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PLASTIC RECONSTRUCTIVE AND REGENERATIVE SURGERY

Abstract

Summary Emerging, albeit controversial, evidence indicates that machine perfusion (MP) is associated with a lower risk of acute graft rejection following adult liver transplantation (LT). This research paper examines how MP affects the immunogenicity of liver grafts and the recipient’s immune response, potentially leading to reduced rejection rates. Based on experimental data, preserving the functional phenotype of liver sinusoidal endothelial cells (LSECs) is vital for alloantigen-specific immune responses and tolerance mechanisms. LSECs function as critical regulators of immune responses and utilize unique mechanisms to interact with passenger lymphocytes, provide instructional programming signals, and activate local effector functions of T cells. Despite this mechanistic rationale, the influence of dynamic perfusion on the risk of post-LT graft rejection is overshadowed by the heightened sensitivity of cholangiocytes and hepatocytes to ischemia-reperfusion injury, as well as the intricate orchestration of immune-reactive cell populations within the liver microenvironment. Furthermore, the available evidence stems from studies with insufficient power and limited participant numbers, exhibiting considerable variability in designs, methods, and treatments. Additionally, the clinical significance of the MP-associated reduction in liver graft immunogenicity may remain unnoticed due to the widespread use of tacrolimus-based immunosuppressive regimens and the resulting trend toward minimizing immunosuppression.

Abbreviations

ACR: acute cellular rejection

ATP: adenosine triphosphate

APC: antigen-presenting cell

cDCD: controlled DCD

CINC: cytokine-induced neutrophil chemoattractant-1

CIT: cold ischemia time

CRP: C-reactive protein

DAMPs: damage-associated molecular patterns

DBD: donors after brain death

DCD: donors after circulatory death

DGF: delayed graft function

D-HOPE: dual HOPE

EAD: early allograft dysfunction

ECD: extended criteria donors

ENA-78: epithelial neutrophil-activating protein-78

ET: endothelin

FMN: flavin mononucleotide

fWIT: functional warm ischemia time

GM-CSF: granulocyte-macrophage colony-stimulating factor

GRADE: grading of recommendations assessment, development, and evaluation

HBV: hepatitis B virus

HMP: hypothermic machine perfusion

HGMB-1: high mobility group box-protein 1

HOPE: hypothermic oxygenated machine perfusion

IC: ischemic cholangiopathy

ICAM-1: intracellular adhesion molecule-1

IFN-γ: interferon-gamma

IL-1: interleukin-1

IL-6: interleukin-6

IL-8: interleukin-8

IL-12: interleukin-12

IRI: ischemia-reperfusion injury

KC: Kupffer cell

LFA-1: lymphocyte function-associated antigen-1

LSEC: liver sinusoidal endothelial cells

LT: liver transplantation

Mac-1: macrophage-1 antigen

MAIT: mucosa-associated invariant T cell

MIP-2: macrophage inflammatory protein-2

MMF: mycophenolate mofetil

MP: machine perfusion

NADH: nicotinamide adenine dinucleotide-H

NK: natural killer cell

NMP: normothermic machine perfusion

NO: nitric oxide

NRP: normothermic regional perfusion

PAF: platelet-activating factor

PAMPs: pathogen-associated molecular patterns

PD1: programmed death-1

PNF: primary non-function

ROS: reactive oxygen species

SCS: static cold storage

SRR: super-rapid recovery

TAC: tacrolimus

TCMR: T-cell-mediated acute rejection

TCR: T-cell receptor

TGF-β, transforming growth factor-beta

TLR: toll-like receptor

TNF-α: tumor necrosis factor-alpha

TNF-β: tumor-necrosis factor-beta

TXA2: thromboxane A2

uDCD: uncontrolled DCD

WIT: warm ischemia time

INTRODUCTION

Liver transplantation (LT) is one of the most complex procedures in modern medicine, providing a life-saving solution for patients with end-stage liver disease 1. However, this process presents significant challenges, especially in preserving donor organs, preventing transplant-related complications, managing the recurrence of the original disease, and reducing the risk of adverse events associated with immunosuppression (IS) 1. Recent advancements in organ preservation, particularly machine perfusion (MP) techniques, have emerged as promising alternatives to traditional static cold storage (SCS) methods, with the potential to lessen ischemia-reperfusion injury (IRI) and lower the risk of graft dysfunction 2-4 (Fig. 1). Yet, the potential of MP to decrease the risk of allograft rejection is still not fully understood. This research paper examines whether MP affects the immunogenicity of liver grafts and the recipient’s immune response in a manner that reduces rejection rates. Specifically, it examines hypothermic (HMP) and normothermic machine perfusion (NMP) in adult LT involving grafts from brain-dead donors (DBD) or donors after circulatory death (DCD). It also investigates the possible immunological mechanisms behind these effects, such as the modulation of the liver sinusoidal endothelial cell (LSEC) phenotype, the upregulation of a beneficial cytokine profile, the expansion of regulatory T-cell activity, and the reduction of damage-associated molecular patterns (DAMPs). The urgency of this investigation is heightened by the rising demand for donor livers and the reliance on marginal grafts, including those from extended criteria donors (ECDs) and DCDs. These grafts, characterized by their increased vulnerability to IRI due to factors like advanced donor age, steatosis, or circulatory death, underscore the necessity to optimize preservation methods 5. While traditional organ preservation techniques are limited in mitigating IRI, MP provides a dynamic preservation strategy, offering both therapeutic potential and functional assessment of organs before transplantation 5,6. Therefore, MP addresses the need for enhanced preservation methods and has implications for alleviating the organ shortage by increasing the viability of marginal grafts 2-6

The paper synthesizes evidence from the existing literature to provide a comprehensive overview of MP’s role in influencing graft rejection rates. It also identifies gaps in current knowledge and suggests directions for future investigations, particularly emphasizing the need to incorporate immunologic studies into research on MP.

HISTORICAL PERSPECTIVE

The clinical impact of acute rejection

Although most cases of early acute cellular rejection (ACR) have a limited impact on the long-term survival of adult liver graft recipients and can be reversed with increased immunosuppression (IS), several risk factors have been identified, including younger recipient age, HLA-DR mismatch, longer cold ischemic time (CIT), and older donor age; however, their predictive ability varies across studies 7-9. The liver possesses unique immunobiological traits, as it contains numerous resident immune cells, which foster a pro-tolerogenic environment while maintaining effective immune surveillance against infections and cancer cells 10,11. This enables the transplanted liver to both shield itself from the host immune response and decrease alloreactivity against other organs transplanted simultaneously 11.

T-cell-mediated acute cellular rejection (TCMR) is the most common form of allograft rejection, mainly occurring within the first three months after liver transplantation (LT) 10-12. The incidence of TCMR varies from 10% to 30% in studies with clinically driven biopsies and can reach up to 64% in cohorts with protocol liver biopsies 13. Late rejection episodes are usually associated with poor adherence to immunosuppressive medications 11. The occurrence and severity of TCMR have significantly declined in recent decades due to the development of more effective immunosuppressive drugs 10-13. Most TCMR cases are histologically mild, and alloimmune-mediated graft loss is uncommon 10. However, monitoring late episodes of TCMR is essential, as they tend to be more severe and less responsive to high doses of steroids 14.

