Gerry McEntee

Resident human hepatitis lymphocytes are phenotypically different from circulating lymphocytes

BACKGROUND/AIMS Murine and human studies have documented the existence of subpopulations of lymphocytes in particular tissues that differ phenotypically and functionally from those in peripheral blood and may mature locally. Since little is known about lymphocyte subpopulations in the normal human liver, we have analysed the surface phenotypes of lymphocytes isolated from liver specimens taken from 15 donors at the time of liver transplantation, and compared these with those of peripheral blood lymphocytes. METHODS Hepatic lymphocytes were prepared by mechanical dissociation and enzymatic digestion of liver tissue. The cells were stained with a panel of monoclonal antibodies (CD3, CD4, CD8, CD19, CD56, gammadeltaTCR, alphabetaTCR, CD8alpha-chain, CD8alphabeta dimer), and analysed by flow cytometry. In situ characterisation of hepatic lymphocytes was by haematoxylin and eosin staining of fixed liver sections and by immunohistochemical staining for common leukocyte antigen and CD3. RESULTS Significant numbers of hepatic T lymphocytes were localised to the portal tracts and parenchyma of normal liver specimens. Flow cytometry revealed that the CD4/CD8 ratio (1:3.5) was consistently reversed compared with that in peripheral blood (2:1). Other lymphocyte populations identified include double positive CD3+CD4+CD8+ cells which accounted for a mean of 5.5% (range 3-11.6%) of hepatic CD3+ cells compared with 1.3% in blood (range 0.7-3.6%; p < 0.007), and double negative CD3+ CD4-8- cells (14.5%; range 2.7-29% compared with 5.0%; range 2.1-10.8%, p < 0.02). Over 15% (range 6.8-34%) of all hepatic CD3+ cells expressed a gammadeltaTCR compared to 2.7% (range 0.9-4.7%) of CD3+ peripheral blood lymphocytes (p < 0.004) and almost 50% of these coexpressed CD8. The CD8 alpha-chain was expressed without the beta-chain (CD8alpha+beta-) by 15.4% (range 4-29.1%) of hepatic T cells, but this phenotype was undetectable among peripheral blood lymphocytes (p < 0.009). Cells expressing both the T cell marker CD3 and the natural killer cell marker CD56 constituted 31.6% (range 14-54%) of all hepatic CD3+ lymphocytes but were rarely present amongst peripheral blood lymphocytes (0-6%; p < 0.0001). CONCLUSIONS These data are the first to describe and quantify unconventional T lymphocyte subpopulations in the normal adult human liver which may have specialised functions in regional immune responses and which may differentiate locally. These findings have important implications for our understanding of hepatic immunoregulation and the pathogenic mechanisms involved in viral and immune-mediated liver disease and allograft rejection.

Natural T cells in the human liver: cytotoxic lymphocytes with dual T cell and natural killer cell phenotype and function are phenotypically heterogenous and include Vα24-JαQ and γδ T cell receptor bearing cells

Abstract The adult liver contains lymphocytes with a unique phenotypic distribution compared to blood and other organs. We have characterized a human lymphocyte population that exhibits dual T cell and natural killer (NK) cell phenotype and function, denoted natural T (NT) cells, in nine normal adult liver specimens. Flow cytometry revealed that up to 55% (mean 27%) of hepatic (but + lymphocytes expressed CD56, CD161 and/or one or more of the killer inhibitory receptors (KIR) p58.1, p58.2, p70 and CD94. NK function was attributed to the CD3 + CD56 + cells by the demonstration that hepatic, but not peripheral, CD3 + lymphocytes could be induced to lyse NK-sensitive K562 target cells, while CD56 − cells from both compartments could not. Three color flow cytometric analysis of fresh hepatic cells indicated that CD3 + CD56 + NT cells can be either CD8 + , CD4 + or CD4 − CD8 − , they express αβ or γδ T cell receptors (TCR) and CD161 and KIRs, but rarely CD16. Hepatic NT cells predominantly express the mature/activated CD45RO and CD56 dim phenotypes. Analysis of mRNA production by isolated NT cells indicated a preferential usage of the invariant CD1-restricted Vα24-JαQ TCR. The presence of such large numbers of chronically activated NT cells provides compelling evidence that the liver has unique immunoregulatory functions.

