The future of Liver Transplantation.

Over the last 50 years, liver transplantation has evolved into a procedure routinely performed in many countries worldwide. Those able to access this therapy frequently experience a miraculous risk-benefit ratio since, particularly if they face imminently life threatening disease. Over the decades, the success of liver transplantation, with dramatic improvements in early post-transplant survival, has aggressively driven demand. However, in spite of the emergence of living donors to augment deceased donors as a source of organs, supply has lagged far behind demand. As a result, rationing has been an unfortunate focus in recent decades. Recent shifts in the epidemiology of liver disease combined with transformative innovations in liver preservation suggest that the underlying premise of organ shortage may erode in the foreseeable future. Focus will sharpen on improving equitable access while mitigating constraints related to workforce training, infrastructure for organ recovery and rehabilitation, and their associated costs. Research efforts in liver preservation will undoubtedly blossom with the aim of optimizing both the timing and conditions of transplantation. Coupled with advances in genetic engineering, regenerative biology, and cellular therapies, the portfolio of innovation, both broad and deep, offers promise that, in the future, liver transplantation will not only be broadly available to those in need but also represent a highly durable life-saving therapy.

Cellular uptake and in vivo distribution of mesenchymal-stem-cell-derived extracellular vesicles are protein corona dependent.

Extracellular vesicles (EVs) derived from mesenchymal stem cells are promising nanotherapeutics in liver diseases due to their regenerative and immunomodulatory properties. Nevertheless, a concern has been raised regarding the rapid clearance of exogenous EVs by phagocytic cells. Here we explore the impact of protein corona on EVs derived from two culturing conditions in which specific proteins acquired from media were simultaneously adsorbed on the EV surface. Additionally, by incubating EVs with serum, simulating protein corona formation upon systemic delivery, further resolved protein corona-EV complex patterns were investigated. Our findings reveal the potential influences of corona composition on EVs under in vitro conditions and their in vivo kinetics. Our data suggest that bound albumin creates an EV signature that can retarget EVs from hepatic macrophages. This results in markedly improved cellular uptake by hepatocytes, liver sinusoidal endothelial cells and hepatic stellate cells. This phenomenon can be applied as a camouflage strategy by precoating EVs with albumin to fabricate the albumin-enriched protein corona-EV complex, enhancing non-phagocytic uptake in the liver. This work addresses a critical challenge facing intravenously administered EVs for liver therapy by tailoring the protein corona-EV complex for liver cell targeting and immune evasion.

Time since liver transplant and immunosuppression withdrawal outcomes: Systematic review and individual patient data meta-analysis.

BACKGROUND & AIMS: Successful immunosuppression withdrawal (ISW) is possible for a subfraction of liver transplant (LT) recipients but the factors that define the risk of ISW failure are largely unknown. One candidate prognostic factor for ISW success or operational tolerance (OT) is longer time between LT and ISW which we term "pre-withdrawal time". To clarify the impact of pre-withdrawal time span on subsequent ISW success or failure, we conducted a systematic review with meta-analysis. METHODS: We systematically interrogated the literature for LT recipient ISW studies reporting pre-withdrawal time. Eligible articles from Embase, Medline, and the Cochrane Central Register of Controlled Trials were used for backward and forward citation searching. Pre-withdrawal time individual patient data (IPD) was requested from authors. Pooled mean differences and time-response curves were calculated using random-effects meta-analyses. RESULTS: We included 17 studies with 691 patients, 15 of which (620 patients) with IPD. Study-level risk of bias was heterogeneous. Mean pre-withdrawal time was greater by 427 days [95% confidence interval (CI) 67-788] in OT compared to non-OT patients. This increase was potentiated to 799 days (95% CI 369-1229) or 1074 days (95% CI 685-1463) when restricting analysis to adult or European study participants. In time-response meta-analysis for adult or European ISW candidates, likelihood of OT increased by 7% (95% CI 4-10%) per year after LT (GRADE low- and moderate-certainty of evidence, respectively). CONCLUSIONS: Our data support the impact of pre-withdrawal time in ISW decision-making for adult and European LT recipients. PROSPERO REGISTRATION: CRD42021272995.

