Mapping new mechanisms: Hypothermic oxygenated perfusion attenuates the immune response after human liver transplantation

New protective effects of machine perfusion techniques are being discovered at an impressive pace, with routine implementation in clinical practice. Beyond the ongoing need for reliable viability assessments, a primary objective of these techniques is to enhance organ function and modulate the immune response to reduce posttransplant complications. In this issue of Liver Transplantation, Elgosbi et al1 demonstrated relevant potential immunoregulatory effects of hypothermic oxygenated perfusion (HOPE). In this study, the authors from King’s College Hospital in London, UK, evaluated 27 liver grafts randomly assigned to end-ischemic HOPE (n = 14) or static cold storage (n = 13). The molecular profile of the recipient's immune response was analyzed together with HOPE perfusate. The authors found fewer donor-liver–derived mononuclear cells in HOPE perfusates, containing a lower number of plasmacytoid dendritic cells. After the transplant, the number of circulating regulatory T cells increased significantly in the HOPE group, compared with recipients of cold-stored liver grafts. At 3 months after transplantation, recipients exhibited an additional reduction of donor-specific CD8+ T cells in the HOPE group (Figure 1).1 This study reveals key mechanisms that may explain why liver and kidney recipients treated with HOPE experience fewer episodes of acute cellular rejection (ACR), even when receiving high-risk grafts.2FIGURE 1: Hepatic transcriptional profiles and systemic T-cell responses after liver transplantation. The article by Elgosbi et al1 in this issue of Liver Transplantation compared livers transplanted after SCS or HOPE. Hepatic flush before transplantation mobilized lower leukocyte numbers in HOPE-perfused livers than after SCS only. Known mechanisms of HOPE-mediated organ protection include mitochondrial reconditioning, particularly through the metabolism of accumulated succinate and reloading of ATP stores. This translated into a post-reperfusion upregulated mRNA signature of the ROS pathway only in SCS livers. Analysis of systemic immunity 2 weeks after transplantation showed an increase in Treg numbers in the HOPE cohort, which were identified as donor-specific. At 3 months after transplantation, alloreactive CD8 T cells were increased in the SCS cohort, leading to the conclusion of decreased immunogenicity of donor livers after HOPE. Italics indicate previously published mechanisms.6 Abbreviations: CD8+, CD8+ cytotoxic lymphocyte; HOPE, hypothermic oxygenated perfusion; IRI, ischemia-reperfusion injury; ROS, reactive oxygen species; SCS, static cold storage; Treg, regulatory T cell.HOPE has emerged as an effective preservation technique for liver grafts before transplantation. The beneficial effects of HOPE on various posttransplant outcomes have been repeatedly demonstrated by several clinical studies. Beneficial effects were shown with a significant reduction of early allograft dysfunction, major complications, and nonanastomotic strictures; notably, HOPE was also found to significantly improve graft survival at 1 year.3 The protective mechanisms are now better understood thanks to a growing number of experimental studies. These studies show that toxic levels of succinate, which build up during warm and cold ischemia, are metabolized during hypothermic oxygenation, allowing ATP to be replenished. The key is the slow succinate metabolism limiting reactive oxygen species release from mitochondrial complex I.4 As a result, the cells are better equipped for rewarming and reoxygenation under normothermic conditions, as observed during and after the transplantation of HOPE-treated livers.5 Cold oxygenation reduces ischemia-reperfusion injury, which is driven by a proinflammatory cascade triggered by dysfunctional mitochondria.6 The observed immune-modulatory effects of HOPE are a downstream consequence of less active KCs and endothelial cells and a lower number of graft infiltrating T cells. Correspondingly, a reduced number of circulating and activated T cells in recipient blood was described.1 Various clinical studies demonstrated a significantly lower ACR rate following end-ischemic HOPE compared with cold storage alone.2 The authors demonstrate here that HOPE confers potentially impactful changes in systemic adaptive immunity in the longer-term posttransplant setting. The systemic, immunologically tolerogenic nature of transplanted liver allografts was well recognized already in the first series of experimental liver transplantations, and even confers immunologic protection for organs transplanted in combination with the liver.7 However, organs from extended-criteria donors, such as after circulatory death, with relevant macrosteatosis or advanced age, are more immunogenic and associated with less systemic tolerance and higher rejection rates.8 The present study demonstrates the mitigation of posttransplant immunogenicity through HOPE, even with the use of extended-criteria donor livers.1 Importantly, such effects were observed despite the performance of HOPE after a median static cold storage of 556 (IQR: 523–648) minutes and median perfusion duration of 121 (IQR: 110–154) minutes. The observed reduction of ischemia-reperfusion injury–associated inflammation in HOPE-treated organs appears to be a logical explanation for the observed changes in immune regulatory responses after transplantation1 and serves as a possible explanation for the phenomenon of decreased ACR rates following HOPE.2,3 In the normothermic setting, machine perfusion has been demonstrated to lower the number of natural killer cells and hepatic proinflammatory neutrophils, decrease the expression of key genes characterizing the inflammatory state of monocytes/macrophages, and elevate the expression level of markers associated with a macrophage’s tolerogenic phenotype.9,10 The authors’ contribution to the field of posttransplant immunology is noteworthy as it opens new avenues in the understanding of the immunological response following the transplantation of machine-perfused grafts. The innovative finding that HOPE modulates the donor-specific T-cell response following transplantation is highly relevant. Calcineurin inhibitors, the gold standard of immunosuppression, interact with T cells by decreasing the activity of gene coding for IL-2 and related cytokines, thus preventing ACR. The double effect of HOPE together with low-dose calcineurin inhibitors has been shown to be effective in reducing ACR after transplant in experimental models.11 It is possible that HOPE and tacrolimus have synergic effects in controlling the donor-specific T-cell response. Although this has not been specifically investigated, in their study, the authors found a significantly lower incidence of ACR in the HOPE group,1 despite similar tacrolimus levels compared with the cold storage cohort, thus corroborating previous findings from other large studies.2 As machine perfusion continues to be increasingly utilized, this landmark study will pave the way for future research by offering a robust framework to better understand the downregulation of immune response during HOPE and the trend toward systemic tolerogenic recipient responses. Future studies are of interest to confirm these findings, especially in grafts with elevated ischemia-reperfusion injury–related innate immune response, combined with recipients prone to have an active immune system also in the context of their underlying liver disease. By improving organ preservation and reducing the likelihood of adverse immune reactions, machine perfusion holds the potential to significantly increase the success rates of transplant surgeries and patient outcomes. This continuous progress underscores the importance of refining these technologies to meet clinical demands.