Detection of type 2-like T-helper cells in hepatitis C virus infection: Implications for hepatitis C virus chronicity

Detection of Type 2–Like T-Helper Cells in Hepatitis C Virus Infection: Implications for Hepatitis C Virus Chronicity SUN-LUNG TSAI,1 YUN-FAN LIAW,1 MING-HUEI CHEN,1 CHAO-YUAN HUANG,1

One striking clinical feature of hepatitis C virus (HCV) infection is that more than 50% of patients with acute hepatitis C will develop chronic infection. To investigate its possible mechanisms, we examined the activation of type 2–like T-helper (Th2-like) cells relating to the development of chronicity. Peripheral blood CD4/ T-cell proliferation and cytokine secretion in response to a panel of recombinant HCV antigens including core (C22), envelope 1 (E1), E2, nonstructural (NS) protein 4 (C100), fusion protein of NS3 and NS4 (C200), and NS5 were assayed in 17 patients with acute hepatitis C. All six patients with self-limited disease had a significant CD4/ T-cell proliferation to C22, E1, C100, C200, and NS5, running parallel with the antigen-stimulated secretion of interleukin (IL)-2 and interferon gamma (IFN-g), but not with interleukin (IL)-4 and IL-10, indicating predominant Th1 responses. Among the remaining 11 patients who developed chronicity, 6, 2, and 9 cases showed a specific CD4/ T-cell response to C22, C100, and C200, respectively, and the responses were significantly lower than those of cases with recovery in terms of stimulation index (SI) (P õ .05) and of antigen-stimulated IL-2 and IFN-g production. Importantly, IL-4 and IL-10 (Th2 responses) were detectable, and C22-specific Th2-like T-cell clones could be generated from patients with chronicity. The data suggested that activation of Th2 responses in acute hepatitis C patients may play a role in the development of chronicity. (HEPATOLOGY 1997;25: 449-458.) Hepatitis C virus (HCV)1 is an RNA virus responsible for the majority of posttransfusion non-A, non-B hepatitis.1,2 One important clinical feature of HCV infection is the high frequency of chronicity. More than half of the patients with acute infection will develop chronic hepatitis that may lead to a protracted clinical course including cirrhosis and hepatocellular carcinoma.3-5 In contrast, less than 10% of patients with acute hepatitis B virus infection will become chronic. This distinct difference implies that different survival strategies of the viruses and/or different host immune responses to

Abbreviations: HCV, hepatitis C virus; Th, T-helper cell; IL, interleukin; IFN-g, interferon gamma; PBMC, peripheral blood mononuclear cells; ALT, alanine transaminase; RTPCR, reverse-transcription polymerase chain reaction; SOD, superoxide dismutase; SI, stimulation index. From the 1Liver Research Unit, Chang Gung Memorial Hospital and Chang Gung Medical College, Taipei, Taiwan; and 2Chiron Corporation, Emeryville, CA. Received March 18, 1996; accepted September 24, 1996. Supported in part by grants from the Department of Health (DOH85-HR-522), the National Science Council (NSC83-0419-B-182-004 MH, NSC85-2331-B-182-026 MH), Executive Yuan, and the Prosperous Foundation, Taipei, Taiwan. Address reprint requests to: Yun-Fan Liaw, M.D., Liver Research Unit, Chang Gung Memorial Hospital, Chang Gung Medical College, 199, Tung Hwa North Road, Taipei, Taiwan 105. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2502-0033$3.00/0

AND

GEORGE C. KUO2

the viruses may exist. HCV genome contains a hypervariable region 1 at the upstream end of the envelope 2/nonstructural 1 (E2/NS1) gene segment. It has been suggested that the high mutation rate in hypervariable region 1 may generate variants to escape host immune surveillance, leading to persistent infection.6-11 However, this does not explain why, under the same host immune pressure, the amino acid substitution rate in the hypervariable region 1 is reduced when acute hepatitis C patients become persistent HCV carriers.11 An alternative explanation proposes that inadequate immunological host responses to HCV infection is the cause of chronicity.6 Yet no data support this hypothesis because no immune defect has been found in either posttransfusion or sporadic hepatitis C patients. In view of the observation that the hepatitis seen in HCV infection is not dependent on the humoral immune response,12 we therefore speculate that certain novel cellular immune mechanisms may be involved in the HCV pathogenesis and chronicity. A major advance in understanding the regulation of specific immune responses to infectious agents is the identification of two subpopulations of murine CD4/ T-helper lymphocytes, termed T-helper type 1 (Th1) and Th2, based on cytokine secretion profiles.13 Th1 cells promote cellular immunity and cytotoxic T-lymphocyte responses, and they produce predominantly interleukin (IL)-2, interferon gamma (IFN-g), and tumor necrosis factor b. Th2 cells favor development of humoral immunity and produce IL-4, IL-5, IL-10, or IL-13. A similar dichotomy exists among mature human CD4/ T-helper cells.14 The T-cell clones producing both Th1and Th2-type cytokines are designated as Th0 clones.15 In human diseases and animal models, evidence has been generated that cellular immune responses promoted by the Th1-related cytokines are associated with resistance to infection, while Th2-related cytokines exert negative immunoregulatory functions. An imbalance of Th1 and Th2 cytokine production may be related to disease progression in infectious diseases.14,16 Romagnani et al.16 reported that the majority of CD4/ T-cell clones derived by phytohemagglutinin stimulation of single T cells from the liver of chronic hepatitis C patients exhibit cytolytic activity and produce predominantly Th1-type cytokines. We have also demonstrated a predominant Th1 type of cytokine secretion by peripheral blood mononuclear cells (PBMC) responding to a nonstructural protein of HCV in chronic hepatitis C patients.17 HCV-specific Th0- and/or Th2like clones, on the other hand, are also reported to compartmentalize in the diseased liver.16,18 The roles of these Th0- or Th2-like cells in HCV pathogenesis and in the evolution from acute to chronic infection have not yet been elucidated. To address this issue, we investigated whether the activation of Th2 responses could be involved in causing chronicity of HCV infection, as reminiscences of the findings in patients infected by Mycobacterium leprae,19,20 and in model systems infected with parasites21,24 and retrovirus.22-24 PATIENTS AND METHODS Subjects and Serological Tests. The subjects studied were 17 pa-