Decrease in hepatic CD56+ T cells and Vα24+ natural killer T cells in chronic hepatitis C viral infection

BACKGROUND/AIMS The intrahepatic immune system is likely to play a key role in determining the outcome of hepatitis C virus (HCV) infection. The hepatic lymphocyte repertoire is characterised by high CD8/CD4 T cell ratios and large numbers of gamma delta T cells, natural killer (NK) cells, NK T cells and NK receptor-positive T cells. It is not known which of these populations contribute to immunity against HCV or immune pathology. METHODS To explore the relative contributions of lymphocyte subpopulations, we have compared the intrahepatic lymphocyte repertoires and cytokine expression in 13 patients with mild chronic hepatitis C infection, 14 with end-stage hepatitis C cirrhosis and five histologically normal livers by flow cytometry and immunohistochemistry. RESULTS CD4(+) T cells bearing alpha beta T cell receptors (TCR) were significantly expanded in livers with chronic HCV infection while CD56(+) alpha beta T cells and V alpha 24 TCR-positive T cells were significantly depleted. Expanded CD4(+)T cells were predominantly Th1 cells, producing interferon-gamma but not interleukin-4. CONCLUSIONS Failure to resolve HCV infection may be due to deficient innate and/or memory immune responses, while Th1 cells may mediate immune pathology.

Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterisation

Murine and human studies have demonstrated that the normal liver contains significant numbers of resident lymphocytes that have functions distinct from those found in blood and other organs. To characterize these cells requires the isolation of viable lymphocytes that can be analysed by flow cytometry and in functional assays. The techniques classically used to isolate single cell suspensions of hepatic lymphocytes for phenotypic and functional studies involve mechanical and/or enzymatic dissociation of liver tissue. The aim of this study was to determine the effect of these procedures on surface molecule expression and lymphocyte function and to optimise an isolation technique that minimises these effects. Mechanical homogenisation of liver tissue alone resulted in low viable lymphocyte yields but these were improved by the combined use of mechanical and enzymatic techniques. A mean yield of 2.3 x 10(6) lymphocytes with a mean viability was 88.8% was obtained from 200 mg wedge biopsy samples of normal adult human liver using a combination of gentle mechanical dissociation followed by digestion with collagenase type IV and DNase I. These cells were suitable for phenotypic characterisation by flow cytometry. They also retained their ability to grow in vitro, to respond to cytokines and activation stimuli, to mediate cytotoxic killing of target cells, and to produce inflammatory and regulatory cytokines.

Selective reduction of natural killer cells and T cells expressing inhibitory receptors for MHC class I in the livers of patients with hepatic malignancy.

Abstract. Natural killer (NK) and CD56+ T cells are thought to play a central role in antitumour immunity. Their cytolytic activities are controlled by a variety of receptors including CD94 and killer immunoglobulin-like receptors (KIR), which bind to major histocompatibility complex (MHC) class I molecules on target cells and mediate cell activation or inhibition. We have examined the numbers, phenotypes and antitumour cytotoxic functions of hepatic NK and CD56+ T cells isolated from 22 patients with hepatic malignancy and 19 healthy donors. Flow cytometry revealed that NK cell numbers were increased among hepatic mononuclear cells in malignancy compared to histologically normal livers (mean: 38% vs 27%; P=0.03), but CD56+ T cell numbers were not (28% vs 27%). NK cells and CD56+ T cells from tumour-bearing livers exhibited lymphokine-activated killing of K562 targets and T cell receptor-mediated lysis of P815 cells. The expression of CD94 and the KIR isotypes CD158a, CD158b and KIR3DL1 by CD56+ T cells and NK cells was significantly and consistently reduced in tumour-bearing livers compared to healthy livers (P<0.05 in all cases). Simultaneous ligation of CD158a, CD158b and KIR3DL1 caused an overall partial inhibition of CD56+ T cell cytotoxic activity, suggesting that the observed reductions in KIR+ cell numbers in malignancy are likely to lead to enhanced cytotoxicity. Our results suggest that, while hepatic CD56+ T cells are not expanded in malignancy, downregulation of KIR and CD94 expression may be a mechanism by which the hepatic immune system can be activated to facilitate tumour rejection.