Static cold storage-associated events

While SCS is the traditional method for preserving liver grafts before transplantation, it has several limitations that can increase the risk of graft dysfunction and rejection 15 (Fig. 1). The basis of this preservation technique is to induce hypothermic conditions that reduce cellular activity, theoretically decreasing the consumption of mitochondrial ATP substrate stores while increasing anaerobic metabolism 15. Although SCS slows metabolic processes and reduces the risk of ischemic injury during the liver’s period without a blood supply, it introduces various physiological challenges that can affect graft quality and increase the risk of rejection 15,16. The most significant risk associated with SCS is IRI (Fig. 1). IRI results from initial oxygen deprivation followed by the restoration of normal oxygenated blood flow to the liver and involves the dysregulation of the healthy phenotype of all liver cellular components, leading to substantial cellular damage, inflammation, and oxidative stress, which may heighten the risk of ACR (Fig. 1) 16,17. Although the liver’s microenvironment is normally hypoxic, it is particularly vulnerable to IRI due to its complex vascular structure and high metabolic demands 17. IRI can cause hepatocyte and cholangiocyte injury, endothelial dysfunction, and activation of the inflammation/innate immune system, shifting to a pro-inflammatory, pro-thrombotic, pro-apoptotic, and vasoconstrictive phenotype, thereby increasing the risk of early graft dysfunction, delayed graft function (DGF), and rejection (Figs. 1,2) 16. DGF may lead to prolonged inflammation and innate immune activation, further increasing the risk of ACR or cholestasis 16-18.

Hepatocyte injury

Most initial changes in anoxic hepatocytes occur within the mitochondria (Figs. 1,2) 19. The absence of O2 immediately disrupts electron flow, causing the respiratory chain to become reduced. As mitochondria can no longer accept electrons from substrates, there is a decline in pyridine nucleotides, leading to an increased intracellular NADH/NAD+ ratio. The disruption of oxidative phosphorylation leads to cellular ATP depletion, accelerated glycolysis, increased lactate production, and alterations in ion balance (H+, Na+, and Ca2+), all of which severely harm hepatocytes. Reperfusion injury primarily results from the generation of toxic reactive oxygen species (ROS) when oxygen is reintroduced to ischemic tissues. ROS are produced from both intracellular and extracellular sources, with mitochondria being the primary source in liver cells 19. In turn, ROS and toxins accumulated in mitochondria during oxygen deprivation are released upon reoxygenation, leading to the breakdown of cellular organelles and the production of DAMPs, such as high-mobility group box-1 protein (HMGB-1). These DAMPs promote inflammation by activating antigen-presenting cells (APCs), including dendritic cells, resident immune cells, and Kupffer cells (KC) 19.

IRI-INDUCED DYSREGULATION OF THE LIVER SINUSOIDAL MICROENVIRONMENT

Liver sinusoidal endothelial cells (LSEC) form the lining of the hepatic sinusoids, lacking an organized basal membrane, with their cytoplasm penetrated by open fenestrations, leading to a discontinuous hepatic microvascular endothelium (Figs. 2,3) 20. LSEC plays a vital role in protecting and regulating vascular homeostasis, inflammation, vascular tone, toxic clearance, and immune response 16,20. LSECs are particularly susceptible to IRI and experience notable changes during cold storage, including disruptions in plasma membranes, vacuolization of the nuclear membrane, and rounding of cell shape 16. These changes contribute to impaired graft microcirculation, platelet activation, persistent vasoconstriction, increased expression of adhesion molecules, oxidative stress, activation of KCs, neutrophil infiltration, and hepatocyte death (Tab. I) 16,21.

Several mechanisms contribute to the disruption of the sinusoidal microenvironment during IRI (Tab. I) 16. The shortage of energetic substrates, combined with an imbalance between low nitric oxide (NO) bioavailability and increased production of endothelin (ET) and thromboxane A2 (TXA2), causes the narrowing of the sinusoidal lumen, leading to microcirculatory dysfunction. The decreased NO levels in the liver result from both reduced production and increased scavenging by higher levels of ROS, which ultimately worsen IRI injury by affecting neutrophil adhesion, platelet aggregation, and contraction of hepatic stellate cells (HSC) 16. Interestingly, the absence of biomechanical stimuli during SCS has been shown to deteriorate LSEC protective phenotype by downregulating the expression of the transcription factor Kruppel-like Factor 2 (KLF2), which regulates the transcription of a variety of protective genes including the endothelial synthase of NO (eNOS), the anti-thrombotic molecule thrombomodulin, or the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) 16.

Activation of inflammation/innate immune response

DAMPs released by hepatic cells trigger the activation of KCs 22,23 (Tabs. I,II, Fig. 2). In turn, activated KCs significantly increase their release of ROS and pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interferon-γ (IFN-γ), and interleukin-12 (IL-12) 24,25. Both TNF-α and IL-1 enhance the expression of Mac-1 (CD11b/CD18) adhesion proteins on neutrophils and stimulate IL-8 production, further promoting neutrophil chemotaxis within the parenchyma 26. IL-1 may stimulate ROS release from neutrophils, which in turn increases TNF-α synthesis by KCs 27. TNF-α promotes the expression of intracellular adhesion molecule-1 (ICAM-1) on the intraluminal side of LSEC, aiding neutrophil rolling, binding, and parenchymal extravasation 27. Additionally, TNF-α induces P-selectin expression in LSEC, which is essential for neutrophil recruitment 28. It has been shown that TNF-α enhances the release of several molecules, including interleukin-6 (IL-6), macrophage inflammatory protein-2 (MIP-2), epithelial neutrophil-activating protein-78 (ENA-78), cytokine-induced neutrophil chemoattractant-1 (CINC), and various CXC motif chemokines (including CXL-1, -2, and -3) 16. Furthermore, IL-1 and TNF-α recruit and activate CD4+ T lymphocytes, which produce granulocyte-macrophage colony-stimulating factor (GM-CSF), INF-γ, and tumor necrosis factor-beta (TNF-β). These cytokines enhance KC activation, facilitating neutrophil recruitment and adhesion to the liver sinusoids 29.

Platelets play a crucial role in IRI and produce and release several factors involved in inflammation, hepatic cell injury, and regeneration (Fig. 2) 30. These include platelet-activating factor (PAF), cytokines, and growth factors – such as nitric oxide (NO), TGF-β, serotonin – and calcium-dependent proteases (calpains). PAF primes neutrophils for ROS generation, further amplifying the neutrophil response 16. Platelets also adhere to hepatic sinusoids and induce programmed LSEC death through NO production, which ultimately leads to the formation of the potent toxicant peroxynitrite (ONOO-) 30. Peroxynitrite is a reactive nitrogen species created by the reaction between nitric oxide (NO) and superoxide anion (O2-). It can modify many biomolecules, including amino acids, proteins, enzymes, and cofactors. Excessive reactive nitrogen species production causes nitrosative stress, leading to structural damage and the loss of cell function, which further amplifies inflammatory and innate immune responses 31.

To note, hepatocytes and LSECs demonstrate differential sensitivity to ischemia, with hepatocytes more sensitive to warm ischemia and LSECs to cold ischemia (Fig. 1) 32. Although most hepatocytes remain viable for 48h after cold preservation and warm reperfusion in rats, LSECs rapidly suffer from severe damage in prolonged cold ischemia (> 6h) (up to 50% of them become non-viable cells) 32. However, considering the intimate cellular crosstalk between LSECs and hepatocytes, prolonged cold storage-induced LSEC injury may adversely impact hepatocyte viability and vice versa. Hepatocytes from cold-stored livers exhibit disturbances in key hepatocellular functions, such as solute transport and drug metabolism, which contribute to liver graft dysfunction 33.

