Immunopathogenesis of Hepatitis B Virus Recurrence After Liver TRANSPLANTATION1

8. Sindhi R, Landmark J, Shaw BW, et al. Combined liver/smallbowel transplantation using a blood group compatible but non-identical donor. Transplantation 1996; 61: 1782.

9. de Ville de Goyet J, Hausleithner V, Reding R, Lerut J, JanssenM, Otte JB. Impact of innovative techniques on the waiting listand results in pediatric liver transplantation. Transplantation1993; 56: 1130.

10. Reyes J, Fishbein T, Mazariegos G, Abu-Elmagd A. Reduced-sizeorthotopic composite liver-intestinal allograft. Transplanta-tion 1998; 66: 489.

11. Kiyosaki H, Robayashi E, Toyama N, Miyata M. Segmentalsmall bowel transplantation in rat: comparison of lipid absorp-tion between ileal and jejunal grafts. J Parenter Enteral Nutr1996; 20: 67.

12. Rahman MS, Taguchi T, Yamada T, Suita S. Morphological andfunctional comparison of jejunal and ileal segmental grafts in

rat intestinal transplantation: long-term results. TransplantProc 1996; 28: 2551.

13. Boudjema K, Cinqualbre J, Simeoni U, et al. Transplantation enbloc du foie, de l’estomac, du pancreas et de l’intestin grele chezun tout-petit. Chirurgie 1991; 117: 860.

14. Xenos ES, Khan F, Nery J, Romero R, Mocros J, Tzakis A.Cadaveric small-bowel/split liver transplantation in a child.Transpl Int 1999; 12: 63.

15. Nery JR, Weppler D, DeFaria W, Liu P, Romero R, Tzakis AG. Isthe graft too big or too small? Technical variations to overcomesize incongruity in visceral organ transplantation. TransplantProc 1998; 30: 2640.

Received 17 May 1999.Accepted 21 July 1999.

0041-1337/00/6904-559/0TRANSPLANTATION Vol. 69, 559–568, No. 4, February 27, 2000Copyright © 2000 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A.

IMMUNOPATHOGENESIS OF HEPATITIS B VIRUS RECURRENCEAFTER LIVER TRANSPLANTATION1

GEORGE MARINOS,2,3 SIEGBERT ROSSOL,2,4 PATRIZIA CARUCCI,2,5 PHILIP Y. N. WONG,2,6

PETER DONALDSON,2 MUNTHER J. HUSSAIN,7,8 DIEGO VERGANI,7,8 BERNARD C. PORTMANN,2

ROGER WILLIAMS,2,8 AND NIKOLAI V. NAOUMOV2,8,9

Institute of Liver Studies, Department of Immunology, King’s College School of Medicine and Dentistry, London SE5, andInstitute of Hepatology, Department of Medicine, University College London, London WC1E, England

Background and aims. Hepatitis B virus (HBV) re-currence after orthotopic liver transplantation is as-sociated with inflammatory graft changes, despite im-munosuppression and donor/recipient HLA mismatch.We investigated whether immune mechanisms are in-volved in the pathogenesis of hepatitis B after livertransplantation.

Methods. The virus-specific T helper (Th) cell re-sponse, activation of Th1/Th2 subpopulations, donor/recipient HLA, and expression of tumor necrosis fac-tor (TNF)-a/TNF receptors were determined in 28patients who underwent transplantation for HBV-re-lated cirrhosis (17 with HBV recurrence and 11 with-out recurrence) in comparison to 30 nontransplantpatients with chronic hepatitis B.

Results. Orthotopic liver transplantation recipientswith HBV recurrence showed significant hepatitis Bcore antigen-specific T-cell proliferation, compara-ble to nontransplant patients, which was not presentin transplant recipients without recurrence. In ad-dition, hepatic and serum interleukin (IL)-2, inter-feron-g, and TNF-a were enhanced, without changesin IL-4 and IL-10. Phenotypically, hepatic infiltratesin allografts with HBV recurrence were comprisedof CD41 lymphocytes and macrophages with a cor-relation between interferon-g– and TNF-a–produc-ing cells and the degree of necroinflammatory activ-ity. There was a marked up-regulation of both TNF-areceptors, significantly greater than in nontrans-plant patients.

Conclusions. These findings suggest that despite im-munosuppression, HLA class I-independent immunemechanisms have a significant pathogenic role in liverdamage associated with HBV recurrence after livertransplantation.

1 Siegbert Rossol was supported by the Alexander von HumboldtFoundation, Bonn, Germany. Patrizia Carucci was supported by theAssociazione Italiana Copev, Milan, Italy. Part of the work wassupported by grant 64/95 from the Faculty of Clinical Medicine,Mannheim, Germany.

2 Institute of Liver Studies, King’s College School of Medicine andDentistry.

3 Current affiliation: Prince of Wales Hospital, Sidney, Australia.4 Current affiliation: Department of Gastroenterology, Mannheim,

Germany.5 Current affiliation: Department of Gastroenterology, Ospedale

Molinette, Turin, Italy.6 Current affiliation: Auckland Hospital, Auckland, New Zealand.7 Department of Immunology, King’s College School of Medicine

and Dentistry.8 Institute of Hepatology, Department of Medicine, University

College London.9 Address correspondence to: Dr. Nikolai V. Naoumov, MD, Insti-

tute of Hepatology, University College London Medical School,69–75, Chenies Mews, London WC1E, 6HX, England. E-mail:[email protected].

MARINOS ET AL.February 27, 2000 559

Hepatitis B virus (*) recurrence after orthotopic livertransplantation (OLT) for end-stage cirrhosis remains a fre-quent complication (1, 2) and is associated with various de-grees of liver inflammation and progressive graft damage(3–5). The pathogenesis of hepatitis B in the liver graft is stillunclear. In the nontransplantation setting, there is a largebody of evidence that demonstrates that HBV-related liverdamage is immune mediated (6). Strong HLA class I- andclass II-restricted T-cell responses to HBV antigens arepresent in patients with acute self-limited hepatitis B,whereas these responses are weak or absent in patients withchronic HBV infection (7). Cytotoxic T lymphocytes (CTL),targeting HBV peptides presented within the HLA class Imolecules, are believed to represent the major effector mech-anism responsible for the control of HBV replication in in-fected hepatocytes (8–10). In the liver transplantation set-ting, the host-virus interactions are modified by twoimportant factors. First, the patients receive immunosup-pression that may enhance HBV replication and viral proteinexpression in the liver (11–13), and will modulate the im-mune reactions in the host (14). Second, because successfulliver transplantation does not require HLA matching be-tween donor and recipient, the pathogenesis of liver graft dam-age after viral recurrence is unlikely to involve HLA class I-re-stricted CTL recognition of viral gene products on the infectedhepatocytes (because the recipient’s lymphocytes and the do-nor’s hepatocytes have different HLA class I antigens).

