Machine Perfusion of Extended Criteria Donor Organs: Immunological Aspects

Due to higher vulnerability and immunogenicity of extended criteria donor (ECD) organs used for organ transplantation (Tx), the discovery of new treatment strategies, involving tissue allorecognition pathways, is important. The implementation of machine perfusion (MP) led to improved estimation of the organ quality and introduced the possibility to achieve graft reconditioning prior to Tx. A significant number of experimental and clinical trials demonstrated increasing support for MP as a promising method of ECD organ preservation compared to classical static cold storage. MP reduced ischemia–reperfusion injury resulting in the protection from inadequate activation of innate immunity. However, there are no general agreements on MP protocols, and clinical application is limited. The objective of this comprehensive review is to summarize literature on immunological effects of MP of ECD organs based on experimental studies and clinical trials.


INTRODUCTION
The remarkable evolution of solid organ transplantation (Tx) has led to improved overall outcomes for patients with terminal organ dysfunction. However, ischemia-reperfusion injury (IRI) in combination with early immune activation remains a significant challenge limiting the potential of this therapy (1,2). IRI depends on several factors, including primary condition of the graft and length of cold and warm ischemia time (CIT and WIT). It additionally determines the extent of the inflammatory response and increases immunogenicity and the degree of microcirculatory perfusion failure during reperfusion resulting in early allograft dysfunction or primary non-function (3,4). As a link between the degree of IRI and activation of innate immunity (5) has been proposed, the discovery of new treatment strategies including tissue allorecognition pathways (Figure 1) has gained importance, especially in the era of extended criteria donor (ECD) organ Tx. The direct pathway starts with recipient CD4 and CD8 T cells recognizing endogenous alloantigens presented by donor human leukocyte antigen (HLA) molecules on the surface of donor antigen-presenting cells (APCs) after their migration from the graft to the recipient's lymph nodes. This process is initiated by the massive release of pro-inflammatory cytokines from damaged cells during IRI (4). On the other hand, the indirect allorecognition relies on recipient-derived APCs, which ingest, process, and present alloantigens (typically HLA antigens) in the context of recipient HLA, for self-restricted recognition by recipient T cells (6,7). In the semi-direct pathway, recipient APCs acquire donor HLA molecules that present alloantigens directly to recipient T cells (8). Direct allorecognition alone can result in acute rejection, even without indirect mechanisms. Furthermore, depletion of donor immune cells from an organ prior to Tx may prevent rejection (9).
For more than 50 years, static cold storage (SCS) was the gold standard method for organ preservation until the interest in the concept of organ machine perfusion (MP) was renewed (10). To date, a significant number of experimental and clinical trials were published demonstrating increasing support of MP as a more physiologic method of solid organ preservation compared to SCS (11)(12)(13)(14)(15).
MP is a promising tool to reduce the gap between organ demand and supply that is resulting in a dramatic prolongation in waiting times and associated with increased morbidity and mortality for patients on the waiting list for Tx (16). In an effort to counter this trend, organ allografts that would have previously been deemed unsuitable are nowadays more frequently used for Tx (12) including donation after circulatory death (DCD) and ECD (aged ≥ 60 years or aged 50-59 years with vascular comorbidities) organs (12,17,18). Older donor organs have higher immunogenicity, mediated by poorer monocyte clearance of damaged necrotic cells, and therefore recipients may require a more intense immunosuppression in the early period after Tx (19)(20)(21)(22). Knowing about the ECD grafts' increased risk for poor function or failure (23)(24)(25), implementation of new storage techniques, such as MP, paved the way for better characterization of organ quality and the possibility for graft reconditioning before Tx to improve organ vulnerability and immunogenicity (10,26).
Currently, there are no general agreements on MP protocols, and clinical application is limited due to the lack of randomized clinical trials comparing the different MP strategies. The objective of this comprehensive review is to summarize literature on MP of ECD organs and discuss arising immunological aspects based on experimental studies and clinical trials.

