Perforin inhibition protects from lethal endothelial damage during fulminant viral hepatitis.

CD8 T cells protect the liver against viral infection, but can also cause severe liver damage that may even lead to organ failure. Given the lack of mechanistic insights and specific treatment options in patients with acute fulminant hepatitis, we develop a mouse model reflecting a severe acute virus-induced CD8 T cell-mediated hepatitis. Here we show that antigen-specific CD8 T cells induce liver damage in a perforin-dependent manner, yet liver failure is not caused by effector responses targeting virus-infected hepatocytes alone. Additionally, CD8 T cell mediated elimination of cross-presenting liver sinusoidal endothelial cells causes endothelial damage that leads to a dramatically impaired sinusoidal perfusion and indirectly to hepatocyte death. With the identification of perforin-mediated killing as a critical pathophysiologic mechanism of liver failure and the protective function of a new class of perforin inhibitor, our study opens new potential therapeutic angles for fulminant viral hepatitis.

Isolated vascular "v" lesions in liver allografts: How to approach this unusual finding.

According to the Banff criteria for kidney allografts, isolated vascular or "v" lesions are defined as intimal inflammation, age-inappropriate fibro-intimal hyperplasia, or both, without the presence of associated interstitial T cell-mediated rejection (TCMR). In general, these lesions portend a worse outcome for kidney allografts, particularly in those where the "v" lesions are identified in patients with coexistent donor specific antibodies (DSA) or later after transplantation. Although affected arteries are rarely sampled in liver allograft biopsies, we identified nine patients at a mean of 1805 days posttransplantation and compared these to matched controls. Almost half (4 of 9) of the study patient biopsies showed inflammatory arteritis associated with focal or diffuse C4d positivity, which was not observed in matched controls. One "v" lesion patient progressed to rejection-related graft failure and two developed moderate/severe TCMR in subsequent biopsies, whereas only one rejection episode occurred in follow-up biopsies, and no rejection-related deaths or graft failures were detected in controls. In conclusion, patients with liver allograft isolated "v" lesions should undergo further evaluation and closer follow-up for impending TCMR and/or underlying co-existent chronic antibody-mediated rejection (AMR).

The bridge between transplantation and regenerative medicine: Beginning a new Banff classification of tissue engineering pathology.

The science of regenerative medicine is arguably older than transplantation-the first major textbook was published in 1901-and a major regenerative medicine meeting took place in 1988, three years before the first Banff transplant pathology meeting. However, the subject of regenerative medicine/tissue engineering pathology has never received focused attention. Defining and classifying tissue engineering pathology is long overdue. In the next decades, the field of transplantation will enlarge at least tenfold, through a hybrid of tissue engineering combined with existing approaches to lessening the organ shortage. Gradually, transplantation pathologists will become tissue-(re-) engineering pathologists with enhanced skill sets to address concerns involving the use of bioengineered organs. We outline ways of categorizing abnormalities in tissue-engineered organs through traditional light microscopy or other modalities including biomarkers. We propose creating a new Banff classification of tissue engineering pathology to standardize and assess de novo bioengineered solid organs transplantable success in vivo. We recommend constructing a framework for a classification of tissue engineering pathology now with interdisciplinary consensus discussions to further develop and finalize the classification at future Banff Transplant Pathology meetings, in collaboration with the human cell atlas project. A possible nosology of pathologic abnormalities in tissue-engineered organs is suggested.

2016 Comprehensive Update of the Banff Working Group on Liver Allograft Pathology: Introduction of Antibody-Mediated Rejection.

The Banff Working Group on Liver Allograft Pathology reviewed and discussed literature evidence regarding antibody-mediated liver allograft rejection at the 11th (Paris, France, June 5-10, 2011), 12th (Comandatuba, Brazil, August 19-23, 2013), and 13th (Vancouver, British Columbia, Canada, October 5-10, 2015) meetings of the Banff Conference on Allograft Pathology. Discussion continued online. The primary goal was to introduce guidelines and consensus criteria for the diagnosis of liver allograft antibody-mediated rejection and provide a comprehensive update of all Banff Schema recommendations. Included are new recommendations for complement component 4d tissue staining and interpretation, staging liver allograft fibrosis, and findings related to immunosuppression minimization. In an effort to create a single reference document, previous unchanged criteria are also included.