tients with acute hepatitis C, defined by the following criteria: no

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history of liver disease; acute onset of malaise or jaundice and a rise in serum alanine transaminase (ALT) levels of at least 10 times the upper limit of normal; presence of HCV RNA detected by nested reverse-transcription polymerase chain reaction (RT-PCR) in the acute phase serum samples; de novo seroconversion of HCV antibodies during or after the acute episode25; and absence of other causes of hepatitis. Their demographic data are shown in Table 1. The hepatitis A, B, and D markers, including total or immunoglobulin M class antibody against hepatitis A virus, hepatitis B surface antigen and antibody, immunoglobulin M class antibody against hepatitis B core antigen, hepatitis B e antigen and antibody, and antibody against hepatitis delta virus were tested using commercially available radioimmunoassay kits (HAVAB or HAVAB-M, Ausria-II, Ausab, Corab-M, HBeAg-RIA and anti-d, Abbott Laboratories, North Chicago, IL). Serum HCV antibodies was assayed using a third-generation enzyme immunoassay (Murex anti-HCV, Version III, Murex Diagnostics Limited, Dartford, England). Antibodies to human immunodeficiency virus were assayed by EIA (GENELAVIA MIXT test kit, SANOFI Diagnostics Pasteur, Inc., Chaska, MN). The presence of serum HCV RNA was detected by nested RT-PCR using a set of primers within the 5* nontranslated region described previously.26 Typing of HCV was performed by PCR with type-specific primers within the core region described by Okamoto et al.27 HLA typing on PBMC was performed by standard serological techniques and DR haplotypes are shown in Table 1. None of the subjects studied had concurrent infections with the hepatitis A, B, or D viruses, or the human immunodeficiency virus. No immunosuppressive therapy was given before or during the study. Recovery of acute HCV infection was defined as normalization of serum ALT levels in association with clearance of serum HCV RNA within 6 months after acute hepatitis. In contrast, those having persistent elevation of serum ALT levels and/or having the presence of serum HCV RNA for more than 6 months were considered to have chronic evolution. To compare the patterns of CD4/ T-cell responses to HCV antigens with that of acute hepatitis C patients, 12 cases with chronic hepatitis C were included as the control group. Expression and Purification of HCV Recombinant Proteins. The polypeptide antigens of HCV virus (Simmonds genotype 1a,28 Okamoto group I27) were prepared as described previously.1,29 The preparations of these HCV proteins, including putative core or nucleocapsid antigen (C22), envelope 1 (E1), envelope 2 (E2), major part of the nonstructural (NS) protein 4 (C100), fusion protein of NS3 and NS4 (C200), and NS5, were described briefly in the following: The HCV proteins C22, E1, E2, C200, C100, and NS5 were expressed as Cterminal fusions with human superoxide dismutase (SOD) in yeast (Saccbaromyces cerevisiae) using methods similar to those described earlier for the expression of the C100-3 antigen.1 Yeast extracts were prepared by agitation with glass beads in 0.05 mol/L Tris-HCl buffer (pH 7.5) containing 0.1 mol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L ethylenediaminetetraacetic acid. The insoluble fractions were obtained by centrifugation at 10,000g for 20 minutes at 47C and washed twice with buffer described above and once with 0.05 mol/L glycine-NaOH buffer (pH 10) containing 0.1 mol/L NaCl and 0.5% Triton X100. Subsequently, the SOD Core, SOD E1, or SOD E2 in the washed protein aggregates was solubilized in 0.05 mol/L Tris-HCl (pH 7.5) containing 5 mmol/L dithiothreitol, 1 mmol/ L ethylenediaminetetraacetic acid, and 6 mol/L urea (buffer A). The solubilized fraction was applied on an S-Sepharose Fast Flow column (Pharmacia, Uppsala, Sweden) equilibrated with buffer A. The column was washed with buffer A, and the absorbed proteins were eluted with linear gradient of NaCl (0 to 1 mol/L). The SOD Core (C22), SOD (E1), or SOD (E2) was eluted at about 0.5 mol/L NaCl. Then, the SOD NS3/4, SOD NS4, or SOD NS5 in the washed insoluble fractions was solubilized by heating in a boiling water bath for 2 minutes in 0.02 mol/L sodium phosphate buffer (pH 6.8) containing 10 mmol/L dithiothreitol, 1 mmol/L ethylenediaminetetraacetic acid, and 2% sodium dodecyl sulfate. The SOD NS3/4, SOD NS4, or SOD NS5 in the solubilized protein aggregates was fractionated on a Sephacryl S-300 High Resolution Column (Pharmacia) equilibrated and run with 0.02 mol/L sodium phosphate buffer (pH 6.8) containing 2 mmol/L dithiothreitol, 1 mmol/L ethylenediaminetetraacetic acid, and 0.1% sodium dodecyl sulfate. After purification, all the HCV antigens were ú90% pure. Additionally, they were dialyzed against 0.1% phoshate-buffered saline before being used to stimulate CD4/ T cells. To exclude the possibility that some of the CD4/ T-cell responses observed might be induced by nonspecific responses to SOD

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or contaminants present in the antigen preparations, we used the proliferative responses to yeast extracts, as well as recombinant SOD expressed in yeast that have been described previously,1 as controls for the specificity of the T-cell response. Media. The media used were either free RPMI (RPMI 1640 medium supplemented with 25 mmol/L HEPES, 2 mmol/L L-glutamine, 1% sodium pyruvate, 0.05 mmol/L 2-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2.5 mg/mL fungizone), or complete medium (free RPMI supplemented with 10% heat-inactivated fetal bovine serum [all from GIBCOBRL Laboratories, Grand Island, NY]). Proliferative Assay. To avoid the possibility of antigen-stimulated cytokine production by non-CD4/ T cells, including CD8/ T cells,30,31 CD4/ T cells were affinity-purified from PBMC using Dynabeads M450 CD4 and DETACHaBEAD (Dynal International, Oslo, Norway) according to the manufacturer’s instructions. Used as feeder cells or antigen-presenting cells, the CD4-depleted PBMC were treated with 50 mg/mL mitomycin C (Kyowa Hakko Kogyo Co., Tokyo, Japan) for 45 minutes, and followed by three washes with free RPMI. Using 96-well flat-bottomed microculture plates (Nunc, Roskilde, Denmark), the CD4/ T cells at a density of 1 1 105 cells per well in complete medium were cocultured with CD4-depleted, mitomycin Ctreated PBMC (2 1 105 cells per well). Each of the HCV test antigens or the SOD control antigen was added to the well at a final concentration of 1 mg/mL. All the proliferative assays were performed in triplicate, and all the proliferative responses and antigen-stimulated cytokine production were measured when peak serum ALT levels were detected during the acute phase of hepatitis. The cells were incubated for 7 days at 377C in a humidified 5% CO2 incubator, and then 1.0 mCi per well of [3H]-thymidine (specific activity, 2 Ci/mmol; Amersham, England) was added to each well 18 hours before harvesting with an automatic cell harvester (Micro96 Harvester, Skatron Instruments AS, Lier, Norway). [3H]-Thymidine incorporation was measured with a model 1450 MicroBeta PLUS liquid scintillation counter (Wallac, Turku, Finland) using ScintiStrip (Wallac) without the addition of liquid scintillation cocktail. The data were expressed as stimulation indexes (SI Å mean counts per minute of antigen-stimulated cultures per mean counts per minute of medium-control cultures). Cytokine Secretion and Expression. Another set of cultures in triplicate were performed concurrently with the same condition as the proliferative assay. Also, one set of cultures in duplicate in the absence of HCV antigens were assayed as controls for spontaneous cytokine production by CD4/ T cells from both groups of patients. Supernatants of the antigen-stimulated and non–antigen-stimulated CD4/ T-cell cultures were collected after 72 hours of incubation, and enzyme immunoassays for cytokines were performed. Cytokine assays included IL-2 (IL-2 Test kit, T Cell Diagnostics, Cambridge, MA), IL-4 (Cytoscreen US Human IL-4 ELISA kit, BioSource International, Camarillo, CA), IL-10 (Cytoscreen US Human IL-10 ELISA kit, BioSource International), and IFN-g (IFN-g-EASIA, Medgenix Diagnostics, Fleurus, Belgium). The assay sensitivities of IL-2, IL4, IL-10, and IFN-g were 59 pg/mL, 0.39 pg/mL, 0.21 pg/mL, and 0.03 IU/mL, respectively. To correlate with cytokine secretion, antigen-stimulated messenger RNA expression of cytokines was detected by RT-PCR. Briefly, after the supernatants were removed for cytokine assays, the antigen-stimulated CD4/ T cells were harvested for total RNA extraction with standard acid guanidinium thiocyanate-phenol-chloroform and isopropanol precipitation. Single-stranded complementary DNA was synthesized by reverse transcription using random hexamer (Promega, Madison, WI) and Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD) according to the manufacturer’s instructions. The complementary DNA was diluted and then subjected to PCR performed in 50 mL reaction containing 5 mL of the diluted cDNA product, 50 mmol/L of each deoxynucleoside triphosphate, 1 unit of Taq DNA polymerase (Roche Molecular System, Branchburg, NJ), 5 mL of 101 PCR buffer (Roche), and 100 ng of each primer shown in Table 2. The reaction mixture was overlaid with mineral oil, and 35 cycles (denaturation at 947C for 60 seconds; annealing at 557C for actin, IL-2, and IL-4, and at 487C for IL-10 and IFN-g for 60 seconds; and extension at 727C for 90 seconds) of PCR were performed in a thermal cycler (Perkin Elmer-Cetus, Emeryville, CA). After amplification, 10 mL of the PCR product was analyzed by electrophoresis in 1.8% agarose gel, and the DNA fragments were visualized after staining with ethidium bromide. The authenticity of the amplified products was confirmed

