**8.4. Coagulation**

are recognized by pre-existing natural antibodies found in human serum. One subset of these antigens is the swine leukocyte antigens (SLA), which are the physical and functional equivalent of the human leukocyte antigens (HLA). Much like the case for human allotransplant, the SLA genes are highly diverse and individual patients will have a variable level of crossreactive antibodies in their serum for a given set of SLA genes [27]. A separate group of xenoantigens are glycan molecules, such as Gal alpha (1,3) Gal and Neu5Gc, which are expressed

Although specific induced antibodies are produced by B cells as part of the adaptive response, the presence of pre-existing antibodies in human serum contributes to the innate response. The specific reasons for the existence of these human natural antibodies are not entirely clear. In the case of glycan structures, one hypothesis is that the molecules are related to those found in pathogens, and that the natural antibodies are cross-reactive to each. Alternately, consumption of porcine materials in the human diet may induce antibody formation. Regardless of the specific source in human serum, xenotransplantion of porcine cells and tissues in humans leads to binding of these pre-existing natural antibodies, activation of complement and even-

Several approaches have been taken to address xenoantigens, including cross-matching donors and recipients for reduced immunoreactivity, removal or modification of the xenoantigen from the donor pig, or the reduction of the ability of the antibodies to induce the complement cascade. In the first case, typing of patients and porcine donors to find the best matches would be very similar to the current system used for determining allotranplant cross-reactivity [29]. Use of gene targeting or editing technologies can eliminate the genes encoding SLA or the enzymes required for expression of the relevant glycan. This has been proven to be highly effective for ablating the genes GGTA1 (the gene encoding alpha 1,3-galactosyltransferase essential for Gal alpha (1,3) Gal), CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase critical for Neu5Gc biosynthesis) and B4GALNT2 (beta 1,4 N-acetylgalactosam inyltransferase). In each case, the elimination of the glycan leads to greatly reduced recognition of porcine cells by natural antibodies in human serum, and reduction in complementmediated destruction [28]. Unfortunately, as the number of antibody targets increases there is a risk that one or more of the xenoantigens alone or in combination may have essential functions which cannot be eliminated without damaging the development or function of the pig. Therefore, efforts to introduce more subtle mutation in SLA which remove immunogenic epitopes while leaving critical antigen-presentation functions intact, or even replacement of

The second approach, which is often used in combination with the first, is to reset the threshold at which the complement cascade is activated, making it more difficult for the binding of human natural antibodies to targets on porcine cells to induce the complement cascade. There are a series of "complement regulatory proteins" (CRPs), such as CD46, CD55 and CD59, expressed on the cell surface which prevent complement activation by the inadvertent non-specific binding of human antibody to human cells [30]. By overexpressing one or more of the CRP molecules on the porcine endothelium, the amount of antibody binding required for complement activation is increased, which reduces the amount of antibody-mediated cell

in porcine, but not human, cells [28].

SLA with HLA, may be more effective.

destruction due to human natural antibodies [31].

tual destruction of target cells carrying the xenoantigens.

338 Organ Donation and Transplantation - Current Status and Future Challenges

Inflammation and vascular leakage, due to loss of endothelial barrier function, both induce coagulation, which normally is required to repair localized endothelial damage. In the case of xenotransplantion, the attack of the endothelium is rapidly occurring at multiple sites, therefore, coagulation spreads throughout the blood vessels in the xeno-organ and can overcome the normal control mechanism. The thrombosis produced by the procoagulant environment leads to occlusion of the vessels within the graft, known as thrombotic microangiopathy (TM). The lack of blood flow results in hypoxia and tissue damage and necrosis, further complicating transplant function. The relatively greater amount of endothelial injury and coagulation in xenotransplant therefore creates more frequent and extensive TM and contributes to the more rapid destruction of the graft [32].

In addition to physiological pathways induced by human innate immune responses, there are non-physiological activities caused by mismatches between porcine and human constituents of the coagulation cascade [33]. For example, porcine von Willebrand factor (vWF) has been shown to bind more avidly to the human GP1b receptor and activate human platelets, leading to coagulation and rapid loss of platelets from the circulation [34]. Ongoing efforts seek to engineer porcine vWF to eliminate the inappropriate interactions with GP1b, while maintaining normal coagulative phenotypes. In addition, porcine proteins which provide positive and negative feedback to control the coagulation cascade do not function as efficiently upon the human coagulation targets, leading to dysregulation of the cascade. The targeting the porcine genome to express human regulatory proteins in porcine cells has been shown to help control human coagulation in response to exposure to the modified porcine materials [35].