MACHINE PERFUSION OF EXTENDED CRITERIA DONOR KIDNEY GRAFTS
It seems that MP for Tx of ECD kidneys is associated with decreased IRI resulting in improved outcome compared to SCS ( Table 1). Whereas most studies on MP in ECD kidneys reported positive effects on the graft, only a few studies reported inconclusive results (40,48).

Hypothermic Machine Perfusion Techniques
A DCD porcine kidney HMP model demonstrated improved graft outcome (27,41,42), particularly concerning the chronic effects of IRI by protecting against chronic immune response by reducing the epithelial to mesenchymal transition (27). Epithelial to mesenchymal transition plays an important role in the genesis of fibroblasts in the course of interstitial fibrosis (27,52). Furthermore, oxygenated HMP showed superior outcome rates compared to non-oxygenated HMP (41). The significantly reduced occurrence of typical signs for chronic graft loss, like chronic inflammation or interstitial fibrosis, confirmed an improvement in recovery from IRI (41). Lately, the use of an extracellular oxygen transporter was investigated. M101 (hemoglobin of the marine worm) was associated with improved effects of HMP upon recovery and late graft outcome, shown by the nearly absent infiltration of mast cells resulting in reduced levels of fibrosis in the kidney (42). Extracellular oxygen carriers may logistically, rheologically, and immunologically be superior to packed red blood cells, but need further investigation. Studies on human DCD and ECD kidneys supported the superiority of HMP over SCS (32,43,45,46). Reznik et al. (43) found a considerably lower number of complications and negative effects, like acute rejection, correlated with HMP kidneys retrieved from DCD donors. Another study in ECD kidneys (Nyberg Score class C or D) demonstrated an association of HMP with lower levels of early inflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-2, and IL-1β] in perfusion solution compared to SCS (32). HMP also affected the expression of hypoxia-related genes [i.e., hypoxiainducible factor (HIF)-1α] (46). This may limit interstitial fibrosis and tubular atrophy, improving long-term outcomes in kidney Tx. ECD kidneys profited most by application of HMP (46).

Normothermic Machine Perfusion Techniques
In a pig study, reduced graft immunogenicity was achieved by initiating an inflammatory cytokine storm [especially IL-6, interferon leading to a donor-derived leukocyte mobilization and removal prior to kidney Tx (1). The authors proposed that migration of donor leukocytes in conjunction with the secretion of an IL-6, IFN-γ, and CXCL-8 storm leads to direct allorecognition and activates the recipient immune response following Tx (1). Short-term NMP of cold-stored human ECD kidneys did not reduce the incidence of acute rejection, while the rate of delayed graft function improved significantly (5.6 vs. 36.2%) (45). More recently, Weissenbacher et al. (49) was able to maintain the quality of ECD kidneys for up to 24 h, hence buying time for viability assessment, improving the feasibility to exploit this important source of donor organs using the NMP technique.
Although the primary results are encouraging, more research focusing on the reduction of immunogenicity of ECD organs is needed.

MACHINE PERFUSION OF EXTENDED CRITERIA DONOR LIVER GRAFTS
Currently, there is no general consensus on the standardized pretreatment of ECD livers in order to improve Tx outcomes (53). Experimental and clinical studies of MP of ECD livers are summarized in Table 2.