However, the potential to mount antigen-specific HLAclass II-restricted cellular immune responses in liver trans-plant recipients, independent of host-donor HLA class IImismatching, remains because the liver graft is repopulatedby antigen-presenting cells and mononuclear cells from therecipient within weeks after liver transplantation (15–17). Inthe nontransplantation setting, activation of the HLA classII-restricted CD41 T helper (Th) cells has been shown, par-ticularly in association with hepatitis flares in HBV infection(18–21). Interferon (IFN)-g and tumor necrosis factor(TNF)-a have also been implicated in HBV-mediated liverdamage. Transgenic mice that overexpress HBV envelopepolypeptides develop the characteristic histological appear-ance of ground glass hepatocytes and are selectively sensi-tized to destruction by IFN-g (22). TNF-a production is in-variably elevated in chronic hepatitis B (23–25), and there isa close association between enhanced hepatocellular expres-sion of TNF-a receptors (TNFR) and the degree of necroin-flammatory activity in all forms of viral hepatitis (26), inparticular chronic active hepatitis B (CAHB) (27).

The aim of this study was to define whether immune mech-anisms have a role in the pathogenesis of liver graft damageafter HBV recurrence. We examined the effect of donor/re-cipient matching for the MHC antigens on the severity ofdisease recurrence in the graft, as well as the involvement of

the following immune reactions: (i) the virus-specific Th cellresponse, directed against recombinant hepatitis B core an-tigen (HBcAg), which reflects the activation of HBV-specificHLA class II-restricted Th cells; (ii) the activation of Th1/Th2lymphocyte subsets, by measuring serum levels of interleu-kin (IL)-2, IFN-g, TNF-a, IL-4, and IL-10; (iii) the phenotypecharacteristics and activation of liver graft-infiltrating lym-phocytes; and (iv) the hepatocellular expression and serumlevels of the TNFR, because modulation of their cellularexpression represents one level of control of TNF-a respon-siveness (28) and their expression correlates with hepaticinflammation and hepatocytolysis (27).

MATERIALS AND METHODS

Patients. The study examined 28 adults with chronic HBV infec-tion (24 male, 4 female) who underwent OLT for end-stage, hepatitisB surface antigen (HBsAg)-positive cirrhosis and survived for morethan 90 days after transplantation. These liver transplant recipientswere recruited to the study during a period between January 1993and December 1995. Twelve of these 28 patients also had chronichepatitis delta virus (HDV) infection before transplantation. Pa-tients were divided into two groups: 11 patients (group 1) had noevidence of HBV recurrence, and 17 patients (group 2) had eitherHBV alone (n510) or HBV and HDV (n57) recurrence. There was nodifference in the interval between liver transplantation and the timeof study between these two groups (mean interval 28.5614.3 and31.6623.7 months after transplantation for groups 1 and 2, respec-tively). In group 2, the mean time between liver transplantation andHBV recurrence (seropositivity for HBsAg) was 3.962.8 months. Thepatients with HDV and HBV reinfection had similar serum aspar-tate aminotransferase (AST) levels compared with the OLT patientswith HBV reinfection only: 132.7692.7 IU/ml and 119.9664.4 IU/ml,respectively.

Thirty nontransplant patients with CAHB (group 3) were alsostudied. These patients were all seropositive for HBsAg, HBeAg, andHBV DNA and negative for anti-HBe; they had elevated serum ASTand moderate to severe necroinflammatory changes in the liver(Table 1). Sera and peripheral blood mononuclear cells from 45HBsAg-negative healthy controls with normal liver function testswere also studied.

All patients included in this study were negative for markers ofhepatitis C and human immunodeficiency virus infection. The 28OLT patients (groups 1 and 2) were HBsAg positive at the time oftransplantation. None of the 11 patients from group 1 were seropos-itive for HBV DNA before transplantation, whereas 8 of 17 patientsfrom group 2 were HBV DNA seropositive before transplantation(mean pretransplantation HBV DNA513.3619.8 pg/ml). Hyperim-mune hepatitis B immunoglobulin was started in all patients imme-diately postoperatively with 5000 IU, intravenously on alternatedays for 7 days, and maintained at 2000 IU intramuscularly toensure the anti-HBs level above 100 IU/L. All 28 post-OLT patientsreceived triple-drug immunosuppression therapy of cyclosporine,azathioprine, and prednisolone initially after OLT, with the pred-nisolone being gradually tapered down and completely stopped. Atthe time of this study, all liver transplant recipients were on immu-nosuppression without prednisolone. There was no significant differ-ence in the number of patients suffering an episode of acute rejectionbetween the patients of groups 1 and 2 (5 of 11 and 7 of 17, respec-tively). No patient received OKT3 as either induction therapy orantirejection treatment.

Histological assessment. Paired liver specimens and serum sam-ples were examined in 25 OLT patients (17 with HBV recurrence and8 without recurrence) and 30 nontransplant patients with chronichepatitis B. Liver histology was assessed blindly, with respect toHBV serology and serum AST (Dr. B. Portmann) on formalin-fixedparaffin sections. Portal tract inflammation, periportal piecemeal

* Abbreviations: APAAP, alkaline phosphatase anti-alkalinephosphatase; AST, aspartate aminotransferase; CAHB, chronic ac-tive hepatitis B; CTL, cytotoxic T lymphocytes; H.A.I., histologicalactivity index; HBcAg, hepatitis B core antigen; HBeAg, hepatitis Be antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis Bvirus; HDV, hepatitis delta virus; IFN, interferon; IL, interleukin;mAbs, monoclonal antibodies; OLT, orthotopic liver transplantation;PBMC, peripheral blood mononuclear cells; s, soluble; SI, stimula-tion index; TBS, Tris-buffered saline; Th, T helper; TNF, tumornecrosis factor; TNFR, tumor necrosis factor-alpha receptor.

TRANSPLANTATION560 Vol. 69, No. 4

necrosis, and lobular inflammation were each graded on a 0–4 scaleand a histological activity index (H.A.I.) was calculated, with amaximum value of 12, according to the classification guidelines ofDesmet et al. (29).

Major histocompatibility antigen profiles. The donor and recipi-ent MHC profiles were determined in all 28 transplant patients. TheMHC profile included HLA class I (HLA-A and -B) and class II(HLA-DR and -DQ) antigens. Most patients were mismatched at oneor more class I and II MHC loci because no attempt was made tomatch prospectively the donor and recipient. HLA class I typing wasdetermined by standard complement-dependent microcytotoxicityassay, HLA-DR typing was determined by a combination of serologyand either restriction-length polymorphism analysis or polymerasechain reaction-based oligotyping, and HLA-DQ typing was by poly-merase chain reaction-based oligotyping only (30). Mismatch scores(0–2) were assigned for the individual loci A, B, DR, and DQ; thesedata were combined to derive the HLA class I (A and B) mismatchscore (0–4) and the HLA class II (DR and DQ) mismatch score (0–4).An overall HLA mismatch score was calculated by combining A, B,DR, and DQ data, with 0 representing no mismatch at any of the fourloci tested and eight representing complete mismatch at all four loci.