Functional Immune Anatomy of the Liver-As an Allograft.

The liver is an immunoregulatory organ in which a tolerogenic microenvironment mitigates the relative "strength" of local immune responses. Paradoxically, necro-inflammatory diseases create the need for most liver transplants. Treatment of hepatitis B virus, hepatitis C virus, and acute T cell-mediated rejection have redirected focus on long-term allograft structural integrity. Understanding of insults should enable decades of morbidity-free survival after liver replacement because of these tolerogenic properties. Studies of long-term survivors show low-grade chronic inflammatory, fibrotic, and microvascular lesions, likely related to some combination of environment insults (i.e. abnormal physiology), donor-specific antibodies, and T cell-mediated immunity. The resultant conundrum is familiar in transplantation: adequate immunosuppression produces chronic toxicities, while lightened immunosuppression leads to sensitization, immunological injury, and structural deterioration. The "balance" is more favorable for liver than other solid organ allografts. This occurs because of unique hepatic immune physiology and provides unintended benefits for allografts by modulating various afferent and efferent limbs of allogenic immune responses. This review is intended to provide a better understanding of liver immune microanatomy and physiology and thereby (a) the potential structural consequences of low-level, including allo-antibody-mediated injury; and (b) how liver allografts modulate immune reactions. Special attention is given to the microvasculature and hepatic mononuclear phagocytic system.

Proposed Diagnostic Criteria for Chronic Antibody-Mediated Rejection in Liver Allografts.

Donor-specific alloantibodies (DSA) can cause acute antibody-mediated rejection (AMR) in all solid organ allografts. However, long-term outcome in patients with posttransplant DSA needs further study. We retrospectively evaluated prospectively collected paired serum, tissue, and data on 45 matched DSA- positive [DSA+; mean florescence intensity (MFI) ≥10,000] and -negative (DSA-) recipients of a primary liver-only allograft from January 2000 to April 2009. Blinded histopathologic evaluation demonstrated that DSA+ versus DSA- patients were more likely to have subtle inflammation and unique patterns of fibrosis, despite normal or near-normal liver function tests. Stepwise multivariable modeling developed a score (putatively named the chronic AMR [cAMR] score) that included interface activity, lobular inflammation, portal tract collagenization, portal venopathy, sinusoidal fibrosis, and hepatitis C virus status. The score was developed (c = 0.811) and cross-validated (c = 0.704) to predict allograft failure. Two cutoffs were employed to optimize sensitivity and specificity (80% each); a value >27.5 predicted 50% 10-year allograft failure. We propose chronic AMR as a potential new entity defined by (1) a high cAMR score, (2) DSA, and (3) elimination of other potential causes of a similar injury pattern. In conclusion, cAMR score calculation identified liver allograft recipients with DSA at highest risk for allograft loss, although independent validation is needed.

Elevated ST2 Distinguishes Incidences of Pediatric Heart and Small Bowel Transplant Rejection.

Elevated serum soluble (s) suppressor of tumorigenicity-2 is observed during cardiovascular and inflammatory bowel diseases. To ascertain whether modulated ST2 levels signify heart (HTx) or small bowel transplant (SBTx) rejection, we quantified sST2 in serially obtained pediatric HTx (n = 41) and SBTx recipient (n = 18) sera. At times of biopsy-diagnosed HTx rejection (cellular and/or antibody-mediated), serum sST2 was elevated compared to rejection-free time points (1714 ± 329 vs. 546.5 ± 141.6 pg/mL; p = 0.0002). SBTx recipients also displayed increased serum sST2 during incidences of rejection (7536 ± 1561 vs. 2662 ± 543.8 pg/mL; p = 0.0347). Receiver operator characteristic (ROC) analysis showed that serum sST2 > 600 pg/mL could discriminate time points of HTx rejection and nonrejection (area under the curve [AUC] = 0.724 ± 0.053; p = 0.0003). ROC analysis of SBTx measures revealed a similar discriminative capacity (AUC = 0.6921 ± 0.0820; p = 0.0349). Quantitative evaluation of both HTx and SBTx biopsies revealed that rejection significantly increased allograft ST2 expression. Pathway and Network Analysis of biopsy data pinpointed ST2 in the dominant pathway modulated by rejection and predicted tumor necrosis factor-α and IL-1ß as upstream activators. In total, our data indicate that alloimmune-associated pro-inflammatory cytokines increase ST2 during rejection. They also demonstrate that routine serum sST2 quantification, potentially combined with other biomarkers, should be investigated further to aid in the noninvasive diagnosis of rejection.

