Virus-Specific Lymphokine Production Differs Quantitatively but Not Qualitatively in Acute and Chronic Hepatitis B Infection

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1 Virology 261, (1999) Article ID viro , available online at on Virus-Specific Lymphokine Production Differs Quantitatively but Not Qualitatively in Acute and Chronic Hepatitis B Infection Maria-Christina Jung,*,,1 Bertram Hartmann, Jörn-Tilman Gerlach, Helmut Diepolder,*, Rudolf Gruber, Winfried Schraut, Norbert Grüner, Reinhart Zachoval,* Robert Hoffmann,*, Teresa Santantonio, Martin Wächtler, and Gerd Rudolf Pape*, *Department of Medicine II, Klinikum Großhadern, University of Munich, Marchioninistrasse 15, Munich, Germany; Institute for Immunology, University of Munich, Goethestrasse 31, Munich, Germany; Clinica Malattie Infettive, Universita degli studi di Bari, Bari, Italy; and Medical Department IV, Schwabinger Krankenhaus, Munich, Germany Received January 12, 1999; returned to author for revision February 3, 1999; accepted June 3, 1999 Cytokines that are secreted as a response to viral antigen not only have direct antiviral properties but also crucially influence immune reactions determining the outcome of infection. As an advantageous alternative to the study of cytokines present in the supernatants of antigen-specific T cell clones and lines, we have used ELISPOT assays to determine the number of interferon- (IFN- )- and IL4-producing cells generated by peripheral blood mononuclear cells from patients with acute hepatitis B (AHB) and chronic hepatitis B (CHB) infection in response to HBcAg in a short-term culture (48 h). In response to HBcAg IFN- was predominantly produced. In contrast to the results obtained in acute hepatitis B, the typical lymphokine pattern in CHB was characterized by a weak or absent antigen-specific IFN- production. A predominance of IL-4-producing cells was not observed in either AHB or CHB. A significant number of IFN- -producing cells was usually detectable during phases of viral elimination and the quality of the lymphokine response seemed to be epitope independent. Comparison of the results obtained in proliferation assays and ELISPOT assays clearly shows that lymphokine production upon stimulation with viral protein is totally independent of T cell proliferation and more sensitively reflects antiviral reactivity Academic Press INTRODUCTION A vigorous, polyclonal, and multispecific cytotoxic and helper T cell response to the hepatitis B virus is readily detectable in the peripheral blood of patients with acute hepatitis B but is weak or undetectable in patients with chronic infection. This T cell response is believed to be responsible for the elimination or control of the hepatitis B virus (Ferrari et al., 1991; Guidotti et al., 1994; Jung et al., 1995; Nayersina et al., 1991; Rehermann et al., 1995; Tsai et al., 1992). The importance of a HLA class II restricted T helper cell response is further supported by the association between the HLA DRB1 allele 1302 and selflimited acute hepatitis B (Thursz et al., 1995). CD4 T helper cells exert their function via lymphokines. According to their lymphokine pattern they have been classified into Th1 and Th2 subsets, where interferon- (IFN- ), IL-2 and TNF represent the Th1-type response, which stimulates cellular immune reactions, and IL-4, IL-5, IL-6, and IL-10 are lymphokines of the Th2-type response, which supports B cell development and enhances antibody production. The question of preferential induction 1 To whom correspondence and reprint requests should be addressed. Fax: Maria-Christina.Jung@med.2. med.uni-muenchen.de. of either T cell subset in HBV infection has not been extensively examined. Cellular immune responses seem to prevail in acute hepatitis (Jung et al., 1991), whereas chronically infected patients demonstrate significantly greater antibody responses. Studies in mice suggest that the preferential recognition of hepatitis B nucleocapsid antigens by Th1 or Th2 cells is epitope and MHC dependent (Milich et al., 1995). Elegant experiments in transgenic mice have revealed the superior role of IFN- and TNF in the antiviral T cell response to HBV (Guidotti et al., 1994, 1996) and studies in human demonstrate a predominance of Th0 in the liver and of Th1 cells in the peripheral blood (Bertoletti et al., 1997; Penna et al., 1997). The techniques used so far in human do not allow a quantitative analysis of lymphokine-producing cells. In addition the cytokine profile of T cell clones and cell lines may not be representative due to the influence of longterm culture, and the measurement of cytokines in supernatants may be hampered by the binding of cytokines to cell-bound or soluble receptors (Engelberts et al., 1991). The ELISPOT assay used in the present paper has important advantages, which contribute to a more complete and perhaps biologically more relevant picture of the antiviral cellular immune response in HBV infection: In the first place it allows quantitative assessment of /99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved.