#### **8.5. Innate immune cells**

Macrophages and neutrophils are two of the earliest host cell types to infiltrate xeno-organs. Both cell types are instrumental in the phagocytosis and destruction of pathogens during infection. During a xenorejection response, the damaged porcine cells release a variety of DAMPs which are recognized by the human innate cells, inducing phagocytic functions which further damage the xeno-organ and increasing production of additional proinflammatory and other immune mediators which attract more innate immune cells [36, 37].

Similar to the molecular mismatch described above for vWF and coagulation, macrophages express the SIRPA receptor, which must interact with the surface receptor CD47 to prevent the target cell destruction by the macrophage. Thus, the CD47 receptor expressed on the cell surface binds to SIRPA to instruct the macrophage not to consume the target cell. In the case of porcine CD47, the interaction with human SIRPA appears to be unproductive and cannot inhibit the macrophage activity. Expression of the human form of CD47 in porcine cells has been shown to greatly reduce human macrophage activity directed against the porcine cells [38].

NK cells are functionally analogous to cytolytic T cells, and even share some mechanistic pathways for targeted cell destruction. NK cells express a collection of stimulatory and inhibitory receptors on the cell surface, which engage conserved targets on the surface of target cells. The balance of activation and inhibition via combinatorial signaling determines whether the NK cells are stimulated to kill or ignore the target cell. The target cell receptors, such as HLA-E, may be perturbed by pathogens or tumorigenesis, which is detected by the NK cells and the target cells eliminated [39].

complexes, with only a small subset of TCRs binding to a given complex. Should a given CD8 T cell be activated by the HLA-peptide complex, it will express a series of cytolytic molecules which kill the target cell. This system works due to the efficient T cell selection mechanisms applied during T cell development. After initial production of a rearranged TCR, the nascent T cell is tested in the thymus for inappropriate reactivity against cellular antigens. If the T cell survives the selection process, it exits to the body and theoretically will only be activated when it encounters and antigen that does not naturally exist in body, such as a peptide from

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Extracellular antigens can be any molecule taken up by a cell from its environment and degraded in lysosomes intracellularly. The resulting peptides are then loaded onto HLA class II molecules which, unlike HLA class I, are expressed on only a subset of immune-related cells. The class II HLA-peptide complex is recognized by a different T cell subset expressing TCRs with the co-receptor CD4. The CD4 T cell subset also undergoes thymic selection as observed with CD8 T cells, to eliminate recognition of self-antigens [44]. However, CD4 T cells can be induced to create different phenotypes once they specifically recognize class II HLA-peptide complexes. A large variety of T cell subsets have been described, including production of helper T cells, which participate in the activation of B cells for the production of antigen-specific antibodies, or regulatory T cells, which act to inhibit the immune response [45]. The choice of outcomes is driven by the soluble mediators, such as cytokines, found in the local environment, and the collection of co-receptors expressed on the antigen presenting cells. HLA itself is a significant direct contributor to rejection responses outside of its role in antigen presentation. As described above, T cells are selected for lack of recognition of self-antigens. This not only includes the recognition of self-peptides bound to HLA molecules, but of the HLA molecules themselves. Normally, T cells bearing TCRs with inappropriately high affinity for binding HLA molecules, even in the absence of peptide, are eliminated early in T cell development. Because the human T cells have not been exposed to, or selected by, the class I or II swine lymphocyte antigens (SLA), a subset of human TCRs will bind to SLA and induce strong T cell activation, regardless of the peptide presented in the SLA [46]. As porcine cells are attacked by the human immune system, donor peptides are efficiently presented by human cells via HLA to human T cells as part of the normal human adaptive response. Conversely, depending upon the organ transplanted, there can also be donor T cells and antigen presenting cells transferred which result in donor immune responses against the host tissues, referred to as graft versus host disease (GvHD) [47]. In all cases, the immune cells are responding normally, but in the setting of xenotransplantation can be extremely pathogenic

a pathogenic organism, or a mutant peptide from an oncogenic cell [43].

due to the artificially high concentration of immunogenic targets present.