THE IMMUNE FUNCTIONS OF LSECS

LSECs are vital regulators of immune responses in the liver due to their strategic position within liver sinusoids and their large surface area for interactions with passenger leukocytes and other resident immune cells (Fig. 3, Tab. II) 34. Compared to professional APCs like dendritic cells, LSECs use unique mechanisms to interact with passenger lymphocytes, transmit instructional signals, and activate local effector functions of antigen-specific CD8 T cells (Tab. II, Fig. 3). Recognizing these specific immune features of LSECs is crucial for understanding the role of MP-based strategies in modulating liver immune functions.

Four immunologic functions are relevant to the immunomodulatory role of LSECs. They are affected during IRI: immune cell residency, antigen uptake, T-cell activation, and the induction of regulatory or quiescent T cells (Tab. II, Fig. 3).

IMMUNE CELL RESIDENCY

LSECs play a crucial role in liver immune-modulatory functions and exhibit immunomodulatory properties (Tab. II, Fig. 3). The liver sinusoids separate the intravascular space from hepatocytes while providing a platform for various immune cell populations to reside in the liver 34. Consequently, specialized natural killer (NK) cell populations, innate lymphocytes – including so-called MAIT (mucosa-associated invariant T) cells – and myeloid cells (such as neutrophils and Kupffer cells) adhere to the surface of LSECs to establish liver residency 34. The expression of chemokine receptors CXCR6 and CX3CR1 appears to facilitate immune cell retention in the liver sinusoids, working alongside the production of CXCL16 and CX3CL1 by LSECs 35. Since these receptor-ligand interactions are not unique to the liver, it remains an open question whether there are liver-specific recruitment processes 34.

SCAVENGER FUNCTION

LSECs have endocytic uptake functions and surpass professional APC populations, such as dendritic cells or myeloid cells, in their ability to uptake circulating antigens (Fig. 3, Tab. II) 36. Additionally, the low perfusion pressure in the liver sinusoids facilitates the endocytic uptake of blood-borne molecules by LSECs. LSECs also express pattern recognition receptors, such as Toll-like receptors (TLRs), located on the plasma membrane at the cell surface or within endocytic compartments. This highly efficient scavenger function, combined with TLR expression, enables LSECs to act as sentinel cells, instructing parenchymal hepatocytes to initiate the liver’s acute phase response through IL-6 secretion. In turn, IL-6-activated hepatocytes enhance systemic innate immune effector functions by supplying increased levels of complement, C-reactive protein (CRP), and other acute-phase proteins 34.

INHIBITION OF T-CELL ACTIVATION AND INDUCTION OF REGULATORY/QUIESCENT T CELLS

Beyond scavenger receptors, LSECs also play a key role in tolerance induction through C-type lectin receptors such as L-SIGN and LSECtin (Fig. 3, Tab. II) 37,38. These receptors participate in the uptake of molecules containing mannose residues and aid in the removal of pathogens from the bloodstream. The T-cell surface molecule CD44, found on activated T cells, has been identified as a natural ligand for LSECtin. The interaction between LSECtin and CD44 inhibits T-cell activation, proliferation, and effector function, emphasizing the importance of LSECtin expression in regulating local T-cell responses.

LSECs also inhibit the antigen-specific activation of CD8 T cells in the liver (Fig. 3, Tab. II) 34,39. T-cell activation by antigen-presenting dendritic cells is suppressed when LSECs are in direct contact with T cells 34. This role of LSECs on other APC populations in the liver can be understood through the diversion of the crucial signaling molecule lymphocyte function-associated antigen-1 (LFA-1) on T cells away from the antigen-presenting cell to LSECs, thus depriving the immunological synapse of a key signal component 40. Therefore, it is likely that these two veto mechanisms (i.e., the interaction of LSEC with CD44 and LFA-1) prevent local antigen-specific activation of CD8 T cells and the execution of effector functions 34.

Because LSECs express only low levels of co-stimulatory molecules and do not produce IL-12, which is essential for inducing effector functions in cells, they do not promote the differentiation of naive CD4 T cells into helper CD4 T cells (Fig. 3, Tab. II) 41,42. The outcome of antigen-specific stimulation of naive CD4 T cells by antigen-presenting LSECs, through transforming growth factor-beta (TGF-β) or Notch-dependent signaling pathways, is their differentiation into T cells with regulatory potential 34,43. It is important to note that the liver does not contain many regulatory T cells, identified by the transcription factor Foxp3; instead, these cells are recruited during inflammation by similar chemokines that attract effector T cells 44. However, LSEC-induced regulatory T cells may not always express Foxp3, which could lead to an underestimation of the number of regulatory T cells in the liver 45.

Finally, in the slow flow of liver sinusoids, the recruitment of antigen-specific naïve CD8+ T cells by LSCE cross-antigen presentation often leads to the formation of quiescent T cells (Fig. 3, Tab. II) 46. LSEC-induced retention of antigen-specific MHC-I–restricted CD8+ T cells begins a complex dialogue resulting in mutual activation, characterized by increased expression of MHC I and B7H1 on LSECs and PD1 on CD8 T cells 47. Co-inhibitory signaling through the B7H1-PD1 axis causes CD8 T cells to revert to a quiescent state 47. In this state, LSEC-activated CD8 T cells do not perform effector functions, secrete cytokines, or display cytotoxic activities against tumor cells 18.

MACHINE PERFUSION IN LIVER TRANSPLANTATION

HYPOTHERMIC PERFUSION

MP has emerged as a significant advancement in LT, offering a transformative approach to organ preservation and optimization 49,50. Techniques used in studies of ACR include HMP (both HOPE and D-HOPE), NMP, and normothermic regional perfusion (NRP). HOPE is recognized for its ability to improve graft preservation by reducing IRI and maintaining mitochondrial function 50-64. By lowering oxidative stress and inflammation, HOPE effectively mitigates the harmful effects that often compromise graft functionality 50-58, particularly in the context of DCD liver grafts 58. This technology is especially beneficial for ECD livers, which are more vulnerable to IRI due to factors like advanced donor age and steatosis 59. HOPE’s mechanism of preserving mitochondrial integrity while decreasing ROS production may also directly and indirectly influence the risk of post-transplant ACR. By interrupting the cascade connecting innate and adaptive immune responses, HMP could alter antigen-specific effector T-cell responses, potentially making the liver graft less reactive or more tolerogenic. Additionally, the targeted regulation of metabolic activity during preservation helps maintain energy balance and improves the graft’s postoperative performance. Liver graft dysfunction early after transplant is a known factor that increases the risk of ACR 7,12. Although the data on HOPE’s impact on LT outcomes is promising for reducing graft dysfunction and ischemic cholangiopathies 56-58, a more detailed understanding of its effect on post-transplant ACR remains a critical area for further research, along with investigating how donor quality (DCD versus DBD), recipient health, and perfusion protocols influence current clinical outcomes.

NORMOTHERMIC PERFUSION

NMP offers additional benefits by keeping the donor liver in a metabolically active state 49,65. This method enables real-time assessments of viability, therapeutic interventions, and improved tissue preservation 65-68. By maintaining continuous metabolic activity, parameters such as bile production and enzyme levels can be monitored, which helps determine the liver’s functional viability before transplantation and lowers the risks associated with using suboptimal grafts 49. NMP’s ability to support glycogen repletion, regenerate ATP stores, and stabilize coagulation pathways by reducing hyperfibrinolysis leads to better post-transplant results, especially with marginal grafts 67. Its capacity to maintain normothermic conditions minimizes immune activation and may improve rejection rates 49. However, like HOPE, there is a need for standardized protocols to ensure consistency and reproducibility, particularly when applying specific therapies such as stem cell delivery or anti-inflammatory agents 67,69-71.

