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Recurrence of hepatitis C virus after loss of virus-specific CD4+ T-cell response in acute hepatitis C
GASTROENTEROLOGY 1999;117:933–941
Recurrence of Hepatitis C Virus After Loss of Virus-Specific CD4ⴙ T-Cell Response in Acute Hepatitis C J. TILMAN GERLACH,*,‡ HELMUT M. DIEPOLDER,*,‡ MARIA–CHRISTINA JUNG,*,‡ NORBERT H. GRUENER,* WINFRIED W. SCHRAUT,* REINHART ZACHOVAL,‡ ROBERT HOFFMANN,‡ C. ALBRECHT SCHIRREN,‡ TERESA SANTANTONIO,§ and GERD R. PAPE*,‡ *Institute for Immunology and ‡Medical Department II, Klinikum Großhadern, University of Munich, Germany; and §Clinica Malattie Infettive, Policlinico, Universita degli Studi Bari, Bari, Italy
See editorial on page 1012. Background & Aims: The prospective comparison of patients with acute hepatitis C virus (HCV) who spontaneously clear the virus with those who cannot achieve viral elimination and progress to chronic hepatitis offers the unique opportunity to analyze natural mechanisms of viral elimination. Methods: We studied the HCV-specific CD4ⴙ T-cell response in 38 patients with acute HCV and correlated the clinical course with the antiviral immune response. The individual HCV-specific T-cell response was assessed in a proliferation assay ( 3H-thymidine uptake) and an enzyme-linked immunospot assay. Results: Patients were classified according to their clinical course and pattern of CD4ⴙ T-cell responses in 3 categories: first, patients mounting a strong and sustained antiviral CD4ⴙ/Th1ⴙ T-cell response who cleared the virus (HCV RNA–negative; n ⴝ 20); second, patients who were unable to mount an HCV-specific CD4ⴙ T-cell response and developed chronic disease (n ⴝ 12); and third, patients who initially displayed a strong CD4ⴙ T-cell response and eliminated the virus (HCV PCR–negative) but subsequently lost this specific T-cell response (n ⴝ 6). The loss of the HCV-specific CD4ⴙ T-cell response was promptly followed by HCV recurrence. Conclusions: The results indicate that a virus-specific CD4ⴙ/Th1ⴙ T-cell response that eliminates the virus during the acute phase of disease has to be maintained permanently to achieve long-term control of the virus. The induction and/or maintenance of virus-specific CD4ⴙ T cells could represent a promising therapeutic approach in HCV infection.
epatitis C virus (HCV) infection is now recognized as one of the most important causes of chronic liver disease. HCV can cause acute icteric hepatitis but most frequently leads to chronic infection after an inapparent onset of disease.1,2 Chronic infection contributes to the development of liver cirrhosis, hepatocellular carcinoma,
H
and end-stage liver disease in patients with chronic HCV (cHCV) infection and is one of the leading indications for liver transplantation in the Western world.3 The mechanisms responsible for the high rate of chronicity are unknown at present, and the lack of an appropriate animal model requires the investigation of infected humans. Because patients with clinically symptomatic acute HCV clear the virus in 40%–60% of cases,4–6 this situation offers the unique opportunity to compare 2 groups of patients, those who achieve viral elimination vs. those with virus persistence, to provide insight into mechanisms leading to viral elimination. Several observations, including the intrahepatic presence of virus-specific T lymphocytes,7 the surge in viremia under immunosuppression,8 autoimmune features, and the deterioration of disease in human immunodeficiency virus (HIV) coinfection,9,10 suggest that immune-mediated mechanisms play a crucial role in viralassociated liver damage and possibly also in viral elimination in HCV infection. Although studies of the humoral immune response have revealed antibodies to all viral antigens during the course of hepatitis, no antiviral antibody conferring immunity to HCV has been detected to date.11 Moreover, the decrease of antibody levels during successful interferon (IFN)-␣ therapy argues against a decisive role of antibodies during viral clearance.12 Thus, the cellular immune response seems to be of central importance for the outcome of acute HCV infection. CD4⫹ T lymphocytes have immunoregulatory functions through their secretion of lymphokine profiles (Th1/Th2) that support either cellular mechanisms (IFN-␥ Abbreviations used in this paper: cHCV, chronic hepatitis C virus; ELISPOT, enzyme-linked immunospot; HIV, human immunodeficiency virus; IFN, interferon; IL, interleukin; PBMC, peripheral blood mononuclear cell; PCR, polymerase chain reaction; SI, stimulation index. r 1999 by the American Gastroenterological Association 0016-5085/99/$10.00
934 GERLACH ET AL.
and interleukin [IL]-2; Th1) or antibody production (IL-4, IL-5, and IL-6; Th2) and display direct antiviral effects. Studies of the T-cell responses in patients with acute hepatitis C infection have shown a close association of a polyclonal, multispecific proliferative CD4⫹ T-cell response, directed mainly against the nonstructural proteins of the virus, with viral clearance and resolution of disease.4,5,13 However, because there is no appropriate animal model it is not known if virus elimination is achieved by specific T cells alone and if the maintenance of this T-cell response is decisive for the long-term outcome of infection. Our prospective follow-up of the CD4⫹ T-cell response in patients with acute hepatitis C during their initial phase of disease and thereafter revealed 3 groups: (1) patients mounting a strong and sustained CD4⫹ T-cell response who cleared the virus and remained HCV RNA negative during long-term follow up; (2) patients who were unable to develop such T-cell responses and did not achieve viral clearance but developed a chronic course of disease; and (3) most interestingly, patients who initially had strong CD4⫹ T-cell responses to eliminate the virus but subsequently lost this specific T-cell response. The loss of the T-cell response was followed promptly by HCV recurrence.
Materials and Methods
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Table 1. Characteristics of Patients With Acute Hepatitis C Self-limited course Patients n Age (yr ) Median Range Sex Male Female Peak ALT (U/L) Source of infection Unknown Sexual Iatrogenic Intravenous drugs Immunoglobulins Genotype (classification of Simmonds) 1a 1b 2a/b 3 4 Not determined Follow-up (mo) Mean Range
20
Chronic course 18
41 (20–70)
30 (15–60)
6 14 1136
10 8 1072
7 1 6 5 1
6 2 6 3 1
3 8 2 1 0 6
3 8 2 2 1 2
21 6–49
20 6–36
NOTE. Thirty-eight consecutive patients with acute hepatitis C were investigated according to their clinical outcome, i.e., self-limited (n ⫽ 20) vs. chronic (n ⫽ 18) course. No statistically significant differences between the 2 groups were found for age, sex, peak ALT level, source of infection, or genotype of the virus.
Patients and Controls Thirty-eight patients with acute hepatitis C (22 women and 16 men; mean age, 35 years) were included (Table 1). The diagnosis of acute infection was based on the following criteria: (1) elevated values of serum alanine aminotransferase (ALT) at least 20 times above the upper limit of normal; (2) seroconversion to anti–HCV-positive antibody status by second- or third-generation enzyme-linked immunosorbent assay or recombinant immunoblot assay II, respectively; (3) positive polymerase chain reaction (PCR) for HCV RNA (Amplicor; Roche Molecular Diagnostics Systems, Branchburg, NJ) in at least the first serum sample; and (4) a history of sudden onset of liver disease in previously healthy individuals. Patients were scheduled to be seen at least once in the acute symptomatic phase, after 3 and 6 months, and then in 6-month intervals. Potential causes of acute hepatitis such as other forms of viral hepatitis, autoimmune hepatitis, alcoholic liver disease, toxins, or metabolic etiology were ruled out. Patients with hepatitis B virus or HIV coinfection were excluded from the study. In comparison to patients with acute hepatitis C, 30 consecutive nontreated patients with established chronic HCV infection (positive HCV RNA and elevated ALT levels for at least 6 months) were analyzed with respect to their immunologic, biochemical, and serological features. Peripheral blood mononuclear cells (PBMCs) of 10 healthy donors served as negative controls for HCVspecific proliferative responses and secretion of cytokines.
HCV Proteins and Peptides Recombinant proteins for antigenic stimulation of CD4⫹ T lymphocytes were kindly provided by Chiron (Emeryville, CA) and comprised the following antigens: c22 (core), c33c (NS3), c100 (NS4), NS5, and the c200 (NS3 ⫹ NS4). All antigens were expressed as COOH-terminal fusion proteins with human superoxide dismutase in yeast. Yeast and superoxide dismutase were tested as controls in each proliferation assay for unspecific stimulation. Purity of antigens ranged between 80% and 90%.
Isolation of PBMCs and Proliferation Assay PBMCs were isolated on Ficoll-Isopaque gradients (Pharmacia, Uppsala, Sweden) at several time points during the course of acute hepatitis and were washed 3 times in phosphatebuffered saline (PBS). PBMCs were incubated at 5 ⫻ 104/well in 96 U-bottom plates (Costar, Cambridge, MA) for 5 days in the presence of HCV proteins (1 µg/mL) in 150 µL RPMI 1640 medium (GIBCO, Grand Island, NY) containing 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% human AB serum. On day 5, cultures were labeled by incubation for 16 hours with 2 µCi 3H-thymidine (sp act, 80 µCi/mmol; Amersham, Buckinghamshire, England). Then cells were collected and washed on filters (Dunn, Asbach, Germany) using a cell harvester (Skatron,
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Sterling, VA), and the amount of radiolabel incorporated into DNA was estimated by a beta counter (LKB/Pharmacia, Uppsala, Sweden). Triplicate cultures were assayed routinely, and the results are expressed as mean counts per minute (cpm). The stimulation index (SI) was calculated as the ratio between cpm obtained in the presence of antigen to that obtained without antigen, and SI ⱖ3 was considered significant.