The positive impact of the COVID 19 pandemic on organ utilisation in liver transplantation.

Background: As the world recovers from the aftermath of devastating waves of an outbreak, the ongoing Coronavirus disease 2019 pandemic has presented a unique perspective to the transplantation community of ''organ utilisation'' in liver transplantation, a poorly defined term and ongoing hurdle in this field. To this end, we report the key metrics of transplantation activity from a high-volume liver transplantation centre in the United Kingdom over the past two years. Methods: Between March 2019 and February 2021, details of donor liver offers received by our centre from National Health Service Blood & Transplant, and of transplantation were reviewed. Differences in the activity before and after the outbreak of the pandemic, including short term post-transplant survival, have been reported. Results: The pandemic year at our centre witnessed a higher utilisation of Donation after Cardiac Death livers (80.4% vs. 58.3%, p = 0.016) with preserved United Kingdom donor liver indices and median donor age (2.12 vs. 2.02, p = 0.638; 55 vs. 57 years, p = 0.541) when compared to the pre-pandemic year. The 1- year patient survival rates for recipients in both the periods were comparable. The pandemic year, that was associated with increased utilisation of Donation after Cardiac Death livers, had an ischaemic cholangiopathy rate of 6%. Conclusions: The pressures imposed by the pandemic led to increased utilisation of specific donor livers to meet patient needs and minimise the risk of death on the waiting list, with apparently preserved early post-transplant survival. Optimum organ utilisation is a balancing act between risk and benefit for the potential recipient, and technologies like machine perfusion may allow surgeons to increase utilisation without compromising patient outcomes.

Dysregulated anti-oxidant signalling and compromised mitochondrial integrity negatively influence regulatory T cell function and viability in liver disease.

BACKGROUND: Dysregulated inflammatory responses and oxidative stress are key pathogenic drivers of chronic inflammatory diseases such as liver cirrhosis (LC). Regulatory T cells (Tregs) are essential to prevent excessive immune activation and maintain tissue homeostasis. While inflammatory cues are well known to modulate the function and stability of Tregs, the extent to which Tregs are influenced by oxidative stress has not been fully explored. METHODS: The phenotypic and functional properties of CD4+CD25+CD127lo/- Tregs isolated from patients with LC were compared to healthy controls (HC). Treg redox state was investigated by characterizing intracellular reactive oxygen species (ROS), NADPH oxidase-2 (Nox2) activity, mitochondrial function, morphology, and nuclear factor-erythroid 2-related factor (Nrf2) antioxidant signalling. The relevance of Nrf2 and its downstream target, Heme-oxygenase-1 (HO-1), in Treg function, stability, and survival, was further assessed using mouse models and CRISPR/Cas9-mediated HO-1 knock-out. FINDINGS: Circulating Tregs from LC patients displayed a reduced suppressive function, correlating with liver disease severity, associated with phenotypic abnormalities and increased apoptosis. Mechanistically, this was linked to a dysregulated Nrf2 signalling with resultant lower levels of HO-1, enhanced Nox2 activation, and impaired mitochondrial respiration and integrity. The functional deficit in LC Tregs could be partially recapitulated by culturing control Tregs in patient sera. INTERPRETATION: Our findings reveal that Tregs rely on functional redox homeostasis for their function, stability, and survival. Targeting Treg specific anti-oxidant pathways may have therapeutic potential to reverse the Treg impairment in conditions of oxidative damage such as advanced liver disease. FUNDING: This study was funded by the Wellcome Trust (211113/A/18/Z).

Liver transplantation immunology: Immunosuppression, rejection, and immunomodulation.

