Proteoforms in Peripheral Blood Mononuclear Cells as Novel Rejection Biomarkers in Liver Transplant Recipients

Biomarker profiles of acute rejection in liver transplant recipients could enhance the diagnosis and management of recipients. Our aim was to identify diagnostic proteoform signatures of acute rejection in circulating immune cells, using an emergent “top‐down” proteomics methodology. We prepared differentially processed and cryopreserved cell lysates from 26 nonviral liver transplant recipients by molecular weight–based fractionation and analyzed them by mass spectrometry of whole proteins in three steps: (i) Nanocapillary liquid chromatography coupled with high‐resolution tandem mass spectrometry; (ii) database searching to identify and characterize intact proteoforms; (iii) data processing through a hierarchical linear model matching the study design to quantify proteoform fold changes in patients with rejection versus normal liver function versus acute dysfunction without rejection. Differentially expressed proteoforms were seen in patients with rejection versus normal and nonspecific controls, most evidently in the cell preparations stored in traditional serum‐rich media. Mapping analysis of these proteins back to genes through gene ontology and pathway analysis tools revealed multiple signaling pathways, including inflammation mediated by cytokines and chemokines. Larger studies are needed to validate these novel rejection signatures and test their predictive value for use in clinical management.     

Liver Allograft Provides Immunoprotection for the Cardiac Allograft in Combined Heart–Liver Transplantation

When transplanted simultaneously, the liver allograft has been thought to have an immunoprotective role on other organs; however, detailed analyses in simultaneous heart–liver transplantation (SHLT) have not been done to date. We analyzed patient outcomes and incidence of immune‐mediated injury in 22 consecutive SHLT versus 223 isolated heart transplantation (IHT) recipients between January 2004 and December 2013, by reviewing 3912 protocol‐ and indication‐specific cardiac allograft biopsy specimens. Overall survival was similar (86.4%, 86.4%, and 69.1% for SHLT and 93.3%, 84.7%, and 70.0% for IHT at 1, 5, and 10 years; p = 0.83). Despite similar immunosuppression, the incidence of T cell–mediated rejection (TCMR) was lower in SHLT (31.8%) than in IHT (84.8%) (p < 0.0001). Although more SHLT patients had preexisting donor‐specific HLA antibody (22.7% versus 8.1%; p = 0.04), the incidence of antibody‐mediated rejection was not different in SHLT compared with IHT (4.5% versus 14.8%, p = 0.33). While the left ventricular ejection fraction was comparable in both groups at 5 years, the incidence and severity of cardiac allograft vasculopathy were reduced in the SHLT recipients (42.9% versus 66.8%, p = 0.03). Simultaneously transplanted liver allograft was associated with reduced risk of TCMR (odds ratio [OR] 0.003, 95% confidence interval [CI] 0–0.02; p < 0.0001), antibody‐mediated rejection (OR 0.04, 95% CI 0–0.46; p = 0.004), and cardiac allograft vasculopathy (OR 0.26, 95% CI 0.07–0.84; p = 0.02), after adjusting for other risk factors. These data suggest that the incidence of alloimmune injury in the heart allograft is reduced in SHLT recipients.     

Using Molecular Phenotyping to Guide Improvements in the Histologic Diagnosis of T Cell–Mediated Rejection

Recognition that some lesions typical of T cell–mediated rejection (TCMR) also occur in antibody‐mediated rejection requires revision of the histologic TCMR definition. To guide this process, we assessed the relative importance of various lesions and the performance of new histology diagnostic algorithms, using molecular TCMR scores as histology‐independent estimates of true TCMR. In 703 indication biopsies, random forest analysis and logistic regression indicated that interstitial infiltrate (i‐lesions) and tubulitis (t‐lesions) were the key histologic predictors of molecular TCMR, with arteritis (v‐lesions) having less importance. Histology predicted molecular TCMR more accurately when diagnoses were assigned by strictly applying the Banff rules to the lesion scores and redefining isolated v‐lesion TCMR. This improved prediction from area under the curve (AUC) 0.70 with existing rules to AUC 0.80. Further improvements were achieved by introducing more categories to reflect inflammation (AUC 0.84), by summing the lesion scores (AUC 0.85) and by logistic regression (AUC 0.90). We concluded that histologic assessment of TCMR can be improved by placing more emphasis on i‐ and t‐lesions and incorporating new algorithms for diagnosis. Nevertheless, some discrepancies between histologic and molecular diagnoses persist, partially due to the inherent nonspecificity of i‐ and t‐lesions, and molecular methods will be required to help resolve these cases.     