HBcAg-specific CD41 T-cell response. Peripheral blood mononu-clear cells (PBMCs) were separated from fresh, heparinized venousblood by standard density gradient centrifugation with Lymphoprep(Nyegaard, Oslo, Norway). The interphase layer of cells was washedthree times in buffered RPMI 1640 medium (Gibco BRL, Glasgow,UK) supplemented with 2 mmol/l L-glutamine, 100 mg/ml streptomy-cin, 200 U/ml penicillin, and 2 mg/ml amphotericin B. The PBMCswere resuspended at 23106 cells/ml in buffered RPMI 1640 supple-mented with 10% (vol/vol) heat-inactivated human AB serum (lotnumber 1652; Gemini Bio-Products Inc., Calabasas, CA).

Because the proliferation assay (see below) was performed withPBMCs freshly isolated from heparinized venous blood, these wereonly available for the assay from 10 of the 11 patients from group 1,and 9 of the 17 patients from group 2. PBMCs were available from all30 CAHB patients (group 3) and from all 45 HBsAg-negative healthycontrols.

Freshly isolated PBMCs (23105 cells/well) were cultured in 96-well, sterile microtiter plates (Nunclon; Gibco BRL) for 6 days at37°C in the presence or absence of 1 mg/ml recombinant HBcAg(Wellcome Research Laboratories, Beckenham, Kent, UK), 0.5 mg/mltetanus toxoid (positive control, T cell recall antigen; ConnaughtLaboratories Ltd., Ontario, Canada) as previously described (20). Inaddition, lymphocytes were cultured for 3 days in the presence orabsence of 0.1 mg/ml monoclonal anti-human CD3 (positive control,pan T cell stimulant; Sera-Lab, Sussex, UK). Each well was pulsedwith 0.5 mCi tritiated-thymidine (Amersham, Buckinghamshire,UK) 16 hr before harvesting. The amount of radiolabel incorporatedinto DNA was measured by a direct beta-counter (Matrix 9600;Packard Instruments, Groningen, The Netherlands). The prolifera-tion assay was performed in 12 replicates, and the results wereexpressed as the mean counts per minute for the 12 replicates. Thestimulation index (SI) was calculated by dividing the mean countsper minute for the antigen-stimulated cultures by that of controlcultures (PBMCs with complete medium only). An SI of greater than

two times the mean plus 2 SD for the 45 healthy controls (SI ofgreater than 2.5) is considered significant.

To determine the HLA restriction of the PBMC proliferative re-sponse to recombinant HBcAg, blocking experiments with monoclo-nal anti-HLA antibodies were performed. Monoclonal antibodies(mAbs) recognizing HLA class I (W6/32) and HLA class II determi-nants to DR (L243), DP (B7/21), DQw1 (4D4), DQw2 (C1A2), andDQw3 (1VD12) were each added separately to the PBMC and anti-gen mixture, at three different final dilutions of 1/6, 1/12, and 1/24.

Cytokine measurement. Serum concentrations of IFN-g, IL-2,IL-4, IL-10, and the soluble (s) receptors TNFR-p55 and TNFR-p75were measured by enzyme-linked immunoassays. Serum IFN-g,IL-2, and IL-4 levels were measured with a sandwich ELISA, eachusing two mAbs directed to different epitopes (IFN-g mAbs clones 68and 69 peroxidase-conjugated antibody (POD); IL-2 mAbs clones3D5/7B1 and 13A6 POD; IL-4 mAbs clones 5 and 4F2 POD; Dr. H.Gallati, Hoffmann-La Roche, Basel, Switzerland), as previously de-scribed (20, 31). IL-10 was determined with a commercially availableELISA (Medgenix, Fleurus, Belgium) according to the manufactur-er’s instructions. The minimum detectable IL-10 concentration was 5pg/ml. The concentrations of sTNFRs were measured in diluted sera(1/5) by an enzyme-linked binding assay with a TNF-a horseradishperoxidase conjugate as detecting agent, as described previously(27). Standard curves were generated with serial dilutions of humanrecombinant sTNFR-p55 or -p75 (standard curve range 0–5 ng/mland 0–50 ng/ml, respectively). The detection limit was 60 pg/ml forboth receptors, with inter- and intraassay coefficients of variation of3–6% and 5–9%. The assays showed no cross-reactions to cytokines(500 pg/ml of recombinant TNF-a, IL-1a, IL-1b, and IL-6) or othercytokine receptors (IFN-gR, IL-6R, IL-2R).

Immunohistochemical analysis of the liver graft. The expressionof cellular IFN-g, TNF-a, HLA-DR, TNFR-p55, and -p75, as well asCD3, CD4, CD8, and CD68-bearing mononuclear cells were analyzedsemiquantitatively in normal human liver tissue and in 25 frozenliver biopsy specimens of OLT patients (17 with and 8 withouthepatitis B recurrence). The cryostat sections were prepared fromfresh liver biopsy specimens, which were embedded in optimal cut-ting temperature compound, snap-frozen in liquid nitrogen-cooledisopentane, and stored at 270°C. Immunohistochemical staining formacrophages was performed on paraffin-embedded liver biopsy tis-sue from 25 OLT and 30 CAHB patients. Two normal liver biopsyspecimens obtained from cut-down liver specimens of transplantdonors (male 26, female 20) were studied as controls.

Phenotypic markers and lymphocyte activation. Immunostainingfor CD3, CD4, CD8, and HLA-DR was with commercially availableprimary mAbs (Dako Ltd., High Wycombe, UK) using the three-stepindirect alkaline phosphatase anti-alkaline phosphatase (APAAP)detection system on cryostat sections. The sections were incubatedwith the primary mAb (CD3 1/50 dilution, CD4 1/25 dilution, CD81/40 dilution, HLA-DR 1/50) for 1 hr at room temperature. Thesecondary and tertiary antibodies were alkaline phosphatase-conju-gated rabbit anti-mouse antibody (1/50 dilution) and mouse APAAPcomplex (1/100 dilution), respectively, (Dako Ltd.). The incubationwith each of these antibodies was carried out for 30 min at room

TABLE 1. Clinical, biochemical, and histological profile of the four groups of patients and controls

Control Group 1 Group 2 Group 3

Liver transplant recipients withoutHBV recurrence

Liver transplant recipients withHBV recurrence

Nontransplant patientswith CAHB

Patient number 45 11 17 30Age 30.866.9 42.667.2 44.869.3 35.5613.3Survival (days) after OLT 12176454 13686802H.A.I. 1.460.5 6.462.7 7.162.3Serum AST (IU/ml) 22.366.1 21.666.4 127.5678.2 130.86112.6Serum HBV DNA (pg/ml) 0 539.46535.8 118.86176.1


temperature. The alkaline phosphatase reaction was developed us-ing naphthol-phosphate buffer (pH 9.5) and nitro-blue tetrazolium.

Expression of TNF-a and IFN-g was detected in cryostat liversections using a two-step immunoperoxidase detection system aspreviously described (32). The anti-TNF-a mAb was donated by Dr.A. Meager (National Institute for Biological Standards and Control,Mill Hill, UK), and the anti-IFN-g mAb was a gift of Dr. H. Gallati.