Outcomes after liver transplantation have continuously improved over the past decades, but long-term survival rates are still lower than in the general population. The liver has distinct immunological functions linked to its unique anatomical configuration and to its harbouring of a large number of cells with fundamental immunological roles. The transplanted liver can modulate the immunological system of the recipient to promote tolerance, thus offering the potential for less aggressive immunosuppression. The selection and adjustment of immunosuppressive drugs should be individualised to optimally control alloreactivity while mitigating toxicities. Routine laboratory tests are not accurate enough to make a confident diagnosis of allograft rejection. Although several promising biomarkers are being investigated, none of them is sufficiently validated for routine use; hence, liver biopsy remains necessary to guide clinical decisions. Recently, there has been an exponential increase in the use of immune checkpoint inhibitors due to the unquestionable oncological benefits they provide for many patients with advanced-stage tumours. It is expected that their use will also increase in liver transplant recipients and that this might affect the incidence of allograft rejection. Currently, the evidence regarding the efficacy and safety of immune checkpoint inhibitors in liver transplant recipients is limited and cases of severe allograft rejection have been reported. In this review, we discuss the clinical relevance of alloimmune disease, the role of minimisation/withdrawal of immunosuppression, and provide practical guidance for using checkpoint inhibitors in liver transplant recipients.

Impact of liver failure on the circulating extracellular vesicle miRNA repertoire.

BACKGROUND & AIMS: Cell-derived small extracellular vesicles (sEVs) participate in cell-cell communication via the transfer of molecular cargo including selectively enriched microRNAs (miRNAs). Utilizing advances in sEV isolation and characterization, this study investigates the impact of liver injury and dysfunction on the circulating EV-miRNA profile. METHODS: High-throughput screening of 799 sEV-miRNAs isolated from plasma was performed in patients across a spectrum of liver disorders including compensated and decompensated chronic liver disease, acute-on-chronic liver failure (ACLF), and acute liver failure, in addition to healthy controls and those with severe sepsis. miRNA levels were compared with clinical and biochemical parameters, composite scores of liver disease, and patient outcomes. RESULTS: miRNA screening revealed the degree of hepatic dysfunction to be the main determinant of changes in circulating sEV-miRNA profile, with liver-specific miRNA-122 being among the most highly dysregulated in severe injury. Principal components analyses of the 215 differentially expressed miRNAs showed differing profiles, particularly among those with acute liver injury and ACLF. A distinct profile of dysregulated miRNA, but not circulating cytokines, was shown to characterize ACLF, with four consensus miRNAs identified-miR-320e, miR-374-5p, miR-202-3p, and miR-1910-5p. High miR-320e was associated with poorer 90-day survival (p = 0.014) and regulated the functional gene targets IK, RPS5, MANBAL, and PEBP1. CONCLUSIONS: This first comprehensive analysis to the best of our knowledge of patients with varying degrees and stages of liver failure demonstrates miRNA profiles specifically within the sEV compartment to be significantly altered in progressive liver disease and highlights the diagnostic and prognostic potential of sEV-miRNA in ACLF while also establishing downstream gene targets.

Low dose interleukin-2 selectively expands circulating regulatory T cells but fails to promote liver allograft tolerance in humans.