Cyclosporine Does Not Prevent Microvascular Loss in Transplantation but Can Synergize With a Neutrophil Elastase Inhibitor,Elafin, to Maintain Graft Perfusion During Acute Rejection

The loss of a functional microvascular bed in rejecting solid organ transplants is correlated with fibrotic remodeling and chronic rejection; in lung allografts, this pathology is predicted by bronchoalveolar fluid neutrophilia which suggests a role for polymorphonuclear cells in microcirculatory injury. In a mouse orthotopic tracheal transplant model, cyclosporine, which primarily inhibits T cells, failed as a monotherapy for preventing microvessel rejection and graft ischemia. To target neutrophil action that may be contributing to vascular injury, we examined the effect of a neutrophil elastase inhibitor, elafin, on the microvascular health of transplant tissue. We showed that elafin monotherapy prolonged microvascular perfusion and enhanced tissue oxygenation while diminishing the infiltration of neutrophils and macrophages and decreasing tissue deposition of complement C3 and the membrane attack complex, C5b‐9. Elafin was also found to promote angiogenesis through activation of the extracellular signal‐regulated kinase (ERK) signaling pathway but was insufficient as a single agent to completely prevent tissue ischemia during acute rejection episodes. However, when combined with cyclosporine, elafin effectively preserved airway microvascular perfusion and oxygenation. The therapeutic strategy of targeting neutrophil elastase activity alongside standard immunosuppression during acute rejection episodes may be an effective approach for preventing the development of irreversible fibrotic remodeling.     

Exogenous Lipocalin 2 Ameliorates Acute Rejection in a Mouse Model of Renal Transplantation

Lipocalin 2 (Lcn2) is rapidly produced by damaged nephron epithelia and is one of the most promising new markers of renal injury, delayed graft function and acute allograft rejection (AR); however, the functional importance of Lcn2 in renal transplantation is largely unknown. To understand the role of Lcn2 in renal AR, kidneys from Balb/c mice were transplanted into C57Bl/6 mice and vice versa and analyzed for morphological and physiological outcomes of AR at posttransplantation days 3, 5, and 7. The allografts showed a steady increase in intensity of interstitial infiltration, tubulitis and periarterial aggregation of lymphocytes associated with a substantial elevation in serum levels of creatinine, urea and Lcn2. Perioperative administration of recombinant Lcn2:siderophore:Fe complex (rLcn2) to recipients resulted in functional and morphological amelioration of the allograft at day 7 almost as efficiently as daily immunosuppression with cyclosporine A (CsA). No significant differences were observed in various donor–recipient combinations (C57Bl/6 wild‐type and Lcn2^?/?, Balb/c donors and recipients). Histochemical analyses of the allografts showed reduced cell death in recipients treated with rLcn2 or CsA. These results demonstrate that Lcn2 plays an important role in reducing the extent of kidney AR and indicate the therapeutic potential of Lcn2 in transplantation.

     

Orthogonal Comparison of Molecular Signatures of Kidney Transplants With Subclinical and Clinical Acute Rejection: Equivalent Performance Is Agnostic to Both Technology and Platform

