Suzanne Norris

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.

The impact of diabetes mellitus on fibrosis progression in patients transplanted for hepatitis C.

Despite the recognition of numerous factors for aggressive hepatitis C virus (HCV) recurrence after liver transplantation (LT) our understanding of this phenomenon is incomplete. We tested the hypothesis that diabetes mellitus (DM) was implicated. One hundred sixty‐three patients undergoing primary LT for HCV from 1990 to 2004 were evaluated and biopsies were scored according to the modified Ishak score. Severe recurrence of HCV was defined as a fibrosis score ≥4 within 6 years of LT. Risk factors assessed included recipient, donor and transplant variables. Fifty‐four patients (33.1%) had a fibrosis score ≥4 at the end of the study period. Factors associated with progression to severe fibrosis was donor age (p = 0.008) especially donor age >55 (p = 0.038, HR 2.43), pre‐LT DM (p = 0.039, HR 2.68) and DM post‐LT (p = 0.004, HR 3.28). The combination of receiving a liver from a donor older than 55 years and having DM post‐LT was associated with an 8.38‐fold risk of progression to severe fibrosis (p = 0.000124) when compared to patients not diabetic post‐LT who received livers from donors aged <55 years. These data indicate that diabetic status is one of the more important variables determining the severity of HCV recurrence and is synergistic with donor age. This observation may provide an additional management opportunity to modify the impact of HCV recurrence.

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.

Outcomes of liver transplantation in HIV‐infected individuals: The impact of HCV and HBV infection

Liver transplantation (LT) in human immunodeficiency virus (HIV)‐positive individuals is considered to be an experimental therapy with limited reported worldwide experience, and little long‐term survival data. Published data suggest that the short‐term outcome is encouraging in selected patients. Here, we report our experience in 14 HIV‐infected liver allograft recipients, and compare outcomes between those coinfected with hepatitis C virus (HCV) and the non‐HCV group. A total of 14 HIV‐infected patients (12 male, 2 female, age range 26‐59 years) underwent LT between January 1995 and April 2003. Indications for LT were HCV (n = 7), hepatitis B virus (HBV; n = 4), alcohol‐induced liver disease (n = 2), and seronegative hepatitis (n = 1); 3 patients presented with acute liver failure. At LT, CD4 cell counts (T‐helper cells that are targets for HIV) ranged from 124 to 500 cells/μL (mean 264), and HIV viral loads from <50 to 197,000 copies/mL. Nine of 12 patients were exposed to highly active antiretroviral therapy (HAART) before LT. In the non‐HCV group (n = 7), all patients are alive, all surviving more than 365 days (range 668‐2,661 days). No patient has experienced HBV recurrence, and graft function is normal in all 7 patients. However, 5 of 7 HCV‐infected patients died after LT at 95‐784 days (median 161 days). A total of 4 patients died of complications due to recurrent HCV infection and sepsis, despite antiviral therapy in 3 of them. A total of 3 patients experienced complications relating to HAART therapy. In conclusion, outcome of LT in HIV‐infected patients with HBV or other causes of chronic liver disease indicates that LT is an acceptable therapeutic option in selected patients. However, longer follow‐up in larger series is required before a conclusive directive can be provided for HCV / HIV coinfected patients requiring LT. (Liver Transpl 2004;10:1271–1278.)

Thiopurine methyltransferase phenotype and genotype in relation to azathioprine therapy in autoimmune hepatitis

BACKGROUND/AIMS Toxicity and efficacy of azathioprine is governed partly by the activity of thiopurine methyltransferase (TPMT). Azathioprine has been used for many years, with corticosteroids or alone, for the treatment of autoimmune hepatitis (AIH) but no studies of TPMT phenotype and genotype in relation to response to the drug in AIH have been published. METHODS Erythrocyte TPMT activities were measured by a radioincorporation assay in 72 consecutive outpatients with AIH, 53 of whom were genotyped for the commonest defective alleles in Europeans (TPMT*3A, *3B and *3C) by restriction fragment length polymorphism analysis. RESULTS TPMT activities were significantly lower in patients intolerant of azathioprine (group I, n=15) than in those who sustained remission on azathioprine alone (group II, n=28; P=0.003) and those who tolerated azathioprine but continued to require corticosteroids (group III, n=29; P<0.0001), and were higher in group III than in group II (P=0.034). Ten patients with defective alleles (all heterozygotes) had significantly lower TPMT activities (P=0.002). However, in 25% there was discordance between phenotype and/or genotype and response to azathioprine. CONCLUSIONS TPMT phenotyping or genotyping may be advisable before institution of azathioprine therapy in AIH but neither approach invariably predicts response to the drug.

Hepatitis C and HIV-1 coinfection

Hepatitis C virus (HCV) has emerged as the cause of the second major epidemic of viral infection after human immunodeficiency virus (HIV) within the past two decades, and coinfection of HIV and HCV represents a growing problem for the future. This article reviews the current evidence on the epidemiology and clinical implications of an interaction between HIV-1 and HCV infection, and the current status of the management of patients with combined infection.