Because HLA matching is part of organ selection in allotransplantation, a frequent question is whether introduction of human HLA in place of porcine SLA would help overcome rejection. Although SLA ablation may be helpful in averting antibody-dependent damage, this approach does not resolve some of the challenges related to antigen presentation in xenorejection responses [29]. It is true that the human T cell binding directly to pig SLA could be eliminated by substitution of SLA with HLA, however, the HLA genes are highly polymorphic, hence the need to HLA match human patients. This means that for a given patient, a donor pig would need to be engineered to specifically express the HLA homologous to that patient, which would be limiting given the timelines necessary for production and validation

In the case of xenotransplantation, the porcine cell receptors, although expressed normally, are not sufficiently well-conserved with their human counterparts and thus cannot inhibit NK cell attack. By expressing on porcine cells the human versions of receptors which stimulate the inhibitory receptors on NK cells, the damage may be averted. With careful genetic modification, the normal mechanisms for detection of infection or other dysfunction may be maintained, allowing normal NK functions while eliminating the xeno-specific destruction [40, 41].

#### **8.6. Resolution of innate immune responses**

There are a variety of mechanisms used to resolve innate immune reactions. Many of the soluble mediators of innate immunity have extremely short half-lives which allows them to dissipate quickly. In addition, immune receptors become increasingly desensitized to further stimulation during the course of the innate response, reducing reactions. A variety of negative regulators are also produced to further inhibit the innate effectors. All of these mechanisms are in place to prevent over-reaction of the immune system and the destruction that it can cause once the pathogenic threat has been eliminated [42]. In the case of a xenorejection response, however, the "threat" that is recognized comes from every porcine cell and thus the innate response is never fully resolved without intervention. It may be possible to take advantage of these resolution mechanisms to create porcine cells with an enhanced ability to curtail or end a human inflammatory response through careful genetic modification.

#### **8.7. The adaptive immune system and xenorejection**

The adaptive immune system is comprised of cellular and antibody components which recognize pathogens and develop highly specific responses, which can increase in specificity and effectiveness over time and exposure. The adaptive immune response also creates immunological "memory" to allow more rapid reactions should similar pathogens be encountered in the future. Because of the time required to develop specific responses, the adaptive immune system generally becomes more critical after the initial innate immune response [21].

#### **8.8. Antigen presentation and T cells**

Antigen presentation is a crucial mechanistic part of the adaptive immune response and plays a major role in the decision between immunity and tolerance for a given target. There are two main routes for antigen presentation to the immune system, reflective of the different classes of pathogen antigens, intracellular or extracellular.

Intracellular antigens, either natural cellular proteins or those derived from viral or bacterial infection of cells, are enzymatically cleaved into peptides which bind to the ubiquitously expressed class I human lymphocyte antigens (HLA). The peptide-HLA class I complex is displayed on the cell surface where it can be surveyed by the binding of cytolytic T cells expressing T cell receptors (TCR) and CD8 co-receptors on the T cell surface. Similar to antibodies, TCRs are assembled combinatorially, creating a diversity of specificities for HLA-peptide complexes, with only a small subset of TCRs binding to a given complex. Should a given CD8 T cell be activated by the HLA-peptide complex, it will express a series of cytolytic molecules which kill the target cell. This system works due to the efficient T cell selection mechanisms applied during T cell development. After initial production of a rearranged TCR, the nascent T cell is tested in the thymus for inappropriate reactivity against cellular antigens. If the T cell survives the selection process, it exits to the body and theoretically will only be activated when it encounters and antigen that does not naturally exist in body, such as a peptide from a pathogenic organism, or a mutant peptide from an oncogenic cell [43].

the NK cells are stimulated to kill or ignore the target cell. The target cell receptors, such as HLA-E, may be perturbed by pathogens or tumorigenesis, which is detected by the NK cells

In the case of xenotransplantation, the porcine cell receptors, although expressed normally, are not sufficiently well-conserved with their human counterparts and thus cannot inhibit NK cell attack. By expressing on porcine cells the human versions of receptors which stimulate the inhibitory receptors on NK cells, the damage may be averted. With careful genetic modification, the normal mechanisms for detection of infection or other dysfunction may be maintained, allowing normal NK functions while eliminating the xeno-specific destruction [40, 41].