BACKGROUND & AIMS: CD4+CD25+Foxp3+ regulatory T cells (Tregs) are essential to maintain immunological tolerance and have been shown to promote liver allograft tolerance in both rodents and humans. Low-dose IL-2 (LDIL-2) can expand human endogenous circulating Tregs in vivo, but its role in suppressing antigen-specific responses and promoting Treg trafficking to the sites of inflammation is unknown. Likewise, whether LDIL-2 facilitates the induction of allograft tolerance has not been investigated in humans. METHODS: We conducted a clinical trial in stable liver transplant recipients 2-6 years post-transplant to determine the capacity of LDIL-2 to suppress allospecific immune responses and allow for the complete discontinuation of maintenance immunosuppression (ClinicalTrials.gov NCT02949492). One month after LDIL-2 was initiated, those exhibiting at least a 2-fold increase in circulating Tregs gradually discontinued immunosuppression over a 4-month period while continuing LDIL-2 for a total treatment duration of 6 months. RESULTS: All participants achieved a marked and sustained increase in circulating Tregs. However, this was not associated with the preferential expansion of donor-reactive Tregs and did not promote the accumulation of intrahepatic Tregs. Furthermore, LDIL-2 induced a marked IFNγ-orchestrated transcriptional response in the liver even before immunosuppression weaning was initiated. The trial was terminated after the first 6 participants failed to reach the primary endpoint owing to rejection requiring reinstitution of immunosuppression. CONCLUSIONS: The expansion of circulating Tregs in response to LDIL-2 is not sufficient to control alloimmunity and to promote liver allograft tolerance, due, at least in part, to off-target effects that increase liver immunogenicity. Our trial provides unique insight into the mechanisms of action of immunomodulatory therapies such as LDIL-2 and their limitations in promoting alloantigen-specific effects and immunological tolerance. CLINICAL TRIALS REGISTRATION: The study is registered at ClinicalTrials.gov (NCT02949492). IMPACT AND IMPLICATIONS: The administration of low-dose IL-2 is an effective way of increasing the number of circulating regulatory T cells (Tregs), an immunosuppressive lymphocyte subset that is key for the establishment of immunological tolerance, but its use to promote allograft tolerance in the setting of clinical liver transplantation had not been explored before. In liver transplant recipients on tacrolimus monotherapy, low-dose IL-2 effectively expanded circulating Tregs but did not increase the number of Tregs with donor specificity, nor did it promote their trafficking to the transplanted liver. Low-dose IL-2 did not facilitate the discontinuation of tacrolimus and elicited, as an off-target effect, an IFNγ-orchestrated inflammatory response in the liver that resembled T cell-mediated rejection. These results, supporting an unexpected role for IL-2 in regulating the immunogenicity of the liver, highlight the need to carefully evaluate systemic immunoregulatory strategies with investigations that are not restricted to the blood compartment and involve target tissues such as the liver.

Next-generation pathology detection of T cell-antigen-presenting cell immune synapses in human liver allografts.

BACKGROUND AND AIMS: In otherwise near-normal appearing biopsies by routine light microscopy, next-generation pathology (NGP) detected close pairings (immune pairs; iPAIRs) between lymphocytes and antigen-presenting cells (APCs) that predicted immunosuppression weaning failure in pediatric liver transplant (LTx) recipients (Immunosuppression Withdrawal for Stable Pediatric Liver Transplant Recipients [iWITH], NCT01638559). We hypothesized that NGP-detected iPAIRs enrich for true immune synapses, as determined by nuclear shape metrics, intercellular distances, and supramolecular activation complex (SMAC) formation. APPROACH AND RESULTS: Intralobular iPAIRs (CD45 high lymphocyte-major histocompatibility complex II + APC pairs; n = 1167, training set) were identified at low resolution from multiplex immunohistochemistry-stained liver biopsy slides from several multicenter LTx immunosuppression titration clinical trials (iWITH; NCT02474199 (Donor Alloantigen Reactive Tregs (darTregs) for Calcineurin Inhibitor (CNI) Reduction (ARTEMIS); Prospective Longitudinal Study of iWITH Screen Failures Secondary to Histopathology). After excluding complex multicellular aggregates, high-resolution imaging was used to examine immune synapse formation ( n = 998). By enriching for close intranuclear lymphocyte-APC distance (mean: 0.713 µm) and lymphocyte nuclear flattening (mean ferret diameter: 2.1), SMAC formation was detected in 29% of iPAIR-engaged versus 9.5% of unpaired lymphocytes. Integration of these morphometrics enhanced NGP detection of immune synapses (ai-iSYN). Using iWITH preweaning biopsies from eligible patients ( n = 53; 18 tolerant, 35 nontolerant; testing set), ai-iSYN accurately predicted (87.3% accuracy vs. 81.4% for iPAIRs; 100% sensitivity, 75% specificity) immunosuppression weaning failure. This confirmed the presence and importance of intralobular immune synapse formation in liver allografts. Stratification of biopsy mRNA expression data by immune synapse quantity yielded the top 20 genes involved in T cell activation and immune synapse formation and stability. CONCLUSIONS: NGP-detected immune synapses (subpathological rejection) in LTx patients prior to immunosuppression reduction suggests that NGP-detected (allo)immune activity usefulness for titration of immunosuppressive therapy in various settings.