Detection of tissue macrophages. Macrophage staining was per-formed on paraffin-embedded sections using a primary mAb CD68(DAKO-CD68) in a three-step indirect streptavidin-biotin immuno-peroxidase detection system. After dewaxing and rehydrating thesections, the endogenous peroxidase was blocked with methanol and1% H2O2 for 15 min. The tissue was then partially digested with0.05% protease (Protease type XXIV; Sigma Chemical Co., Dorset,UK) in Tris-buffered saline (TBS) for 10 min. Nonspecific bindingwas blocked with 10% normal rabbit serum/1% bovine serum albu-min in 0.05 M TBS, pH 7.6. The mAb CD68 was applied at a 1/100dilution in 1% bovine serum albumin/TBS, and the sections wereincubated for 45 min at 37°C. The biotin-labeled rabbit anti-mousewas applied at 1/400 dilution in 1% bovine serum albumin/TBS for 30min at room temperature, followed by preformed streptavidin-bio-tinylated horseradish peroxidase complex, 1/200 dilution for 30 minat room temperature. The streptavidin-biotin immunoperoxidase re-action was developed with diaminobenzidine hydrochloride withMayer’s hematoxylin counterstaining.

TNFR expression. TNFR-p55 and TNFR-p75 expression in theliver graft was determined using primary mAbs in a three-stepindirect APAAP procedure on cryostat sections, as previously de-scribed (27). The sections were incubated overnight at 4°C witheither anti-TNFR-p55 mAb (htr-9) or anti-TNFR-p75 mAb (utr-1),kindly provided by Dr. M. Brockhaus (Hoffmann-La Roche). The sec-ondary and tertiary antibodies were as for the CD3/CD4/CD8 immuno-staining. The alkaline phosphatase reaction was developed using naph-thol and Fast Red TR salt. The specificity of the immunoreactivity of thetwo mAbs (htr-9 and utr-1) was validated by using primary mAbs withdifferent specificities (anti-rubella and anti-p53 protein) on cryostatsections from the same patients and by staining cryostat sections ofnormal human liver with both htr-9 and utr-1 mAbs.

Hepatitis serology. HBsAg, HBeAg, anti-HBe, anti-HDV, and an-ti-HCV were tested using commercially available assays (AbbottDiagnostics, Maidenhead, UK; Sorin Biomedical, Saluggia, VC, It-aly; and UBI HCV EIA, United Biochemical Inc., Lake Success, NY).Serum HBV DNA was measured using a solution hybridizationassay (Genostics; Abbott Diagnostics).

Statistical analysis. Nonparametric tests were used to analyzethe results obtained. The Mann-Whitney U test was used to comparedata between two groups. The nonparametric Wilcoxon’s matchedpairs test was used to compare two sets of data in the same group.Correlation analysis was performed using the Spearman’s rank-regression coefficient. The chi-square test was used to compare theprevalence or distribution of two variables in the same group.

RESULTS

Liver biopsy specimens from all OLT patients with HBVrecurrence (n517) showed some degree of necroinflammatoryactivity, ranging from mild portal infiltrates to panacinarcollapse (mean H.A.I.56.462.8), whereas the graft histologyin patients without HBV recurrence (n58) showed no orminimal hepatic inflammation (mean H.A.I.51.460.5;P,0.01, Mann-Whitney U test). The histological diagnosis inthe 17 OLT patients with HBV recurrence included mildchronic hepatitis (n54); moderate-severe chronic hepatitis(n56); cirrhosis (n55); and fulminant hepatitis with panaci-nar collapse (n52). The mean H.A.I. for the liver biopsy speci-mens from the nontransplant patients with CAHB was7.162.3. In OLT patients with HBV recurrence, there was no

correlation between the level of serum HBV DNA and thedegree of liver graft damage as assessed by H.A.I. and serumAST.

Lymphocyte proliferation. In the HBsAg-negative healthycontrols, the proliferative response to tetanus toxoid was21.9619.2 (mean SI 6 SD); 48.8629.7 to anti-human CD3;and 1.460.5 to recombinant HBcAg. The antigen-specific T-cell proliferative response in OLT patients was slightly lowerthan healthy controls or nontransplant patients, but therewas no significant difference in the SI for tetanus toxoid andanti-CD3 between normal controls and groups 1, 2, and 3;mean SI for tetanus toxoid and for anti-human CD3 was13.6615.4 and 55.9648.1 for group 1, 11.764.4 and42.0625.6 for group 2, and 26.8619.9 and 81.4649.7 forgroup 3. The proliferative response to recombinant HBcAgwas 1.660.6 for group 1, 3.762.5 for group 2, and 3.860.8 forgroup 3 (P,0.01, Mann-Whitney U test; Fig. 1). In group 2, 7of the 9 patients tested had a significant proliferative re-sponse against recombinant HBcAg (SI . 2.5) as opposed tonone of 10 patients from group 1 (P,0.01, Fisher exact test).The PBMC proliferative response to recombinant HBcAg wassignificantly inhibited only by the antibody to HLA-DR in aconcentration-dependent manner (.90% inhibition at a 1/6dilution), thus confirming that the PBMC proliferative re-sponse was HLA class II restricted.

Donor-recipient HLA matching. Among the 28 transplantrecipients from groups 1 and 2, a zero mismatch (i.e., acomplete HLA match) was found in 24% and 27% at theHLA-A locus, 7% and 9% at the HLA-B locus, 0% and 0% atthe HLA-DR locus, and 6% and 9% at the HLA-DQ locus,respectively. Among the 17 OLT patients with HBV recur-rence, there was no relationship between the degree of mis-match at individual A, B, DR, or DQ loci, the combined classI or class II mismatch scores, or the total mismatch score forall four loci with either H.A.I. or AST (P.0.1, Spearman rankcorrelation). In OLT patients with HBV recurrence, therewas no correlation between the degree of HLA class II match-ing and the PBMC proliferative response to recombinantHBcAg (P.0.1, Wilcoxon’s matched pairs test).

Serum cytokine levels. The serum levels of the two Th1cytokines, IL-2 (ng/ml) and IFN-g (pg/ml), were significantly

FIGURE 1. HBcAg-specific PBMC proliferative response. PBMC pro-liferative response against recombinant HBcAg among the threegroups of patients and controls (C). Group 1: liver transplant pa-tients without HBV recurrence; group 2: liver transplant patientswith HBV recurrence; group 3: nontransplant patients with chronicactive hepatitis B. * Significantly higher than controls and group 1(P,0.01, Mann-Whitney U test).


higher in patients with HBV recurrence after liver transplan-tation (group 2) in comparison to OLT patients with no HBVrecurrence (group 1) and healthy controls (P,0.01, Mann-Whitney U test; Fig. 2). The serum levels of these two cyto-kines did not differ significantly between liver transplantpatients and nontransplant patients with hepatitis B. Therewas no significant difference in the serum levels of the Th2cytokines, IL-4 and IL-10, between OLT patients with orwithout recurrent HBV infection and the normal controls.The serum levels of both sTNFR-p55 (ng/ml) and sTNFR-p75(ng/ml) were markedly elevated in liver transplant patientswith HBV recurrence in comparison to OLT patients withoutrecurrence and healthy controls (P,0.01; Fig. 3). Further-more, both sTNFR-p55 and -p75 were significantly higher inthe OLT patients with hepatitis B recurrence than in thenontransplant patients with CAHB.