The role of donor-specific HLA alloantibodies in liver transplantation.

The impact of donor-specific HLA alloantibodies (DSA) on short- and long-term liver transplant outcome is not clearly defined. While it is clear that not all levels of allosensitization produce overt clinical injury, and that liver allografts possess some degree of alloantibody resistance, alloantibody-mediated adverse consequences are increasingly being recognized. To better define the current state of this topic, we assembled experts to provide insights, explore controversies and develop recommendations for future research on the consequences of DSA in liver transplantation. This article summarizes the proceedings of this inaugural meeting. Several insights emerged. Acute antibody-mediated rejection (AMR), although rarely diagnosed, is increasingly understood to overlap with T cell-mediated rejection. Isolated liver allograft recipients are at increased risk of early allograft immunologic injury when preformed DSA are high titer and persist posttransplantation. Persons who undergo simultaneous liver-kidney transplantation are at risk of renal AMR when Class II DSA persist posttransplantation. Other under-appreciated DSA associations include ductopenia and fibrosis, plasma cell hepatitis, biliary strictures and accelerated fibrosis associated with recurrent liver disease. Standardized DSA testing and diagnostic criteria for both acute and chronic AMR are needed to distil existing associations into etiological processes in order to develop responsive therapeutic strategies.

Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

"Plasma cell hepatitis" in liver allografts: identification and characterization of an IgG4-rich cohort.

Plasma cell hepatitis (PCH), also known as "de novo autoimmune" hepatitis, is an increasingly recognized, but suboptimally named and poorly understood, category of late allograft dysfunction strongly resembling autoimmune hepatitis (AIH): They share plasma-cell-rich necro-inflammatory activity on biopsy, autoantibodies and steroid responsiveness, but overlap with rejection is problematic. A retrospective study of clinical, serological, histopathological and IgG4 immunohistological features of PCH (n = 20) in liver allograft recipients, native liver AIH (n = 19) and plasma-cell-rich renal allograft rejection (n = 20) showed: (1) high frequency (44%) of HLA-DR15; (2) less female predominance (p = 0.03) and (3) n = 9/20 PCH recipients showed >25 IgG4+ plasma cells/high-power field (IgG4+ PCH) versus AIH (n = 1/19, p = 0.008) or plasma-cell-rich kidney rejection (n = 2/20, p = 0.03). The IgG4+ PCH (n = 9) subgroup showed lower alanine transaminase (ALT) (p < 0.01) and aspartate transaminase (AST) (p < 0.05) at index biopsy but (a) higher plasma cell number/percentage, (b) more aggressive-appearing portal/periportal and perivenular necro-inflammatory activity and (c) more severe portal/periportal fibrosis than IgG4- PCH (n = 11). Significant demographic, histopathologic and plasma cell phenotype differences between PCH and AIH suggest distinct pathogenic mechanisms for at least the IgG4+ PCH subgroup likely representing an overlap between allo- and auto-immunity. IgG4+ PCH was associated with fibrosis, but also highly responsive to increased immunosuppression.

Regulatory dendritic cell infusion prolongs kidney allograft survival in nonhuman primates.

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5-10 × 10(6) /kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on Day -2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n = 6) and 113.5 days (p < 0.05) in DCreg-treated animals (n = 6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95(+) T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.

Allograft outcomes in outbred mice.

Inbreeding depression and lack of genetic diversity in inbred mice could mask unappreciated causes of graft failure or remove barriers to tolerance induction. To test these possibilities, we performed heart transplantation between outbred or inbred mice. Unlike untreated inbred mice in which all allografts were rejected acutely (6-16 days posttransplantation), untreated outbred mice had heterogeneous outcomes, with grafts failing early (<4 days posttransplantation), acutely (6-24 days) or undergoing chronic rejection (>75 days). Blocking T cell costimulation induced long-term graft acceptance in both inbred and outbred mice, but did not prevent the early graft failure observed in the latter. Further investigation of this early phenotype established that it is dependent on the donor, and not the recipient, being outbred and that it is characterized by hemorrhagic necrosis and neutrophilic vasculitis in the graft without preformed, high titer antidonor antibodies in the recipient. Complement or neutrophil depletion prevented early failure of outbred grafts, whereas transplanting CD73-deficient inbred hearts, which are highly susceptible to ischemia-reperfusion injury, recapitulated the early phenotype. Therefore, outbred mice could provide broader insight into donor and recipient determinants of allograft outcomes but their hybrid vigor and genetic diversity do not constitute a uniform barrier to tolerance induction.