Selective decrease of donor-reactive Tregs after liver transplantation limits Treg therapy for promoting allograft tolerance in humans.

Promoting immune tolerance to transplanted organs can minimize the amount of immunosuppressive drugs that patients need to take, reducing lifetime risks of mortality and morbidity. Regulatory T cells (Tregs) are essential for immune tolerance, and preclinical studies have shown their therapeutic efficacy in inducing transplantation tolerance. Here, we report the results of a phase 1/2 trial (ARTEMIS, NCT02474199) of autologous donor alloantigen-reactive Treg (darTreg) therapy in individuals 2 to 6 years after receiving a living donor liver transplant. The primary efficacy endpoint was calcineurin inhibitor dose reduction by 75% with stable liver function tests for at least 12 weeks. Among 10 individuals who initiated immunosuppression withdrawal, 1 experienced rejection before planned darTreg infusion, 5 received darTregs, and 4 were not infused because of failure to manufacture the minimal infusible dose of 100 × 106 cells. darTreg infusion was not associated with adverse events. Two darTreg-infused participants reached the primary endpoint, but an insufficient number of recipients were treated for assessing the efficacy of darTregs. Mechanistic studies revealed generalized Treg activation, senescence, and selective reduction of donor reactivity after liver transplantation. Overall, the ARTEMIS trial features a design concept for evaluating the efficacy of Treg therapy in transplantation. The mechanistic insight gained from the study may help guide the design of future trials.

IL-2 availability regulates the tissue specific phenotype of murine intra-hepatic Tregs.

CD4+CD25+Foxp3+ Tregs are known to acquire tissue-specific features and exert cytoprotective and regenerative functions. The extent to which this applies to liver-resident Tregs is unknown. In this study, we aimed to explore the phenotypic and functional characteristics of adult murine liver resident Tregs during homeostasis. Additionally, we investigated their role in ameliorating liver inflammation and tissue damage. Quantification of Foxp3+CD4+CD25+ cells comparing different tissues showed that the liver contained significantly fewer resident Tregs. A combination of flow cytometry phenotyping and microarray analysis of intra-hepatic and splenic Tregs under homeostatic conditions revealed that, although intra-hepatic Tregs exhibited the core transcriptional Treg signature, they expressed a distinct transcriptional profile. This was characterized by reduced CD25 expression and increased levels of pro-inflammatory Th1 transcripts Il1b and Ifng. In vivo ablation of Tregs in the Foxp3-DTR mouse model showed that Tregs had a role in reducing the magnitude of systemic and intra-hepatic inflammatory responses following acute carbon tetrachloride (CCl4) injury, but their absence did not impact the development of hepatocyte necrosis. Conversely, the specific expansion of Tregs by administration of IL-2 complexes increased the number of intra-hepatic Tregs and significantly ameliorated tissue damage following CCl4 administration in C57BL/6 mice. The cytoprotective effect observed in response to IL-2c was associated with the increased expression of markers known to regulate Treg suppressive function. Our results offer insight into the transcriptome and complex immune network of intra-hepatic Tregs and suggest that strategies capable of selectively increasing the pool of intra-hepatic Tregs could constitute effective therapies in inflammatory liver diseases.

Acute Antibody-mediated rejection in liver transplantation: Impact and applicability of the Banff working group on liver allograft pathology 2016 criteria.