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TABLE 1. Demographic Data of Patients With Acute Hepatitis C and Their CD4/ T-Cell Responses to HCV Antigens, Lymphokine Secretion, and PCR Analysis

Patients

PCR Analysis‡

Peak Case Age ALT HCV No. (y) Sex (IU/mL)* Genotype

Acute HCV infection A1 with recovery

33

M

973

III(2a)

HCV Antigens

DR6,DR9

A2

26

F

601

II(1b)

DR2,DR4

A3

51

F

1,048

II(1b)

DR6,DR10

A4

61

F

1,090

II(1b)

DR5,DR9

A5

74

F

437

II(1b)

DR2,DR6

A6

60

F

1,841

II(1b)

DR6,—

Acute HCV infection B1 with chronic evolution

54

F

609

II(1b)

DR2,DR4

AID

B2

66

M

1,037

II(1b)

DR2,DR5

B3

56

M

749

II(1b)

DR2,DR5

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SI†

IFN-g (IU/mL)

IL-4 (pg/mL)

C22

7.3

22.1

õ0.39

/

/

0

0

E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22

3.3 2.1 3.2 4.5 4.0 0.6 10.3 6.2 2.3 3.4 7.5 4.3 0.7 7.9 3.6 3.4 5.2 5.0 4.5 0.9 11.2 3.5 4.2 8.5 17.1 6.8 0.77 12.0 4.1 3.2 7.3 4.2 7.1 1.0 11.0 3.4 2.5 12.5 13.0 10.1 0.92 5.1

12.2 9.0 13.3 30.5 20.5 õ0.03 33.3 11.8 9.7 3.4 26.3 16.9 õ0.03 24.0 11.9 9.9 4.5 27.3 20.3 õ0.03 42.1 12.8 20.5 15.3 35.6 30.7 õ0.03 45.7 12.1 10.2 10.5 16.3 21.5 õ0.03 40.3 13.0 10.1 9.7 32.7 20.5 õ0.03 12.3

õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 0.49 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 õ0.39 7.55

/ / / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0 /

/ / / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0 /

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 / 0 0 0 0 0 0 0 0 0 0 0 0 0 /

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 /

E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD

1.3 2.8 1.3 3.8 3.4 1.3 4.3 1.4 3.0 3.0 4.0 1.4 1.2 3.8 1.5 2.4 1.5 5.2 2.8 1.3

2.6 2.1 2.4 13.5 10.1 õ0.03 10.9 0.05 0.18 õ0.03 14.0 õ0.03 õ0.03 9.6 4.0 2.7 1.6 7.9 2.6 õ0.03

1.5 2.4 3.1 8.0 5.9 õ0.39 6.64 õ0.39 2.9 1.3 7.8 õ0.39 õ0.39 5.89 1.2 3.3 7.7 1.2 2.1 õ0.39

/ / / / / 0 / 0 / / / 0 0 / / / / / / 0

/ / / / / 0 / 0 0 0 / 0 0 / / / / / / 0

/ / / / / 0 / 0 / / / 0 0 / / / / / / 0

/ / / / / 0 / 0 / / / 0 0 / / / / 0 / 0

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IL-2 IFN-g IL-4 IL-10

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Follow-up Blood (mo) Transfusion

21

No

20

No

9

No

10

No

8

Yes

26

No

13

Yes

14

No

9

No

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Patients

PCR Analysis‡

Peak Case Age ALT No. (y) Sex (IU/mL)*

HCV Genotype

B4

29

F

1,110

II(1b)

DR4,DR9

B5

40

M

2,135

II(1b)

DR2,DR9

B6

55

F

1,520

I(1a)/II(1b)

B7

61

M

1,827

II(1b)/III(2a) DR4,— /IV(2b)

DR2,—

B8

74

M

1,012

IV(2b)

DR9,—

B9

67

M

1,215

II(1b)

DR4,—

B10

60

M

1,667

B11

59

F

1,337

II(1b)/III(2a) DR5,—

II(1b)

DR4,DR8

HCV Antigens

SI†

IFN-g (IU/mL)

IL-4 (pg/mL)

C22 E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22

2.2 1.2 2.5 1.7 3.5 2.9 1.4 8.1 1.2 1.5 1.7 3.5 2.8 0.9 6.5 1.3 2.1 1.8 4.2 2.7 1.4 2.5

5.1 2.4 3.0 3.3 7.3 10.6 õ0.03 19.9 2.4 2.3 8.3 10.5 3.9 õ0.03 18.5 2.3 2.1 2.4 8.4 2.7 õ0.03 6.3

5.42 2.5 6.1 8.7 7.7 2.1 õ0.39 24.18 1.5 2.3 8.3 3.5 2.0 õ0.39 12.00 1.3 1.7 5.0 8.5 3.0 õ0.39 0.99

/ / / / / / 0 / / / / / / 0 / / / / / / 0 /

/ / / / / / 0 / / / / / / 0 / / / / / / 0 /

/ / / / / / 0 / / / / / / 0 / / / / / / 0 /

/ / / / / / 0 / / / / / / 0 / / / / / / 0 0

E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD C22 E1 E2 C100 C200 NS5 SOD

3.4 2.2 1.9 5.0 3.1 1.3 1.7 2.6 2.1 3.0 4.7 3.0 1.2 3.4 2.7 3.0 3.8 5.3 2.9 1.0 2.0 1.3 1.5 2.7 2.8 3.1 1.5 1.3 1.4 1.6 2.9 2.7 3.3 1.3

1.7 1.9 3.9 12.7 10.0 õ0.03 0.7 2.1 2.0 11.2 9.8 11.5 õ0.03 8.1 1.8 3.3 13.9 11.2 8.7 õ0.03 5.0 1.3 1.6 6.7 8.6 4.5 õ0.03 0.1 0.8 0.9 8.5 6.7 10.1 õ0.03