Characteristics of autoimmune hepatitis in patients who are not of European Caucasoid ethnic origin

Background: Significant diversity in disease severity has been identified for autoimmune disorders among different ethnic groups but there is a lack of data on autoimmune hepatitis (AIH) in populations other than those of European Caucasoid (EC) or Japanese extraction. Aims: To assess the clinical features, response to therapy, and eventual outcome in AIH patients of non-EC ethnicity. Methods: A retrospective review of a regularly updated database of patients with AIH referred to liver outpatient clinics at Kings College Hospital, London, since 1983. Results: Twelve patients were identified (10 female; six African, five Asian, one Arabic; median age at presentation 30 years (range 12–58)) who satisfied international criteria for type 1 (11 cases) or type 2 (one case) AIH. Nine (75%) had cholestatic serum biochemistry and three (25%) had mild biliary changes on liver biopsy without definitive features of primary biliary cirrhosis or cholangiographic evidence of primary sclerosing cholangitis. Four showed a complete biochemical response to standard prednisolone with or without azathioprine therapy, three partial, and five no response. Four have required liver transplantation for intractable disease. By comparison with 180 EC patients with definite AIH attending during the same period, the non-EC patients were younger (p<0.05), presented with cholestatic biochemistry (p=0.014), and morphological biliary features more frequently (p<0.0005) and showed a poorer initial response to standard therapy (p<0.0005). Conclusions: Clinical expression of AIH in non-EC patients seems to differ in important respects from that in EC or Japanese patients. Management of such patients is challenging and may require alternative or more aggressive treatment strategies.

Expansion of innate CD5pos B cells expressing high levels of CD81 in hepatitis C virus infected liver.

BACKGROUND/AIMS Association of hepatitis C virus (HCV) with increased autoantibodies, mixed cryoglobulinaemia, non-Hodgkins B-cell lymphoma and increased peripheral innate (CD5(pos)) B cells suggests a role for B-lymphocytes in the pathogenesis of HCV-infection. METHODS Flow cytometry was used to estimate CD5(pos) B cell levels and CD81 co-expression in chronic HCV infection. Viral load was assessed using PCR. RESULTS We demonstrate expansion of innate B cells in HCV-infected liver from patients with fibrosis score less than stage II (39%, % of total B cells, P=0.002) and end stage HCV cirrhosis (20%, P<0.05) compared with normal liver (8%). Expression of CD81, a signal transducing molecule and putative HCV receptor, was significantly increased on peripheral blood CD5(pos) B cells compared with conventional B cells (P=0.0001). Higher levels of CD81 on CD5(pos) B cells were more dramatic in the liver of HCV-infected individuals. However, no significant difference was observed in the viral load of CD5(pos)CD81(High) B cells and CD5(neg)CD81(Low) B cells. CONCLUSIONS Increased expression of CD81 on innate B cells, a population that is expanded in the livers and peripheral blood of chronic HCV-infected patients, suggests a role in viral specific activation and clonal proliferation in chronic HCV infection.

Transforming growth factor-beta 1, activin and follistatin in patients with hepatocellular carcinoma and patients with alcoholic cirrhosis.

Background: Transforming growth factor-beta 1 (TGF- β 1) exerts an inhibitory effect on DNA synthesis in hepatocytes. Activin, through different mechanisms, also exhibits an apoptotic effect on hepatocytes. Follistatin antagonizes the actions of activin. Methods: Patients with hepatocellular carcinoma (HCC, n = 20), patients with alcoholic cirrhosis ( n = 12), patients with cirrhosis due to other causes ( n = 5) and normal controls ( n = 19) were studied. TGF- β 1, activin and follistatin concentrations in blood and ascites were measured by ELISA. Results: All three groups of patients had significantly higher serum levels of total TGF- β 1, activin and follistatin compared to those of controls. In patients with HCC, the total TGF β 1 level correlated negatively with tumour size ( r =-0.644, P = 0.001). The activin level correlated with alkaline phosphatase (ALP) level ( r = 0.374, P = 0.046). The follistatin level correlated with the ALP level ( r = 0.404, P = 0.026), and the glutamyl transpeptidase level ( r = 0.457, P = 0.01). In patients with alcoholic cirrhosis, serum activin correlated with the Child-Pugh score ( r = 0.601, P = 0.01). The levels of the cytokines in ascites ( n = 16) did not correlate with the corresponding levels in serum. Conclusions: Serum levels of total TGF- β 1, activin and follistatin were elevated in patients with hepatocellular carcinoma and in patients with alcoholic cirrhosis. Apoptosis of tumour cells may be reduced by a subsequent decrease in serum TGF- β 1 levels when the tumours expand in size. Activin and follistatin were associated with tumour activity, as both correlated with ALP and/or GGT levels. Further studies are required to define the exact relationships between these cytokines, the dynamics of tumour growth and their significance in cirrhosis.