SEQUENTIAL HYPOTHERMIC AND NORMOTHERMIC PERFUSION

The integration of sequential HMP and NMP offers an alternative method for preserving ECD livers 72,73. This approach combines the advantages of both hypothermic and normothermic conditions, tailored to the specific preservation needs of high-risk grafts 73. Laboratory analyses show that enzyme levels (AST, ALT), bilirubin, and graft function remain stable across groups using advanced perfusion techniques, demonstrating the reliability and safety of these methods in transplant applications 73. Sequential approaches improve graft preparation by applying different perfusion conditions, potentially reducing early allograft dysfunction (EAD) and enhancing post-transplant survival 73. While promising, challenges related to implementing sequential methods, such as the technical complexities of dual-system setups, must be addressed to enable broader clinical adoption and research on the immunologic benefits they provide.

NORMOTHERMIC REGIONAL PERFUSION

The role of NRP in reducing the risk of post-transplant ACR has been addressed only minimally. The need to lower the higher complication rates associated with transplantation from DCD liver grafts, especially when using uncontrolled DCD (uDCD), has led to the adoption of stricter donor and graft selection criteria in some contexts and the use of NRP in others to overcome the limitations of super rapid retrieval (SRR) 74,75. Many retrospective studies have shown that NRP treatment effectively prevents IC compared to SRR 76, but randomized trials are lacking. A recent retrospective, observational cohort study compared LT from controlled DCD (cDCD) donors recovered by NRP (=106) vs SRR (136) 77. EAD was higher in the SRR group (56.1% vs 36.4%; p = 0.007), while IC-free survival improved in NRP recipients, and patient and graft survival were similar between groups 77. However, the inflammatory and immunomodulatory effects of NRP alone or combined with hypothermic perfusion techniques before and after SCS have been limitedly studied 78. In a rodent LT model, DBD livers exhibited minimal inflammation during the 2-hour NRP period. In contrast, DCD livers with 30- and 60-minute donor WIT experienced greater mitochondrial damage and inflammation, indicated by increased perfusate lactate, flavin mononucleotide (FMN), and high mobility group box protein-1 (HMGB-1) levels, leading to subsequent TLR activation during NRP. D-HOPE, compared to NRP, resulted in significantly less mitochondrial complex I injury and inflammation 78. Overall, these findings indicate that DCD donation is linked to mitochondrial and DAMP-related inflammatory injuries in grafts, which can be partially reversed when O2 is administered hypothermically before organ procurement 78.

DONOR TYPE

Both DBD and DCD trigger systemic inflammatory responses through different pathways and can cause organ injury and dysfunction 79,80. Brain death is associated with a systemic inflammatory response like sepsis-induced cytokine release, marked by elevated levels of pro-inflammatory cytokines, including IL-6, IL-8, and IL-10, compared to DCD donors 81. The duration of brain death may influence the severity of the inflammatory response; however, longer durations do not necessarily lead to more severe inflammatory reactions in abdominal organs 80. Brain death also activates the complement and coagulation cascades, leading to a pro-thrombotic state that can further affect graft survival 79-81. The implications of brain death on the risk of post-transplant immune activation are mainly mediated by SCS, with prolonged cold ischemia time (CIT), older donor age, and liver graft steatosis contributing to cell injury and the activation of native and acquired immune responses 7-9.

DCD is generally associated with lower pro-inflammatory gene expression and cytokine activation, leading to a milder inflammatory response compared to DBD 82. However, DCD may impact organ viability due to the activation of the IRI cascade associated with prolonged warm ischemia times (WIT) when compared to DBD. Given the heightened sensitivity of cholangiocytes to oxygen deprivation overall, and WIT, compared to hepatocytes and LSECs, the introduction of MP has provided clinical benefits in patients at high risk of post-transplant biliary lesions 58.

Both HMP and NMP can reduce inflammatory pathways caused by DBD and DCD. However, the complexity of current clinical situations may explain the conflicting evidence about which technique is better at lowering the risk of post-transplant ACR in liver grafts (Tab. III). Additionally, the choice of MP strategy is often based on the transplant center’s experience and primarily aims to improve early graft dysfunction and protect the biliary tree from injury 83.

CRITICAL APPRAISAL OF LITERATURE DATA

Despite existing evidence on the benefits of both HMP 56,58 and NMP 65 regarding specific LT outcomes, particularly in the context of ECD and DCD 58, published studies exhibit significant heterogeneity, as previously discussed in surveys 83, systematic reviews 84, and meta-analyses 56,84. There is notable variability in indications related to donors and recipients, as well as in techniques (such as hypothermic, normothermic, or sequential combination) and technical parameters (perfusion pressure, flow, duration, perfusate composition, and oxygenation regimen), highlighting the need to determine the best scenarios for each method 83. Moreover, the assessment of graft viability, utilization criteria, and post-transplant outcome measures remain subjects of ongoing debate 83. Importantly, ACR has seldom been investigated in most reports 84, mainly due to reduced interest in liver graft rejection in clinical practice, the availability of advanced immunosuppressive drugs, and the relatively limited impact of ACR on graft and patient outcomes 11. Finally, unlike immunosuppressive medications, whose clinical use depends heavily on pre-clinical research, MP has often been adopted by LT centers worldwide based on clinician discretion before the completion of sufficiently powered randomized trials 56. As a result, evidence on MP mainly derives from single-center series, with only a small fraction of published experiences coming from randomized controlled trials (RCTs) in the international literature 56,84-89. Even among RCTs, the risk of bias – stemming from donor and patient selection, the randomization process, deviations from intended interventions, missing outcome data, outcome measurement, and the selection of reported results– can vary considerably 58,84-89.

THE IMMUNOLOGICAL EFFECTS OF MACHINE PERFUSION AND THE REPORTED EVIDENCE

The current body of research provides strong evidence that MP technologies, including HOPE, D-HOPE, and NMP, offer significant advantages in LT. These methods not only improve donor liver preservation but also enhance recipient outcomes by reducing IRI and IRI-related complications like EAD and IC. However, the clinical evidence on the role of MP in modulating post-LT antigen-specific immune responses still requires more robust data (Tab. IV, Fig. 4).

To date, no adequately powered study on the risk of ACR in adult liver transplantation (LT) has been published in the literature. Therefore, the following considerations regarding the role of MP in reducing the risk of liver graft rejection are presented in a narrative format. The main mechanism behind HOPE’s biological effects involves mitigating or reversing mitochondrial damage caused by oxygen deprivation and reinfusion. This leads to increased cellular energy storage and decreased accumulation of succinate and nucleotide metabolites 51. Additionally, it lowers the release of ROS, pro-inflammatory cytokines, and DAMPs, ultimately reducing the severity of IRI metabolic cascades (Tab. IV, Fig. 2) 51. As demonstrated in experimental models of rodent LT, reduced activation of the innate immune response extends to the adaptive immune system, evidenced by lower expression of costimulatory signals such as CD40 on Kupffer cells (KCs) and CD80 on liver sinusoidal endothelial cells (LESCs), fewer activated CD4+ T cells (CD154+ and CD28+ T cells), and decreased levels of pro-inflammatory IL-2 and IL-17 52. In an experimental rat model of LT rejection, 1-hour HOPE combined with low-dose tacrolimus (TAC) successfully prevented ACR at 4 weeks, similar to full-dose TAC 90. However, this model faces challenges in applying findings to clinical settings, as the proposed full-dose (1 mg/kg) and low-dose (0.3 mg/kg) TAC regimens are ten times higher than those used in human LT 90.