2.0 3.8 8.8 9.6 5.9 õ0.39 0.41 1.5 1.7 4.5 9.4 6.0 õ0.39 1.23 2.1 1.8 8.6 8.0 6.1 õ0.39 1.11 1.1 1.7 4.9 7.7 6.1 õ0.39 0.40 2.0 1.5 3.9 6.5 6.4 õ0.39

/ / / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0

/ / / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0 0 0 / / / / 0

/ / / / / 0 / / 0 / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0

/ / / / / 0 0 / / / / / 0 / / / / / / 0 / / / / / / 0 / / / / / / 0

IL-2 IFN-g IL-4 IL-10

Follow-up Blood (mo) Transfusion

10

No

8

No

17

Yes

18

No

15

No

8

No

17

No

13

Yes

NOTE. CD4/ T-cell proliferation and cytokine responses in 17 cases (A1Ç6,B1Ç11) with acute HCV infection. * Normal range of serum levels ° 36 IU/mL. † SI in response to each of HCV antigens; 1 mg/mL in the culture and the assay of each case was performed at the time when peak serum ALT level was detected. ‡ PCR products visible in the ethidium bromide–staining agarose gels (/), and no band visible (0).

by Southern blotting and hybridization with specific oligonucleotide probes shown in Table 2. Limiting Dilution T-Cell Cloning. To further verify the activation of Th2-like or, at least, Th0-like cells in acute hepatitis C patients with chronic evolution, limiting dilution T-cell cloning was performed from C22-stimulated CD4/ T-cell cultures of five patients with chro-

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nicity and of two patients with recovery. In the first-stage culture, CD4/ T cells in six wells (1 1 105 cells per well) of a 96-well flatbottomed microtiter tray in a final volume of 200 mL per well, the following were added: 1) 1 1 105 cells per well of autologous CD4depleted, mitomycin C–treated PBMC as antigen-presenting cells; 2) 1 mg/mL of C22 polypeptide; and 3) 20 ng/mL of recombinant

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TABLE 2. Primers Used for RT-PCR Detection of Lymphokines and Actin IL-2

Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe Forward Reverse Probe

IL-4

IL-10

IFN-g

Actin

5*-TAC AGG ATG CAA CTC CTG TCT TGC ATT GCA-3* (61-90)* 5*-GTT GCT GTC TCA TCA GCA TAT TCA CAC ATG-3* (445-416) 5*-CAG TGT CTA GAA GAA GAA CTC AAA CCT CTG-3* (276-305) 5*-CTG CTA GCA TGT GCC GGC AAC TTT GTC CAC-3* (103-132) 5*-TAT GAT GAG TTA TAA ATA TAT AAA TAC TTA-3* (592-563) 5*-GAA GCC AAC CAG AGT ACG TTG GAA AAC TTC TTG-3* (442-474) 5*-CTG AGA ACC AAG ACC CAG ACA TCA-3* (323-356) 5*-AAC TCC TGA CCT CAA GTG ATC CAC-3* (1272-1249) 5*-CTC TGA TAC CTC AAC CCC CA-3* (701-720) 5*-GCA GGT CAT TCA GAT GTA G-3* (247-265) 5*-TAA TTA GTC AGA AAA CAA AGG-3* (850-830) 5*-TGC AGA GCC AAA TTG TCT CC-3* (332-351) 5*-AAC CCC AAG GCC AAC CGC GAG AAG ATG ACC-3* (372-401) 5*-GGT GAT GAC CTG GCC GTC AGG CAG CTC GTA-3* (788-759) 5*-GCC CTG GAC TTC GAG CAA GAG ATG GCC ACG-3* (699-728)

385 bp†

490 bp

950 bp

603 bp

416 bp

Abbreviation: bp, base pairs. * Location of nucleotide sequences. † Size of PCR products in bp.

human IL-2 (R & D Systems, Minneapolis, MN). After incubation at 377C in a humidified 5% CO2 atmosphere for 3 days, C22-stimulated T lymphocytes were harvested, counted, and then cloned by limiting dilutions at 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 cells per well in 96-well flat-bottomed trays. The plating was duplicate on each cell number, and thus there were 12 plates for each case. In each well, we added 1 mg/mL of phytohemagglutinin (Sigma Chemical Co., St. Louis, MO), 20 ng/mL of recombinant human IL-2, 1 ng/mL of recombinant human IL-4 (R & D Systems), and 2 1 105 cells of mitomycin C–treated autologous B-lymphoblastoid cell line established by Epstein-Barr virus transformation of PBMC. The purpose of the addition of IL-4 to the cultures is to enhance the growth of Th2-like cells that may have been activated at the first-stage culture. The growing cells were expanded with IL-2–containing medium.

patients. HLA-DR5 antigen was positive in 1 patient with recovery and 3 patients with chronic evolution, whereas HLA-DR6 was positive in 4 of the 6 cases with recovery (64%), but in none of the 11 cases with chronicity (P Å .006, Fisher’s test). There was no significant difference in peak serum ALT levels between two groups (P ú .5, Mann-Whitney U test). CD4/ T-Cell Responses in Acute Hepatitis C Patients. During the acute phase of hepatitis, CD4/ T cells from 12, 7, 4, 8, 15, and 10 of 17 patients proliferated significantly (SI ¢ 3.0) in response to C22, E1, E2, C100, C200, and NS5, respectively (Fig. 1). None of the CD4/ T cells responded to the

Screening of C22-Specific T-Cell Clones and Flow Cytometry Analysis. Growing T-cell lines were tested for the C22-specific prolifera-

tion. The cloned T-cell lines (2 1 104 cells per well) in triplicate were cocultured with mitomycin C–treated autologous B-lymphoblastoid cell line (2 1 105 cells per well) and 1 mg/mL of C22 polypeptide and SOD or yeast extract as controls for 72 hours. Then, supernatant was collected for lymphokine assays, and positive proliferative response (SI ¢ 5.0) was ascertained in the rescreened clones. For confirmation of CD4/ T-cell phenotype, the responsive cloned T-cell lines were analyzed by flow cytometry (FACSort, Becton-Dickinson, San Jose, CA) by two-colored direct immunofluoresence method using monoclonal antibodies of the Leu series described previously.32 Clonality Analysis by T-Cell Receptor b-Gene Rearrangement. Genomic DNA was isolated from 107 cells of T-cell lines by standard phenol and chloroform extraction. Approximately, 10 mg of DNA was digested overnight with 60 U of restriction enzyme BamHI (Boehringer Mannheim, Mannheim, Germany). DNA fragments were size-separated overnight in 0.8% agarose gel and were then transferred onto nylon membranes (Hybond-N; Amersham) according to the manufacturer’s instructions. The filter was prehybridized overnight in a 427C water bath, and then was hybridized with a randomly primed 32Plabeled DNA probe for the constant region of the T-cell receptor b gene. After washing at appropriate stringency, the filter was autoradiographed. DNAs of a hepatoblastoma cell line (HepG2), of a T-cell lymphoma, and of a B-lymphoblastoid cell line were used as the nonlymphoid-negative, lymphoid-positive, and lymphoid-negative controls, respectively. The probe used is a 0.4-kb human complementary DNA fragment, which includes the constant region of the T-cell receptor b2 region of T-cell receptor b (Oncogene Science, Manhasset, NY). Close homology of the constant region of the T-cell receptor b2 and b1 allows detection of both regions during Southern hybridization.33 RESULTS Development of Chronicity. Of the 17 patients with acute

hepatitis C shown in Table 1, 6 had a recovery (35%) and 11 developed chronicity (65%). Okamoto’s group II (Simmonds’ genotype 1b) was the predominant type in both groups of

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FIG. 1. Stimulation index. CD4/ T-cell responses of 17 cases with acute HCV infection and of 12 cases with chronic hepatitis C. Dashed line indicates an SI Å 3.0, above or equal to which the response is significant.