There was a significant correlation between the serumlevels of IFN-g, sTNFR-p55, and -p75 with serum AST(r50.67, 0.56, and 0.55, respectively, P,0.01, Spearmanrank correlation; Fig. 4). H.A.I. showed a significant correla-tion with the serum levels of IFN-g, sTNFR-p55, and -p75(r50.53, 0.70, and 0.76, respectively, P,0.01, Spearmanrank correlation; Fig. 5).

Characterization of mononuclear infiltrates in the livergraft and cytokine expression. In the biopsy specimens frompatients with HBV recurrence (group 2), there was a signif-icant increase in the mononuclear cell infiltration of the liver,both in the portal tracts and within the liver lobules, com-pared with the posttransplantation biopsy specimens with-out HBV recurrence (group 1), which had no intralobularlymphocytes and only minimally expanded portal tracts. Themononuclear cells in the portal tract were predominantlyCD3-positive T cells. In patients with HBV recurrence, theportal tract T cells were predominantly CD4 positive in 16 of

the 17 biopsy specimens studied (Fig. 6, top and bottom).There was a marked expression of HLA-DR antigen by al-most all mononuclear cells within the portal tracts of thesepatients, indicating that the CD4 T cells are activated.

In normal liver biopsy specimens, the macrophages andKupffer cells (CD68-positive cells) were star-shaped or elon-gated cells with long processes, found evenly scattered withinthe liver lobules. In the liver biopsy specimens of patientsfrom groups 2 and 3, these were increased in both numberand size. The CD68-positive cells in the patients with a highH.A.I. were enlarged and had increased cytoplasm. Therewas a marked increase in the number and size of macro-phages in the liver biopsy specimens of OLT recipients withHBV recurrence compared with the OLT patients with norecurrence (Fig. 7, top). In two patients with fulminant hep-atitis and panacinar collapse, almost 50% of liver paren-chyma was replaced by CD68-positive macrophages (Fig. 7,bottom).

IFN-g and TNF-a expression. The two normal liver speci-mens studied showed no staining for either IFN-g or TNF-a.No IFN-g– or TNF-a–positive hepatocytes were seen in anyof the liver biopsy specimens examined. IFN-g immunostain-ing was seen in mononuclear cells within the portal tractcells and in the liver lobules (Fig. 8, top). TNF-a was detectedpredominantly in mononuclear cells in the periportal areaalong the limiting plate (Fig. 8, bottom). The IFN-g andTNF-a immunostainings in the liver biopsy specimens wereeach graded on a 0–3 scale giving a value of 0 for no staining(equivalent to the immunoreactivity of normal tissue); 11minimal staining (i.e., ,5% of the portal tract cells were

FIGURE 2. Serum levels of IL-2 and IFN-g. Serum levels of IL-2 andIFN-g among the three groups of patients and controls (C). Group 1:liver transplant patients without HBV recurrence; group 2: livertransplant patients with HBV recurrence; group 3: nontransplantpatients with chronic active hepatitis B. * Significantly higher thancontrols and group 1 (P,0.01 Mann-Whitney U test).

FIGURE 3. Serum levels of sTNFR-p55 and -p75. Serum levels ofsTNFR-p55 and -p75 among the three groups of patients studied andcontrols (C). *Significantly higher than controls and group 1 (P,0.01Mann-Whitney U test). * * Significantly higher than controls, group1, and group 3 (P,0.01 Mann-Whitney U test).


positively stained); 21 moderate staining (between 5–10% ofthe portal tract cells were positively stained plus occasional[one to five] positively stained cells within the liver lobules);and 31 abundant staining (.10% positively stained portaltract cells plus scattered [.5] positively stained cells withinthe liver lobules).

IFN-g was expressed in all of the biopsy specimens withHBV recurrence, mean grade of 1.8360.83, compared with0.560.53 for the biopsy specimens without HBV recurrence(P,0.01, Mann-Whitney U test). TNF-a was detected in allposttransplantation biopsy specimens with HBV recurrenceand in four of eight posttransplantation biopsy specimenswithout HBV recurrence, mean grade of 2.0860.79 and0.560.53, respectively (P,0.01, Mann-Whitney U test).Within group 2, there was a close correlation between thedegree of IFN-g and TNF-a immunostaining (r50.85,P,0.001, Spearman rank correlation).

TNFR expression. Normal liver tissue showed no or veryweak expression of both receptors on hepatocytes, bile ductepithelium, sinusoidal epithelial cells, and lymphocytes. En-hanced expression of the TNFRs in the specimens with a high

necroinflammatory activity was demonstrated predomi-nantly by the hepatocytes and to a lesser degree by the portaltract lymphocytes. The TNFR immunostaining in the liverbiopsy specimens was assessed using a scoring system, giv-ing a value of 11 for less than 20% of hepatocytes stainedwith a faint intensity of expression (equivalent to the immu-noreactivity of normal tissue); 21 between 20% and 50%hepatocytes stained with faint to mild intensity; 31 between50% to 75% hepatocytes stained with moderate to strongintensity; and 41 for more than 75% hepatocytes stainedwith strong intensity. The mean hepatocellular expression ofTNFR-p55 and -p75 in the liver biopsy specimens was1.160.3 and 1.460.5, respectively, for group 1; 2.660.7 and2.960.8, for group 2; and 1.660.6 and 2.760.3, for group 3.There was a strong correlation between the hepatocellularexpression of both TNFR-p55 and -p75 and the H.A.I. (r50.8and 0.84, respectively, P,0.001, Spearman rank correlation;Fig. 9) and their corresponding serum sTNFR levels (r50.85and 0.88, respectively, P,0.001, Spearman rank correlation).

FIGURE 4. Association between AST and serum levels of IFN-g, sT-NFR-p55, and -p75. Correlation between serum AST and the serumlevels of IFN-g, sTNFR-p55, and -p75 among the patients with HBVrecurrence after liver transplantation (n517, Spearman rank corre-lation).

FIGURE 5. Association between histological activity and serum lev-els of IFN-g, sTNFR-p55, and -p75. Correlation between the H.A.I.and the serum levels of IFN-g, sTNFR-p55, and -p75 among thepatients with HBV recurrence after liver transplantation (n515,Spearman rank correlation).