A recent study by Elgosbi et al. compared 14 patients treated with MP to 13 patients who underwent SCS and participated in two randomized controlled trials assessing SCS against HOPE (the HOPE and D-HOPE trials, which used either DBD or DCD, respectively) 91. Patients who received livers preserved with SCS showed a significantly higher incidence of early ACR (i.e., within the first month after LT) compared to those who received livers treated with HOPE (46% vs 7%, p = 0.03) 91. Using intracellular cytokine staining and an activation-induced marker assay to simultaneously count antigen-specific effector and regulatory T cells (Tregs) 92, the authors showed that HOPE-treated patients had an increase in CD4+Foxp3+CD127lo cells two weeks after transplantation, due to the preferential expansion of alloreactive Treg clones. This explained the reduced expansion of alloreactive CD8+ T cells seen in the HOPE group compared to recipients of SCS livers 91. The transcriptomic analysis (RNAseq) on liver tissue samples from HOPE-treated grafts revealed upregulation of genes related to cellular responses to heat and hypoxia, as well as overexpression of genes involved in acute inflammatory responses, T-helper cell differentiation, and neutrophil homeostasis and angiogenesis 91. After reperfusion, HOPE was also linked to the downregulation of genes that induce oxidative stress and apoptosis 91. In contrast, SCS-treated livers showed downregulation of genes related to the positive regulation of metabolic processes 91.

Besides the numerical limits of the recruited population, several issues suggest that these data should be interpreted carefully. The incidence of ACR in SCS-treated patients is somewhat higher (46%) than reported in cohorts with clinically diagnosed rejection 11. The trials’ primary endpoints did not emphasize ACR, and the patient count was not based on statistical hypotheses regarding ACR risk. While the diagnosis of ACR depended on histology and the Banff classification system, the criteria for liver biopsies were not specified, and data on rejection severity were not provided. Additionally, the IS protocol referenced by the authors included variability, such as the use of basiliximab or mycophenolate mofetil (MMF) for patients with worsening renal function, which could have affected results. The two patient groups had similar median TAC exposure during the first two weeks post-transplant, but TAC exposure levels in rejecting patients were not compared to those without rejection. Lastly, despite expressing favorable tolerogenic transcriptomic signatures based on methods from a previous publication 92, the authors did not investigate any evidence of clinical tolerance – at least operational – over a median follow-up of 4.6 years 91.

A recent systematic review by Maspero et al. reported a reduced incidence of ACR after MP compared to SCS by combining data from eight international studies, which included six studies on HOPE and one each on NMP and NRP (Tab. III) 93. In the pooled analysis, the overall ACR rate was 11% (95% CI, 7%-15%) after MP and 18% (13%-24%) after SCS, which was statistically significant 89. ACR was significantly lower after HOPE compared with SCS (OR: 0.54, 95% CI, 0.29-1, p = 0.05) and overall after MP compared with SCS alone (OR: 0.55, 95% CI, 33-91, p = 0.02) 93. In a subgroup analysis of studies that included only DCD grafts, the ACR rate was significantly lower with MP compared to SCS (OR: 0.43, 95% CI: 0.20-0.91, p = 0.03). This effect was also maintained when considering only studies on HOPE with DCD grafts (OR: 0.37, 95% CI, 0.14–1, p = 0.05) 93.

Although compelling, the paper has faced criticism. Liang et al. pointed out that the systematic review missed several studies, including one HOPE study, three NMP studies, and three involving NRP, despite accounting for the data extraction time 94. Although there was considerable variability in study designs, donor types, and modes of MP, the incidence of ACR in the studies not included in the review was higher than SCS in one HOPE study and two NMP studies 94. Neil et al. 95 reported that their prior experience with NMP for repeat LT might have been included in the systematic review except for one patient, and their results contrasted with those of Maspero et al., with NMP patients experiencing ACR in up to 61.9% of cases 96. Given the characteristics of the included studies, Gu et al. recommended using trial sequential analysis to determine whether the number of participants and trials was adequate to draw conclusions and to enhance the review with an assessment of the evidence level according to the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) 97.

Inspired by the findings of Maspero et al., Liang et al. presented the results of a further systematic analysis, which included 7 HOPE studies, 4 NMP studies, and 4 NRP studies (Tab. III) 98. Their work indicated that HOPE and NRP, but not NMP, may be linked to a reduction in ACR compared to SCS, with this effect observed only in DCD grafts 98. HOPE did not demonstrate protective effects against decreasing ACR in the ECD-DBD or DBD subgroups, while NMP showed no advantages for ACR in DCD 98.

All the reviews published so far in the literature feature small sample sizes and significant heterogeneity in study design, organ perfusion protocols, clinical characteristics, and perioperative management (Fig. 4) 93-98. Since the original studies were not designed or powered to detect ACR, the diagnosis of ACR was often unclear, complicating the reporting of ACR events. Only a few studies reported ACR, and the diagnostic criteria varied without a standard biopsy-proven ACR. Therefore, conclusions about the reduction of ACR after LT by MP should be drawn cautiously (Fig. 4) 98.

BALANCING SCIENTIFIC EVIDENCE AND CLINICAL SIGNIFICANCE

Although the basic mechanisms of ACR in liver grafts treated with SCS have been extensively described – especially regarding the modes of alloantigen presentation (direct versus indirect), the role of liver-resident APCs and LSECs, and the chemokine environment that supports both innate and adaptive immune responses in the liver sinusoids 11 — the pathogenesis of ACR in liver grafts treated with MP remains poorly understood 98. Based on the available evidence in the relevant literature, we propose that ACR should be comparable in SCS-treated and MP-treated grafts. However, this assumption remains unproven, as essential information on the type, severity, and mode of ACR presentation is lacking across studies on MP 93-98. This can be attributed to the reduced impact of ACR on liver graft prognosis in the current era of TAC-based regimens, along with the increasing focus of MP studies on the mechanisms of graft dysfunction. Considerations regarding MP’s role in modulating graft and recipient alloreactivity are rooted in acquired knowledge of IRI-associated inflammation and the subsequent activation of an antigen-specific T cell-mediated immune response, which may then worsen the ACR cascade 99. However, MP also contributes to changes in the mechanistic processes of IRI that require further analysis 98,100.

GRAFT IMMUNODEPLETION AND BLUNTING OF INFLAMMATION – GREATER BENEFITS FOR THE BILIARY COMPARTMENT

Unlike in SCS, where grafts are immersed in perfusion solutions and minimal passenger (i.e., graft-derived) immune cell circulation is typically expected, in dynamic perfusion techniques, inflammatory passenger cells circulate within the graft depending on the cell type (mononuclear cells, T cells, B cells, NK cells, etc.), the graft (e.g., kidney versus liver), the perfusion method (HMP versus NMP), MP’s pressure and flow settings, the duration of perfusion (short versus prolonged), and the use of blood, nutrients, and O2 carriers 100,101. In experimental studies of ex vivo lung and kidney perfusion, many passenger leukocytes mobilize and extravasate into the perfusate, affecting (i.e., downregulating) graft immunogenicity 100-102. Data indicating the mobilization of passenger leukocytes into the perfusate have also been published in liver NMP 103,104. The dynamics of inflammatory cell mobilization in kidney grafts during NMP exhibit significant cellular diapedesis of T cells, B cells, NK cells, and monocytes from the kidney into the circuit during perfusion, accompanied by high concentrations of IFN-γ and cell-free donor DNA 100. In the context of LT, the migration of donor passenger T cells from the donor liver allograft into the recipient’s circulation was demonstrated before the clinical use of MP 105.