Enzyme-Linked Immunospot Assay The enzyme-linked immunospot (ELISPOT) assay was performed as described previously.14 Briefly, nitrocellulose bottom 96-well Millititer HA plates (Millipore Co., Bedford, MA) were coated under sterile conditions with 100 µL of the monoclonal antibody of interest (anti–IFN-␥, anti–IL-4; Mabtech AB, Stockholm, Sweden) at a concentration of 15 µg/mL in PBS and incubated overnight at 4°C. Excessive antibodies were removed by 4 successive washings with PBS containing 0.05% Tween. The coated wells were filled with 100 µL of RPMI medium containing 2 ⫻ 105 PBMCs in duplicates together with HCV-derived proteins (1 µg/mL), tetanus toxoid (10 µg/mL), or phytohemagglutinin (5 µg/mL) and incubated undisturbed for 48 hours at 37°C in a humidified atmosphere with 5% CO2. After 48 hours of incubation, cytokine secretion reached peak levels (data not shown) and antigen-specific stimulation was stopped. PBMCs were removed by washing the plates 4 times with PBS. One hundred microliters of a biotinconjugated monoclonal antibody (Mabtech) was added to each well at a concentration of 1 µg/mL and incubated for 4 hours at room temperature. The plates were rinsed 4 times with PBS and exposed to 100 µL streptavidin–alkaline phosphatase (Mabtech AB) for 1 hour. Unbound conjugate was removed by washing thoroughly with PBS, and finally 100 µL of 5-bromo4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate solution (Bio-Rad Laboratories, Richmond, CA) was added and incubated until the appearance of blue spots in the wells (30–60 minutes). The color reaction was stopped by extensive washings, and after drying, the number of spots was scored using a dissection microscope. Secretion of cytokines was optimal after 48 hours of incubation. Stimulation with the mitogen phytohemagglutinin and tetanus toxoid as specific antigen served as positive controls for both cytokines. To compare antigen-specific T-cell activation detected in the proliferation assay and in the corresponding ELISPOT assay, a stimulatory index (SIE) was defined: SIE was considered significant if the index, calculated as the ratio of IFN-␥–producing cells in the presence of antigen and the control wells, was ⱖ3.
Depletion of CD4ⴙ and CD8ⴙ T Cells In 4 patients with acute hepatitis C, proliferation and ELISPOT assays were performed before and after magnetic immunoseparation of CD4⫹ or CD8⫹ T-cell fractions, respectively. PBMCs (20 ⫻ 106) were suspended in 160 µL PBS supplemented with 0.5% bovine serum albumin and 2 mmol/L EDTA and were stained with 40 µL paramagnetic microbeads conjugated to a monoclonal mouse anti-human anti-CD4 or
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anti-CD8 antibody (Micro Beads; Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 minutes at 4°C. Then cells were washed and resuspended in 500 µL buffer. Separation of positively stained T cells (CD4⫹ or CD8⫹) was done in a depletion column AS (Miltenyi Biotec) placed in a magnetic field of a suitable separator (reduction of stained T lymphocytes ⱖ95%). The unstained cell fractions were collected, respectively, and washed twice before they were tested for HCVspecific proliferation and cytokine release in the ELISPOT assay.
Statistical Analysis Results are presented as means and/or medians. Comparisons of stimulation index and number of IFN-␥–secreting cells of patients with self-limited course vs. chronic course of disease were done by the Mann–Whitney rank sum test.
Results Clinical Outcome and Patient Characteristics A total of 38 patients diagnosed with acute hepatitis C were included in the study (Table 1). In all patients, clinical outcome (self-limited vs. chronic evolution) was determined after a follow-up of more than 6 months after onset of disease (start of observation). Mean follow-up in patients with self-limited hepatitis was 21 months (range, 8–49) and in patients with chronic evolution 20 months (range, 6–36). The peak ALT level in the acute phase did not differ between patients with self-limited (mean ALT, 1136 U/L) vs. those with chronic course (mean ALT, 1072 U/L). In 20 patients (53%), HCV RNA became undetectable by repeatedly performed nested PCR and serum ALT levels normalized in this group within the first 6 months after onset of disease. Because these patients displayed no evidence of clinical symptoms or abnormal biochemical serum parameters and remained HCV RNA–negative by PCR during the entire follow-up, they were considered to have a self-limited course of disease. The time between onset of disease and normalization of ALT, combined with loss of serum HCV RNA, was 8 weeks (range, 4–41 weeks; median, 8 weeks). Eighteen patients (47%) who had persistently elevated levels of ALT and/or persistent HCV RNA in serum for more than 6 months after onset of disease evolved to chronic hepatitis. Seven of the 18 patients were treated with IFN-␣. At the end of observation, 2 of the treated patients had achieved sustained response with viral elimination (negative PCR) and normal ALT extending more than 6 months after end of therapy. With regard to the clinical characteristics, patients with a self-limited course of disease seemed to be older (41 vs. 30 years; P ⫽ NS), although the difference did not
936 GERLACH ET AL.
Figure 1. Proliferative CD4⫹ T-cell response of the first sample in the acute phase of disease to recombinant HCV proteins (HCV-NS3, -NS4, -NS5, and -core) of PBMCs from 38 patients with acute hepatitis C. Patients are grouped according to the final outcome of disease in self-limited hepatitis C (SL, n ⫽ 20) and patients with chronic evolution (C, n ⫽ 18). Results are shown as SI ⫽ 3H-thymidine incorporation of antigen-stimulated PBMCs (cpm)/unstimulated control (cpm). Values ⱖ3 are considered significant. All patients with self-limited disease displayed a significant proliferative T-cell response against at least 1 of the viral proteins, while patients with chronic evolution mounted no or only transient antiviral T-cell responses. NS3 and NS4 revealed the most frequent (see Table 2) and most vigorous responses. In 4 patients, the proliferative response against NS5 was not tested in the first sample.
reach statistical significance (Table 1). The self-limited group (female, n ⫽ 14; male, n ⫽ 6) consisted of more women than the group of patients progressing to chronic disease (female, n ⫽ 8; male, n ⫽ 10; t test, P ⫽ NS). The mode of transmission was heterogeneous in both groups but did not differ significantly and did not seem to influence the clinical outcome of hepatitis C. Intravenous drug abuse and sporadic infection of unknown origin were the most common modes of transmission. Seven patients developed acute HCV infection within 4 months after medical procedures or dental treatment without having any other known risk factor. There was no significant difference in the genotypes of the infecting virus in the 2 groups (Table 1). Proliferative T-Cell Responses CD4⫹
T-cell responses in the acute phase of acute HCV. In both groups (self-limited course of disease
and chronic hepatitis), the strongest proliferative responses were found during the first 6 months after onset of disease (SI, chronic 2.4 ⫹ 1.85 vs. self-limited 11.3 ⫹ 10.7; P ⫽ 0.0031; number of tests per patient, self-limited, 4.1 ⫾ 4.7; chronic, 3.6 ⫾ 3.1; P ⫽ NS). The difference in CD4⫹ HCV-specific proliferative responses between both groups was statistically significant
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in the first proliferative response after onset of disease (Figure 1) and throughout the entire period of follow-up. All patients with a self-limited course of disease mounted a significant proliferative CD4⫹ T-cell response against at least 1 of the 4 tested viral proteins (mean, 2.8 proteins per patient) during the first 6 months of disease. The most immunogenic proteins were the nonstructural proteins, which led to a significant stimulation of T lymphocytes in 70%–85% of patients, followed by the core protein with a significant proliferative response in 45% (Table 2). Proliferative response to HCV proteins was significantly reduced after depletion of CD4⫹ T cells but not after depletion of CD8⫹ T cells compared with unfractioned PBMCs (Figure 2). Patients with chronic evolution of disease displayed significantly lower levels of antiviral T-cell activity in percentage of patients and in strength of stimulation indices against each tested viral protein (Table 2 and Figure 1). Recurrence of HCV RNA after loss of HCVspecific CD4ⴙ T-cell response. Six (33%) of the 18
patients with chronic evolution mounted a transient significant antiviral proliferative CD4⫹ T-cell response, and HCV RNA (tested by nested PCR) became undetectable in serum. The subsequent loss of virus-specific T-cell response in this group was immediately followed by reappearance of HCV RNA in all 6 patients after a time period of up to 10 months after onset of disease (range, 5–10 months; median, 6.6 months; mean, 7.0 months). Table 2. Significant Proliferative CD4⫹ T-Cell Responses (PBMC) Against HCV Proteins During the First 6 Months in Patients With Acute Hepatitis C
Core n % NS3 n % NS4 n % NS5 n % ⱖ1 Antigen recognized n %
Self-limited course (n ⫽ 20)
Chronic course (n ⫽ 18)
9 45
2 11
14 70
4 22
17 85
6 33
15 75
6 33
20 100
6 33
NOTE. Number and percentage of patients with significant SIs (SI ⱖ 3) against recombinant HCV proteins within 6 months after onset of disease. All patients with self-limited course show a significant T-cell response against different viral proteins. Patients with chronic evolution had no (n ⫽ 12) or only transient (n ⫽ 6) significant T-cell responses against the viral proteins (number of tests: self-limited, 4.1 ⫾ 4.7; chronic evolution, 3.6 ⫾ 3.1; P ⫽ NS).
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Figure 2. (A ) Proliferative response ( 3H-thymidine uptake) and (B ) secretion of IFN-␥ (ELISPOT assay) were assessed in patients with acute hepatitis C (n ⫽ 4) upon stimulation with HCV-specific antigen (C200) in CD4⫹- or CD8⫹-depleted PBMCs and in unfractionated PBMCs. Representative results of 1 patient. Depletion of CD4⫹ T cells but not depletion of CD8⫹ T cells resulted in a significant decrease of antigen-specific proliferation and IFN-␥ secretion.