There are a variety of mechanisms used to resolve innate immune reactions. Many of the soluble mediators of innate immunity have extremely short half-lives which allows them to dissipate quickly. In addition, immune receptors become increasingly desensitized to further stimulation during the course of the innate response, reducing reactions. A variety of negative regulators are also produced to further inhibit the innate effectors. All of these mechanisms are in place to prevent over-reaction of the immune system and the destruction that it can cause once the pathogenic threat has been eliminated [42]. In the case of a xenorejection response, however, the "threat" that is recognized comes from every porcine cell and thus the innate response is never fully resolved without intervention. It may be possible to take advantage of these resolution mechanisms to create porcine cells with an enhanced ability to curtail

The adaptive immune system is comprised of cellular and antibody components which recognize pathogens and develop highly specific responses, which can increase in specificity and effectiveness over time and exposure. The adaptive immune response also creates immunological "memory" to allow more rapid reactions should similar pathogens be encountered in the future. Because of the time required to develop specific responses, the adaptive immune

Antigen presentation is a crucial mechanistic part of the adaptive immune response and plays a major role in the decision between immunity and tolerance for a given target. There are two main routes for antigen presentation to the immune system, reflective of the different classes

Intracellular antigens, either natural cellular proteins or those derived from viral or bacterial infection of cells, are enzymatically cleaved into peptides which bind to the ubiquitously expressed class I human lymphocyte antigens (HLA). The peptide-HLA class I complex is displayed on the cell surface where it can be surveyed by the binding of cytolytic T cells expressing T cell receptors (TCR) and CD8 co-receptors on the T cell surface. Similar to antibodies, TCRs are assembled combinatorially, creating a diversity of specificities for HLA-peptide

system generally becomes more critical after the initial innate immune response [21].

or end a human inflammatory response through careful genetic modification.

and the target cells eliminated [39].

340 Organ Donation and Transplantation - Current Status and Future Challenges

**8.6. Resolution of innate immune responses**

**8.7. The adaptive immune system and xenorejection**

**8.8. Antigen presentation and T cells**

of pathogen antigens, intracellular or extracellular.

Extracellular antigens can be any molecule taken up by a cell from its environment and degraded in lysosomes intracellularly. The resulting peptides are then loaded onto HLA class II molecules which, unlike HLA class I, are expressed on only a subset of immune-related cells. The class II HLA-peptide complex is recognized by a different T cell subset expressing TCRs with the co-receptor CD4. The CD4 T cell subset also undergoes thymic selection as observed with CD8 T cells, to eliminate recognition of self-antigens [44]. However, CD4 T cells can be induced to create different phenotypes once they specifically recognize class II HLA-peptide complexes. A large variety of T cell subsets have been described, including production of helper T cells, which participate in the activation of B cells for the production of antigen-specific antibodies, or regulatory T cells, which act to inhibit the immune response [45]. The choice of outcomes is driven by the soluble mediators, such as cytokines, found in the local environment, and the collection of co-receptors expressed on the antigen presenting cells.

HLA itself is a significant direct contributor to rejection responses outside of its role in antigen presentation. As described above, T cells are selected for lack of recognition of self-antigens. This not only includes the recognition of self-peptides bound to HLA molecules, but of the HLA molecules themselves. Normally, T cells bearing TCRs with inappropriately high affinity for binding HLA molecules, even in the absence of peptide, are eliminated early in T cell development. Because the human T cells have not been exposed to, or selected by, the class I or II swine lymphocyte antigens (SLA), a subset of human TCRs will bind to SLA and induce strong T cell activation, regardless of the peptide presented in the SLA [46]. As porcine cells are attacked by the human immune system, donor peptides are efficiently presented by human cells via HLA to human T cells as part of the normal human adaptive response. Conversely, depending upon the organ transplanted, there can also be donor T cells and antigen presenting cells transferred which result in donor immune responses against the host tissues, referred to as graft versus host disease (GvHD) [47]. In all cases, the immune cells are responding normally, but in the setting of xenotransplantation can be extremely pathogenic due to the artificially high concentration of immunogenic targets present.

Because HLA matching is part of organ selection in allotransplantation, a frequent question is whether introduction of human HLA in place of porcine SLA would help overcome rejection. Although SLA ablation may be helpful in averting antibody-dependent damage, this approach does not resolve some of the challenges related to antigen presentation in xenorejection responses [29]. It is true that the human T cell binding directly to pig SLA could be eliminated by substitution of SLA with HLA, however, the HLA genes are highly polymorphic, hence the need to HLA match human patients. This means that for a given patient, a donor pig would need to be engineered to specifically express the HLA homologous to that patient, which would be limiting given the timelines necessary for production and validation of genetically-modified pigs. In addition, in the normal situation human cells display human peptides in the HLA, the overwhelming majority of which will be conserved between a human donor and recipient, and thus much less likely to induce a response. If pigs were engineered to express human HLA which is perfectly matched to the patient, the donor porcine cells could now be significantly more efficient at displaying porcine peptides to the human immune system and more rapidly induce T cell activation. Therefore, introduction of human HLA in place of porcine SLA may not provide a benefit without additional engineering.