Hypothermic Machine Perfusion Techniques
In several studies in DCD rat models, a reduction in IRI in liver tissue was evident after HMP when compared to SCS (28,29,54,56,57,61,62). This finding was confirmed in large domestic animal studies (64,71). Hypothermic oxygenated    perfusion (HOPE) treatment of DCD and severely fatty livers significantly decreased IRI of hepatocytes by reducing the activation of Kupffer and endothelial cells (29,70). Moreover, HOPE successfully suppressed the recipient's immune system, blunting the alloimmune pathway (28,29). This was evident by decreased Kupffer and endothelial cell activation induced by initial anti-oxidative effects and damage-associated molecular pattern (DAMP) release as a consequence of HOPE treatment and liver Tx (28). Furthermore, T cell infiltration in liver grafts as well as blood levels of circulating activated T cells decreased (28). A short time (1 h) of reconditioning of DCD rat and porcine livers using HMP after up to 16 h of SCS showed improvements in organ quality (58,64). Long-term (24 h) HMP of DCD rat livers markedly reduced HLA class II antigen expression on post-sinusoidal venular endothelium compared to SCS (55). Bae et al. (33) found that supplementation of HMP perfusion solution with the antioxidant, vitamin E, reduced inflammatory cytokine levels [IL-6, TNF-α, and monocyte chemoattractant protein (MCP)-1], involved in alloimmune response, in graft tissue. The addition of metformin to HMP preservation solution reduced liver IRI, with significant protective effects on livers, especially in aged rats (69). Furthermore, HMP significantly reduced pro-inflammatory cytokine expression (TNF-α, IL-1β, and IL-8) (34). The attenuation of those cytokines affects many downstream pathways, including a reduced expression of chemokines and adhesion molecules such as intercellular adhesion molecule (ICAM)-1, MCP-1, P-selectin, and others. This effect subsequently decreases the level of neutrophil activation and inevitable leukocyte migration to stressed cell sites, leading to improved overall outcome rates in human livers (34). In another study, HOPE protected DCD livers from initial IRI, leading to improved graft function preventing intrahepatic biliary complications; however, acute rejection rate remained similar (16 vs. 12%) when compared to SCS (75).

Subnormothermic/Normothermic Machine Perfusion Techniques
SNMP and NMP significantly ameliorated hepatic damage in DCD livers compared to SCS in animal models (31,39,60,65,68,72). In a porcine model of liver MP, prolonged periods of NMP facilitate a reduction in hepatic steatosis (63), while the supplementation of perfusate with defatting agents significantly reduced the intracellular fat content of perfused rat livers within a few hours (59). Efficient vasodilation was found to be important in order to improve the effectiveness in the preservation of DCD livers during NMP (66). Olschewski et al. (60) compared HMP to SNMP and SCS, demonstrating beneficial effects on the initial organ function, structural integrity of the sinusoidal endothelium, and hepatocellular damage when DCD rat livers were perfused using SNMP. Furthermore, SNMP was associated with lower IRI when compared to SCS (74), while prolonged NMP additionally eliminated the neutrophil infiltrate in grafts (76). Another study of ECD livers showed superiority of COR over HMP, SNMP, and SCS (38). When comparing NMP to SNMP and SCS, NMP was most efficient in terms of recovery of DCD livers (67). Avoiding initial hypothermia did not improve liver graft quality in a porcine DCD model of NMP (73). Recently, the first randomized controlled trial showed a 50% reduction in liver graft injury, despite a 50% decrease in the number of discarded organs and a 54% increased mean preservation time after a period of NMP compared to SCS (∼36% of grafts were DCD). However, they found no significant difference in bile duct complications, graft, or patient survival (78). The currently ongoing VITTAL trial aims to improve the suitability of non-transplantable livers in the UK by monitoring their function during NMP followed by Tx of the sufficiently improved graft (79,80). We expect that the results of this novel approach could improve consistency and increase the usage of ECD liver grafts without compromising recipient safety.

MACHINE PERFUSION OF EXTENDED CRITERIA DONOR LUNG GRAFTS
Experimental and clinical studies of ECD lungs and MP are compiled in Table 3.

Hypothermic Machine Perfusion Techniques
Short-term HMP could resuscitate ischemically damaged DCD lungs and ameliorate IRI. In a canine model of MP, HMP improved the ATP production by the mitochondrial electron transport chain, leading to a significant decrease in oxidative damage and production of pro-inflammatory cytokines (IL-6 and TNF-α) after reperfusion compared to SCS (81). Moreover, shortterm HMP washed out residual microthrombi in the donor lungs. All of those factors are important for Tx outcomes, including the reduction of the immunological rejection rate.