This study was aimed to examine the clinical utility and impact of the 2016 Banff criteria for acute antibody-mediated rejection (acute AMR) in patients with liver transplantation. Among adult patients with donor-specific antibody (DSA) assays performed between 2015 and 2020, cases with proved DSA (mean fluorescent index >2000) and matched liver biopsy available were reviewed. Among 55 patients identified, 28 (51%) had class I DSA, 45 (82%) had class II DSA and 18 (33%) had both. Mild, moderate and severe microvasculitis were observed in 11 (20%), 2 (4%) and 1 (2%) case, respectively. Diffuse immunoreactivity to C4d on portal microvascular endothelia was confirmed in 5 cases (9%), which met the criteria of definite (n = 2) or suspicious for acute AMR (n = 3). Cases of acute AMR more commonly had class I DSA (100% vs. 46%; p = 0.027) or both class I and II DSA (80% vs. 28%; p = 0.018) than cases of non-acute AMR. One case of pure acute AMR with veno-occlusion was successfully treated with plasma exchange. The remaining 4 cases had features of combined acute AMR/T cell-mediated rejection (TCMR), and two progressed to ductopenic rejection within 3 weeks. In conclusion, only 9% of DSA-positive patients met the Banff criteria for acute AMR, necessitating careful morphological and immunohistochemical assessments of the allograft biopsies according to the proposed standards. Combined acute AMR/TCMR was more common than isolated acute AMR, and additional AMR in TCMR cases may be associated with rapid progression to ductopenic rejection.

Exaggerated inflammatory response to bacterial products in decompensated cirrhotic patients is orchestrated by interferons IL-6 and IL-8.

Cirrhosis-associated immune dysfunction (CAID) contributes to disease progression and organ failure development. We interrogated immune system function in nonseptic compensated and decompensated cirrhotic patients using the TruCulture whole blood stimulation system, a novel technique that allows a more accurate representation than traditional methods, such as peripheral blood mononuclear cell culture, of the immune response in vivo. Thirty cirrhotics (21 decompensated and 9 compensated) and seven healthy controls (HCs) were recruited. Whole blood was drawn directly into three TruCulture tubes [unstimulated to preloaded with heat-killed Escherichia coli 0111:B4 (HKEB) or lipopolysaccharide (LPS)] and incubated in dry heat blocks at 37°C for 24 h. Cytokine analysis of the supernatant was performed by multiplex assay. Cirrhotic patients exhibited a robust proinflammatory response to HKEB compared with HCs, with increased production of interferon-γ-induced protein 10 (IP-10) and IFN-λ1, and to LPS, with increased production of IFN-λ1. Decompensated patients demonstrated an augmented immune response compared with compensated patients, orchestrated by an increase in type I, II, and III interferons, and higher levels of IL-1ß, IL-6, and IL-8 post-LPS stimulation. IL-1ß, TNF-α, and IP-10 post-HKEB stimulation and IP-10 post-LPS stimulation negatively correlated with biochemical markers of liver disease severity and liver disease severity scores. Cirrhotic patients exposed to bacterial products exhibit an exaggerated inflammatory response orchestrated by IFNs, IL-6, and IL-8. Poststimulation levels of a number of proinflammatory cytokines negatively correlate with markers of liver disease severity raising the possibility that the switch to an immunodeficient phenotype in CAID may commence earlier in the course of advanced liver disease. NEW & NOTEWORTHY Decompensated cirrhotic patients, compared with compensated patients, exhibit a greater exaggerated inflammatory response to bacterial products orchestrated by interferons, IL-6, and IL-8. Postbacterial product stimulation levels of a number of pro-inflammatory cytokines negatively correlate with liver disease severity biomarkers and liver disease severity scores raising the possibility that the switch to an immunodeficient phenotype in cirrhosis-associated immune dysfunction may commence earlier in the course of advanced liver disease.

The molecular phenotypes of injury, steatohepatitis, and fibrosis in liver transplant biopsies in the INTERLIVER study.