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lower than those stimulated by HCV antigens (P õ .001, paired t test). The antigen-stimulated secretion of cytokines was consistent with messenger RNA expression as detected by RT-PCR. The data of CD4/ T cell response to each of the HCV antigens in each case are shown in Table 1. As demonstrated in Fig. 3, case B2, who had a specific CD4/ Tcell proliferation to C22, E2, C100, and C200, but not to SOD, showed a relevant messenger RNA expression of IL-2, IFNg, IL-4, and IL-10, respectively. Predominance of Th1 Responses and Presence of Th0 or Th2 Responses in Chronic Hepatitis C. The pattern of CD4/ T-cell

responses to HCV antigens in acute hepatitis C patients with evolving chronicity was similar to that of chronic hepatitis C patients. (Fig. 1). Furthermore, the antigen-stimulated production of both IL-2/IFN-g (higher levels; Th1 response) and IL-4/IL10 (lower levels; Th2 response) was also detectable (data not shown). Th2-Like Clones Can Be Generated From Patients Developing Chronicity. C22-stimulated cultures of cases A3, A4,

and B2 through B6 were expanded, and growing T cells were cloned by the limiting dilution method. From each case, among 12 microculture trays (96 wells 1 12 plates Å 1,152 total wells), C22-specific T-cell lines could be identified in 30,

FIG. 2. Cytokine secretion of CD4/ T cells from patients with acute HCV infection in response to HCV antigens and spontaneous cytokine production without antigen stimulation. The data were expressed as means { SEM. (j) Antigen-stimulated cytokine production by CD4/ T cells from acute HCV patients with recovery (n Å 6) and ( ) spontaneous cytokine production. (h) Antigen-stimulated cytokine production by CD4/ T cells from acute HCV patients with chronicity (n Å 11) and ( ) spontaneous cytokine production.

SOD alone (Table 1). Likewise, none of the CD4/ T cells from all patients responded to the yeast extracts (data not shown). Furthermore, mitomycin C–treated PBMCs from all patients were cultured under the same conditions. None of these cultures proliferated in response to any of the HCV proteins (data not shown), and no cytokines were detectable in these mitomycin C–treated cells (data not shown). All six patients with self-limited hepatitis had a significant CD4/ T-cell proliferation to C22, E1, C100, C200, and NS5. Except for one case (case A5), the proliferation ran parallel with the antigenstimulated secretion of IL-2 and IFN-g, but not with IL-4 and IL-10 (Table 1 and Fig. 2). In a follow-up study 3 months later, CD4/ T cells of case A5 showed a predominant Th1-type cytokine response to all the HCV antigens and IL-4 became undetectable (data not shown). In contrast, only 6, 1, 1, 2, 9, and 4 of 11 patients with chronic evolution exhibited a significant response to each of the HCV antigens, respectively. The responses were lower than those of cases with recovery in terms of SI (P õ .05) (Table 1 and Fig. 1). The levels of antigen-stimulated IL-2 and IFN-g production were also lower (P õ .05) (Fig. 2). Importantly, using an ultrasensitive enzyme-linked immunosorbent assay system, antigenstimulated IL-4 and IL-10 production was detectable in patients developing chronicity but was virtually undetectable in patients with recovery (Fig. 2). As a control, the data of spontaneous cytokine production by CD4/ T cells cultured with mitomycin C–treated PBMC as antigen-presenting cells in the absence of antigen stimulation are shown in Fig. 2. The levels of spontaneous cytokine production were significantly

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FIG. 3. RT-PCR analysis of cytokine expression by CD4/ T cells in response to HCV antigens in a patient with acute hepatitis C who developed chronic hepatitis on follow-up (Table 1, case B2). Both Th1-type and Th2-type cytokine expression could be detected in HCV antigen-stimulated CD4/ T-cell cultures, but could not be detected in SOD or yeast extract-stimulated cultures (data not shown).

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TABLE 3. Cytokine Secretion Profiles of C22-Specific Cloned T-Cell Lines Generated From Acute Hepatitis C Patients Patient*

No. of TCCs

Disease Status

A3 A4 Total

Recovery Recovery

30 27 57

B2 B3 B4 B5 B6 Total

Chronic Chronic Chronic Chronic Chronic

60 72 81 49 55 317

evolution evolution evolution evolution evolution

Th0 (%)†

Th1 (%)†

Th2 (%)†

2 (7%) 0 2 (4%)

28 (93%) 27 (100%) 55 (96%)

0 0 0‡

24 27 29 21 30 131

(40%) (38%) (36%) (43%) (55%) (41%)

33 41 40 23 18 155

(55%) (57%) (49%) (47%) (33%) (49%)

3 4 12 5 7 31

(5%) (5%) (15%) (10%) (12%) (10%)‡

Abbreviation: TCC, T-cell clone. * The patient numbers are the same as that of cases in Table 1. † TCCs producing IFN-g but not IL-4 were defined as Th1; TCCs producing IL-4 but not IFN-g were defined as Th2; and TCCs producing both IFN-g and IL-4 were referred to as Th0. ‡ x2 Å 26.97, P ° .005, x2 test with progression analysis.

27, 60, 72, 81, 49, and 55 wells, respectively (Table 3). All cloned T-cell lines shown in Table 3 were tested to be C22specific; none responded to SOD or yeast extracts. Th2-like cloned T-cell lines could be generated from patients with chronic evolution but could not be identified from patients with recovery (P õ .005, x2 test with progression analysis), although in much lower frequency compared with Th1-like cloned T-cell lines. The frequency of Th0-like cloned T-cell lines was also significantly higher in patients developing chronic hepatitis than in patients with recovery. Proliferative response of one representative Th2-like clone, designated T2, is demonstrated in Fig. 4. Clonality analysis of T2 clone by Southern blot hybridization of gene coding for the b-chain of the T-cell receptor revealed two homogeneous restriction fragments, indicating a clonal rearrangement (Fig. 5). A total of 12 additional Th2-like clones (1, 1, 7, 1, and 2 clones from patients B2 through B6, respectively) had been analyzed in a similar way. All of them showed a clonal rearrangement (data not shown).

