**8.1. The innate immune system and xenorejection**

The innate immune system is evolutionarily ancient, with related mechanisms found in both plants and animals. The innate immune system consists of relatively invariant mechanisms for the identification of pathogens, and, although less specific, is extremely rapid and strong in response. The rapidity of the innate immune system provides an immediate barrier to pathogen infiltration and infection of the host, limiting the pathogen burden and giving the adaptive immune system time to develop more specific responses [24].

The use of physical barriers is one of the most critical elements of innate immunity. Although organ transplant bypasses the skin as a protective layer, the endothelium of the blood vessels, which connect the organ to the host circulatory system, remains as the main interface between the human hematopoietic system and xeno-organ tissues. As such, many of the immediate mechanisms of the innate response are greatly influenced by the interactions between the human immune cells and the porcine endothelial cells. Once the human innate immune system attacks the porcine endothelium, the barrier function is quickly lost, followed by rapid influx of human immune cells, pro-inflammatory infiltrates and edema, and then necrosis and destruction of the xeno-organ. It is important to note that the endothelium is an extremely active part of the immune response, which responds to soluble factor and cellular interactions to induce a variety of immune and inflammatory responses. Therefore, any efforts to improve the engraftment of xeno-organs must take into account the functional role of the endothelium in regulating the rejection response [25].

#### **8.2. Inflammation**

an adaptive immune response via germinal center reactions, typically days to weeks. Much like natural antibodies, the induced antibodies recognize components of the xeno-organ and, similar to HAR cause activation of the endothelial cells and their destruction via the complement system. The specific antibody binding also attracts multiple elements of the cellular immune system, such as NK cells and phagocytes, creating further damage of the target tissues and secreting soluble factors, such as cytokines and chemokines, which further enhance immune responses. ACR includes predominantly cellular responses to the graft, such as T cell activation, which occur within days to weeks of organ transplant. Although ACR is well-established in allotransplant, the importance of ACR in the xenorejection response is not entirely clear. This may be due to either the more rapid activation of hematopoietic populations in HAR and AVR/AHXR compared with allotransplantation. However, some groups have proposed that reduction of the HAR and AVR/AHXR in earlier stages of xenorejection would unmask ACR which would otherwise be unnoticed amidst the earlier more pathogenic responses. In either case, ACR is expected to be substantially similar between allo- and xenotransplantation and thus more readily controlled by immunosuppressive drugs already in use for allotransplant. CR is longer term, occurring within months or even years after transplantation. CR can be due to complications due to other immune activity, such as infection, or escape of humoral or cellular responses from immunosuppressive drug control. CR is well-understood in allotrans-

336 Organ Donation and Transplantation - Current Status and Future Challenges

plant and effective treatments are available for control and reversal of CR.

reveals the next, but as each layer is removed the overall size may be diminished.

**8. Innate and adaptive immunity in xenotransplantation**

clinical trials.

HAR and AVR/AHXR are the most unique and most critical to address in xenotransplantation. These earlier reactions can greatly enhance later reactions, with some of the mechanistic elements of the xenorejection response initiated even before the transplant surgery itself occurs. Therefore, it is essential to control the initiating events as early as possible in order to reduce the course of later responses. Much like the layers of an onion, removing one layer

The latter two responses, ACR and CR, are mechanistically similar between xeno- and allorejection responses [23]. Use of currently-available immunosuppressive drugs are believed to be able to control both responses as evidenced by the extensive data from allotransplants in humans. However, the speed and violence of the HAR and AVR reactions against xenoorgans can greatly accelerate and strengthen ACR and CR. Thus, even well-established treatments for allorejection may need to be reviewed as xenotransplantation proceeds toward

The immune system has two inter-related arms; the innate and the adaptive immune systems, both of which contribute to the rejection of xenotransplanted cells, tissues and organs. Although often described as separate, the systems have a large network of connections which are interdependent, and thus are not completely distinct. Both systems utilize multiple mechanisms to protect the host, creating a series of defense layers of increasing specificity. When functioning properly, a given layer may not be 100% efficient, but in aggregate will capture the overwhelming majority of pathogens. In addition, the ability to detect subtle differences between highly Inflammation is one of the earliest innate responses, driven by pattern recognition receptors found on human immune cells which recognize damage-associated molecular patterns (DAMPs). The binding and signaling of DAMPs causes the immediate secretion of proinflammatory mediators, such as cytokines and chemokines, which attract additional innate immune cells and induce a variety of local responses which would be highly beneficial during an infection but destructive to xeno-organs. For example, vasodilation and increased vascular permeability, which would normally allow host immune cells greater access to tissue to rapidly eliminate pathogens, instead causes the xeno-tissue to be more quickly infiltrated by human innate immune cells, which in turn leads to more inflammation and destruction. Similarly, there are blood-borne proteinaceous biochemical cascades activated by inflammation, such as the coagulation and the complement systems, which further degrade xeno-organ function and survival [26].

#### **8.3. Xenoantigens**

The genes encoded by the porcine genome can encode proteins that are substantially different from their human counterparts or may carry post-translational modifications which are not present in humans. Interestingly, some of these molecules, referred to as "xenoantigens", 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 in porcine, but not human, cells [28].

**8.4. Coagulation**

more rapid destruction of the graft [32].

**8.5. Innate immune cells**

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

Xenotransplantation

339

http://dx.doi.org/10.5772/intechopen.76502

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].

tory and other immune mediators which attract more innate immune cells [36, 37].

greatly reduce human macrophage activity directed against the porcine cells [38].

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 proinflamma-

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

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

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 eventual destruction of target cells carrying the xenoantigens.

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 SLA with HLA, may be more effective.

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 destruction due to human natural antibodies [31].