Although biochemical parameters normalized within 2 months after onset of disease in 3 of the 6 patients, ALT levels of the other 3 remained at the upper limit or just above the normal range during the entire observation period. Significant antiviral CD4⫹ T-cell proliferation in this group was strongly associated with undetectable HCV RNA in patients’ sera. The comparison of proliferative T-cell response of HCV RNA–positive vs. –negative samples in this subgroup of patients with evolving chronic hepatitis revealed a significant difference in stimulation indices of RNA-positive vs. RNA-negative samples (Table 3). The initial strength of antiviral T-cell response did not differ between patients with relapse or self-limited disease. None of the other patients with chronic evolution (n ⫽ 12) mounted a sustained CD4⫹ HCV-specific T-cell response. The course of viral and biochemical parameter and the corresponding T-cell responses of 2 representative patients are exemplified in Figure 3. In all patients with a self-limited course of disease, a strong and sustained proliferative CD4⫹ T-cell response against at least 1 viral protein could be detected during the first 6 months after onset of symptoms, whereas 66% of patients with evolving chronic hepatitis had no significant antiviral T-cell response at any time during follow-up. During treatment with IFN-␣, HCV RNA became undetectable and ALT level returned to the normal range in 6 patients.
ACUTE HEPATITIS C 937
Two patients achieved sustained viral elimination after therapy (n ⫽ 2) and mounted a significant T-cell response against NS3 (first patient, SINS3 ⫽ 7.6) or NS3 and NS4 (second patient, SINS3⫹NS4 ⫽ 5.3) during treatment. Two transient responders who achieved biochemical remission without virus-specific T-cell responses had a breakthrough before therapy was terminated. Healthy controls and patients with cHCV. In comparison to patients with acute hepatitis C, the proliferative T-cell responses of 30 untreated patients with chronic hepatitis C and of 10 healthy controls was assessed. Healthy controls mounted no (0/10) significant proliferative response to any HCV-specific antigen (proliferative stimulation index of healthy controls (mean: SINS3 ⫽ 1.3; SINS4 ⫽ 1.0; SINS5 ⫽ 0.9; SIcore ⫽ 1.2), and patients with cHCV displayed no proliferative (0/30) response to the NS3, NS4, and NS5 region of HCV (proliferative SI of patients with chronic hepatitis C; mean: SINS3 ⫽ 1.4; SINS4 ⫽ 1.3; SINS5 ⫽ 1.0). In 3 patients (3/30) with cHCV infection, significant proliferative T-cell responses against the core protein (proliferative SI of 3 responders: mean, SIcore ⫽ 3.8) could be detected. ELISPOT Assay The significance of CD4⫹ T cells for HCV-specific IFN-␥ secretion was shown in the ELISPOT assay by depletion assays in patients with acute hepatitis C (n ⫽ 4). After depletion of CD4⫹ T cells, but not after depletion of CD8⫹ T cells, antigen-specific secretion of IFN-␥ was significantly decreased (Figure 3). HCV-specific secretion of cytokines (IFN-␥ and IL-4) was assessed in 20 patients with acute HCV (10 patients with chronic evolution vs. 10 patients with self-limited course) and in 10 healthy controls (Figure 4). Stimulation indices ⱖ3 were considered significant. Stimulation of Table 3. Proliferative T-Cell Responses (SI) Against HCV Protein (C200) in Patients With Transient Viral Clearance Months after onset of disease
1–3
4–6
7–12
12–18
18–24
Pt. 1 Pt. 2 Pt. 3 Pt. 4 Pt. 5 Pt. 6
0.9 6.3 21.2 3.8 3.6 4.2
5.2 6.4 3.8 5.4 9.1 5.8
2.1 9.3 1.9 1.3 1.4 0.5
1.0 0.9 — — — —
— 0.8 0.9 0.7 — 2.5
NOTE. In patients with transient viral clearance (Pt. 1 to Pt. 6) during acute hepatitis C, RNA in serum was undetectable (RNA-negative samples in bold) only as long as HCV-specific CD4⫹ T-cell responses reached significant levels (SI ⱖ 3). After the loss of significant T-cell responses, HCV RNA becomes positive again. Differences in T-cell responses were significant between RNA-positive and RNA-negative samples by the Mann–Whitney rank sum test (P ⫽ 0.004).
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PBMCs was low in healthy subjects (SIE ⫽ 1.1 ⫾ 0.4) and in patients with chronic evolution (SIE ⫽ 1.85 ⫾ 1.4), PBMCs of patients with a self-limited disease (SIE ⫽ 14.6 ⫾ 17.8) displayed significantly higher stimulation indices for IFN-␥–secreting cells (chronic vs. self-limited course; P ⫽ 0.025) (Figure 4). These differences in number and SIE of IFN-␥–secreting cells in patients with acute HCV could be confirmed in longterm follow-up of patients with different clinical courses of acute hepatitis. In the self-limited course, high numbers of IFN-␥–secreting cells were maintained through the entire follow-up, whereas patients with a chronic course of disease did not show significant numbers of IFN-␥–secreting T cells after HCV-specific stimulation.
Discussion In this study we prospectively compared HCVspecific CD4⫹ T-cell responses of patients with acute self-limited hepatitis C vs. those with evolving chronic hepatitis. Our results provide new information on the decisive role of CD4⫹ T-cell responses in the elimination and long-term control of HCV. By using the largest cohort of patients published to date, we confirm data of previous studies of patients with acute hepatitis C.
Figure 3. Follow-up of HCV-RNA and ALT level (upper part of each graph), antiviral CD4⫹ proliferative T-cell responses (middle part of each graph), and number of spots representing IFN-␥–secreting cells/ 2 ⫻ 105 PBMCs in the ELISPOT assay (lower part of each graph) after stimulation with recombinant HCV protein (C200) in 2 representative patients with acute hepatitis C. (A ) Patient with a self-limited course of acute hepatitis C and sustained vigorous proliferative T-cell responses against the viral proteins c33c (NS3), c100 (NS4), and c200 (NS3 and NS4). HCV RNA turns negative by week 8 and remains undetectable throughout the entire follow-up. Initially elevated ALT level falls to normal values in the acute phase. The increased number of IFN-␥– secreting cells upon antigen-specific stimulation is maintained during follow-up. (B ) Patient with initially significant (SI ⱖ 3) antiviral CD4⫹ T-cell responses and transient viral clearance. Loss of significant T-cell responsiveness is promptly followed by recurrence of HCV RNA. The precursor frequency of IFN-␥–secreting cells upon antigen-specific stimulation in the ELISPOT assay decreases during follow-up.
2 ⫻ 105 PBMCs with mitogen (phytohemagglutinin) or tetanus toxoid (10 µg/mL) for 48 hours revealed a significant number of cells producing IFN-␥ and IL-4, whereas stimulation with recombinant HCV proteins induced only the secretion of IFN-␥ without IL-4 secretion, indicating Th1-dominated cytokine profiles. Although the mean stimulation index for IFN-␥ of
Figure 4. Stimulatory index of HCV-specific T lymphocytes in the first ELISPOT assay within 6 months after onset of disease (SIE ⫽ number of spots after antigen-specific stimulation/number of spots in the control) for IFN-␥ secretion after incubation of 2 ⫻ 105 PBMCs with viral protein (c200; 1 µg/mL) in healthy controls (n ⫽ 10), patients with acute hepatitis C and chronic evolution (n ⫽ 10), or patients with self-limited course of disease (n ⫽ 10). Differences are significant for healthy controls vs. self-limited course (**P ⫽ 0.020) and chronic evolution vs. self-limited course (*P ⫽ 0.025).