Banff 2011 Meeting report: new concepts in antibody-mediated rejection.

The 11th Banff meeting was held in Paris, France, from June 5 to 10, 2011, with a focus on refining diagnostic criteria for antibody-mediated rejection (ABMR). The major outcome was the acknowledgment of C4d-negative ABMR in kidney transplants. Diagnostic criteria for ABMR have also been revisited in other types of transplants. It was recognized that ABMR is associated with heterogeneous phenotypes even within the same type of transplant. This highlights the necessity of further refining the respective diagnostic criteria, and is of particular significance for the design of randomized clinical trials. A reliable phenotyping will allow for definition of robust end-points. To address this unmet need and to allow for an evidence-based refinement of the Banff classification, Banff Working Groups presented multicenter data regarding the reproducibility of features relevant to the diagnosis of ABMR. However, the consensus was that more data are necessary and further Banff Working Group activities were initiated. A new Banff working group was created to define diagnostic criteria for ABMR in kidneys independent of C4d. Results are expected to be presented at the 12th Banff meeting to be held in 2013 in Brazil. No change to the Banff classification occurred in 2011.

Re-examination of the lymphocytotoxic crossmatch in liver transplantation: can C4d stains help in monitoring?

C4d-assisted recognition of antibody-mediated rejection (AMR) in formalin-fixed paraffin-embedded tissues (FFPE) from donor-specific antibody-positive (DSA+) renal allograft recipients prompted study of DSA+ liver allograft recipients as measured by lymphocytotoxic crossmatch (XM) and/or Luminex. XM results did not influence patient or allograft survival, or cellular rejection rates, but XM+ recipients received significantly more prophylactic steroids. Endothelial C4d staining strongly correlates with XM+ (<3 weeks posttransplantation) and DSA+ status and cellular rejection, but not with worse Banff grading or treatment response. Diffuse C4d staining, XM+, DSA+ and ABO- incompatibility status, histopathology and clinical-serologic profile helped establish an isolated AMR diagnosis in 5 of 100 (5%) XM+ and one ABO-incompatible, recipients. C4d staining later after transplantation was associated with rejection and nonrejection-related causes of allograft dysfunction in DSA- and DSA+ recipients, some of whom had good outcomes without additional therapy. Liver allograft FFPE C4d staining: (a) can help classify liver allograft dysfunction; (b) substantiates antibody contribution to rejection; (c) probably represents nonalloantibody insults and/or complete absorption in DSA- recipients and (d) alone, is an imperfect AMR marker needing correlation with routine histopathology, clinical and serologic profiles. Further study in late biopsies and other tissue markers of liver AMR with simultaneous DSA measurements are needed.

Digital transplantation pathology: combining whole slide imaging, multiplex staining and automated image analysis.

Conventional histopathology is the gold standard for allograft monitoring, but its value proposition is increasingly questioned. "-Omics" analysis of tissues, peripheral blood and fluids and targeted serologic studies provide mechanistic insights into allograft injury not currently provided by conventional histology. Microscopic biopsy analysis, however, provides valuable and unique information: (a) spatial-temporal relationships; (b) rare events/cells; (c) complex structural context; and (d) integration into a "systems" model. Nevertheless, except for immunostaining, no transformative advancements have "modernized" routine microscopy in over 100 years. Pathologists now team with hardware and software engineers to exploit remarkable developments in digital imaging, nanoparticle multiplex staining, and computational image analysis software to bridge the traditional histology-global "-omic" analyses gap. Included are side-by-side comparisons, objective biopsy finding quantification, multiplexing, automated image analysis, and electronic data and resource sharing. Current utilization for teaching, quality assurance, conferencing, consultations, research and clinical trials is evolving toward implementation for low-volume, high-complexity clinical services like transplantation pathology. Cost, complexities of implementation, fluid/evolving standards, and unsettled medical/legal and regulatory issues remain as challenges. Regardless, challenges will be overcome and these technologies will enable transplant pathologists to increase information extraction from tissue specimens and contribute to cross-platform biomarker discovery for improved outcomes.