We performed orthogonal technology comparisons of concurrent peripheral blood and biopsy tissue samples from 69 kidney transplant recipients who underwent comprehensive algorithm‐driven clinical phenotyping. The sample cohort included patients with normal protocol biopsies and stable transplant (sTx) function (n = 25), subclinical acute rejection (subAR, n = 23), and clinical acute rejection (cAR, n = 21). Comparisons between microarray and RNA sequencing (RNA‐seq) signatures were performed and demonstrated a strong correlation between the blood and tissue compartments for both technology platforms. A number of shared differentially expressed genes and pathways between subAR and cAR in both platforms strongly suggest that these two clinical phenotypes form a continuum of alloimmune activation. SubAR is associated with fewer or less expressed genes than cAR in blood, whereas in biopsy tissues, this clinical phenotype demonstrates a more robust molecular signature for both platforms. The discovery work done in this study confirms a clear ability to detect gene expression profiles for sTx, subAR, and cAR in both blood and biopsy tissue, yielding equivalent predictive performance that is agnostic to both technology and platform. Our data also provide strong biological insights into the molecular mechanisms underlying these signatures, underscoring their logistical potential as molecular diagnostics to improve clinical outcomes following kidney transplantation.     

Pretransplant Numbers of CD16+ Monocytes as a Novel Biomarker to Predict Acute Rejection After Kidney Transplantation: A Pilot Study

Acute rejection is one of the major immunological determinants of kidney graft function and survival. Early biomarkers to predict rejection are lacking. Emerging evidence reveals a crucial role for the monocyte/macrophage lineage cells in the pathogenesis of rejection. We hypothesized that higher pretransplant numbers of proinflammatory CD16+ monocytes can predict rejection. The study cohort consisted of 104 kidney transplant recipients (58 with no rejection and 46 with biopsy‐proven rejection) and 33 healthy persons. Posttransplant median follow‐up time was 14.7 mo (interquartile range 0.3–34 mo). Pretransplantation blood samples were analyzed by flow cytometry for monocyte immunophenotypes. Groups were compared by Cox regression models for the occurrence of acute rejection. We documented a significantly increased absolute number of pretransplant CD16+ monocytes in patients who developed biopsy‐proven rejection after transplantation compared with those with no rejection (hazard ratio [HR] 1.60, 95% CI 1.28–2.00, p < 0.001) and healthy persons (HR 1.47, 95% CI 1.18–1.82, p < 0.001). In parallel, significantly fewer absolute numbers of CD16? monocytes were observed at pretransplant time points in rejectors versus nonrejectors (HR 0.74, 95% CI 0.58–0.94, p < 0,014). A higher pretransplant number of CD16+ monocytes is significantly associated with a higher risk of acute rejection after kidney transplantation.     

Donor‐Specific HLA Antibodies in Living Versus Deceased Donor Liver Transplant Recipients

With less ischemia, improved donor selection and controlled procedures, living donor liver transplantation (LDLT) might lead to less HLA donor‐specific antibody (DSA) formation or fewer adverse outcomes than deceased donor liver transplantation (DDLT). Using the multicenter A2ALL (Adult‐to‐Adult Living Donor Liver Transplantation Cohort Study) biorepository, we compared the incidence and outcomes of preformed and de novo DSAs between LDLT and DDLT. In total, 129 LDLT and 66 DDLT recipients were identified as having serial samples. The prevalence of preformed and de novo DSAs was not different between DDLT and LDLT recipients (p = 0.93). There was no association between patient survival and the timing (preformed vs. de novo), class (I vs. II) and relative levels of DSA between the groups; however, preformed DSA was associated with higher graft failure only in DDLT recipients (p = 0.01). De novo DSA was associated with graft failure regardless of liver transplant type (p = 0.005) but with rejection only in DDLT (p = 0.0001). On multivariate analysis, DSA was an independent risk factor for graft failure regardless of liver transplant type (p = 0.017, preformed; p = 0.002, de novo). In conclusion, although similar in prevalence, DSA may have more impact in DDLT than LDLT recipients. Although our findings need further validation, future research should more robustly test the effect of donor type and strategies to mitigate the impact of DSA.     

