12066
    CrossRef - PubMed
41. Cozzi E, White DJ. The generation of transgenic pigs as potential organ donors for humans. Nat Med. 1995;1(9):964-966. doi:10.1038/nm0995-964
    CrossRef - PubMed
42. Zhou H, Hara H, Cooper DKC. The complex functioning of the complement system in xenotransplantation. Xenotransplantation. 2019;26(4):e12517. doi:10.1111/xen.12517
    CrossRef - PubMed
43. Liu F, Liu J, Yuan Z, et al. Generation of GTKO Diannan miniature pig expressing human complementary regulator proteins hCD55 and hCD59 via T2A peptide-based bicistronic vectors and SCNT. Mol Biotechnol. 2018;60(8):550-562. doi:10.1007/s12033-018-0091-6
    CrossRef - PubMed
44. Iwase H, Hara H, Ezzelarab M, et al. Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts. Xenotransplantation. 2017;24(2). doi:10.1111/xen.12293
    CrossRef - PubMed
45. Ide K, Wang H, Tahara H, et al. Role for CD47-SIRPalpha signaling in xenograft rejection by macrophages. Proc Natl Acad Sci U S A. 2007;104(12):5062-5066. doi:10.1073/pnas.0609661104
    CrossRef - PubMed
46. Yan JJ, Koo TY, Lee HS, et al. Role of human CD200 overexpression in pig-to-human xenogeneic immune response compared with human CD47 overexpression. Transplantation. 2018;102(3):406-416. doi:10.1097/TP.0000000000001966
    CrossRef - PubMed
47. Tena A, Kurtz J, Leonard DA, et al. Transgenic expression of human CD47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation. Am J Transplant. 2014;14(12):2713-2722. doi:10.1111/ajt.12918
    CrossRef - PubMed
48. Tena AA, Sachs DH, Mallard C, et al. Prolonged survival of pig skin on baboons after administration of pig cells expressing human CD47. Transplantation. 2017;101(2):316-321. doi:10.1097/TP.0000000000001267
    CrossRef - PubMed
49. Hoek RM, Ruuls SR, Murphy CA, et al. Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science. 2000;290(5497):1768-1771. doi:10.1126/science.290.5497.1768
    CrossRef - PubMed
50. Wang Y, Yang HQ, Jiang W, et al. Transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting. Transgenic Res. 2015;24(2):199-211. doi:10.1007/s11248-014-9833-9
    CrossRef - PubMed
51. Phelps CJ, Ball SF, Vaught TD, et al. Production and characterization of transgenic pigs expressing porcine CTLA4-Ig. Xenotransplantation. 2009;16(6):477-485. doi:10.1111/j.1399-3089.2009.00533.x
    CrossRef - PubMed
52. Parisi F, Squitieri C, Marcelletti C. [Heart transplantation in childhood: heredity of Baby Fae]. G Ital Cardiol. 1996;26(3):353-355.
    CrossRef - PubMed
53. Martens GR, Reyes LM, Li P, et al. Humoral reactivity of renal transplant-waitlisted patients to cells from GGTA1/CMAH/B4GalNT2, and SLA class I knockout pigs. Transplantation. 2017;101(4):e86-e92. doi:10.1097/TP.0000000000001646
    CrossRef - PubMed
54. Griffith BP, Goerlich CE, Singh AK, et al. Genetically modified porcine-to-human cardiac xenotransplantation. N Engl J Med. 2022;387(1):35-44. doi:10.1056/NEJMoa2201422
    CrossRef - PubMed
55. Mohiuddin MM, Singh AK, Scobie L, et al. Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report. Lancet. 2023;402(10399):397-410. doi:10.1016/S0140-6736(23)00775-4
    CrossRef - PubMed