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Obviously, patients with acute HCV infection, who show clinically apparent disease with symptoms such as jaundice, fever, and malaise, represent a distinct group. This clinical picture probably reflects a strong antiviral response in these individuals, which is followed by virus elimination in more than 50% of patients. The results are in line with the respective subgroups in studies of the Centers for Disease Control and Prevention in which only 30% of infected individuals developed symptomatic disease and up to 85% of patients evolved chronic disease.1 In the present study only patients with symptomatic disease were included. Infected individuals with self-limited or chronic courses displayed no differences in the mode of infection and genotype of the infecting virus. In the group with self-limited hepatitis, patients tended to be older and the percentage of females was higher than in the group with evolving chronic hepatitis, but these differences were not statistically significant. In 7 patients, the only anamnestic source of infection was a medical procedure, suggesting a possible nosocomial transmission. The most significant difference between both groups was found in the HCV-specific proliferative CD4⫹ T-cell responses. All self-limited infections, regardless of the genotype of infecting virus, displayed a proliferative response against at least 1 viral protein. In contrast, 66% of patients with evolving chronic hepatitis did not respond to any protein at any time. The lack of antiviral CD4⫹ T-cell responses in the acute phase of hepatitis was unequivocally followed by chronic evolution of disease. A striking finding was that 30% of patients with evolving chronic hepatitis in the acute phase were capable of mounting a significant transient virus-specific CD4⫹ T-cell response. In these patients, initial T-cell responses were lost at variable time points (5–10 months) during follow-up, resulting in chronic hepatitis. The clinical implication of this finding and the need for antiviral treatment suggest a careful follow-up of patients with acute symptomatic hepatitis C for a prolonged period. Only as long as virus-specific CD4⫹ T cells were detectable in the peripheral blood of these patients, HCV RNA was absent by means of nested PCR, possibly reflecting the attempt of the individual’s cellular immune system to combat the virus. The loss of T-cell responsiveness was immediately followed by recurrence of HCV RNA in the patients’ serum. The inverse correlation of antiviral CD4⫹ T-cell response and detectable HCV RNA was observed during follow-up of all patients, who temporarily or permanently cleared the virus. Of course, the only exception of this rule was found in the very first sample at onset of disease, which was characterized by the presence of a significant T-cell response, positive HCV
ACUTE HEPATITIS C 939
RNA, and increased ALT levels. Although HCV RNA as determined by PCR was absent for up to 9 months after onset of disease in patients with transient CD4⫹ T-cell responses, permanent viral clearance was unlikely in the face of continuous marginal increase of ALT levels in 3 of the 6 patients. Although only peripheral blood lymphocytes were analyzed, the elevated serum ALT, evidencing an inflammatory process within the liver, in the presence and absence of peripheral HCV-specific CD4⫹ T-cell responses, contradicts the opinion that virus-specific T cells are only found in peripheral blood if inflammatory activity in the liver is low. If T-cell responsiveness in these patients reflects a compartmentalization or recruitment of virus-specific T cells rather than a general antiviral immune response, ALT in the serum should behave inversely. The cytokine pattern of peripheral HCV-specific T cells was confined to a Th1 profile, which is also predominantly found in liver-infiltrating lymphocytes.15 Our finding in freshly isolated PBMCs tested in the ELISPOT assay confirms previous results in T-cell clones from patients with acute HCV infection without the influence of long-term culture.13 Depletion experiments showed that IFN-␥ secretion in response to stimulation with viral proteins was accomplished by CD4⫹ T cells and ruled out a significant participation of CD8⫹ T cells in IFN-␥ secretion by a possible leakage in the MHC class II antigen-processing pathway within antigenpresenting cells after stimulation with viral proteins. Although there was no functional shift of virus-specific T cells from a Th1 to Th2 cytokine profile during progression to chronicity, the number of HCV-specific CD4⫹ T lymphocytes with a Th1 cytokine profile decreased during follow-up in patients with chronic evolution of hepatitis as defined by means of the sensitive ELISPOT assay. In contrast, patients with self-limited disease maintained a high number of HCV-specific IFN-␥– secreting cells over the entire period of observation, which was associated with long-lasting viral control. The inverse correlation of antiviral CD4⫹ T-cell responses and HCV RNA supports the hypothesis of viral control mediated by CD4⫹ T helper cells. The important contribution of CD4⫹ T lymphocytes to the development of sustained immunity has been evidenced in experimental infection of animals with viruses such as herpes, lymphocytic choriomeningitis virus, and influenza16–19 and in tumor immunity.20 Although cytotoxic CD8⫹ T cells in these infections may achieve virus elimination in the acute phase without T helper cells, CD4⫹ T lymphocytes are mandatory for a persistent CD8⫹ cytotoxic T-cell response and depletion of CD4⫹ T cells in the acute phase results in chronic infection.21 Cytotoxic CD8⫹
940 GERLACH ET AL.
T-cell responses have not been studied in acute HCV to date, but viral replication in the presence of cytotoxic T-cell responses in cHCV, in which antiviral CD4⫹ T-cell responses are weak or absent, indicates that effective HCV elimination is not mediated by CD8⫹ T cells alone.22–24 The virus-specific CD4⫹ T-cell response in HCV infection may persist as long as 20 years, as we have observed in women who were accidentally infected by a contaminated lot of anti-D immunoglobulin and displayed, in about 75%, a strong HCV-specific T-cell response. In contrast, women from this cohort with evolving chronic hepatitis did not show antiviral CD4⫹ T-cell activity (Gerlach et al., unpublished data, October 1997). In acute hepatitis B, which shows a self-limited course in almost all cases, similar long-lasting CD4⫹ T-cell responses in the presence of minute amounts of hepatitis B virus detected by nested PCR have been described.25 The importance of HCV-specific CD4⫹ T cells in viral control is also suggested in HCV/HIV coinfection, which in the late stages of acquired immunodeficiency syndrome with low CD4⫹ T-cell counts often leads to a rapid deterioration of liver disease.9,26 The abrogation of the HCV-specific Th1-dominated T-cell responsiveness, together with the breakdown of viral control, as reflected in the reappearance of virus in serum, could represent an important mechanism for viral persistence. While the present work highlights the importance of a sustained vigorous CD4⫹ T-cell response for viral control in HCV, it cannot delineate the causes of its absence or abrogation, a status discussed as virusspecific T-cell tolerance. The literature gives examples of potential mechanisms27,28 that may lead to T-cell unresponsiveness in viral infection, e.g., anergy,29 T-cell exhaustion,30 altered peptide binding,31 and others, but provides no data on the degree of importance and contribution of each factor to the observed tolerant T-cell status in viral hepatitis. The characterization of such mechanisms, however, will help to develop therapeutic strategies designed to overcome cHCV infection. In summary, our data suggest that a vigorous antiviral CD4⫹ T-cell response in the early phase of acute hepatitis C is not only required for virus elimination leading to a self-limited course of disease, but also that an antiviral CD4⫹ T-cell response in the late phase is a prerequisite for achieving long-term viral control. We could show consistently that the abrogation of the early CD4⫹/Th1dominated immune response in acute hepatitis C is associated with immediate recurrence and persistence of hepatitis C virus.
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References 1. Alter MJ. Epidemiology of hepatitis C. Hepatology 1997;26(suppl 1):62S–65S. 2. Hoofnagle JH. Hepatitis C: the clinical spectrum of disease. Hepatology 1997;26(suppl 1):15S–20S. 3. Di Bisceglie AM, Simpson LH, Lotze MT, Hoofnagle JH. Development of hepatocellular carcinoma among patients with chronic liver disease due to hepatitis C viral infection. J Clin Gastroenterol 1994;19:222–226. 4. Diepolder HM, Zachoval R, Hoffmann RM, Wierenga EA, Santantonio T, Jung MC, Eichenlaub D, Pape GR. Possible mechanism involving T-lymphocyte response to nonstructural protein 3 in viral clearance in acute hepatitis C virus infection. Lancet 1995;346: 1006–1007. 5. Missale G, Bertoni R, Lamonaca V, Valli A, Massari M, Mori C, Rumi MG, Houghton M, Fiaccadori F, Ferrari C. Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response. J Clin Invest 1996;98:706–714. 6. Tsai SL, Liaw YF, Chen MH, Huang CY, Kuo GC. Detection of type 2-like T-helper cells in hepatitis C virus infection: implications for hepatitis C virus chronicity. Hepatology 1997;25:449–458. 7. Koziel MJ, Dudley D, Wong JT, Dienstag J, Houghton M, Ralston R, Walker BD. Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis. J Immunol 1992;149:3339–3344. 8. Gruber A, Lundberg L-G, Bjo¨rkholm M. Reactivation of chronic hepatitis C after withdrawal of immunosuppressive therapy. J Intern Med 1993;234:223–225. 9. Eyster ME, Diamondstone LS, Lien JM, Ehmann WC, Quan S, Goedert JJ. Natural history of hepatitis C virus infection in multitransfused hemophiliacs: effect of coinfection with human immunodeficiency virus. The Multicenter Hemophilia Cohort Study. J Acquir Immune Defic Syndr Hum Retrovirol 1993;6:602–610. 10. Thomas DL, Shih JW, Alter HJ, Vlahov D, Cohn S, Hoover DR, Cheung L, Nelson KE. Effect of human immunodeficiency virus on hepatitis C virus infection among injecting drug users. J Infect Dis 1996;174:690–695. 11. Krawczynski K, Alter MJ, Tankersley DL, Beach M, Robertson BH, Lambert S, Kuo G, Spelbring JE, Meeks E, Sinha S, Carson DA. Effect of immune globulin on the prevention of experimental hepatitis C virus infection. J Infect Dis 1996;173:822–828. 12. Yoshioka K, Kakumu S, Hayashi H, Shinagawa T, Wakita T, Ishikawa T, Itoh Y, Takayanagi M. Anti-hepatitis C antibodies in patients with chronic non-A, non-B hepatitis: relation to disease progression and effect of interferon alpha. Am J Gastroenterol 1991;86:1495–1499. 13. Diepolder HM, Gerlach JT, Zachoval R, Hoffmann RM, Jung MC, Wierenga EA, Scholz S, Santantonio T, Houghton M, Southwood S, Sette A, Pape GR. Immunodominant CD4⫹ T-cell epitope within nonstructural protein 3 in acute hepatitis C virus infection. J Virol 1997;71:6011–6019. 14. elGhazali GE, Paulie S, Andersson G, Hansson Y, Holmquist G, Sun JB, Olsson T, Ekre HP, Troye Blomberg M. Number of interleukin 4– and interferon gamma–secreting human T cells reactive with tetanus toxoid and the mycobacterial antigen PPD or phytohemagglutinin: distinct response profiles depending on the type of antigen used for activation. Eur J Immunol 1993;23:2740– 2745. 15. Bertoletti A, D’Elios MM, Boni C, DeCarli M, Zignego AL, Durazzo M, Missale G, Penna A, Fiaccadori F, Del Prete G, Ferrari C. Different cytokine profiles of intrahepatic T cells in chronic hepatitis B and hepatitis C virus infections. Gastroenterology 1997;112:193–199. 16. Topham DJ, Tripp RA, Sarawar SR, Sangster MY, Doherty PC. Immune CD4⫹ T cells promote the clearance of influenza virus
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from major histocompatibility complex class II ⫺/⫺ respiratory epithelium. J Virol 1995;70:1288–1291. Planz O, Ehl S, Furrer E, Horvath E, Brundler MA. A critical role for neutralizing-antibody–producing B cells, CD4(⫹) T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: implications for adoptive immunotherapy of virus carriers. Proc Natl Acad Sci USA 1997;94: 6874–6879. Manickan E, Rouse RJ, Yu Z, Wire WS, Rouse BT. Genetic immunization against herpes simplex virus. Protection is mediated by CD4⫹ T lymphocytes. J Immunol 1995;155:259–265. Cardin RD, Brooks JW, Sarawar SR, Doherty PC. Progressive loss of CD8⫹ T cell-mediated control of a gamma-herpes virus in the absence of CD4⫹ T cells. J Exp Med 1996;184:863–871. Ossendorp F, Mengede E, Camps M, Filius R, Melief CJ. Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors. J Exp Med 1998;187:693–702. Matloubian M, Concepcion RJ, Ahmed R. CD4⫹ T cells are required to sustain CD8⫹ cytotoxic T-cell responses during chronic viral infection. J Virol 1994;68:8056–8063. Rehermann B, Chang KM, McHutchison J, Kokka F, Houghton M, Rice CM, Chisari FV. Differential cytotoxic T-lymphocyte responsiveness to the hepatitis B and C viruses in chronically infected patients. J Virol 1996;70:7092–7102. Koziel MJ, Dudley D, Afdhal N, Choo Q-L, Houghton M, Ralston R, Walker BD. Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes recognize epitopes in the core and envelope proteins of HCV. J Virol 1993;67:7522–7532. Koziel M, Dudley D, Afdhal N, Grakoui A, Rice CM, Choo Q-L, Houghton M, Walker BD. HLA class I–restricted cytotoxic T lymphocytes specific for hepatitis C virus. J Clin Invest 1995;96: 2311–2321. Penna A, Artini M, Cavalli A, Levrero M, Bertoletti A, Pilli N, Chisari FV, Rehermann B, Del Prete G, Fiaccadori F, Ferrari C. Long-lasting memory T cell responses following self-limited acute hepatitis B. J Clin Invest 1996;98:1185–1194.