Risk Assessment in High‐ and Low‐MELD Liver Transplantation

Allocation of liver grafts triggers emotional debates, as those patients, not receiving an organ, are prone to death. We analyzed a high–Model of End‐stage Liver Disease (MELD) cohort (laboratory MELD score ≥30, n = 100, median laboratory MELD score of 35; interquartile range 31–37) of liver transplant recipients at our center during the past 10 years and compared results with a low‐MELD group, matched by propensity scoring for donor age, recipient age, and cold ischemia time. End points of our study were cumulative posttransplantation morbidity, cost, and survival. Six different prediction models, including donor age x recipient MELD (D‐MELD), Difference between listing MELD and MELD at transplant (Delta MELD), donor‐risk index (DRI), Survival Outcomes Following Liver Transplant (SOFT), balance‐of‐risk (BAR), and University of California Los Angeles–Futility Risk Score (UCLA‐FRS), were applied in both cohorts to identify risk for poor outcome and high cost. All score models were compared with a clinical‐oriented decision, based on the combination of hemofiltration plus ventilation. Median intensive care unit and hospital stays were 8 and 26 days, respectively, after liver transplantation of high‐MELD patients, with a significantly increased morbidity compared with low‐MELD patients (median comprehensive complication index 56 vs. 36 points [maximum points 100] and double cost [median US$179 631 vs. US$80 229]). Five‐year survival, however, was only 8% less than that of low‐MELD patients (70% vs. 78%). Most prediction scores showed disappointing low positive predictive values for posttransplantation mortality, such as mortality above thresholds, despite good specificity. The clinical observation of hemofiltration plus ventilation in high‐MELD patients was even superior in this respect compared with D‐MELD, DRI, Delta MELD, and UCLA‐FRS but inferior to SOFT and BAR models. Of all models tested, only the BAR score was linearly associated with complications. In conclusion, the BAR score was most useful for risk classification in liver transplantation, based on expected posttransplantation mortality and morbidity. Difficult decisions to accept liver grafts in high‐risk recipients may thus be guided by additional BAR score calculation, to increase the safe use of scarce organs.     

Graft‐Infiltrating Macrophages Adopt an M2 Phenotype and Are Inhibited by Purinergic Receptor P2X7 Antagonist in Chronic Rejection

Macrophages exhibit diverse phenotypes and functions; they are also a major cell type infiltrating chronically rejected allografts. The exact phenotypes and roles of macrophages in chronic graft loss remain poorly defined. In the present study, we used a mouse heart transplant model to examine macrophages in chronic allograft rejection. We found that treatment of C57BL/6 mice with CTLA4 immunoglobulin fusion protein (CTLA4‐Ig) prevented acute rejection of a Balb/c heart allograft but allowed chronic rejection to develop over time, characterized by prominent neointima formation in the graft. There was extensive macrophage infiltration in the chronically rejected allografts, and the graft‐infiltrating macrophages expressed markers associated with M2 cells but not M1 cells. In an in vitro system in which macrophages were polarized into either M1 or M2 cells, we screened phenotypic differences between M1 and M2 cells and identified purinergic receptor P2X7 (P2x7r), an adenosine triphosphate (ATP)–gated ion channel protein that was preferentially expressed by M2 cells. We further showed that blocking the P2x7r using oxidized ATP (oATP) inhibited M2 induction in a dose‐dependent fashion in vitro. Moreover, treatment of C57BL/6 recipients with the P2x7r antagonist oATP, in addition to CTLA4‐Ig treatment, inhibited graft‐infiltrating M2 cells, prevented transplant vasculopathy, and induced long‐term heart allografts survival. These findings highlight the importance of the P2x7r–M2 axis in chronic rejection and establish P2x7r as a potential therapeutic target in suppression of chronic rejection.     

Sinusoidal obstruction syndrome as a manifestation of acute antibody-mediated rejection after liver transplantation

Antibody-mediated rejection (AMR) after liver transplantation is uncommon but, when present, manifests as graft dysfunction. We report the case of a 54-year-old woman who developed portal hypertension with pleural effusion and ascites secondary to sinusoidal obstruction syndrome (SOS) due to acute AMR following an ABO-matched liver transplantation for autoimmune cirrhosis and hepatocellular carcinoma. Initial immunosuppression comprised basiliximab, decreasing prednisone, tacrolimus, and mycophenolate mofetil. After 1 month, she presented with the massive pleural effusion, slight ascites, and normal liver tests. After excluding common causes of pleural effusion, we performed a liver biopsy that showed atypical rejection with the involvement of large centrilobular veins partially occluded by marked endotheliitis and lax fibrosis suggestive of SOS. Direct immunofluorescence study of C4d showed diffuse endothelial sinusoidal staining, and de novo donor-specific anti-human leukocyte antigen antibodies were detected in his blood. Thus, we diagnosed AMR focused on centrilobular veins and initiated treatment with defibrotide, steroid pulses, and diuretics. However, this was ineffective, and the pleural effusion only resolved when plasmapheresis and intravenous immunoglobulin were started. This case shows that AMR can cause SOS with portal hypertension and present with a pleural effusion, and as such, it should be suspected after excluding other more common causes of effusion.     