DISCUSSION

The results from this study demonstrate that OLT recipi-ents with HBV recurrence are able to mount a significantHBV-specific, HLA class II-restricted T-cell response despitethe immunosuppression. This response seems to be of theTh1 type, associated with a significant local production andthe systemic release of IFN-g and TNF-a, which correlatewith the necroinflammatory activity in the liver allograft.Moreover, there is a strong up-regulation of hepatocellularTNFR, which endows the hepatocytes with an augmentedresponsiveness to TNF-a. Thus, these data indicate that HLAclass I-independent immune mechanisms play a significantrole in the pathogenesis of HBV-induced liver damage in theliver allograft.

Activation of HLA class II-restricted CD41 cells is pivotalin the development and augmentation of the effector immunereactions (CTL, humoral, and cytokine responses) directed toHBV-infected hepatocytes in the nontransplantation setting(18–20, 33). The presence of a HBcAg-specific PBMC prolif-erative response after liver transplantation was first demon-strated in a single case with fulminant HBV recurrence (34).According to our results, liver transplant recipients are ca-pable of mounting an effective HLA class II-restricted CD41

proliferative response to a variety of T-cell immunogens, andthe responses to tetanus toxoid and anti-CD3 in these pa-

tients seem to be similar to those of the nontransplant pa-tients and controls. Importantly, the present study showsthat an HLA class II-restricted CD41 proliferative responseto recombinant HBcAg, similar to the nontransplant CAHBpatients, is observed only in OLT recipients with HBV recur-rence but not in transplant recipients without HBV recur-rence. This clearly indicates the presence of an expanded andactivated population of antigen-specific CD41 cells in caseswith HBV recurrence, rather than a memory response fromthe pretransplantation HBV infection. Analysis of the effectof donor/recipient HLA class I or II matching in our studyrevealed no association with the development or degree ofliver damage in the liver allograft. These results are consis-tent with another study that examined the effect of HLAmatching in the evolution of HBV disease in 45 liver trans-plant recipients (3). Furthermore, recently we found that thedegree of donor/recipient HLA match has no effect on theseverity of graft damage and graft survival in patients withHCV recurrence after OLT (35). Two earlier studies havesuggested that HLA class I matching between donor andrecipient correlates with the severity of hepatitis in recurrentHBV infection after liver transplantation; the number ofpatients examined, however, was small and one study ana-lyzed HBV and HCV infection together (36, 37).

FIGURE 6. Expression of CD4 and CD8 in the portal tract infiltrate.(Top) In patients with HBV recurrence after liver transplantation,the portal tract lymphocytes were predominantly CD4 positive (darkstained cells). (Bottom) In the same patients, a minority of the portaltract lymphocytes were CD8 positive.

FIGURE 7. Expression of CD68 in transplant patients. (Top) In livergrafts of patients with no HBV recurrence and minimal histologicalchanges, the CD68-positive cells were star shaped or elongated withlong processes. (Bottom) In the two patients with HBV recurrenceand fulminant hepatitis B, almost 50% of liver parenchyma wasreplaced by CD68-positive macrophages, which were increased inboth number and size.


The finding that the intrahepatic infiltrates in HBV-rein-fected allografts are predominantly composed of CD41 Tlymphocytes supports further their involvement in the patho-genesis of liver graft damage. The combination of enhancedIL-2, IFN-g, and TNF-a responses in the absence of a signif-icant IL-4 and IL-10 response, defines the activated CD4 cellsas predominantly of the Th1 subset (38).

A close correlation was observed between hepatic IFN-gand TNF-a expression, and the serum levels of IFN-g, withthe intensity of the necroinflammatory activity, thus suggest-ing that these cytokines may have a significant role in thedevelopment of liver graft damage after HBV reinfection. Theability of IFN-g and TNF-a, which work both independentlyand synergistically, to cause cell damage is well documented(22, 39–41). This effect may be particularly relevant in post-transplantation patients with HBV recurrence because of themassive viral antigen expression within hepatocytes (42), asa direct result of the immunosuppressive treatment (13).HBsAg-positive hepatocytes have been shown to be selec-tively vulnerable to the actions of these two cytokines in theHBsAg transgenic mouse model, with liver cell destructionbeing abrogated by the administration of anti-TNF-a mAbsor greatly reduced if antibodies to IFN-g are used instead(22). Two additional pathways may contribute to cytokine-mediated liver graft damage. First, IFN-g and TNF-a have

chemotactic functions for leukocytes to the site of inflamma-tion with recruitment of inflammatory cells and enhance-ment of the T-cell reactivity (43, 44). Second, the exposure toeither IFN-g or TNF-a has been shown to induce hepaticnitric oxide synthase activity, resulting in potentiation ofintracellular oxidative stress, inhibition of energy metabo-lism, and cytotoxicity of hepatocytes. In addition, IFN-g actssynergistically with TNF-a to induce nitric oxide synthaseactivity in human monocytes/macrophages and release ofnitric oxide (45, 46). A possible role of this mechanism issupported by our observation of a marked increase of CD68-positive macrophages in the liver graft of OLT patients withHBV recurrence in comparison to transplant recipients withno recurrence.

OLT patients with HBV recurrence have an enhanced hep-atocellular expression of both TNFRs, associated with in-creased serum levels of both sTNFRs. The serum levels ofsTNFR-p55 and -p75 in the OLT patients with HBV reinfec-tion were significantly higher than in nontransplant patientswith CAHB, despite a comparable H.A.I. between the twogroups of patients. The mechanism for the enhanced expres-sion of TNFR in OLT patients with hepatitis B is not clear,but this may be implicated in the rapid progression of HBV-induced liver damage in liver transplant patients, leading to

FIGURE 8. Hepatic expression of IFN-g and TNF-a. (Top) NumerousIFN-g-positive cells in a patient with severe hepatitis associatedwith HBV recurrence after liver transplantation. IFN-g expression ispresent diffusely within the cytoplasm of positive mononuclear cells.(Bottom) TNF-a expression within mononuclear cells, predominantlyalong the periphery of portal tracts.

FIGURE 9. Correlation between TNF receptor expression and histo-logical activity. Hepatocellular expression of TNFR-p55 and -p75among the OLT patients with HBV recurrrence in relation to histo-logical activity. There was a strong correlation between hepaticexpression of both TNFR-p55 and -p75 and the H.A.I. (r50.8 and0.84, respectively, P,0.001, Spearman rank correlation).


cirrhosis in some cases within 3–5 years, unlike the course ofHBV infection in nontransplant patients. The strong up-regulation of the expression of both TNFRs, which in ourstudy correlates with the degree of hepatic inflammation,endows the receptor-bearing hepatocytes with an augmentedresponsiveness for the TNF-a effects leading to cytotoxicityand apoptosis (47).

