Confronted with the growing gap between the number of patients on the wait list and the limited number of organs available for transplantation, transplant centres worldwide have steadily increased the utilization of livers from extended criteria donors (ECD). Whereas this practice constantly pushes the limits of what is acceptable for transplantation carrying the risk of inferior outcomes, some ECD grafts display excellent survival rates (1, 2). However, the identification of risk determinants for ECD organs is still complicated by the fact that the impact of seemingly obvious clinical parameters, such as chronological donor age, has been analysed in several studies with contradictory conclusions (3, 4). Therefore, it remains a challenge to define alternative approaches allowing the discrimination between ECD livers, which can be considered for transplantation and those needing to be excluded. In this regard, both the donor risk index (DRI) and in particular the Eurotransplant Donor-Risk-Index (ET-DRI) have been suggested as valuable instruments to score donor liver quality (5). Although useful, the validity of these scores remains limited in several facets (6). Consequently, the necessity persists to define alternative parameters as a metric for liver graft quality. In our previous studies on renal pre-implantation (zero-hour) biopsies, we identified a set of candidate genes to be characteristic of intragraft immune activation (7), and most importantly, especially the activating cytotoxicity receptor NKG2D appeared to be predictive for renal allograft function after 12 months post kidney transplantation (8). In this present study, aiming to investigate whether our earlier findings in kidney allografts may also be translated to LT, we analysed defined candidate genes in 88 liver zero-hour biopsies and evaluated their potential to predict allograft function up to 10 years post-transplantation. In addition to NKG2D, we focused on its ligand, the MHC class I poly-peptide-related sequence A (MICA), as well as markers indicating increased donor graft immunogenicity (HLA-DRB), immune activation (CCL19), obesity (Leptin) and alternative Natural Killer (NK) cell receptors (DNAM-1).

To compensate the immanently high wait list mortality, the transplant community is turning to the utilization of marginal and high-risk grafts (1, 20–22). The term “marginal” refers to a not well-defined group of donors with characteristics such as advanced age, displaying steatosis, hepatitis C virus positive serology or donation after circulatory death (DCD) (22). However, these descriptive attributes alone remain insufficient to predict the specific risk of transplanting an individual graft. The analysis of zero-hour biopsies may therefore provide additional information about these organs. Indeed, pre-transplant biopsy retrieval has become a widely accepted standard in many centers worldwide. However, so far, such specimens are mainly used to evaluate the grade of steatosis/fibrosis by microscopic frozen section histology (9).

In a recent study, we illustrated that the potential scope of zero-hour biopsy analysis can by far exceed the information obtained from histology in kidney allografts as we could show that the classical histo-morphologic assessment (Remuzzi biopsy score) could predict allograft outcome, although the predictive value of molecular markers (NKG2D) proved to be significantly superior (8). In this present study, we translated these findings derived from kidney allografts to investigate whether a defined set of inflammatory genes may also be applicable to predict outcomes after LT. In Austria, organs for transplantation are mainly procured from BD donors (23), a circumstance that allowed us to exclusively include BD graft samples and avoid potential biasing effects from a heterogenous BD/DCD study group.

Our analysis demonstrates that intragraft expression of NKG2D, an activating receptor expressed on NK cells and which has also been identified on γδ T, CD8⁺ αβ T, and NKT lymphocytes (24–26), was significantly induced in livers derived from donors with a BMI 25-29.9 kg/m² ( Figure 1 ). Interestingly, in strong contrast to our prior findings in kidney allografts (8), NKG2D expression in liver grafts was not significantly affected by donor age ( Supplemental Figure 1 ). Steatotic livers represent the most common type of “marginal” organs that have been introduced during the last two decades. A precise definition and reproducible method for steatosis quantification is currently solely based on the histological assessment. We therefore analysed the expression of leptin, since leptin has been reported to augment both inflammatory and profibrogenic responses in the liver and plays a role in the pathogenesis of hepatic steatosis (27, 28). However, we were not able to detect an increase of leptin mRNA expression in livers from donors with a BMI 25-29.9 kg/m², nor could we detect an induction in livers with an advanced age. Contrary to a prior study reporting on a possible role of leptin as a biomarker in kidney transplantation (29), our study indicates that leptin has no predictive value in the setting of LT.

Likewise, the expression of the markers CCL19, DNAM1 and HLA-DRB, all of which were chosen due to their relevance in kidney transplantation (7, 8), did not show a significant predictive value for liver graft survival or function.