Normothermic Machine Perfusion Techniques
NMP was able to modulate pro-inflammatory gene expression and reduce pulmonary dysfunction, edema, pro-inflammatory cytokines, and the number of neutrophils in animal DCD lungs (82,83). Moreover, NMP resulted in reduced donor leukocyte transfer into the recipient by inducing mobilization of donor leukocytes into the perfusate and allowing their removal via the leukocyte filter prior to Tx (9). Therefore, reduced donor leukocyte migration to recipient lymph nodes resulted in a reduction of direct allorecognition and T cell priming, diminishing recipient T cell infiltration, the hallmark of acute rejection (9). In a clinical study, NMP showed the capacity to remove donor dendritic cells generating non-classical monocytes, which are directly involved in immune surveillance, from the graft (36). NMP of donor after brain death (DBD) lungs with clinically diagnosed infection significantly reduced bacterial counts in the fluid of the bronchoalveolar lavage and inflammatory injury by decreasing endotoxin levels and key inflammatory mediators [TNF-α, IL-1β, macrophage inflammatory protein (MIP)-1α, MIP-1β] when combined with broad-spectrum antibiotic treatment (35). The administration of mesenchymal stromal cells (MSCs) ameliorated ischemic injury in donor lungs during ex vivo NMP and attenuated the subsequent IRI after Tx (84). The use of MP in reconditioning of ECD donor lungs for Tx is currently under investigation in clinical trials (85,86), with results being expected soon.

MACHINE PERFUSION OF EXTENDED CRITERIA DONOR HEART GRAFTS
Currently, clinical evidence of MP in ECD heart grafts is limited ( Table 4). HMP improved the preservation of DCD heart grafts compared to SCS proven by superior postreperfusion contractility. The underlying mechanisms could include enhanced preservation of the energetic states and superior cellular integrity (87). Recently, Korkmaz-Icöz et al. (88) demonstrated that HMP of aged donor hearts with MSCs protected against myocardial IRI in a rat model.

MACHINE PERFUSION OF EXTENDED CRITERIA DONOR PANCREAS GRAFTS
There is a limited number of studies evaluating the safety and feasibility of ex situ MP for ECD pancreas graft for whole-organ Tx ( Table 5). HMP of porcine DCD pancreas was associated  with a reduction in islet and acinar cell damage, stable perfusion dynamics, and minimal edematous weight change as well as potentially ameliorated endocrine viability and functionality after preservation (89,90). More recent studies in the human pancreas indicated that especially DCD pancreas benefits more from oxygenated HMP compared to SCS alone (91). Even 24 h of HMP of ECD human pancreas-duodenum organs was feasible resulting in no deleterious parenchymal effects (92). Since those studies focused on the results after MP without following Tx, currently, there are no data available about clinical outcomes in this context.

CONCLUSION
MP allows successful utilization of more vulnerable and immunogenic otherwise discarded ECD organs. It has been shown that MP not only reduces the levels of pro-inflammatory cytokines and positively influences gene expression related to hypoxia during reperfusion but also induces donor-derived leukocytes, including dendritic cell-generating non-classical monocytes, mobilization, and removal prior to Tx. Moreover, MP was able to protect against epithelial and Kupffer cell activation and to reduce recipient T cell infiltration of the donor graft. More recently, novel methods such as viral vector delivery during MP to allografts are under investigation (93). This biological modification of the graft prior to Tx may be a future therapeutic strategy to suppress the immune response against the allograft leading to Tx without or at least reduced dose of the systemic immunosuppression that carries the additional risk of infection and malignancy. Many studies have already shown superiority of ECD organ MP over the current standard SCS. However, there are no general agreements on MP protocols, and wider clinical application is limited due to the lack of randomized controlled trials. More trials focusing on immunological pathways in the different MP settings with respect to every single organ are mandatory to get detailed mechanistic insights. This knowledge about various pathways will help us to optimize organ quality after MP of ECD organs and therefore improve Tx outcomes as well as graft and patient survival.