To extend previous molecular analyses of rejection in liver transplant biopsies in the INTERLIVER study (ClinicalTrials.gov #NCT03193151), the present study aimed to define the gene expression selective for parenchymal injury, fibrosis, and steatohepatitis. We analyzed genome-wide microarray measurements from 337 liver transplant biopsies from 13 centers. We examined expression of genes previously annotated as increased in injury and fibrosis using principal component analysis (PCA). PC1 reflected parenchymal injury and related inflammation in the early posttransplant period, slowly regressing over many months. PC2 separated early injury from late fibrosis. Positive PC3 identified a distinct mildly inflamed state correlating with histologic steatohepatitis. Injury PCs correlated with liver function and histologic abnormalities. A classifier trained on histologic steatohepatitis predicted histologic steatohepatitis with cross-validated AUC = 0.83, and was associated with pathways reflecting metabolic abnormalities distinct from fibrosis. PC2 predicted histologic fibrosis (AUC = 0.80), as did a molecular fibrosis classifier (AUC = 0.74). The fibrosis classifier correlated with matrix remodeling pathways with minimal overlap with those selective for steatohepatitis, although some biopsies had both. Genome-wide assessment of liver transplant biopsies can not only detect molecular changes induced by rejection but also those correlating with parenchymal injury, steatohepatitis, and fibrosis, offering potential insights into disease mechanisms for primary diseases.

Non-invasive alloimmune risk stratification of long-term liver transplant recipients.

BACKGROUND & AIMS: Management of long-term immunosuppression following liver transplantation (LT) remains empirical. Surveillance liver biopsies in combination with transcriptional profiling could overcome this challenge by identifying recipients with active alloimmune-mediated liver damage despite normal liver tests, but this approach lacks applicability. Our aim was to investigate the utility of non-invasive tools for the stratification of stable long-term survivors of LT, according to their immunological risk and need for immunosuppression. METHODS: We conducted a cross-sectional multicentre study of 190 adult LT recipients assessed to determine their eligibility to participate in an immunosuppression withdrawal trial. Patients had stable liver allograft function and had been transplanted for non-autoimmune non-replicative viral liver disease >3 years before inclusion. We performed histological, immunogenetic and serological studies and measured the intrahepatic transcript levels of an 11-gene classifier highly specific for T cell-mediated rejection (TCMR). RESULTS: In this cohort, 35.8% of patients harboured clinically silent fibro-inflammatory liver lesions (13.7% had mild damage and 22.1% had moderate-to-severe damage). The severity of liver allograft damage was positively associated with TCMR-related transcripts, class II donor-specific antibodies (DSAs), ALT, AST, and liver stiffness measurement (LSM), and negatively correlated with serum creatinine and tacrolimus trough levels. Liver biopsies were stratified according to their TCMR transcript levels using a cut-off derived from biopsies with clinically significant TCMR. Two multivariable prediction models, integrating ALT+LSM or ALT+class II DSAs, had a high discriminative capacity for classifying patients with or without alloimmune damage. The latter model performed well in an independent cohort of 156 liver biopsies obtained from paediatric liver recipients with similar inclusion/exclusion criteria. CONCLUSION: ALT, class II DSAs and LSM are valuable tools to non-invasively identify stable LT recipients without significant underlying alloimmunity who could benefit from minimisation of immunosuppression. LAY SUMMARY: A large proportion of liver transplant patients with normal liver tests have inflammatory liver lesions, which in 17% of cases are molecularly indistinguishable from those seen at the time of rejection. ALT, class II donor-specific antibodies and liver stiffness are useful in identifying patients with this form of subclinical rejection. We propose these markers as a useful tool to help clinicians determine if the immunosuppression administered is adequate.

Advances in Liver Transplantation: where are we in the pursuit of transplantation tolerance?