Orthotopic liver transplantation for veno-occlusive disease complicating autologous bone marrow transplantation.

BACKGROUND Veno-occlusive disease of the liver is a serious and often life-threatening complication after bone marrow transplantation. Although risk factors for the development of veno-occlusive disease have been postulated, there is no precise method for accurately identifying those patients who are at risk and for whom early intervention and treatment would have maximum potential benefit. Liver transplantation has been advocated as a treatment for veno-occlusive disease in selected patients. METHODS In this report, we describe a patient who underwent liver transplantation for life-threatening veno-occlusive disease after autologous bone marrow transplantation for acute lymphoblastic leukemia. RESULTS Liver engraftment was achieved, but the patient developed Pneumocystis pneumonia, which failed to respond to pentamidine. The patient died 6 months after liver transplantation. CONCLUSIONS While acknowledging the limited experience of orthotropic liver transplantation in this patient population, we suggest its consideration as a feasible, potentially beneficial treatment option in patients with severe veno-occlusive disease after bone marrow transplantation.

Delayed correction of portal hypertension after portal vein conduit arterialization in liver transplantation.

A 55-year-old woman underwent orthotopic liver transplantation for autoimmune chronic active hepatitis. Extensive portal and superior mesenteric venous thrombosis precluded standard portal venous reconstruction and necessitated use of a venous conduit from the recipient splenic vein of the donor liver. Flow through this conduit was poor, however, and to prevent subsequent portal venous thrombosis and graft loss, the conduit was arterialized by end-to-side anastomosis with the recipient hepatic artery. This ensured graft survival but resulted in prehepatic portal hypertension, which required ligation of the arterioportal fistula for 4 months. The patient had a satisfactory outcome.

Selective Necrosectomy for Infected Pancreatic Necrosis

Background: Until recently, a diagnosis of infected pancreatic necrosis (IPN) warranted necrosectomy, which was associated with high morbidity and mortality rates greater than 20%. Preoperative percutaneous drainage delayed the need for necrosectomy with improved outcomes. Methods: In 2008, this institution changed its approach to the management of such cases opting instead for percutaneous drainage with selective deferred necrosectomy. A total of 38 consecutive patients with IPN from January 2008 to December 2014 were included. Results: All 38 underwent percutaneous radiological drainage, and selective necrosectomy was performed on 15 where the infected necrosis did not completely resolve. Twenty-three patients did not require surgery and were managed with pancreatic drain insertion, optimal nutritional support and critical care interventions. Median peak Acute Physiology and Chronic Health Evaluation and Sequential Organ Failure Assessment scores were 10 (range 0-18) and 3 (range 0-10) prior to radiological intervention. Overall mortality was 5% (n = 2). Conclusion: This study demonstrates that radiological-guided drainage of infected pancreatic collections can, in most cases, prove curative and, if not, facilitates delayed surgical intervention with improved outcomes.

Osteoclastic-Type Giant Cell Tumours of the Pancreas: A Homogenous Series of Rare Tumours Diagnosed by Endoscopic Ultrasound

Background: Giant cell tumors (GCT) of the pancreas are a rare form of pancreatic cancer. Although data are limited, clinical outcomes appear to depend largely on histological subtype with osteoclastic tumors carrying a better prognosis. We report on a homogenous series of patients with osteoclastic-type GCTs of the pancreas presenting to a national pancreatico-biliary gastrointestinal oncology center. Methods: Patients underwent endoscopic, radiological and histopathological assessments. Data were collected in relation to consecutive patients presenting with osteoclastic-type tumors of the pancreas and analyzed with survival as a primary end point. Results: Four patients were treated over a 4-year period. Median age was 77 years with equal gender distribution. Median tumor size was 42 mm. Histology was osteoclast-type giant cells in all 4 patients. Two patients underwent surgery with curative intent. Median overall survival was 13.1 months. Conclusion: This is the largest reported series of osteoclast-type histology in GCTs of the pancreas.