MP techniques have much greater potential for mobilizing liver-resident inflammatory cells into the perfusate than SCS. This has been demonstrated for T cells and neutrophils during HOPE 55 and NMP 106. Recent single-cell transcriptome profiling of human donor livers before, during NMP, and after transplantation revealed a significant efflux of passenger leukocytes, mainly neutrophils, in the perfusate 107. Additionally, during NMP, neutrophils shift from a pro-inflammatory state to an aged, chronically activated, and exhausted phenotype, while anti-inflammatory and tolerogenic monocytes and macrophages increase 103. Along with O2 supplementation during perfusion, maintaining the mitochondrial respiration chain, and reducing ROS-driven inflammatory stimulation upon graft reperfusion, the decreased number of liver-resident T cells has been reported to downregulate both innate and adaptive immune responses, leading to less post-transplant cholangiopathy 55 and – presumably – a reduced risk of post-transplant ACR 93,100.

The major liver compartments (i.e., hepatocytes, biliary epithelium, and sinusoidal endothelium) exhibit different sensitivities to O2 deprivation and inflammatory injury, with cholangiocytes being more vulnerable than both hepatocytes and LSECs 32,33,107. As a result, the effect of MP on inflammatory cell mobilization and their transition to an exhausted phenotype is more clearly seen in post-transplant cholangiopathy than in reducing ACR across various donor risk levels 98. This is particularly true for high-risk grafts, especially those involving DCDs (uDCS >> cDCDs), extended donor fWIT, elderly livers, steatosis, and prolonged CIT. All these factors significantly affect the viability of cholangiocytes, followed by hepatocytes 55.

Finally, the current evidence on MP’s effects on IRI, immune cell mobilization, priming, and graft metabolic processes lacks the organ-organ and organ-system crosstalk observed in the recipient’s body. Interactions between multiple organs are essential to ensure the viability and homeostasis of physically separated organs 108. To date, the interactions between grafts and lymphoid organs are not reproducible during dynamic perfusion, yet they play a crucial role in the mechanisms of rejection and tolerance 109.

THE COMPLEXITY OF THE CLINICAL SCENARIO

Despite existing evidence about the role of sinusoidal endothelium and the LSEC phenotype in the liver’s immunomodulatory and tolerogenic profile, the impact of MP on the risk of ACR is likely overshadowed by several factors (Fig. 4). These include the significant role of MP in enhancing cholangiocyte and hepatocyte viability 2-6, the complex orchestration of immune responses – both rejection and tolerance – within the liver graft 7-13, as well as the use of more potent immunosuppressants (TAC-based) 14. Although crucial, the role of LSECs in regulating alloantigen presentation, recognition, and response is not exclusive 34-40; various liver cell populations contribute to innate and adaptive immune responses, influencing the development of graft tolerance 41-48. The widespread use of TAC-based IS has reduced the frequency and severity of rejection episodes, facilitating the adoption of minimization protocols to mitigate calcineurin-inhibitor-related adverse events 14. This has resulted in significant variability in immunosuppressive regimens, shifting from fixed-dose protocols to schedules tailored to individual patient rejection risk profiles 14. Similarly, the broader application of MP has been accompanied by considerable variability in dynamic perfusion techniques, with different practices varying in the type of MP (e.g., HMP versus NMP), duration, pressure, flow regimens, and their combinations 93,98. Finally, current understanding suggests that liver graft tolerance is an organ-specific process achieved through antigen-specific stimulation of liver-resident immune-responsive and immune-modulatory cell populations, along with a specific chemokine microenvironment 45-48. In this context, persistence, rather than elimination, of a biological inflammatory signature is necessary to promote tolerance 45-48.

PROPOSED IMMUNOLOGIC MECHANISMS OF MP

Based on experimental and clinical evidence, the proposed inflammatory and immunologic mechanisms associated with MP are illustrated in Table IV, along with unresolved issues that require clarification for a critical appraisal of the impact of dynamic perfusion on the post-transplant immune response. Exploring these endpoints in clinical practice is challenging due to significant variability in the use of MP and the complexity of current clinical scenarios. Given the low number of RCTs published in the international literature compared to cohort studies and single-center experiences, a preliminary step would be to include protocol biopsies for ACR detection in all studies on MP, focusing on the modification of the LSECs phenotype, T-cell effector functions, and the induction of Treg populations. This requires multidisciplinary involvement of all professionals engaged in LT care.

Several limitations temper the conclusions drawn from this research (Tab. IV). A key constraint is the reliance on secondary data, which introduces variability in donor characteristics, recipient profiles, and perfusion protocols among the studies analyzed 93,98. Methodological inconsistencies across clinical trials – such as variations in perfusion parameters, perfusate compositions, and durations – further complicate the generalizability of these findings to diverse transplant scenarios. Therefore, the results should be interpreted cautiously, and their applicability to broader clinical contexts warrants further investigation.

CONCLUSIONS

Despite a strong mechanistic rationale, evidence supporting the beneficial effects of machine perfusion in reducing the risk of acute liver graft rejection remains debated. This evidence comes from underpowered studies that show significant variation in their design, methodology, and treatments. In clinical practice, restoring or enhancing the biliary and hepatocyte phenotypes is the most crucial outcome of MP preservation strategies, which outperform SCS, especially when using ECD grafts and for donor grafts at risk of ischemic cholangiopathy. Additionally, the impact of dynamic perfusion on post-transplant graft rejection risk is overshadowed by the different thresholds of the liver cell compartments (i.e., biliary, hepatocytic, and sinusoidal) to IRI and the complex coordination of immune-reactive and immune-modulatory cell populations within the liver microenvironment. Experimental and preclinical data highlight the specific immune features of liver sinusoids and LSECs in regulating the liver immune response, and exploring the impact of MP on LSECs could help understand the role of MP-based strategies in modulating liver immune functions. However, designing and conducting specific trials remains a challenging task. Finally, the idea that the mechanisms of acute rejection in liver grafts preserved with MP are the same as in grafts preserved with static cold storage needs more solid evidence.

Conflict of interest statement

The authors declare no conflict of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author contributions

PDS, DP, QL, DC: conceptualized the study; EFK, MG, AR, J D: conducted the literature search; PDS, DP, RD, AR: wrote the preliminary draft; PDS, QL: made critical revisions. All authors prepared the draft and approved the final version.

Ethical consideration

Not applicable

History

Received: July 24, 2025

Accepted: November 10, 2025

Figures and tables

Figure 1. Schematic representation of the cascade of ischemia-reperfusion injury (IRI) events in liver transplantation. The process of liver graft procurement and transplantation involves oxygen deprivation under hypothermic (cold ischemia) and normothermic (warm ischemia) conditions, followed by its restoration. Oxygen deprivation causes damage to the mitochondrial respiration chain and saturation of its reducing capacity. After oxygen restoration post-graft implantation, reactive oxygen species (ROS) are produced both within and outside mitochondria, spreading through intracellular and extracellular spaces, which contributes to cellular dysfunction and cell death. Damage-associated molecular patterns (DAMPs) released from injured cells have strong inflammatory properties, attracting innate and adaptive immune cells and creating a pro-inflammatory environment affecting various liver cell types: hepatocytes, cholangiocytes, and sinusoidal endothelium. The intensity of inflammatory responses depends on the type of ischemia (cold versus warm), its duration, the methods of restoration, and the biological features of the liver graft (such as age, steatosis, fibrosis), leading to different clinical outcomes. Liver cells exhibit varying levels of sensitivity to ischemia-reperfusion injury (IRI), with hepatocytes and cholangiocytes being more susceptible to warm ischemia than liver sinusoidal endothelial cells (LSEC). The activation of the innate immune response triggered by IRI initiates adaptive (antigen-specific) immune reactions, which may explain the higher rates of post-transplant acute rejection seen in some clinical studies. LSEC: liver sinusoidal endothelial cell; ROS: reactive oxygen species.