In nontransplant patients with chronic hepatitis B, antivi-ral immune reactions from the host are not only responsiblefor the HBV-induced liver damage, but they also control viralreplication. A recent study has elegantly demonstrated thatIFN-g and TNF-a, secreted by antigen-specific T cells andantigen-nonspecific macrophages in the liver, activate twovirocidal pathways that eliminate HBV nucleocapsid parti-cles and viral RNA in infected hepatocytes (48). Our datasupport the notion that this is relevant to liver transplantrecipients with HBV reinfection and is illustrated by theevolution of HBV infection, which we observed in fiveHBeAg-positive liver transplant recipients, who had not re-ceived any posttransplantation antiviral treatment (Table 2).In the early phase of HBV recurrence after transplantation,there is a high level of HBV replication. After the induction ofthe host immune response with mononuclear cell infiltrationin the graft, the level of viral replication gradually falls overtime and liver graft damage progresses. These data providefurther evidence, albeit indirect, that in the majority of livertransplant recipients with HBV recurrence the virus is notcytopathic for the infected hepatocytes. Instead, the immunemechanisms induced by viral recurrence will limit to a certainextent the level of HBV replication, at the expense of progres-sive graft damage. Although liver damage was seen only in thepatients with HBV recurrence, there was no correlation be-tween the degree of HBV replication and the extent of necroin-flammatory activity. In a minority of liver transplant recipients,recurrent HBV infection may lead to fibrosing cholestatic hep-atitis, a particular type of rapidly progressive liver graft dam-age with minimal hepatic inflammation (49), but such patientswere not included in the present study.

On the basis of the data obtained in this study, the follow-ing sequence of events in the pathogenesis of liver damage intransplant recipients with HBV recurrence can be suggested.After HBV reinfection of the liver graft, viral peptides arepresented to recirculating T helper cells in association withHLA class II molecules by the host monocytes (macrophages/Kupffer cells), which have repopulated the liver allograftafter transplantation. Recognition of viral antigens by thepreexposed T helper cells leads to expansion and activation ofantigen-specific CD41 lymphocytes, which release IFN-g, the

most potent activator of monocytes/macrophages. Activationof the latter results in the production of proinflammatorycytokines, in particular TNF-a. The two cytokines thereafterdeliver their pathogenic potential, firstly, by recruitment andactivation of nonspecific inflammatory cells to the infectedgraft, thereby further amplifying the entire inflammatoryprocess; secondly, by up-regulating TNFR expression in theliver allograft, thus making the hepatocytes more vulnerableto the cytolytic and apoptotic actions of TNF-a; thirdly, byexerting a direct cytotoxic effect on the HBsAg-expressinghepatocytes; and finally, by inducing local mediators of tissueinjury such as nitric oxide. All these mechanisms can operateindependent of the degree of HLA matching between the hostand donor and the immunosuppression given and in combi-nation contribute to the progressive liver damage seen inpatients with HBV recurrence after transplantation.

Acknowledgments. We thank Dr. H. Gallati and Dr. M. Brock-haus, Pharmaceutical Research-New Technologies, Basel, Switzer-land, for providing mAbs.

REFERENCES

1. O’Grady JG, Smith HM, Davies SE, et al. Hepatitis B virusreinfection after orthotopic liver transplantation: serologicaland clinical implications. J Hepatol 1992; 14: 104.

2. Samuel D, Muller R, Alexander G, et al. Liver transplantation inEuropean patients with the hepatitis B surface antigen.N Engl J Med 1993; 329: 1842.

3. Demetris AJ, Todo S, Van TD, et al. Evolution of hepatitis Bvirus liver disease after hepatic replacement: practical andtheoretical considerations. Am J Pathol 1990; 137: 667.

4. Phillips MJ, Cameron R, Flowers MA, et al. Post-transplantrecurrent hepatitis B viral liver disease: viral-burden, steato-viral, and fibroviral hepatitis B. Am J Pathol 1992; 140: 1295.

5. Konig V, Hopf U, Neuhaus P, et al. Long-term follow-up ofhepatitis B virus-infected recipients after orthotopic livertransplantation. Transplantation 1994; 58: 553.

6. Chisari FV, Ferrari C, Mondelli MU. Hepatitis B virus structureand biology. Microb Pathog 1989; 6: 311.

7. Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis.Annu Rev Immunol 1995; 13: 29.

8. Moriyama T, Guilhot S, Klopchin K, et al. Immunobiology andpathogenesis of hepatocellular injury in hepatitis B virustransgenic mice. Science 1990; 248: 361.

9. Bertoletti A, Ferrari C, Fiaccadori F, et al. HLA class I-restrictedhuman cytotoxic T cells recognize endogenously synthesizedhepatitis B virus nucleocapsid antigen. Proc Natl Acad SciUSA 1991; 88: 10445.

10. Guidotti LG, Ando K, Hobbs MV, et al. Cytotoxic T lymphocytesinhibit hepatitis B virus gene expression by a noncytolytic

TABLE 2. Changes in serum HBV DNA during the follow-up of five HBeAg-positive liver transplant recipients with HBV recurrence

Patient number Posttransplantationfollow-up (mo)

Serum HBV DNA (pg/ml)Liver graft histology

Initial level End of follow-up

1 36 1057 0 Cirrhosis2 60 768 215 CAHa

3 78 200 48 Cirrhosis4b 30 86 21 CAH5b 43 18 5 NA

The initial HBV DNA level was taken within the first 6 months after HBV recurrence.a Abbreviations used in table: CAH, chronic active hepatitis; NA, not available.b These two patients received hepatitis B immunoglobulin prophylaxis, and HBV reinfection occured at 6 and 10 months after the

operation, respectively.


mechanism in transgenic mice. Proc Natl Acad Sci USA 1994;91: 3764.

11. Scullard GH, Smith CI, Merigan TC, Robinson WS, Gregory PB.Effects of immunosuppressive therapy on viral markers inchronic active hepatitis B. Gastroenterology 1981; 81: 987.

12. Lau JYN, Bain VG, Smith HM, Alexander GJM, Williams R.Modulation of hepatitis-B viral-antigen expression by immu-nosuppressive drugs in primary hepatocyte culture. Trans-plantation 1992; 53: 894.

13. McMillan JS, Shaw T, Angus PW, Locarnini SA. Effect of immu-nosuppressive and antiviral agents on hepatitis B virus repli-cation in vitro. Hepatology 1995; 22: 36.

14. Hanson RG, Peters MG, Hoofnagle JH. Effects of immunosup-pressive therapy with prednisolone on B and T lymphocytefunction in patients with chronic type B hepatitis. Hepatology1986; 6: 173.

15. Gassel HJ, Engemann R, Thiede A, Hamelmann H. Replacementof donor Kupffer cells by recipient cells after orthotopic ratliver transplantation. Transplant Proc 1987; 19: 353.

16. Gouw AS, Houthoff HJ, Huitema S, Beelen JM, Gips CH,Poppema S. Expression of major histocompatibility complexantigens and replacement of donor cells by recipient ones inhuman liver grafts. Transplantation 1987; 43: 291.

17. Steinhoff G, Wonigeit K, Sorg C, Behrend M, Mues B, PichlmayrR. Patterns of macrophage immigration and differentiation inhuman liver grafts. Transplant Proc 1989; 21: 398.

18. Ferrari C, Penna A, Bertoletti A, et al. Cellular immune re-sponse to hepatitis B virus-encoded antigens in acute andchronic hepatitis B virus infection. J Immunol 1990; 145: 3442.