Mixed chimerism is one route that has shown significant promise in both allo- and xenotransplant settings. This approach combines the transfer to the recipient of both the organ and hematopoietic cells from the donor. Typically, the patient is pre-treated with radiation or drugs to allow hematopoietic cell engraftment prior to the organ transplant. The combination of hematopoietic cells from host and donor allows cross-tolerance of host immune cells to donor tissue as well as donor immune cells to host tissue. Therefore, the resulting immune system is a combination of the donor and host, or a "mixed chimera," which recognizes the

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A further refinement of mixed chimerism includes transplant of donor thymus into the recipient, allowing selection of host T cells via donor antigen presentation [52], suggesting that tolerance is T cell dependent. A large body of evidence points to the role of regulatory T cells (Treg) as a driver of immune tolerance. Treg cells are antigen-specific but upon binding of the specific HLA-peptide complex on antigen-presenting cells will produce a variety of immune inhibiting and tolerogenic factors. The Treg cells may be derived from either thymus selection (central tolerance) or selection in tissues (peripheral tolerance), with central tolerance believed to be more durable, and the conceptual basis for donor thymus transplantation in mixed chimerism [53]. A critical factor in the maintenance of tolerance is the balance between Treg and effector T cells over time. Any imbalance that increases the number of effector T cells can rapidly lead to immune rejection. If indeed the Treg population is the main active component of immune tolerance, then it may be desirable to specifically bolster the numbers of Treg cells transferred to the recipient to more greatly ensure that the balance is biased firmly toward tolerance. A number of groups have established protocols for the generation of Treg cells that are specific for xeno-organs and tissues through *in vitro* selections and expansions [54]. While this has been shown to have positive effects in allograft tolerance, the durability is variable and, worse, some studies have described conversion of Treg to effector T cells which then contribute to rejection [55]. Despite these concerns, mixed chimerism, with or without Treg supplementa-

donor organ and host tissue as "self" despite the differences in genetic origin [51].

tion, remains a potentially valuable approach to immune tolerance.

**10. Genome engineering to improve xenotransplantation**

address the immunological challenges described in previous sections.

The progress of xenotransplantation research in recent times has closely paralleled the advancement in genome engineering technologies. As the complexity of the engineering toolsets has increased, so too has the complexity of porcine genomic manipulations increased to

Complex mammalian genome engineering has advanced much more rapidly in mice than in virtually any other species, including pigs. The reason for the rapid progress in mice is the availability of embryonic stem (ES) cells which can be maintained in culture for extended periods time and undergo extensive transfection/transduction protocols and drug selection without losing the ability to produce large numbers of fertile progeny via blastocyst injection [56]. Although several labs have made strides in this area, similarly manipulable and viable ES cells are not currently available for routine use in the generation of cloned pigs [57].

Humans possess a number of pre-existing antibodies specific for porcine antigens which can contribute to the xeno-organ damage during HAR. As the donor tissue is damaged, the antigens are released and presented to T cells as described above, causing the activation of helper T cells. These T cells interact with B cells in lymphoid organs, inducing the activation of any B cells which express antibodies specific for the xeno-antigens. This initiates the germinal center reaction, in which antigen-specific B cells rapidly proliferate and mutate their antibody sequences and are then progressively selected for improved antibody function. The resulting B cells expressing the affinity-matured antibodies exit the germinal center and can differentiate further to plasma cells, which act as factories that can produce extraordinarily high levels of serum antibody [48]. These induced antibodies, like natural antibodies, further amplify AVR/AHXR and contribute to the destruction of the xeno-organ.

The *de novo* production of antibodies can be quite rapid and are a risk for the lifetime of the transplant whether for allo- or xenotransplantation. There are a number of drugs available for the control of B cell reactions. One of the most effective approaches is the depletion of B cells using antibody therapeutics such as Rituxan, specific for the CD20 surface molecule [49]. However, the constitutive ablation of host B cells will create long term immunosuppression and could be prohibitively expensive. Although highly related to CR in allotransplant, the B cell responses in xenotransplant are stronger and more challenging and likely to require more stringent therapeutic control.