Figure 2. Schematic depiction of the inflammatory process driven by ischemia-reperfusion injury (IRI) within liver grafts. O2 deprivation causes mitochondrial injury, leading to the release of reactive oxygen species (ROS) in liver cells. Damage-associated molecular patterns (DAMPs) are released due to the toxic effects of ROS on cell organelles and structures. DAMPs activate Kupffer cells (KC), which in turn increase the release of ROS and pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), interferon-gamma (IFN-γ), and interleukin-12 (IL-12). TNF-α and IL-1 enhance the expression of Mac-1 (CD11b/CD18) adhesion proteins on neutrophils and stimulate IL-8 production, promoting neutrophil chemotaxis within the liver sinusoids. The TNF-α-driven expression of intracellular adhesion molecule-1 (ICAM-1) on the intraluminal side of liver sinusoidal endothelial cells (LSEC) facilitates neutrophil and inflammatory cell rolling, binding, and extravasation in the Space of Disse and hepatic plates. Additionally, IL-1 and TNF-α recruit and activate CD4+ T (Th1) lymphocytes, which interact with antigen-presenting cells (APCs), including inflammatory dendritic cells (DCs) and LSECs, promoting MHC-II and MHC-I-restricted antigen-specific immune responses under stimulation of INF-γ, IL-2, and TNF-β. Platelets play a vital role in IRI by releasing platelet-activating factor (PAF), cytokines, and growth factors. PAF primes neutrophils for ROS production, further amplifying the neutrophil response. Activated platelets adhere to LSEC and induce their programmed cell death. DAMP: damage-associated molecular pattern; IFN: interferon; IL: interleukin; KC: macrophage-1 antigen; Kupffer cell; PAF: platelet-activating factor; ROS: reactive oxygen species; SC: stellate cells. TNF: tumor necrosis factor.

Figure 3. Schematic representation of the immunomodulatory functions of liver sinusoidal endothelial cells (LSEC). 1) LSECs help retain inflammatory and immune cells in the liver sinusoids, creating an environment for both innate and adaptive immune responses; 2) LSECs exhibit high scavenger activity via their Toll-like receptors (TLR), aiding in the clearance of intestinal and blood-borne pathogens and molecules; 3A-D) LSECs display unique modulatory functions by: A) inactivating CD4+ T cells through the interaction of the LSECtin receptor with CD44 (on CD4+ T cells); B) diverting the activation of CD8+ T cells from engaging with antigen-presenting cells (APC), such as dendritic cells (DC), via the interaction of the LSECtin receptor with lymphocyte function-associated antigen-1 (LFA-1); C) inducing the formation of antigen-specific regulatory CD4+ T cells by the interaction of MHC-II-restricted antigens with the T cell receptor complex (TCR) in the presence of LSEC-produced interferon-gamma, Notch signaling activation, and the absence of IL-12 co-stimulation (which is poorly expressed on LSECs); D) inducing quiescence in CD8+ T cells due to the combined effect of low blood flow in the sinusoidal space. In the unique microenvironment of liver sinusoids, antigen-specific, MHC-I-restricted cross-presentation by LSECs, in the presence of B7H1-to-programmed death-1 receptor (PD-1) co-stimulation, triggers the generation of quiescent CD8+ T cells. Therefore, LSECs play a vital role in orchestrating the liver microenvironment’s innate and adaptive immune responses against intestinal and blood-borne alloantigens and pathogens. IFN: interferon; KC: Kupffer cells; LFA-1: lymphocyte function-associated antigen-1; LMC: liver monocytic cells; LSEC: liver endothelial sinusoid cells; MAIT: mucosa-associated invariant T cells; NK: natural killer; PD-1: programmed death-1; SC: stellate cell; TCR: T-cell receptor complex; TLR: toll-like receptor.

Figure 4. Summary of pros and cons of the impact of machine perfusion on the risk of acute rejection of the liver graft. Abbreviations: D-HOPE: dual-HOPE; HOPE: hypothermic oxygenated perfusion; MP: machine perfusion; NMP: normothermic machine perfusion; NRP: normothermic regional perfusion.