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evolution. Furthermore, our data showed that C22-specific CD4/ T-cell clones from patients with self-limited infection predominantly secreted Th1-type cytokines, a finding consistent with a recent report by Diepolder et al.,36 in which NS3specific CD4/ T-cell clones from patients with recovery predominantly produce IFN-g (Th1-type cytokine pattern). Also, our data revealed that the CD4/ T-cell responses to HCV antigens are significantly stronger in acute hepatitis C patients with recovery than those who develop chronicity. These results are in agreement with a study in 21 Italian patients that showed that antiviral T-cell–mediated immune responses in terms of SI are more vigorous in patients with self-limited disease than in those who develop a chronic infection.37 The predominance of Th1-type cytokine responses has been reported to play an important role in viral clearance of patients with both acute and chronic hepatitis B.16,40-43 In contrast, a combined Th1-Th2–like response was found in patients with chronic hepatitis C in this study, exhibiting a pattern like that of acute hepatitis C with chronic evolution. This distinction suggested that different mechanisms were involved in causing chronicity between the hepatitis B virus and HCV. In the present study, HCV infection by genotype 1b was not a predisposing factor to develop chronicity. This is conceivable because the rates of HCV chronicity are similar worldwide, irrespective of HCV genotypes in different geographic areas.4,44 In addition, only one case in this study with recovery was positive for HLA-DR5 antigen that has been reported to be genetically correlated with a benign evolution

DISCUSSION

Several observations support an important role of CD4/ T cells in the elimination of HCV infection.29,34-37 However, all these studies are based on assays of PBMC proliferation. Using purified CD4/ T cells in this study, we have demonstrated that (1) Th2- or Th0-like responses exist in chronic hepatitis C patients; (2) the activation of Th2- or Th0-like responses in acute hepatitis C patients correlates with chronic evolution, whereas cases with predominant Th1-like responses result in recovery; (3) the levels of spontaneous cytokine production by CD4/ T cells cultured with mitomycin C–treated PBMC as antigen-presenting cells in the absence of antigen stimulation were significantly lower than those stimulated by HCV antigens. Therefore, the difference in production of cytokines between acute patients with chronic evolution and with self-limited infection is caused by the activation of HCV-specific T cells, but not by the effects of virus nonspecific T cells; (4) Th2-like clones can be generated from acute hepatitis C patients with chronic evolution; and (5) these Th2-like clones showed a clonal rearrangement as revealed by clonality analysis, indicating that they were a clonal expansion of Th2 cells.38,39 Therefore, C22-stimulated CD4/ T-cell cultures expressing both Th1-type and Th2-type cytokine profiles were composed of both Th1-type and Th2type cell populations, in addition to Th0-type cells. These results suggest that activation of Th2 responses to HCV antigens in acute hepatitis C patients is associated with chronic

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FIG. 4. T-cell clone T2. C22-specific proliferation of a Th2 clone T2 . Each datum was a mean of triplicate determinations. For the details of procedure, see text. Ag, HCV polypeptide antigens or SOD protein.

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FIG. 5. Analysis of one Th2 clone T2 . (A) Flow cytometry analysis of Th2 clone T2 . Leu 3 (CD4)-positive cells are ¢ 99%. (B) Clonality analysis by Tcell receptor gene rearrangement of C22-specific Th2 clone T2 . TLM, T-cell lymphoma; B, Epstein-Barr virus–transformed B-cell line; HEG2, hepG2 cell line; R, rearranged restriction fragment; G, germline band.

of the infection in Italy, where genotype 1a is the most prevalent.44,45 Patients of different HLA-DR haplotypes (DR5 vs. DR6) may influence the outcome of infection by different HCV genotypes (1a vs. 1b). More data are needed to validate this observation. The mechanisms of activation of Th2- or Th0-like responses in hepatitis C patients remain unclear. Preliminary reports have revealed that liver-derived, HCV-specific T-cell lines infected by HCV have a tendency to differentiate into Th0/ Th2-like phenotypes.16 Our data have suggested that each of the HCV antigens used may have the potential to stimulate the differentiation of precursor T-helper cells into both Th1 and Th2 cells, or Th0 cells. Because the recombinant HCV antigens used in this study were derived from a single HCV strain (genotype 1a) and most of the patients studied were infected by HCV genotype 1b, the HCV antigen-specific CD4/ T-cell responses may have been underestimated in the present study because of viral heterogeneity. This is reflected by the finding that the response to E2 is the lowest in terms of SI and of cytokine production in both acute and chronic hepatitis C patients. More works are needed to clarify these factors in the immune responses of hepatitis C patients, and to answer the question as to why every HCV antigen behaves similarly in the stimulation of cytokine production. IFN-g, produced by Th1 cells, not only favors the elimination of virus-infected hepatocytes but also increases the severity of hepatitis activity through the IFN-g –mediated enhancement of cellular immunity.46-48 It could even induce a vicious cycle by activating the intrahepatic macrophages and by inducing a delayed-type hypersensitivity response that could lead to fulminant hepatitis, as demonstrated in a hepatitis B surface antigen transgenic mouse model.49 In contrast, Th2-like responses may ameliorate the tissue-damaging effects of immune responses mediated by Th1-like responses, thereby modulating self-inflicted injury through the action

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of IL-4 and IL-10, which counteract the effects of IFN-g on macrophages.50,51 Therefore, our data should not be interpreted as a contradiction to the notion that the quasispecies nature of HCV and the hypermutation in the hypervariable region of the virus are important viral mechanisms for inducing chronicity, because these mechanisms may permit the virus to escape host immune surveillance, including antibody neutralization and cytotoxic T-lymphocyte recognition.6-11,52 Perhaps it is better to consider that the activation of Th2-like responses in acute HCV infection may down-regulate the deleterious effects of Th1 responses and may protect the hosts from lethal liver damage. In other words, it is a protective mechanism to minimize liver cell damage, but at the expense of the potential eradication of the virus, thereby leading to chronicity. In this regard, the activation of Th2-like responses in association with chronicity might provide a possible explanation for the finding that fulminant hepatitis occurs much less frequently in acute HCV infection than in acute hepatitis B virus infection.53-56 It is noted that not even a single case of fulminant hepatitis induced by acute HCV infection was found in a prospective study of posttransfusion non-A, non-B hepatitis in Taiwan.57 Because there are significant discrepancies between biochemical, virological, and histological changes in patients with HCV infection,58,59 the severity of liver damage cannot be judged only by the serum ALT levels. This may explain why there is no significant difference in peak serum ALT levels between the patients with evolving chronicity and the patients with recovery. Clinical observations have shown that autoantibodies and/ or immunological abnormalities, important indicators of Th2like responses, are more frequently found in patients with chronic hepatitis C than in those with non-HCV liver diseases other than autoimmune hepatitis and primary biliary cirrhosis.60-64 Thus, the activation of Th2-like responses can also account in part for the presence of autoantibodies and of certain immunological disorders in chronic hepatitis C patients. In summary, the activation of Th2-like responses in acute HCV infection may play a role in the HCV pathogenesis and may be a useful predictor for chronicity. Acknowledgment: The authors thank Su-Chiung Chu for her secretarial assistance. REFERENCES 1. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359-362. 2. Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362-364. 3. Di Bisceglie AM. Hepatitis C and hepatocellular carcinoma. Semin Liver Dis 1995;15:64-69. 4. Alter MJ, Margolis HS, Krawczynski K, Judson FN, Mares A, Alexander WJ, Hu PY, et al. The natural history of community-acquired hepatitis C in the United States. N Engl J Med 1992;327:1899-1905. 5. Resnick RH, Koff R. Hepatitis C–related hepatocellular carcinoma: prevalence and significance. Arch Intern Med 1993;153:1672-1677. 6. Weiner AJ, Brauer MJ, Rosenblatt J, Richman KH, Tung J, Crawford K, Bonino F, et al. Variable and hypervariable domains are found in the regions of HCV corresponding to the Flavivirus envelope and NS1 proteins and the Pestivirus envelope glycoproteins. Virology 1991;180:842-848. 7. Weiner AJ, Geysen HM, Christopherson C, Hall JE, Marson TJ, Saracco G, Bonino F, et al. Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections. Proc Natl Acad Sci U S A 1992;89:3468-3472. 8. Farci P, Alter HJ, Govindarajan S, Wong DC, Engle R, Lesniewski RR, Mushahwar IK, et al. Lack of protective immunity against reinfection with hepatitis C virus. Science 1992;258:135-140. 9. Martell M, Esteban JI, Quer J, Genesca J, Weiner A, Esteban R, Guardia J, et al. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J Gen Virol 1992;66:3225-3229.