Multiphoton intravital microscopy of the transplanted mouse kidney.

Graft outcomes after kidney transplantation continue to be adversely affected by ischemia-reperfusion injury and rejection. High-resolution, real-time imaging of the transplanted kidney could shed valuable insights into these dynamic processes, but such methodology has not been established. Here we describe a technique for intravital imaging of the transplanted mouse kidney using multiphoton fluorescence microscopy. The technique enabled real-time, high-resolution imaging and quantitation of renal filtration, cell death, leukocyte adhesion and capillary blood flow after transplantation. Using this technique, we found that brief graft ischemia associated with the transplantation procedure led to a rapid decline in renal filtration accompanied by a significant increase in microvascular leakage and renal tubular epithelial cell death within the first 3 h after transplantation. No significant changes in leukocyte adhesion or capillary blood flow were observed during the same time period. This report establishes multiphoton fluorescence microscopy as a sensitive tool for simultaneously studying functional and structural perturbations that occur in the mouse kidney after transplantation and for investigating the migration of leukocytes to the graft.

Adding value to liver (and allograft) biopsy evaluation using a combination of multiplex quantum dot immunostaining, high-resolution whole-slide digital imaging, and automated image analysis.

Various technologies including nucleic acid, protein, and metabolic array analyses of blood, liver tissue, and bile are emerging as powerful tools in the study of hepatic pathophysiology. The entire lexicon of liver disease, however, has been written using classical hematoxylin-eosin staining and light microscopic examination. The authors' goal is to develop new tools to enhance histopathologic examination of liver tissue that would enrich the information gained from liver biopsy analysis, enable quantitative analysis, and bridge the gap between various "-omics" tools and interpretation of routine liver biopsy results. This article describes the progress achieved during the past 2 years in developing multiplex quantum dot (nanoparticle) staining and combining it with high-resolution whole-slide imaging using a slide scanner equipped with filters to capture 9 distinct fluorescent signals for multiple antigens. The authors first focused on precise characterization of leukocyte subsets, but soon realized that the data generated were beyond the practical limits that could be properly evaluated, analyzed, and interpreted visually by a pathologist. Therefore, the authors collaborated with the open source FARSIGHT image analysis project (http://www.farsight-toolkit.org). FARSIGHT's goal is to develop and disseminate the next-generation toolkit of automated image analysis methods to enable quantification of molecular biomarkers on a cell-by-cell basis from multiparameter images. The resulting data can be used for histocytometric studies of the complex and dynamic tissue microenvironments that are of biomedical interest. The authors envisage that these tools will eventually be incorporated into the routine practice of surgical pathology and precipitate a revolution in the specialty.

Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups.

The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v-lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics-technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

Liver transplantation using donation after cardiac death donors: long-term follow-up from a single center.

There is a lack of universally accepted clinical parameters to guide the utilization of donation after cardiac death (DCD) donor livers and it is unclear as to which patients would benefit most from these organs. We reviewed our experience in 141 patients who underwent liver transplantation using DCD allografts from 1993 to 2007. Patient outcomes were analyzed in comparison to a matched cohort of 282 patients who received livers from donation after brain death (DBD) donors. Patient survival was similar, but 1-, 5- and 10-year graft survival was significantly lower in DCD (69%, 56%, 44%) versus DBD (82%, 73%, 63%) subjects (p < 0.0001). Primary nonfunction and biliary complications were more common in DCD patients, accounting for 67% of early graft failures. A donor warm ischemia time >20 min, cold ischemia time >8 h and donor age >60 were associated with poorer DCD outcomes. There was a lack of survival benefit in DCD livers utilized in patients with model for end-stage liver disease (MELD) < or =30 or those not on organ-perfusion support, as graft survival was significantly lower compared to DBD patients. However, DCD and DBD subjects transplanted with MELD >30 or on organ-perfusion support had similar graft survival, suggesting a potentially greater benefit of DCD livers in critically ill patients.