19. Tsai SL, Chen PJ, Lai MY, et al. Acute exacerbations of chronictype B hepatitis are accompanied by increased T cell responsesto hepatitis B core and e antigens. J Clin Invest 1992; 89: 87.

20. Marinos G, Torre F, Chokshi S, et al. Induction of T helper cellresponse to HBcAg in chronic hepatitis B: a major factor inactivation of the host immune response to the hepatitis Bvirus. Hepatology 1995; 20: 1040.

21. Lohr HF, Weber W, Schlaak J, Goergen B, Meyer zum Buschen-felde KH, Gerken G. Proliferative response of CD41 T cells andhepatitis B virus clearance in chronic hepatitis with or withouthepatitis B e-minus hepatitis B virus mutants. Hepatology1995; 22: 61.

22. Gilles PN, Guerrette DL, Ulevitch RJ, Schreiber RD, Chisari FV.HBsAg retention sensitizes the hepatocyte to injury by physi-ological concentrations of interferon-gamma. Hepatology 1992;16: 655.

23. Yoshioka K, Kakumu S, Arao M, Tsutsumi Y, Inoue M. Tumornecrosis factor alpha production by peripheral blood mononu-clear cells of patients with chronic liver disease. Hepatology1989; 10: 769.

24. Yoshioka K, Kakumu S, Arao M, et al. Immunohistochemicalstudies of intrahepatic tumour necrosis factor alpha in chronicliver disease. J Clin Pathol 1990; 43: 298.

25. Sheron N, Lau JYN, Daniels H, et al. Increased production oftumour necrosis factor alpha in chronic hepatitis B virus in-fection. J Hepatol 1991; 12: 241.

26. Volpes R, van den Oord JJ, De Vos R, Desmet VJ. Hepaticexpression of type A and type B receptors for tumor necrosisfactor. J Hepatol 1992; 14: 361.

27. Marinos G, Naoumov NV, Rossol S, et al. Tumor necrosis factorreceptors in patients with chronic hepatitis B virus infection.Gastroenterology 1995; 108: 1453.

28. Tartaglia LA, Goeddel DV. Two TNF receptors. Immunol Today1992; 13: 151.

29. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ.Classification of chronic hepatitis: diagnosis, grading and stag-ing. Hepatology 1994; 19: 1513.

30. Donaldson P, Underhill J, Doherty D, et al. Influence of humanleukocyte antigen matching on liver allograft survival and

rejection. Hepatology 1993; 17: 1008.31. Rossol S, Voth R, Laubenstein HP, et al. Interferon production in

patients infected with HIV-1. J Infect Dis 1989; 159: 815.32. Hussain MJ, Mustafa A, Gallati H, Mowat AP, Mieli Vergani G,

Vergani D. Cellular expression of tumour necrosis factor-alphaand interferon-gamma in the liver biopsies of children withchronic liver disease. J Hepatol 1994; 21: 816.

33. Jung MC, Diepolder HM, Spengler U, et al. Activation of aheterogeneous hepatitis B (HB) core and e antigen-specificCD41 T-cell population during seroconversion to anti-HBe andanti-HBs in hepatitis B virus infection. J Virol 1995; 69: 3358.

34. Missale G, Brems JJ, Takiff H, Pockros PJ, Chisari FV. Humanleukocyte antigen class I-independent pathways may contrib-ute to hepatitis B virus-induced liver disease after liver trans-plantation. Hepatology 1993; 18: 491.

35. Gane EJ, Portmann BC, Naoumov NV, et al. Long-term outcomeof hepatitis C infection after liver transplantation. N EnglJ Med 1996; 334: 815.

36. Calmus Y, Hannoun L, Dousset B, et al. HLA class I matching isresponsible for the hepatic lesions in recurrent viral hepatitisB after liver transplantation. Transplant Proc 1990; 22: 2311.

37. Manez R, Mateo R, Tabasco J, Kusne S, Starzl TE, DuquesnoyRJ. The influence of HLA donor-recipient compatibility on therecurrence of HBV and HCV hepatitis after liver transplanta-tion. Transplantation 1995; 59: 640.

38. Paul WE, Seder RA. Lymphocyte responses and cytokines. Cell1994; 76: 241.

39. Pujol-Borrell R, Todd I, Doshi M, et al. HLA class II induction inhuman islet cells by interferon-gamma plus tumour necrosisfactor or lymphotoxin. Nature 1987; 326: 304.

40. Shinagawa T, Yoshioka K, Kakumu S, et al. Apoptosis in cul-tured rat hepatocytes: the effects of tumour necrosis factoralpha and interferon gamma. J Pathol 1991; 165: 247.

41. Brennan FM, Feldmann M. Cytokines in autoimmunity. CurrOpin Immunol 1992; 4: 754.

42. Lau JY, Bain VG, Davies SE, et al. High-level expression ofhepatitis B viral antigens in fibrosing cholestatic hepatitis.Gastroenterology 1992; 102: 956.

43. Munro JM, Pober JS, Cotran RS. Tumor necrosis factor andinterferon-gamma induce distinct patterns of endothelial acti-vation and associated leukocyte accumulation in skin of Papioanubis. Am J Pathol 1989; 135: 121.

44. Thornhill MH, Wellicome SM, Mahiouz DL, Lanchbury JS, KyanAung U, Haskard DO. Tumor necrosis factor combines withIL-4 or IFN-gamma to selectively enhance endothelial celladhesiveness for T cells: the contribution of vascular cell ad-hesion molecule-1-dependent and -independent binding mech-anisms. J Immunol 1991; 146: 592.

45. Adamson GM, Billings RE. Cytokine toxicity and induction ofNO synthase activity in cultured mouse hepatocytes. ToxicolAppl Pharmacol 1993; 119: 100.

46. Vouldoukis I, Riveros Moreno V, et al. The killing of Leishmaniamajor by human macrophages is mediated by nitric oxideinduced after ligation of the Fc epsilon RII/CD23 surface anti-gen. Proc Natl Acad Sci USA 1995; 92: 7804.

47. Grell M, Zimmermann G, Hulser D, Pfizenmaier K, Scheurich P.TNF receptors TR60 and TR80 can mediate apoptosis via induc-tion of distinct signal pathways. J Immunol 1994; 153: 1963.

48. Guidotti LG, Ishikawa T, Hobbs MV, Matzke B, Schreiber R,Chisari FV. Intracellular inactivation of the hepatitis B virusby cytotoxic T lymphocytes. Immunity 1996; 4: 25.

49. Davies SE, Portmann BC, O’Grady JG, et al. Hepatic histologicalfindings after transplantation for chronic hepatitis B virusinfection, including a unique pattern of fibrosing cholestatichepatitis. Hepatology 1991; 13: 150.

Received 13 January 1999.Accepted 22 July 1999.


Documents

Immunopathogenesis of Hepatitis B Virus Recurrence After Liver TRANSPLANTATION1