Function Signals/pathways Effect
Patency of the sinusoidal lumen ↓ NO↑ET↑ TXA2 Sinusoidal vasoconstriction and microcirculatory dysfunction
↓ KLF2↓ eNOS↓ thrombomodulin↓ Nrf2 Sinusoidal lumen narrowing and pro-thrombotic stimulation
Intra-sinusoidal cell adhesion ↑ ROS Increased neutrophil chemotaxis and adhesion within the sinusoidal lumen, activation of platelet aggregation and HSC contraction
KC activation DAMPsPAMPs DAMP and PAMP-activated KCs release ROS, IL-1, IL-12, TNF-α, and INF-γIL-1 and TNF-α upregulates neutrophils adhesions within sinusoidsTNF-α upregulates ICAM-1 expression on LSECs leading to neutrophils rolling, binding and extravasation in the liver parenchyma
Activation of native and acquired immune response IL-1TNF-αGM-CSFTNF-β Recruitment of neutrophilsActivation of CD4+ T-cellsExtravasation of immune cells into the liver parenchyma
DAMPs: damage-associated molecular patterns; eNOS: endothelial NO synthase; ET: endothelin; GM-CSF: granulocyte-macrophage colony stimulating factor; KLF2: Kruppel-like Factor 2; HSC: hepatic stellate cells; IL-1: inteleukin-1; IL-8: interleukin-8; IL-12: interleukin-12; NO: nitric oxide; Nrf2: nuclear factor erythroid 2-related factor 2; PAMPs: pathogens associated molecular patterns; TNF-α: tumor necrosis factor alpha; TNF-β: tumor necrosis factor beta; TXA2: thromboxane A2.
Table I. Dysregulation of the sinusoidal microenvironment in ischemia-reperfusion injury (IRI). IRI shifts the sinusoidal phenotype to a pro-inflammatory, pro-thrombotic, pro-apoptotic, and vasoconstricted environment (based on references 16-21,23,24,26-30,32-43,45-48).
Function Target cells Receptors/signals Effect
IMMUNE CELL RESIDENCY
Retention of immune cell populations in liver sinusoids 34,35 NK cellsInnate and activated T cellsMAITAPC (KC, DC) CXCR6CX3CR1CXCL16CX3CL1 Combined with the low blood flow in sinusoids, this function allows for immune cell circulation and residency in the liver parenchyma
SCAVENGER FUNCTION
Antigen uptaking 34,36 Blood-borne molecules and pathogens TLR Intestinal and blood borne pathogens and molecules are removed from circulation
INHIBITION OF T-CELL ACTIVATION
Silencing of CD4+ T-cell effector function 37,38 Activated CD4+ T cells LSECtin (LSECs)CD44 (T cells) Inhibition of CD4+ T-cell activation into CD4+ helper
Silencing of CD8+ T-cell activation 34,39,40 CD8+ T cells LFA-1 Inhibition of CD8+ T-cell activation of effector functions
INDUCTION OF T-REG/QUIESCENT T-CELLS
Induction of regulatory T cells through MHC II-restricted antigen presentation 41-45 CD4+ T cells MHC-IIIFN-γTGF-betaNOTCH Due to low expression of MHC-II and the absence of IL-12 production by LSCEs, antigen-specific presentation to T cells results in the generation of regulatory cells
Cross-presentation of soluble antigens on MHC I molecules to CD8+ T cells 46-48 CD8+ T cells MHC-I (on LSECs)B7H1 (on LSECs)PD1 (on T cells) Due to low blood flow in sinusoids, LSEC MHC-I restricted antigen stimulation results in differentiation of quiescent CD8+ T cells
APC: antigen-presenting cell; CD: cluster differentiation; IFN-γ, interferon-gamma; IL-12: interleukin-12; KC: Kupffer cell; LFA-1: lymphocyte function-associated antigen-1; LSEC: liver sinusoidal endothelial cell; MAIT: mucosa-associated invariant T cell; NK: natural killer; PD1: programmed death 1; TLR, toll-like receptor.
Table II. Immunomodulatory functions of liver endothelial sinusoidal cells (LSECs).
Author No. of studies No. of patients ACR Limitations
Maspero M et al. 2023 93 6 HOPE1 NMP1 NRP 226 MP330 SCS Overall11% in MP vs 18% in SCS (p = 0.02)Sub-analysisHOPE is superior to SCS for all types of graftMP is superior to SCS for DCD graftsHOPE is superior to SCS for DCD grafts IS was reported in 6 studiesTAC was used in all studiesBasiliximab was used in 4 studiesOnly 4 studies provided a definition of ACROnly 1 study provided classification of ACR based on RAINo study provided information on treatment of rejection
Liang A et al. 2023 98 7 HOPE4 NMP4 NRP 705 MP1056 SCS Overall11.2% MP vs 23% in SCS (p = 0.31)Sub-analysisHOPE and NRP are superior to SCS with DCD grafts onlyHOPE was not superior to SCS for all types of graftsHOPE was superior to SCS for DCD and ECD-DCD graftsNRP was superior to SCS for DCD graftsHOPE was not superior to SCS for ECD-DBD grafts Limited sample size of included studiesSignificant heterogeneities across studies for design, organ perfusion protocols, patient clinical characteristics and post-operative management.Diagnosis of ACR is often ambiguousACR reporting is often poor
ACR: acute rejection rate; CI: confidence interval; DBD: donation after brain death; DCD: donation after cardiocirculatory death; ECD: extended criteria donors; HOPE: hypothermic oxygenated perfusion; IS: immunosuppression; MP: machine perfusion; NMP: normothermic machine perfusion; NRP: normothermic regional perfusion; RAI: rejection activity index; SCS: standard cold storage; TAC: tacrolimus.
Table III. Summary of two systematic reviews on the impact of machine perfusion (MP) on acute liver graft rejection.
Function Effect Open issues
Dynamic perfusion of the liver preserves the phenotype of liver sinusoids by mechanical and molecular mechanisms The mechanical stimuli during MP may preserve LSEC phenotype upregulating the expression of KLF2 and Nrf2 transcription factors 16This results in preserved production of eNOS and thrombomodulin within the sinusoidal endothelium and in reduced production of oxidant species 16 The most appropriate pressure and flow regimens to reduce LSEC deterioration is currently unknownThe superiority of combined hepatic arterial and portal perfusion (D-HOPE) over portal-only (HOPE) on the sinusoidal milieu and LSEC phenotype awaits elucidation
Oxygenation of graft perfusate Ex-vivo O2 supplementation mitigates the mitochondrial electron chain disruption, ATP depletion, ROS production and DAMP release 22-29,31,50-58This results in reduced intensity of post-reperfusion inflammation and immune response within the sinusoidal spaces, liver parenchyma and biliary tracts 50-58 A partial oxygen pressure (pO2) of at least 70 kPa (500 mmHg) is currently considered necessary to allow for ATP restoration after 1-2 hours of HOPE/D-HOPE 50-58The optimal pO2 to mitigate the immune activation and reduce ACR risk is currently unknownLSECs, hepatocytes and cholangiocytes show different sensitivity thresholds to CIT 32,33
Perfusion temperature Hypothermic perfusion with adequate pO2 improves mitochondrial function versus SCS resulting in diminished accumulation of succinate and reduced nucleotide substrates 50-58Normothermic perfusion preserves cellular and mitochondrial functions and reduces the extent of inflammatory and immune activation 49,65-68 The T-cell dynamics occurring after hypothermic and normothermic perfusion should be elucidated betterPreliminary data suggest that hypothermic perfusion is associated with early expansion of regulatory cells and transcriptomic signatures of cellular responses to acute inflammation injury and downregulation of genes that induce oxidative stress. Longer observation is necessary
Perfusion pressure and flow Perfusion pressure and flow may contribute to immune cell circulation and clearance, shutting down post-reperfusion immune activation 100,102-104,106The transition to an exhausted immune phenotype is particularly relevant to cholangiocytes and post-transplant cholangiopathies 98 MP produce liver-resident inflammatory cells mobilization into the perfusate, but this effect has mainly been described for leukocytes and T cells 55,103,106Immune cell circulation and depletion may be influenced by additional factors, like the perfusate medium composition, use of blood, nutrients and oxygen carriers 100,101The dynamics of mobilization of all inflammatory cell populations during MP require clarification
Donor type Experimental and clinical data suggest that DBD and DCD are associated with activation of pro-inflammatory and pro-thrombotic pathways 79-82Hypothermic O2 administration before procurement (i.e., NRP) is associated with reduced inflammatory activation 78 Matching the type of MP with donor category is currently based on need to assess graft viability prior to transplantation and reduce the risk of ischemic cholangiopathies 83
ACR: acute cellular rejection; CIT: cold ischemia time; D-HOPE: dual HOPE; eNOS: endothelial NO synthase; HOPE: hypothermic oxygenated perfusion; KLF2: Kruppel-like factor 2; LSEC: liver sinusoidal endothelial cell; MP: machine perfusion; Nrf2: nuclear factor erythroid 2-related factor 2; NRP: normothermic regional perfusion; SCS: static cold storage; WIT: warm ischemia time.
Table IV. Proposed immunomodulatory mechanisms of machine perfusion (MP) compared to static cold storage (SCS).

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Paolo De Simone - Liver Transplant Program, University of Pisa Medical School Hospital, Pisa, Italy; Department of Surgical, Medical, Biomolecular Pathology and Intensive Care, University of Pisa, Pisa, Italy. Corresponding author - paolo.desimone@unipi.it

Daniela Peritore - The Italian National Center for Transplantation (Centro Nazionale Trapianti, CNT), Rome, Italy

Daniela Campani - Department of Surgical, Medical, Biomolecular Pathology and Intensive Care, University of Pisa, Pisa, Italy; Department of Pathology, University of Pisa Medical School Hospital, Pisa, Italy

Emanuele Federico Kauffmann - Division of General and Transplant Surgery, University of Pisa Medical School Hospital, Pisa, Italy

Juri Ducci - University of Pisa Medical School Hospital, Pisa, Italy

Michael Ginesini - Division of General and Transplant Surgery, University of Pisa Medical School Hospital, Pisa, Italy

Raffaele Donadio - The Italian National Center for Transplantation (Centro Nazionale Trapianti, CNT), Rome, Italy

Allegra Ripolli - Division of General and Transplant Surgery, University of Pisa Medical School Hospital, Pisa, Italy

Quirino Lai - Department of General and Transplant Surgery, La Sapienza University of Rome, Rome, Italy

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Copyright (c) 2023 European Journal of Transplantation

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De Simone, P., Peritore, D., Campani, D., Kauffmann, E.F., Ducci, J., Ginesini, M. , Donadio, R., Ripolli, A. and Lai, Q. 2025. Is the use of machine perfusion associated with a reduced risk of graft rejection after liver transplantation?. European Journal of Transplantation. (Dec. 2025). DOI:https://doi.org/10.57603/EJT-1545.
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