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26. Eyster ME, Fried MW, DiBisceglie AM, Goedert JJ. Increasing hepatitis C virus RNA levels in hemophiliacs: relationship to human immunodeficiency virus infection and liver disease. Multicenter Hemophilia Cohort Study. Blood 1994;84:1020–1023. 27. Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995;13:29–60. 28. Milich DR, Jones JE, Hughes JL, Maruyama T, Price J, Melhado I, Jirik F. Extrathymic expression of the intracellular hepatitis B core antigen results in T cell tolerance in transgenic mice. J Immunol 1994;152:455–466. 29. Diepolder HM, Jung MC, Wierenga E, Hoffmann RM, Zachoval R, Gerlach TJ, Schotz S, Heavner G, Riethmu¨ller G, Pape GR. Anergic TH1 clones specific for hepatitis B virus (HBV) core peptides are inhibitory to other HBV core-specific CD4⫹ T cells in vitro. J Virol 1996;70:7540–7548. 30. Moskophidis D, Lechner F, Hengartner H, Zinkernagel RM. MHC class I and non-MHC-linked capacity for generating an anti-viral CTL response determines susceptibility to CTL exhaustion and establishment of virus persistence in mice. J Immunol 1994;152: 4976–4983. 31. Bertoletti A, Sette A, Chisari FV, Penna A, Levrero M, De Carli M, Fiaccadori F, Ferrari C. Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells. Nature 1994;369:407–410.
Received October 6, 1999. Accepted July 28, 1999. Address requests for reprints to: Gerd R. Pape, M.D., Institute for Immunology, Goethestrasse 31, D-80336 Mu ¨nchen, Germany. email: [email protected]; fax: (49) 89-591183. Supported by the Bundesministerium fu ¨r Bildung und Forschung and the European community project Biomed 2 PL95-1064 and SFB217. The authors thank Jutta Do ¨hrmann and Carola Steiger for excellent technical assistance and personal encouragement and Professor Dolores Schendel for critical reading of the manuscript.
Recurrence of Hepatitis C Virus After Loss of Virus-Specific CD4ⴙ T-Cell Response in Acute Hepatitis C J. TILMAN GERLACH,*,‡ HELMUT M. DIEPOLDER,*,‡ MARIA–CHRISTINA JUNG,*,‡ NORBERT H. GRUENER,* WINFRIED W. SCHRAUT,* REINHART ZACHOVAL,‡ ROBERT HOFFMANN,‡ C. ALBRECHT SCHIRREN,‡ TERESA SANTANTONIO,§ and GERD R. PAPE*,‡ *Institute for Immunology and ‡Medical Department II, Klinikum Großhadern, University of Munich, Germany; and §Clinica Malattie Infettive, Policlinico, Universita degli Studi Bari, Bari, Italy
See editorial on page 1012. Background & Aims: The prospective comparison of patients with acute hepatitis C virus (HCV) who spontaneously clear the virus with those who cannot achieve viral elimination and progress to chronic hepatitis offers the unique opportunity to analyze natural mechanisms of viral elimination. Methods: We studied the HCV-specific CD4ⴙ T-cell response in 38 patients with acute HCV and correlated the clinical course with the antiviral immune response. The individual HCV-specific T-cell response was assessed in a proliferation assay ( 3H-thymidine uptake) and an enzyme-linked immunospot assay. Results: Patients were classified according to their clinical course and pattern of CD4ⴙ T-cell responses in 3 categories: first, patients mounting a strong and sustained antiviral CD4ⴙ/Th1ⴙ T-cell response who cleared the virus (HCV RNA–negative; n ⴝ 20); second, patients who were unable to mount an HCV-specific CD4ⴙ T-cell response and developed chronic disease (n ⴝ 12); and third, patients who initially displayed a strong CD4ⴙ T-cell response and eliminated the virus (HCV PCR–negative) but subsequently lost this specific T-cell response (n ⴝ 6). The loss of the HCV-specific CD4ⴙ T-cell response was promptly followed by HCV recurrence. Conclusions: The results indicate that a virus-specific CD4ⴙ/Th1ⴙ T-cell response that eliminates the virus during the acute phase of disease has to be maintained permanently to achieve long-term control of the virus. The induction and/or maintenance of virus-specific CD4ⴙ T cells could represent a promising therapeutic approach in HCV infection.
epatitis C virus (HCV) infection is now recognized as one of the most important causes of chronic liver disease. HCV can cause acute icteric hepatitis but most frequently leads to chronic infection after an inapparent onset of disease.1,2 Chronic infection contributes to the development of liver cirrhosis, hepatocellular carcinoma,
H
and end-stage liver disease in patients with chronic HCV (cHCV) infection and is one of the leading indications for liver transplantation in the Western world.3 The mechanisms responsible for the high rate of chronicity are unknown at present, and the lack of an appropriate animal model requires the investigation of infected humans. Because patients with clinically symptomatic acute HCV clear the virus in 40%–60% of cases,4–6 this situation offers the unique opportunity to compare 2 groups of patients, those who achieve viral elimination vs. those with virus persistence, to provide insight into mechanisms leading to viral elimination. Several observations, including the intrahepatic presence of virus-specific T lymphocytes,7 the surge in viremia under immunosuppression,8 autoimmune features, and the deterioration of disease in human immunodeficiency virus (HIV) coinfection,9,10 suggest that immune-mediated mechanisms play a crucial role in viralassociated liver damage and possibly also in viral elimination in HCV infection. Although studies of the humoral immune response have revealed antibodies to all viral antigens during the course of hepatitis, no antiviral antibody conferring immunity to HCV has been detected to date.11 Moreover, the decrease of antibody levels during successful interferon (IFN)-␣ therapy argues against a decisive role of antibodies during viral clearance.12 Thus, the cellular immune response seems to be of central importance for the outcome of acute HCV infection. CD4⫹ T lymphocytes have immunoregulatory functions through their secretion of lymphokine profiles (Th1/Th2) that support either cellular mechanisms (IFN-␥ Abbreviations used in this paper: cHCV, chronic hepatitis C virus; ELISPOT, enzyme-linked immunospot; HIV, human immunodeficiency virus; IFN, interferon; IL, interleukin; PBMC, peripheral blood mononuclear cell; PCR, polymerase chain reaction; SI, stimulation index. r 1999 by the American Gastroenterological Association 0016-5085/99/$10.00
934 GERLACH ET AL.
and interleukin [IL]-2; Th1) or antibody production (IL-4, IL-5, and IL-6; Th2) and display direct antiviral effects. Studies of the T-cell responses in patients with acute hepatitis C infection have shown a close association of a polyclonal, multispecific proliferative CD4⫹ T-cell response, directed mainly against the nonstructural proteins of the virus, with viral clearance and resolution of disease.4,5,13 However, because there is no appropriate animal model it is not known if virus elimination is achieved by specific T cells alone and if the maintenance of this T-cell response is decisive for the long-term outcome of infection. Our prospective follow-up of the CD4⫹ T-cell response in patients with acute hepatitis C during their initial phase of disease and thereafter revealed 3 groups: (1) patients mounting a strong and sustained CD4⫹ T-cell response who cleared the virus and remained HCV RNA negative during long-term follow up; (2) patients who were unable to develop such T-cell responses and did not achieve viral clearance but developed a chronic course of disease; and (3) most interestingly, patients who initially had strong CD4⫹ T-cell responses to eliminate the virus but subsequently lost this specific T-cell response. The loss of the T-cell response was followed promptly by HCV recurrence.