Hiding in Plain Sight: A Case of Ornithine Transcarbamylase Deficiency Unmasked Post–Liver Transplantation

Ornithine transcarbamylase deficiency represents the most common inherited defect of the urea cycle. This enzyme, predominantly found in the liver, plays a crucial role in recycling free ammonia, with deficiencies often leading to fatal complications. Here, we present the case of a 63‐year‐old man with alcoholic cirrhosis who underwent orthotopic liver transplantation, gradual worsening of his mental status, and progressive elevation of ammonia levels. Liver allograft function was deemed normal, raising concern for a donor‐derived metabolic disorder of the urea cycle. Evaluation of the donor patient's blood revealed that the donor was heterozygous for the OTC gene. Posttransplantation changes in mental status should prompt a clinician to consider the most likely causes; however, once these have been ruled out, it is important to consider the less common causes of metabolic derangements. The rarity of these disorders makes expertise of diagnosis, standardization of evaluation, and treatment strategies challenging.     

Allelic and Epitopic Characterization of Intra–Kidney Allograft Anti‐HLA Antibodies at Allograft Nephrectomy

The reasons for the increased incidence of de novo anti–human leukocyte antibody (HLA) donor‐specific antibodies (DSAs) observed after kidney allograft nephrectomy are not fully understood. One advocated mechanism suggests that at graft loss, DSAs are not detected in the serum because they are fixed on the nonfunctional transplant; removal of the kidney allows DSAs to then appear in the blood circulation. The aim of our study was to compare anti‐HLA antibodies present in the serum and in the graft at the time of an allograft nephrectomy. Using solid‐phase assays, anti‐HLA antibodies were searched for in the sera of 17 kidney transplant patients undergoing allograft nephrectomy. No anti‐HLA antibodies were detected in the graft if they were not also detected in the serum. Eleven of the 12 patients who had DSAs detected in their sera also had DSAs detected in the grafts. Epitopic analysis revealed that most anti‐HLA antibodies detected in removed grafts were directed against the donor. In summary, our data show that all anti‐HLA antibodies that were detected in grafts were also detected in the sera. These intragraft anti‐HLA antibodies are mostly directed against the donor at an epitopic level but not always at an antigenic level.     

Left Lobe Auxiliary Liver Transplantation for End‐stage Hepatitis B Liver Cirrhosis

Auxiliary liver transplantation (ALT) for hepatitis B virus (HBV)‐related liver cirrhosis previously showed poor results, because the native liver was a significant source of HBV recurrence and the graft could be rapidly destroyed by HBV infection in an immunosuppressive condition. Four patients with HBV‐related liver cirrhosis were unable to undergo orthotopic liver transplantation because the only available grafts of left lobe were too small. Under entecavir‐based anti‐HBV treatment, they underwent ALT in which the recipient left liver was removed and the small left lobe graft was implanted in the corresponding space. The mean graft weight/recipient weight was 0.49% (range, 0.38%–0.55%). One year after transplantation, the graft sizes were increased to 273% and the remnant livers were decreased to 44%. Serum HBV DNA was persistently undetectable. Periodic graft biopsy showed no signs of tissue injury and negative immunostaining for hepatitis B surface antigen and hepatitis B core antigen. After a mean follow‐up period of 21 months, all patients live well with normal graft function. Our study suggests that ALT for HBV‐related liver cirrhosis is feasible under entecavir‐based anti‐HBV treatment. Successful application of small left livers in end‐stage liver cirrhosis may significantly increase the pool of left liver grafts for adult patients.