Liver transplantation is the ultimate treatment option for end-stage liver disease. Breakthroughs in surgical practice and immunosuppression have seen considerable advancements in survival after transplantation. However, the intricate management of immunosuppressive regimens, balancing desired immunological quiescence while minimizing toxicity has proven challenging. Diminishing improvements in long-term morbidity and mortality have been inextricably linked with the protracted use of these medications. As such, there is now enormous interest to devise protocols that will allow us to minimize or completely withdraw immunosuppressants after transplantation. Immunosuppression withdrawal trials have proved the reality of tolerance following liver transplantation, however, without intervention will only occur after several years at the risk of potential cumulative immunosuppression-related morbidity. Focus has now been directed at accelerating this phenomenon through tolerance-inducing strategies. In this regard, efforts have seen the use of regulatory cell immunotherapy. Here we focus particularly on regulatory T cells, discussing preclinical data that propagated several clinical trials of adoptive cell therapy in liver transplantation. Furthermore, we describe efforts to further optimize the specificity and survival of regulatory cell therapy guided by concurrent immunomonitoring studies and the development of novel technologies including chimeric antigen receptors and co-administration of low-dose IL-2.

Preformed T cell alloimmunity and HLA eplet mismatch to guide immunosuppression minimization with tacrolimus monotherapy in kidney transplantation: Results of the CELLIMIN trial.

Personalizing immunosuppression is a major objective in transplantation. Transplant recipients are heterogeneous regarding their immunological memory and primary alloimmune susceptibility. This biomarker-guided trial investigated whether in low immunological-risk kidney transplants without pretransplant DSA and donor-specific T cells assessed by a standardized IFN-γ ELISPOT, low immunosuppression (LI) with tacrolimus monotherapy would be non-inferior regarding 6-month BPAR than tacrolimus-based standard of care (SOC). Due to low recruitment rates, the trial was terminated when 167 patients were enrolled. ELISPOT negatives (E-) were randomized to LI (n = 48) or SOC (n = 53), E+ received the same SOC. Six- and 12-month BPAR rates were higher among LI than SOC/E- (4/35 [13%] vs. 1/43 [2%], p = .15 and 12/48 [25%] vs. 6/53 [11.3%], p = .073, respectively). E+ patients showed similarly high BPAR rates than LI at 6 and 12 months (12/55 [22%] and 13/66 [20%], respectively). These differences were stronger in per-protocol analyses. Post-hoc analysis revealed that poor class-II eplet matching, especially DQ, discriminated E- patients, notably E-/LI, developing BPAR (4/28 [14%] low risk vs. 8/20 [40%] high risk, p = .043). Eplet mismatch also predicted anti-class-I (p = .05) and anti-DQ (p < .001) de novo DSA. Adverse events were similar, but E-/LI developed fewer viral infections, particularly polyoma-virus-associated nephropathy (p = .021). Preformed T cell alloreactivity and HLA eplet mismatch assessment may refine current baseline immune-risk stratification and guide immunosuppression decision-making in kidney transplantation.

Impact of donor extracellular vesicle release on recipient cell "cross-dressing" following clinical liver and kidney transplantation.

In several murine models of transplantation, the "cross-dressing" of recipient antigen presenting cells (APCs) with intact donor major histocompatibility complex (MHC) derived from allograft-released small extracellular vesicles (sEVs) has been recently described as a key mechanism in eliciting and sustaining alloimmune responses. Investigation of these processes in clinical organ transplantation has, however, been hampered by the lack of sensitivity of conventional instruments and assays. We have employed advanced imaging flow cytometry (iFCM) to explore the kinetics of allograft sEV release and the extent to which donor sEVs might induce cross-dressing following liver and kidney transplantation. We report for the first time that recipient APC cross-dressing can be transiently detected in the circulation shortly after liver, but not kidney, transplantation in association with the release of HLA-bearing allograft-derived sEVs. In liver transplant recipients the majority of circulating cells exhibiting donor HLA are indeed cross-dressed cells and not passenger leukocytes. In keeping with experimental animal data, the downstream functional consequences of the transfer of circulating sEVs harvested from human transplant recipients varies depending on the type of transplant and time posttransplant. sEVs released shortly after liver, but not kidney, transplantation exhibit immunoinhibitory effects that could influence liver allograft immunogenicity.