Materials and Methods
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Table 1. Characteristics of Patients With Acute Hepatitis C Self-limited course Patients n Age (yr ) Median Range Sex Male Female Peak ALT (U/L) Source of infection Unknown Sexual Iatrogenic Intravenous drugs Immunoglobulins Genotype (classification of Simmonds) 1a 1b 2a/b 3 4 Not determined Follow-up (mo) Mean Range
20
Chronic course 18
41 (20–70)
30 (15–60)
6 14 1136
10 8 1072
7 1 6 5 1
6 2 6 3 1
3 8 2 1 0 6
3 8 2 2 1 2
21 6–49
20 6–36
NOTE. Thirty-eight consecutive patients with acute hepatitis C were investigated according to their clinical outcome, i.e., self-limited (n ⫽ 20) vs. chronic (n ⫽ 18) course. No statistically significant differences between the 2 groups were found for age, sex, peak ALT level, source of infection, or genotype of the virus.
Patients and Controls Thirty-eight patients with acute hepatitis C (22 women and 16 men; mean age, 35 years) were included (Table 1). The diagnosis of acute infection was based on the following criteria: (1) elevated values of serum alanine aminotransferase (ALT) at least 20 times above the upper limit of normal; (2) seroconversion to anti–HCV-positive antibody status by second- or third-generation enzyme-linked immunosorbent assay or recombinant immunoblot assay II, respectively; (3) positive polymerase chain reaction (PCR) for HCV RNA (Amplicor; Roche Molecular Diagnostics Systems, Branchburg, NJ) in at least the first serum sample; and (4) a history of sudden onset of liver disease in previously healthy individuals. Patients were scheduled to be seen at least once in the acute symptomatic phase, after 3 and 6 months, and then in 6-month intervals. Potential causes of acute hepatitis such as other forms of viral hepatitis, autoimmune hepatitis, alcoholic liver disease, toxins, or metabolic etiology were ruled out. Patients with hepatitis B virus or HIV coinfection were excluded from the study. In comparison to patients with acute hepatitis C, 30 consecutive nontreated patients with established chronic HCV infection (positive HCV RNA and elevated ALT levels for at least 6 months) were analyzed with respect to their immunologic, biochemical, and serological features. Peripheral blood mononuclear cells (PBMCs) of 10 healthy donors served as negative controls for HCVspecific proliferative responses and secretion of cytokines.
HCV Proteins and Peptides Recombinant proteins for antigenic stimulation of CD4⫹ T lymphocytes were kindly provided by Chiron (Emeryville, CA) and comprised the following antigens: c22 (core), c33c (NS3), c100 (NS4), NS5, and the c200 (NS3 ⫹ NS4). All antigens were expressed as COOH-terminal fusion proteins with human superoxide dismutase in yeast. Yeast and superoxide dismutase were tested as controls in each proliferation assay for unspecific stimulation. Purity of antigens ranged between 80% and 90%.
Isolation of PBMCs and Proliferation Assay PBMCs were isolated on Ficoll-Isopaque gradients (Pharmacia, Uppsala, Sweden) at several time points during the course of acute hepatitis and were washed 3 times in phosphatebuffered saline (PBS). PBMCs were incubated at 5 ⫻ 104/well in 96 U-bottom plates (Costar, Cambridge, MA) for 5 days in the presence of HCV proteins (1 µg/mL) in 150 µL RPMI 1640 medium (GIBCO, Grand Island, NY) containing 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% human AB serum. On day 5, cultures were labeled by incubation for 16 hours with 2 µCi 3H-thymidine (sp act, 80 µCi/mmol; Amersham, Buckinghamshire, England). Then cells were collected and washed on filters (Dunn, Asbach, Germany) using a cell harvester (Skatron,
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Sterling, VA), and the amount of radiolabel incorporated into DNA was estimated by a beta counter (LKB/Pharmacia, Uppsala, Sweden). Triplicate cultures were assayed routinely, and the results are expressed as mean counts per minute (cpm). The stimulation index (SI) was calculated as the ratio between cpm obtained in the presence of antigen to that obtained without antigen, and SI ⱖ3 was considered significant.
Enzyme-Linked Immunospot Assay The enzyme-linked immunospot (ELISPOT) assay was performed as described previously.14 Briefly, nitrocellulose bottom 96-well Millititer HA plates (Millipore Co., Bedford, MA) were coated under sterile conditions with 100 µL of the monoclonal antibody of interest (anti–IFN-␥, anti–IL-4; Mabtech AB, Stockholm, Sweden) at a concentration of 15 µg/mL in PBS and incubated overnight at 4°C. Excessive antibodies were removed by 4 successive washings with PBS containing 0.05% Tween. The coated wells were filled with 100 µL of RPMI medium containing 2 ⫻ 105 PBMCs in duplicates together with HCV-derived proteins (1 µg/mL), tetanus toxoid (10 µg/mL), or phytohemagglutinin (5 µg/mL) and incubated undisturbed for 48 hours at 37°C in a humidified atmosphere with 5% CO2. After 48 hours of incubation, cytokine secretion reached peak levels (data not shown) and antigen-specific stimulation was stopped. PBMCs were removed by washing the plates 4 times with PBS. One hundred microliters of a biotinconjugated monoclonal antibody (Mabtech) was added to each well at a concentration of 1 µg/mL and incubated for 4 hours at room temperature. The plates were rinsed 4 times with PBS and exposed to 100 µL streptavidin–alkaline phosphatase (Mabtech AB) for 1 hour. Unbound conjugate was removed by washing thoroughly with PBS, and finally 100 µL of 5-bromo4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate solution (Bio-Rad Laboratories, Richmond, CA) was added and incubated until the appearance of blue spots in the wells (30–60 minutes). The color reaction was stopped by extensive washings, and after drying, the number of spots was scored using a dissection microscope. Secretion of cytokines was optimal after 48 hours of incubation. Stimulation with the mitogen phytohemagglutinin and tetanus toxoid as specific antigen served as positive controls for both cytokines. To compare antigen-specific T-cell activation detected in the proliferation assay and in the corresponding ELISPOT assay, a stimulatory index (SIE) was defined: SIE was considered significant if the index, calculated as the ratio of IFN-␥–producing cells in the presence of antigen and the control wells, was ⱖ3.
Depletion of CD4ⴙ and CD8ⴙ T Cells In 4 patients with acute hepatitis C, proliferation and ELISPOT assays were performed before and after magnetic immunoseparation of CD4⫹ or CD8⫹ T-cell fractions, respectively. PBMCs (20 ⫻ 106) were suspended in 160 µL PBS supplemented with 0.5% bovine serum albumin and 2 mmol/L EDTA and were stained with 40 µL paramagnetic microbeads conjugated to a monoclonal mouse anti-human anti-CD4 or
ACUTE HEPATITIS C 935
anti-CD8 antibody (Micro Beads; Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 minutes at 4°C. Then cells were washed and resuspended in 500 µL buffer. Separation of positively stained T cells (CD4⫹ or CD8⫹) was done in a depletion column AS (Miltenyi Biotec) placed in a magnetic field of a suitable separator (reduction of stained T lymphocytes ⱖ95%). The unstained cell fractions were collected, respectively, and washed twice before they were tested for HCVspecific proliferation and cytokine release in the ELISPOT assay.
Statistical Analysis Results are presented as means and/or medians. Comparisons of stimulation index and number of IFN-␥–secreting cells of patients with self-limited course vs. chronic course of disease were done by the Mann–Whitney rank sum test.
Results Clinical Outcome and Patient Characteristics A total of 38 patients diagnosed with acute hepatitis C were included in the study (Table 1). In all patients, clinical outcome (self-limited vs. chronic evolution) was determined after a follow-up of more than 6 months after onset of disease (start of observation). Mean follow-up in patients with self-limited hepatitis was 21 months (range, 8–49) and in patients with chronic evolution 20 months (range, 6–36). The peak ALT level in the acute phase did not differ between patients with self-limited (mean ALT, 1136 U/L) vs. those with chronic course (mean ALT, 1072 U/L). In 20 patients (53%), HCV RNA became undetectable by repeatedly performed nested PCR and serum ALT levels normalized in this group within the first 6 months after onset of disease. Because these patients displayed no evidence of clinical symptoms or abnormal biochemical serum parameters and remained HCV RNA–negative by PCR during the entire follow-up, they were considered to have a self-limited course of disease. The time between onset of disease and normalization of ALT, combined with loss of serum HCV RNA, was 8 weeks (range, 4–41 weeks; median, 8 weeks). Eighteen patients (47%) who had persistently elevated levels of ALT and/or persistent HCV RNA in serum for more than 6 months after onset of disease evolved to chronic hepatitis. Seven of the 18 patients were treated with IFN-␣. At the end of observation, 2 of the treated patients had achieved sustained response with viral elimination (negative PCR) and normal ALT extending more than 6 months after end of therapy. With regard to the clinical characteristics, patients with a self-limited course of disease seemed to be older (41 vs. 30 years; P ⫽ NS), although the difference did not
936 GERLACH ET AL.
Figure 1. Proliferative CD4⫹ T-cell response of the first sample in the acute phase of disease to recombinant HCV proteins (HCV-NS3, -NS4, -NS5, and -core) of PBMCs from 38 patients with acute hepatitis C. Patients are grouped according to the final outcome of disease in self-limited hepatitis C (SL, n ⫽ 20) and patients with chronic evolution (C, n ⫽ 18). Results are shown as SI ⫽ 3H-thymidine incorporation of antigen-stimulated PBMCs (cpm)/unstimulated control (cpm). Values ⱖ3 are considered significant. All patients with self-limited disease displayed a significant proliferative T-cell response against at least 1 of the viral proteins, while patients with chronic evolution mounted no or only transient antiviral T-cell responses. NS3 and NS4 revealed the most frequent (see Table 2) and most vigorous responses. In 4 patients, the proliferative response against NS5 was not tested in the first sample.
