**7. Xenotransplant rejection**

Xenorejection is a much more exaggerated and rapid form of the allorejection response. Four overlapping and inter-related reactions occur temporally; hyperacute rejection (HAR), acute vascular rejection/acute humoral xenograft rejection (AVR/AHXR), acute cellular rejection (ACR) and chronic rejection (CR) (**Figure 1**) [22]. Although these processes can be characterized as distinct stages based on histological and clinical data, each is due to highly interconnected pathways and mechanisms that are challenging to separate. In fact, these responses were defined as pathologic observations prior to development of more detailed analyses of the molecular immunology mechanisms.

Husbandry techniques for pigs are extremely well-understood, with large litter sizes and rapid cycle times, allowing production of populations that could overcome organ shortages in a much shorter timeframe than possible with non-human primates. Furthermore, a suite of genome engineering technologies is available for use in pigs to make critical changes to enhance survival and function of the pig organ, while avoiding the human immune rejection response. In fact, the complex engineering approaches now available may actually provide

A variety of academic, clinical and industrial institutions have made substantial progress in recent years in the understanding of the molecular mechanisms of the xenorejection response and the genetic modification of pigs to overcome these mechanisms [18]. Professional organizations are working with the FDA to develop guidelines for clinical use [19], with several groups indicating their intention to initiate clinical trials in the near term with porcine organs [20].

For xenotransplantation to become a viable routine human therapeutic option, there are a number of challenges that still need to be overcome. These challenges fall into two broad categories; the biology of the xenorejection responses and the engineering technologies needed

The immune system is an evolutionarily ancient collection of structures, mechanisms and cells that detect and eliminate harmful organisms from the host. In older texts, the immune system is often described as distinguishing "self" from "non-self," but more recent research demonstrates that there are a variety of roles for the microbiome ("non-self" micro-organisms resident within, on or around the host) in maintaining the health of the host organism. Thus, the host immune system must be able to not only identify and eliminate harmful pathogens, it must also tolerate the presence of a variety of beneficial bacteria, fungi and yeast [21]. Because transplantation of cells, tissues and organs is an unnatural situation created through deliberate medical intervention, the human immune response uses incredible precision to identify even closely related human cells as "non-self" and efficiently removing them, a process referred to as "immune rejection." In general, the strength of the response is proportional to the degree of difference between the host and donor materials, therefore, when exposed to materials from an animal, the rejection response is much faster and stronger, increasing the

Xenorejection is a much more exaggerated and rapid form of the allorejection response. Four overlapping and inter-related reactions occur temporally; hyperacute rejection (HAR), acute vascular rejection/acute humoral xenograft rejection (AVR/AHXR), acute cellular rejection (ACR) and chronic rejection (CR) (**Figure 1**) [22]. Although these processes can be

organs with advantages over even closely-matched human organs.

334 Organ Donation and Transplantation - Current Status and Future Challenges

to restructure the porcine genome to overcome these responses.

**6. The immune system and rejection**

challenge in controlling the immune response.

**7. Xenotransplant rejection**

**5. Current status and challenges for xenotransplantation**

HAR is primarily due to immediate binding of pre-existing host natural antibodies specific for xenoantigens expressed by the donor tissues. Antibody binding can activate the endothelial cells, causing the release of immune activators, as well as inducing the complementmediated destruction of the endothelial layer, reducing the barrier function and allowing host cells to infiltrate the organ. Cell debris released by the damage to the endothelium and products of the complement cascade also stimulate coagulation and the innate inflammatory response. These pathways synergize during rejection to create stronger responses that are more pathogenic and can be less amenable to control.

AVR/AHXR, like HAR, is also mediated by host antibodies. However, instead of pre-existing natural antibodies, AVR/AHXR is often the result of humoral responses which lead to production of antigen-specific antibodies. The AVR/AHXR is delayed due to the time it takes to induce

**Figure 1.** Overview of the xeno-rejection response. The human immune response to xeno-organs initiates within minutes to hours with hyperacute rejection (HAR, upper right), in which pre-existing antibodies (Ab) in human serum bind to xenoantigens (xeno Ag) on the surface of the pig cells. This results in cell destruction and presentation of porcine antigens to human helper (TH) and cytolytic (TC) T cells, as well as groups of pro-inflammatory and pro-immunity cytokines and other soluble mediators. Activation of human helper T cells (TH) stimulates human B cells (B) over the course of days to weeks, resulting in production of induced antibodies (induced Ab) as part of the acute vascular rejection/acute humoral xenograft rejection (AVR/AHXR, lower right) response. These secondary antibodies are often more specific and higher affinity than the pre-existing human serum antibodies, and also cause cellular destruction of the xenograft. in parallel with AVR/AHXR, the acute cellular rejection response (ACR, lower left) is carried out by human NK cells (NK), macrophages (M) and the xenograft-specific cytolytic T cells (TC) recruited to the xeno-organ within days to weeks. The activated cells express a variety of molecules to attack the porcine cells, as well as secrete additional cytokines to recruit more human immune cells. After weeks to months, the human immune response may be again induced to react to the xeno-organ during chronic rejection (CR, upper left), leading to specific antibody (Ab) responses from B cells (B) or cytolytic T cell (TC) destruction. A large collection of cytokines, chemokines, complement and coagulation factors (center) play a key role in regulating the complex set of reactions occurring in every aspect of rejection.

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.

related cells has the potential benefit of identifying and eliminating cells with oncogenic muta-

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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

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

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

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",

tions, preventing tumors before they have a chance to establish themselves [21].

adaptive immune system time to develop more specific responses [24].

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

in regulating the rejection response [25].

**8.2. Inflammation**

function and survival [26].

**8.3. Xenoantigens**

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

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

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 clinical trials.