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WBS: Hepatology

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10. Sakamoto N, Enomoto N, Kurosaki M, Marumo F, Sato C. Sequential change of the hypervariable region of the hepatitis C virus genome in acute infection. J Med Virol 1994;42:103-108. 11. Yamaguchi K, Tanaka E, Higashi K, Kiyosawa K, Matsumoto A, Furuta S, Hasegawa A, et al. Adaptation of hepatitis C virus for persistent infection in patients with acute hepatitis. Gastroenterology 1994;106:13441348. 12. Kumar U, Monjardino J, Thomas HC. Hypervariable region of hepatitis C virus envelope glycoprotein (E2/NS1) in an agammaglobulinemic patient. Gastroenterology 1994;106:1072-1075. 13. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:23482357. 14. Del Prete G, Maggi E, Romagnani S. Human Th1 and Th2 cells: functional properties, mechanisms of regulation, and role in disease. Lab Invest 1994; 70:299-306. 15. Street NE, Schumacher JH, Fong TAT, Bass H, Fiorentino DF, Leverah JA, Mosmann TR. Heterogeneity of mouse helper T cells: evidence from bulk cultures and limiting dilution cloning for precursors of Th1 and Th2 cells. J Immunol 1990;144:1629-1639. 16. Romagnani S. Lymphokine production by human T cells in disease states. Annu Rev Immunol 1994;12:227-257. 17. Tsai SL, Chen PJ, Hwang LH, Kao JH, Huang JH, Chang TH, Chen DS. Immune response to a hepatitis C virus nonstructural protein in chronic hepatitis C virus infection. J Hepatol 1994;21:403-411. 18. Minutello MA, Pileri P, Unutmaz D, Censini S, Kuo G, Houghton M, Brunetto MR, et al. Compartmentalization of T lymphocytes to the site of disease: intrahepatic CD4/ T cells specific for the protein NS4 of hepatitis C virus in patients with chronic hepatitis C. J Exp Med 1993;178:17-25. 19. Yamamura M, Uyemura K, Deans RJ, Weinberg K, Rea TH, Bloon BR, Modlin RL. Defining protective responses to pathogens: cytokine profiles in leprosy lesions. Science 1991;254:277-279. 20. Sieling PA, Abrams JS, Yamamura M, Salgame P, Bloom BR, Rea TH, Modlin RL. Immunosuppressive roles for IL-10 and IL-4 in human infection: in vitro modulation of T cell responses in leprosy. J Immunol 1993; 150:5501-5510. 21. Sher A, Coffman RL. Regulation of immunity to parasites by T cells and T cell-derived cytokines. Annu Rev Immunol 1992;10:385-409. 22. Gazzinelli RT, Makino M, Chattopadhyay SK, Snapper CM, Sher A, Hugin AW, Morse HC. CD4/ Subset regulation in viral infection: preferential activation of Th2 cells during progression of retrovirus-induced immunodeficiency in mice. J Immunol 1992;148:182-188. 23. Kanagawa O, Vaupel BA, Gayama S, Koehler G, Kopf M. Resistance of mice deficient in IL-4 to retrovirus-induced immunodeficiency syndrome (MAIDS). Science 1993;262:240-242. 24. Mosmann TR, Sad S. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 1996;17:138-144. 25. Yeh CT, Han CM, Lo SY, Ou JH, Fan KD, Sheen IS, Chu CM, et al. Early detection of anti-HCc antibody in acute hepatitis C virus (HCV) by Western blot (immunoblot) using a recombinant HCV core protein fragment. J Clin Microbiol 1994;32:2235-2241. 26. Liaw YF, Tsai SL, Chang JJ, Sheen IS, Chien RN, Lin DY, Chu CM. Displacement of hepatitis B virus by hepatitis C virus as the cause of continuing chronic hepatitis. Gastroenterology 1994;106:1048-1053. 27. Okamoto H, Sugiyama Y, Okada S, Kurai K, Akahane Y, Sugai Y, Tanaka T, et al. Typing hepatitis C virus by polymerase chain reaction with typespecific primers: applications to clinical surveys and tracing infectious sources. J Gen Virol 1992;73:673-679. 28. Simmonds P. Variability of hepatitis C virus. HEPATOLOGY 1995;21:570583. 29. Botarelli P, Brunetto MR, Minutello MA, Calvo P, Unutmaz D, Weiner AJ, Choo QL, et al. T-lymphocyte response to hepatitis C virus in different clinical courses of infection. Gastroenterology 1993;104:580-587. 30. Erard F, Wild MT, Garcia-Sanz JA, Le Gros GG. Switch of CD8 T to noncytolytic CD80CD40 cells that make TH2 cytokines and help B cells. Science 1993;260:1802-1805. 31. Llorente L, Richaud-Patin Y, Flor R, Alcocer-Varela J, Wijdenes J, MorelFourrier B, Galanaud P, et al. In vivo production of interleukin-10 by nonT cells in rheumatoid arthritis, Sjo¨gren’s syndrome and systemic lupus erythematosus: a potential mechanism of B lymphocyte hyperactivity and autoimmunity. Arthritis Rheum 1994;37:1647-1655. 32. Liaw YF, Lee CS, Tsai SL, Liaw BW, Chen TC, Sheen IS, Chu CM. Tcell–mediated autologous hepatocytotoxicity in patients with chronic hepatitis C virus infection. HEPATOLOGY 1995;22:1368-1373. 33. Waldmann TA, Davis MM, Bongiovanni KF, Korsmeyer SJ. Rearrangements of genes for the antigen receptor on T-cells as markers of lineage and clonality in human lymphoid neoplasms. N Engl J Med 1985;313:776783. 34. Ferrari C, Valli A, Galati L, Penna A, Scaccglia P, Giuberti T, Schianchi C, et al. T-cell response to structural and nonstructural hepatitis C virus antigens in persistent and self-limited hepatitis C virus infections. HEPATOLOGY 1994;19:286-295. 35. Hoffmann RM, Diepolder HM, Zachoval R, Zwiebel FM, Jung MC, Scholz S, Nitschko H, et al. Mapping of immunodominant CD4/ T lymphocyte

AID

Hepa 0056

/

5P1B$$1104

01-13-97 14:41:40

36.

37.

38.

39. 40.

41. 42. 43.

44. 45. 46. 47. 48. 49.

50. 51.

52.

53.

54.

55.

56. 57.

58.

59.

60.