reach statistical significance (Table 1). The self-limited group (female, n ⫽ 14; male, n ⫽ 6) consisted of more women than the group of patients progressing to chronic disease (female, n ⫽ 8; male, n ⫽ 10; t test, P ⫽ NS). The mode of transmission was heterogeneous in both groups but did not differ significantly and did not seem to influence the clinical outcome of hepatitis C. Intravenous drug abuse and sporadic infection of unknown origin were the most common modes of transmission. Seven patients developed acute HCV infection within 4 months after medical procedures or dental treatment without having any other known risk factor. There was no significant difference in the genotypes of the infecting virus in the 2 groups (Table 1). Proliferative T-Cell Responses CD4⫹
T-cell responses in the acute phase of acute HCV. In both groups (self-limited course of disease
and chronic hepatitis), the strongest proliferative responses were found during the first 6 months after onset of disease (SI, chronic 2.4 ⫹ 1.85 vs. self-limited 11.3 ⫹ 10.7; P ⫽ 0.0031; number of tests per patient, self-limited, 4.1 ⫾ 4.7; chronic, 3.6 ⫾ 3.1; P ⫽ NS). The difference in CD4⫹ HCV-specific proliferative responses between both groups was statistically significant
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in the first proliferative response after onset of disease (Figure 1) and throughout the entire period of follow-up. All patients with a self-limited course of disease mounted a significant proliferative CD4⫹ T-cell response against at least 1 of the 4 tested viral proteins (mean, 2.8 proteins per patient) during the first 6 months of disease. The most immunogenic proteins were the nonstructural proteins, which led to a significant stimulation of T lymphocytes in 70%–85% of patients, followed by the core protein with a significant proliferative response in 45% (Table 2). Proliferative response to HCV proteins was significantly reduced after depletion of CD4⫹ T cells but not after depletion of CD8⫹ T cells compared with unfractioned PBMCs (Figure 2). Patients with chronic evolution of disease displayed significantly lower levels of antiviral T-cell activity in percentage of patients and in strength of stimulation indices against each tested viral protein (Table 2 and Figure 1). Recurrence of HCV RNA after loss of HCVspecific CD4ⴙ T-cell response. Six (33%) of the 18
patients with chronic evolution mounted a transient significant antiviral proliferative CD4⫹ T-cell response, and HCV RNA (tested by nested PCR) became undetectable in serum. The subsequent loss of virus-specific T-cell response in this group was immediately followed by reappearance of HCV RNA in all 6 patients after a time period of up to 10 months after onset of disease (range, 5–10 months; median, 6.6 months; mean, 7.0 months). Table 2. Significant Proliferative CD4⫹ T-Cell Responses (PBMC) Against HCV Proteins During the First 6 Months in Patients With Acute Hepatitis C
Core n % NS3 n % NS4 n % NS5 n % ⱖ1 Antigen recognized n %
Self-limited course (n ⫽ 20)
Chronic course (n ⫽ 18)
9 45
2 11
14 70
4 22
17 85
6 33
15 75
6 33
20 100
6 33
NOTE. Number and percentage of patients with significant SIs (SI ⱖ 3) against recombinant HCV proteins within 6 months after onset of disease. All patients with self-limited course show a significant T-cell response against different viral proteins. Patients with chronic evolution had no (n ⫽ 12) or only transient (n ⫽ 6) significant T-cell responses against the viral proteins (number of tests: self-limited, 4.1 ⫾ 4.7; chronic evolution, 3.6 ⫾ 3.1; P ⫽ NS).
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Figure 2. (A ) Proliferative response ( 3H-thymidine uptake) and (B ) secretion of IFN-␥ (ELISPOT assay) were assessed in patients with acute hepatitis C (n ⫽ 4) upon stimulation with HCV-specific antigen (C200) in CD4⫹- or CD8⫹-depleted PBMCs and in unfractionated PBMCs. Representative results of 1 patient. Depletion of CD4⫹ T cells but not depletion of CD8⫹ T cells resulted in a significant decrease of antigen-specific proliferation and IFN-␥ secretion.
Although biochemical parameters normalized within 2 months after onset of disease in 3 of the 6 patients, ALT levels of the other 3 remained at the upper limit or just above the normal range during the entire observation period. Significant antiviral CD4⫹ T-cell proliferation in this group was strongly associated with undetectable HCV RNA in patients’ sera. The comparison of proliferative T-cell response of HCV RNA–positive vs. –negative samples in this subgroup of patients with evolving chronic hepatitis revealed a significant difference in stimulation indices of RNA-positive vs. RNA-negative samples (Table 3). The initial strength of antiviral T-cell response did not differ between patients with relapse or self-limited disease. None of the other patients with chronic evolution (n ⫽ 12) mounted a sustained CD4⫹ HCV-specific T-cell response. The course of viral and biochemical parameter and the corresponding T-cell responses of 2 representative patients are exemplified in Figure 3. In all patients with a self-limited course of disease, a strong and sustained proliferative CD4⫹ T-cell response against at least 1 viral protein could be detected during the first 6 months after onset of symptoms, whereas 66% of patients with evolving chronic hepatitis had no significant antiviral T-cell response at any time during follow-up. During treatment with IFN-␣, HCV RNA became undetectable and ALT level returned to the normal range in 6 patients.
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Two patients achieved sustained viral elimination after therapy (n ⫽ 2) and mounted a significant T-cell response against NS3 (first patient, SINS3 ⫽ 7.6) or NS3 and NS4 (second patient, SINS3⫹NS4 ⫽ 5.3) during treatment. Two transient responders who achieved biochemical remission without virus-specific T-cell responses had a breakthrough before therapy was terminated. Healthy controls and patients with cHCV. In comparison to patients with acute hepatitis C, the proliferative T-cell responses of 30 untreated patients with chronic hepatitis C and of 10 healthy controls was assessed. Healthy controls mounted no (0/10) significant proliferative response to any HCV-specific antigen (proliferative stimulation index of healthy controls (mean: SINS3 ⫽ 1.3; SINS4 ⫽ 1.0; SINS5 ⫽ 0.9; SIcore ⫽ 1.2), and patients with cHCV displayed no proliferative (0/30) response to the NS3, NS4, and NS5 region of HCV (proliferative SI of patients with chronic hepatitis C; mean: SINS3 ⫽ 1.4; SINS4 ⫽ 1.3; SINS5 ⫽ 1.0). In 3 patients (3/30) with cHCV infection, significant proliferative T-cell responses against the core protein (proliferative SI of 3 responders: mean, SIcore ⫽ 3.8) could be detected. ELISPOT Assay The significance of CD4⫹ T cells for HCV-specific IFN-␥ secretion was shown in the ELISPOT assay by depletion assays in patients with acute hepatitis C (n ⫽ 4). After depletion of CD4⫹ T cells, but not after depletion of CD8⫹ T cells, antigen-specific secretion of IFN-␥ was significantly decreased (Figure 3). HCV-specific secretion of cytokines (IFN-␥ and IL-4) was assessed in 20 patients with acute HCV (10 patients with chronic evolution vs. 10 patients with self-limited course) and in 10 healthy controls (Figure 4). Stimulation indices ⱖ3 were considered significant. Stimulation of Table 3. Proliferative T-Cell Responses (SI) Against HCV Protein (C200) in Patients With Transient Viral Clearance Months after onset of disease
1–3
4–6
7–12
12–18
18–24
Pt. 1 Pt. 2 Pt. 3 Pt. 4 Pt. 5 Pt. 6
0.9 6.3 21.2 3.8 3.6 4.2
5.2 6.4 3.8 5.4 9.1 5.8
2.1 9.3 1.9 1.3 1.4 0.5
1.0 0.9 — — — —
— 0.8 0.9 0.7 — 2.5
NOTE. In patients with transient viral clearance (Pt. 1 to Pt. 6) during acute hepatitis C, RNA in serum was undetectable (RNA-negative samples in bold) only as long as HCV-specific CD4⫹ T-cell responses reached significant levels (SI ⱖ 3). After the loss of significant T-cell responses, HCV RNA becomes positive again. Differences in T-cell responses were significant between RNA-positive and RNA-negative samples by the Mann–Whitney rank sum test (P ⫽ 0.004).
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PBMCs was low in healthy subjects (SIE ⫽ 1.1 ⫾ 0.4) and in patients with chronic evolution (SIE ⫽ 1.85 ⫾ 1.4), PBMCs of patients with a self-limited disease (SIE ⫽ 14.6 ⫾ 17.8) displayed significantly higher stimulation indices for IFN-␥–secreting cells (chronic vs. self-limited course; P ⫽ 0.025) (Figure 4). These differences in number and SIE of IFN-␥–secreting cells in patients with acute HCV could be confirmed in longterm follow-up of patients with different clinical courses of acute hepatitis. In the self-limited course, high numbers of IFN-␥–secreting cells were maintained through the entire follow-up, whereas patients with a chronic course of disease did not show significant numbers of IFN-␥–secreting T cells after HCV-specific stimulation.
Discussion In this study we prospectively compared HCVspecific CD4⫹ T-cell responses of patients with acute self-limited hepatitis C vs. those with evolving chronic hepatitis. Our results provide new information on the decisive role of CD4⫹ T-cell responses in the elimination and long-term control of HCV. By using the largest cohort of patients published to date, we confirm data of previous studies of patients with acute hepatitis C.