In contrast, the most promising candidate turned out to be the NGK2D ligand, the non-HLA antigen MICA. Since MICA, unlike the classical HLA molecules, is not involved in antigen presentation to T cells but via NKG2D recognition interacts with human NK cells, γδ T, mucosal-associated invariant T (MAIT), CD56⁺ T, and CD8⁺ T cells (30), MICA is unique to the extent that it plays a key role in linking the innate and adaptive immune responses in organ transplantation (31). This circumstance has drawn considerable attention in the scientific community, and the role of this non-HLA antigenic target has been extensively studied in the setting of kidney transplantation (31). In contrast, as outlined by recent review articles, the role of MICA in LT currently remains unclear due to a lack of available data (31, 32). To our knowledge, only two prior studies exist assessing the potential influence of MICA on LT outcomes (33, 34). Both investigated anti-MICA antibodies in patients’ sera and only one study investigated MICA expression profiles in liver graft samples. The authors state that they found only weak mRNA levels for MICA in liver cells but no protein or cell surface expression (34). However, in contrast to this study, we used whole liver tissue lysates for a comprehensive mRNA analysis. With this approach, we found a significant correlation of MICA expression with allograft function after 3, 6, 12, 24 and 36 months, which was confirmed in the multivariate analysis. MICA expression revealed to be independent of donor age or BMI, but high expression of MICA revealed to be an independent predictor of long-term allograft survival after 10 years post-transplantation. Importantly, low MICA expression was significantly associated with the occurrence of patient death.

Noteworthy, our observation that an elevated mRNA expression of MICA is associated with improved outcomes after LT seems paradoxical at a first glance. Since MICA represents a ligand for NK and T cell receptors, it could be expected such cells displaying high MICA expression could be rendered for killing by cytotoxic lymphocytes. This mechanism was proven in various tumor cell lines which strongly expressed MICA on the cell surface. However, in a wide range of normal epithelia, MICA was shown to be broadly expressed, but only in distinct intracellular structures with only occasional (<20%) membrane localization in a few normal tissue types (bladder, bronchus, kidney and colon) (35). Accordingly, our experiments show that MICA was expressed in liver tissue samples on an mRNA level, whereas no MICA expression was found on the cell surface. This finding could serve as an explanation why anti-MICA antibodies in the recipient seem to have no clinical significance. Although we did not assess patient sera for anti-MICA antibodies in our patients, the studies by Ciszek et al. and Uzunel et al. (33, 34) report that, against initial expectations, the presence of anti-MICA antibodies in patients’ sera were not associated with deteriorated outcomes.

In response to cellular stress, MICA expression is induced in many cell types, including epithelium or fibroblasts, whereas MICA expression on hepatocytes or Kupffer cells of has not been demonstrated so far. However, it has been shown that levels of MICA/B mRNA positively correlate with the stage of fibrosis, suggesting that MICA/B also contribute to the progression of liver fibrosis (36). In addition, MICA expressing T cells have been shown to be enriched within HBV-infected livers compared with the periphery or to healthy livers (37). Both observations clearly demonstrate that MICA can be up-regulated as a consequence of inflammatory pathology, independent of the cell type. Thus, our results suggest that the detection of MICA mRNA in liver zero-hour biopsies detects sub-clinical changes in the graft, which are predictive for outcomes, but beyond the scope of classical histopathological evaluation.

One possible limitation to this study is that we propose a biomarker which requires several hours for its assessment (e.g. via RT-PCR). In consideration of the immanent time pressure to minimize cold ischemia storage time one might argue that such a marker is simply not feasible for solid organ transplantation. However, regarding the rapid technological advances in ex situ organ preservation, such concerns might soon be a matter of the past. Both, cold (38, 39) as well as warm (40, 41) liver perfusion systems are now able to significantly extend preservation times. Consequently, ECD livers which would have formerly been discarded can now be considered for transplantation after the assessment of graft quality. Therefore, new biomarkers are urgently needed to predict liver function after subsequent transplantation (38). As recently formulated by Verhoeven et al., ideally, such biomarkers could help to enlarge the donor pool by objectively screening liver grafts that initially would be discarded based on their predisposing characteristics (42).

Based on our findings illustrating that MICA gene expression in zero-hour liver biopsies correlates with both, short and long-term graft function, we suggest MICA as a candidate biomarker for future pre-transplant graft evaluation.

The original contributions presented in the study are included in the article/ Supplementary Material , further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethics Committee of the Medical University Innsbruck, Innsbruck, Austria. The patients/participants provided their written informed consent to participate in this study.

TR and KK were responsible for the conception of the project, literature review, data analysis, interpretation of results and write up of the manuscript. HH performed the statistical analyses. HE, JG, HS, PR, SE, MM, VM, MB, RÖ, HZ, and SS contributed to the acquisition of data, data analysis and interpretation of results. All authors contributed to the article and approved the submitted version.

This study was supported by a grant from the Deutsche Forschungsgemeinschaft to KK (Ko-2270/4-1).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.