457

epitopes of hepatitis C virus antigens and their relevance during the course of chronic infection. HEPATOLOGY 1995;21:632-638. Diepolder HM, Zachoval R, Hoffmann RM, Wierenga EA, Santantonio T, Jung MC, Eichenlaub D, et al. Possible mechanism involving T-lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus infection. Lancet 1995;346:1006-1007. Missale G, Bertoni R, Lamonaca V, Valli A, Massari M, Mori C, Rumi MG, et al. Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the antiviral cell-mediated immune response. J Clin Invest 1996;98:706-714. Del Prete GF, De Carli M, Mastromanro C, Macchia D, Biagiotti R, Ricci M, Romagnani S. Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. J Clin Invest 1991;88:346-350. Cogan E, Schandene´ L, Crusiaux A, Cochaux P, Velu T, Goldman M. Clonal proliferation of type 2 helper T cells in a man with the hypereosinophilic syndrome. N Engl J Med 1994;330:535-538. Inoue M, Kakumu S, Yoshioka K, Tsutsumi Y, Wakita T, Arao M. Hepatitis B core antigen-specific IFN-g production of peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection. J Immunol 1989; 142:4006-4011. Ferrari C, Mondelli MU, Penna A, Fiaccadori F, Chisari FV. Functional characterization of cloned intrahepatic, hepatitis B virus nucleoprotein specific helper T cell lines. J Immunol 1987;139:539-544. Maruyama T, McLachlan A, Iino S, Koike K, Kurokawa K, Milich DR. The serology of chronic hepatitis B infection revisited. J Clin Invest 1993;91: 2586-2595. Barnaba V, Franco A, Paroli M, Benvenuto R, De Petrillo G, Burgio VL, Santilio I, et al. Selective expansion of cytotoxic T lymphocytes with a CD4/ CD56/ surface phenotype and a T helper type 1 profile of cytokine secretion in the liver of patients chronically infected with hepatitis B virus. J Immunol 1994;152:3074-3087. Bukh J, Miller RH, Purcell RH. Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes. Semin Liver Dis 1995;15:41-63. Peano G, Menardi G, Ponzetto A, Fenoglio LM. HLA-DR5 antigen: a genetic factor influencing the outcome of hepatitis C virus infection? Arch Intern Med 1994;154:2733-2736. Gilles PN, Guerrette DL, Ulevitch RJ, Schreiber RD, Chisari FV. HBsAg retention sensitizes the hepatocyte to injury by physiological concentration of interferon-g. HEPATOLOGY 1992;16:655-663. Toyonaga T, Hino O, Sugai S, Wakasugi S, Abe K, Shichiri M, Yamamura K-I. Chronic active hepatitis in transgenic mice expressing interferon-g in the liver. Proc Natl Acad Sci U S A 1994;91:614-618. Paul WE, Seder RA. Lymphocyte responses and cytokines. Cell 1994;76: 241-251. Ando K, Moriyama T, Guidotti LG, Wirth S, Schreiber RD, Schlicht HJ, Huang SN, et al. Mechanisms of class I restricted immunopathology: a transgenic mouse model of fulminant hepatitis. J Exp Med 1993;178:15411554. Gazzinelli RT, Oswald IP, James SL, Sher A. IL-10 inhibits parasite killing and nitrogen oxide production by IFN-g –activated macrophages. J Immunol 1992;148:1792-1796. Tanaka T, Hu-Li J, Seder RA, de St Groth BF, Paul WE. Interleukin 4 suppresses interleukin 2 and interferon g production by naive T cells stimulated by accessory cell-dependent receptor engagement. Proc Natl Acad Sci U S A 1993;90:5914-5918. Tsai SL, Yeh CT, Liaw YF, Huang JH, Kuo GC. Rapid emergence of naturally occurring hepatitis C virus (HCV) variants antagonizing cytotoxic T lymphocyte activity in acute HCV infection with chronic evolution [Abstract C68]. Presented at the 3rd International Meeting on Hepatitis C Virus and Related Viruses (Molecular Virology and Pathogenesis): Aug. 28-September 3, 1995, Gold Coast, Australia. Wright TL, Hsu H, Donegan E, Feinstone S, Greenberg H, Read A, Ascher NL, et al. Hepatitis C virus not found in fulminant non-A, non-B hepatitis. Ann Intern Med 1991;115:111-112. Fe´ray C, Gigou M, Samuel D, Reyes G, Bernuau J, Reynes M, Bismuth H, et al. Hepatitis C virus RNA and hepatitis B virus DNA in serum and liver of patients with fulminant hepatitis. Gastroenterology 1993;104:549555. Liang TJ, Jeffers L, Reddy RK, Silva MO, Cheinquer H, Findor A, de Medina M, et al. Fulminant or subfulminant non-A, non-B viral hepatitis: the role of hepatitis C and E viruses. Gastroenterology 1993;104:556-562. Hoofnagle JH, Carithers Jr RL, Shapiro C, Ascher N. Fulminant hepatic failure: summary of workshop. HEPATOLOGY 1995;21:240-266. Wang JT, Wang TH, Lin JT, Sheu JC, Sung JL, Chen DS. Hepatitis C virus in a prospective study of posttransfusion non-A, non-B hepatitis in Taiwan. J Med Virol 1990;32:83-86. Alberti A, Morsica G, Chemello L, Cavalletto D, Noventa F, Pontisso P, Ruol A. Hepatitis C viraemia and liver disease in symptom-free individuals with anti-HCV. Lancet 1992;340:697-698. Bhandari BN, Kim M, Ferrell L, Bacchetti P, Wright TL. Serum hepatitis C virus (HCV) levels and alanine transaminase (ALT) do not correlate with liver histopathology [Abstract]. Gastroenterology 1994; 106: A866. Mishiro S, Hoshi Y, Takeda K, Yoshikawa A, Gotanda T, Takahashi K,

hptas

WBS: Hepatology

458

TSAI ET AL.

HEPATOLOGY February 1997

Akahane Y, et al. Non-A, non-B hepatitis specific antibodies directed at host-derived epitope: implication for an autoimmune process. Lancet 1990; 336:1400-1403. 61. Pawlotsky JM, Ben Hayia M, Andre´ C, Voisin MC, Intrator L, RoudotThoraval F, DeForges L, et al. Immunological disorders in C virus chronic active hepatitis: a prospective case-control study. HEPATOLOGY 1994;19: 841-848. 62. Koskinas J, McFarlane BM, Nouri-Aria KT, Tibbs CJ, Mizokami M, Donaldson PT, McFarlane IG, et al. Cellular and humoral immune reactions

AID

Hepa 0056

/

5P1B$$1104

01-13-97 14:41:40

against autoantigens and hepatitis C viral antigens in chronic hepatitis C. Gastroenterology 1994;107:1436-1442. 63. Lo¨hr HF, Gerken G, Michel G, Braun HB, Zum Bu¨schenfelde KHM. In vitro secretion of anti-GOR protein and anti-hepatitis C virus antibodies in patients with chronic hepatitis C. Gastroenterology 1994;107:1443-1448. 64. Pawlotsky JM, Roudot-Thoraval F, Simmonds P, Mellor J, Yahia MB, Andre´ C, Voisin MC, et al. Extrahepatic immunologic manifestations in chronic hepatitis C and hepatitis C virus serotypes. Ann Intern Med 1995; 122:169-173.

hptas

WBS: Hepatology