Figure 3. Follow-up of HCV-RNA and ALT level (upper part of each graph), antiviral CD4⫹ proliferative T-cell responses (middle part of each graph), and number of spots representing IFN-␥–secreting cells/ 2 ⫻ 105 PBMCs in the ELISPOT assay (lower part of each graph) after stimulation with recombinant HCV protein (C200) in 2 representative patients with acute hepatitis C. (A ) Patient with a self-limited course of acute hepatitis C and sustained vigorous proliferative T-cell responses against the viral proteins c33c (NS3), c100 (NS4), and c200 (NS3 and NS4). HCV RNA turns negative by week 8 and remains undetectable throughout the entire follow-up. Initially elevated ALT level falls to normal values in the acute phase. The increased number of IFN-␥– secreting cells upon antigen-specific stimulation is maintained during follow-up. (B ) Patient with initially significant (SI ⱖ 3) antiviral CD4⫹ T-cell responses and transient viral clearance. Loss of significant T-cell responsiveness is promptly followed by recurrence of HCV RNA. The precursor frequency of IFN-␥–secreting cells upon antigen-specific stimulation in the ELISPOT assay decreases during follow-up.
2 ⫻ 105 PBMCs with mitogen (phytohemagglutinin) or tetanus toxoid (10 µg/mL) for 48 hours revealed a significant number of cells producing IFN-␥ and IL-4, whereas stimulation with recombinant HCV proteins induced only the secretion of IFN-␥ without IL-4 secretion, indicating Th1-dominated cytokine profiles. Although the mean stimulation index for IFN-␥ of
Figure 4. Stimulatory index of HCV-specific T lymphocytes in the first ELISPOT assay within 6 months after onset of disease (SIE ⫽ number of spots after antigen-specific stimulation/number of spots in the control) for IFN-␥ secretion after incubation of 2 ⫻ 105 PBMCs with viral protein (c200; 1 µg/mL) in healthy controls (n ⫽ 10), patients with acute hepatitis C and chronic evolution (n ⫽ 10), or patients with self-limited course of disease (n ⫽ 10). Differences are significant for healthy controls vs. self-limited course (**P ⫽ 0.020) and chronic evolution vs. self-limited course (*P ⫽ 0.025).
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Obviously, patients with acute HCV infection, who show clinically apparent disease with symptoms such as jaundice, fever, and malaise, represent a distinct group. This clinical picture probably reflects a strong antiviral response in these individuals, which is followed by virus elimination in more than 50% of patients. The results are in line with the respective subgroups in studies of the Centers for Disease Control and Prevention in which only 30% of infected individuals developed symptomatic disease and up to 85% of patients evolved chronic disease.1 In the present study only patients with symptomatic disease were included. Infected individuals with self-limited or chronic courses displayed no differences in the mode of infection and genotype of the infecting virus. In the group with self-limited hepatitis, patients tended to be older and the percentage of females was higher than in the group with evolving chronic hepatitis, but these differences were not statistically significant. In 7 patients, the only anamnestic source of infection was a medical procedure, suggesting a possible nosocomial transmission. The most significant difference between both groups was found in the HCV-specific proliferative CD4⫹ T-cell responses. All self-limited infections, regardless of the genotype of infecting virus, displayed a proliferative response against at least 1 viral protein. In contrast, 66% of patients with evolving chronic hepatitis did not respond to any protein at any time. The lack of antiviral CD4⫹ T-cell responses in the acute phase of hepatitis was unequivocally followed by chronic evolution of disease. A striking finding was that 30% of patients with evolving chronic hepatitis in the acute phase were capable of mounting a significant transient virus-specific CD4⫹ T-cell response. In these patients, initial T-cell responses were lost at variable time points (5–10 months) during follow-up, resulting in chronic hepatitis. The clinical implication of this finding and the need for antiviral treatment suggest a careful follow-up of patients with acute symptomatic hepatitis C for a prolonged period. Only as long as virus-specific CD4⫹ T cells were detectable in the peripheral blood of these patients, HCV RNA was absent by means of nested PCR, possibly reflecting the attempt of the individual’s cellular immune system to combat the virus. The loss of T-cell responsiveness was immediately followed by recurrence of HCV RNA in the patients’ serum. The inverse correlation of antiviral CD4⫹ T-cell response and detectable HCV RNA was observed during follow-up of all patients, who temporarily or permanently cleared the virus. Of course, the only exception of this rule was found in the very first sample at onset of disease, which was characterized by the presence of a significant T-cell response, positive HCV
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RNA, and increased ALT levels. Although HCV RNA as determined by PCR was absent for up to 9 months after onset of disease in patients with transient CD4⫹ T-cell responses, permanent viral clearance was unlikely in the face of continuous marginal increase of ALT levels in 3 of the 6 patients. Although only peripheral blood lymphocytes were analyzed, the elevated serum ALT, evidencing an inflammatory process within the liver, in the presence and absence of peripheral HCV-specific CD4⫹ T-cell responses, contradicts the opinion that virus-specific T cells are only found in peripheral blood if inflammatory activity in the liver is low. If T-cell responsiveness in these patients reflects a compartmentalization or recruitment of virus-specific T cells rather than a general antiviral immune response, ALT in the serum should behave inversely. The cytokine pattern of peripheral HCV-specific T cells was confined to a Th1 profile, which is also predominantly found in liver-infiltrating lymphocytes.15 Our finding in freshly isolated PBMCs tested in the ELISPOT assay confirms previous results in T-cell clones from patients with acute HCV infection without the influence of long-term culture.13 Depletion experiments showed that IFN-␥ secretion in response to stimulation with viral proteins was accomplished by CD4⫹ T cells and ruled out a significant participation of CD8⫹ T cells in IFN-␥ secretion by a possible leakage in the MHC class II antigen-processing pathway within antigenpresenting cells after stimulation with viral proteins. Although there was no functional shift of virus-specific T cells from a Th1 to Th2 cytokine profile during progression to chronicity, the number of HCV-specific CD4⫹ T lymphocytes with a Th1 cytokine profile decreased during follow-up in patients with chronic evolution of hepatitis as defined by means of the sensitive ELISPOT assay. In contrast, patients with self-limited disease maintained a high number of HCV-specific IFN-␥– secreting cells over the entire period of observation, which was associated with long-lasting viral control. The inverse correlation of antiviral CD4⫹ T-cell responses and HCV RNA supports the hypothesis of viral control mediated by CD4⫹ T helper cells. The important contribution of CD4⫹ T lymphocytes to the development of sustained immunity has been evidenced in experimental infection of animals with viruses such as herpes, lymphocytic choriomeningitis virus, and influenza16–19 and in tumor immunity.20 Although cytotoxic CD8⫹ T cells in these infections may achieve virus elimination in the acute phase without T helper cells, CD4⫹ T lymphocytes are mandatory for a persistent CD8⫹ cytotoxic T-cell response and depletion of CD4⫹ T cells in the acute phase results in chronic infection.21 Cytotoxic CD8⫹
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T-cell responses have not been studied in acute HCV to date, but viral replication in the presence of cytotoxic T-cell responses in cHCV, in which antiviral CD4⫹ T-cell responses are weak or absent, indicates that effective HCV elimination is not mediated by CD8⫹ T cells alone.22–24 The virus-specific CD4⫹ T-cell response in HCV infection may persist as long as 20 years, as we have observed in women who were accidentally infected by a contaminated lot of anti-D immunoglobulin and displayed, in about 75%, a strong HCV-specific T-cell response. In contrast, women from this cohort with evolving chronic hepatitis did not show antiviral CD4⫹ T-cell activity (Gerlach et al., unpublished data, October 1997). In acute hepatitis B, which shows a self-limited course in almost all cases, similar long-lasting CD4⫹ T-cell responses in the presence of minute amounts of hepatitis B virus detected by nested PCR have been described.25 The importance of HCV-specific CD4⫹ T cells in viral control is also suggested in HCV/HIV coinfection, which in the late stages of acquired immunodeficiency syndrome with low CD4⫹ T-cell counts often leads to a rapid deterioration of liver disease.9,26 The abrogation of the HCV-specific Th1-dominated T-cell responsiveness, together with the breakdown of viral control, as reflected in the reappearance of virus in serum, could represent an important mechanism for viral persistence. While the present work highlights the importance of a sustained vigorous CD4⫹ T-cell response for viral control in HCV, it cannot delineate the causes of its absence or abrogation, a status discussed as virusspecific T-cell tolerance. The literature gives examples of potential mechanisms27,28 that may lead to T-cell unresponsiveness in viral infection, e.g., anergy,29 T-cell exhaustion,30 altered peptide binding,31 and others, but provides no data on the degree of importance and contribution of each factor to the observed tolerant T-cell status in viral hepatitis. The characterization of such mechanisms, however, will help to develop therapeutic strategies designed to overcome cHCV infection. In summary, our data suggest that a vigorous antiviral CD4⫹ T-cell response in the early phase of acute hepatitis C is not only required for virus elimination leading to a self-limited course of disease, but also that an antiviral CD4⫹ T-cell response in the late phase is a prerequisite for achieving long-term viral control. We could show consistently that the abrogation of the early CD4⫹/Th1dominated immune response in acute hepatitis C is associated with immediate recurrence and persistence of hepatitis C virus.
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Received October 6, 1999. Accepted July 28, 1999. Address requests for reprints to: Gerd R. Pape, M.D., Institute for Immunology, Goethestrasse 31, D-80336 Mu ¨nchen, Germany. email: [email protected]; fax: (49) 89-591183. Supported by the Bundesministerium fu ¨r Bildung und Forschung and the European community project Biomed 2 PL95-1064 and SFB217. The authors thank Jutta Do ¨hrmann and Carola Steiger for excellent technical assistance and personal encouragement and Professor Dolores Schendel for critical reading of the manuscript.