2 166 JUNG ET AL. FIG. 1. The median number of IFN- spots/200,000 PBMC in response to an in vitro challenge with HBcAg (1 g/ml) in patients with acute hepatitis B (AHB, n 51) and chronic hepatitis B (CHB, n 59). Solid spots indicate a significant response. The median values have been corrected by subtracting the background medians. Each spot represents the response of one patient. cytokine-producing cells in response to virus-specific antigen. This determination can be performed in a shortterm culture, which excludes the disadvantages of T cell lines and T cell clones. The test can be carried out with relatively low numbers of cells. It does not concentrate exclusively on T cells. Comparison between cytokine production and T cell proliferation could show whether T cell proliferation is accompanied by cytokine production and whether cellular antiviral nonresponsiveness in chronically infected individuals is confined to T cell proliferation. The most important advantage of this approach is the proximity to the biological situation with respect to the frequency of lymphokine-producing cells in different cell populations after a short-term culture with viral antigens. The results of these studies should therefore help to answer the following questions: (a) Which is the predominant cytokine produced as a response to hepatitis B core antigen (HBcAg)? (b) Is there a difference in cytokine production between patients with self-limited infection and those with chronic infection? (c) Compared to the conventional [H 3 ]thymidine uptake assay, is the ELIS- POT assay superior in reflecting cellular antiviral activity? RESULTS Number of cells producing IFN- and IL-4 in response to HBcAg and phytohemagglutin (PHA) in patients with acute and chronic hepatitis B The number of IFN- - and IL-4-producing cells in response to HBcAg and PHA was estimated in acutely infected (n 51) and chronically infected patients (n 59). PHA was used as a positive control. All patients produced IL-4 (mean number of spots/ cells, SEM, n 61) and IFN- (mean number of spots/ cells SEM, n 110) in response to PHA. As expected the number of lymphokine-producing cells was much lower in response to specific antigens. As can be seen in Fig. 1 the majority of patients with acute hepatitis B (60.78% 31/51 patients) showed a significant number of IFN- -producing cells in response to HBcAg (mean number of spots/ cells SEM, ), whereas only 25.42% (15/59) of the chronically infected patients responded to the viral antigen (mean number of sponts/ cells SEM ) (P ). In only a few chronically infected patients did the number of lymphokine-producing cells reach the level obtained in patients with acute hepatitis B infection. To evaluate a possible correlation between the number of positive cells in the ELISPOT assay and transaminases we calculated the Pearson correlation coefficient. Neither in chronically infected patients (R 0.03) nor in acutely infected patients (R 0.06) could we demonstrate a correlation between these parameters (Table 1). In contrast IL-4-secreting cells in response to HBcAg could not be detected in either patients with acute hepatitis (n 27) or those with chronic hepatitis (n 34). That the absence of IL-4 production cannot be explained by the technique used is shown by stimulation of PBMC of acute hepatitis B (AHB) (n 10) and chronic hepatitis B (CHB) (n 13) with tetanus toxoid (TT), which is known to be a potent inductor of IL-4 (Fig. 2). Peripheral blood mononuclear cells (PBMC) from both patient groups are able to produce IL-4 in response to this antigen (TT). Three of ten patients with acute hepatitis and 3/13 patients with chronic hepatitis demon-

3 VIRUS-SPECIFIC CYTOKINE INDUCTION IN HEPATITIS B INFECTION 167 TABLE 1 Correlation of ALT Values and Significant ELISPOT Results in 31 Patients with Acute Hepatitis B (AHB) and 15 Patients with Chronic Hepatitis B (CHB) AHB Number of interferon- spots ALT (U/l) CHB Number of interferon- spots ALT (U/l) strated IL-4-producing cells in response to tetanus toxoid. IL-4 production in response to HBcAg stimulation was absent, whereas the peripheral blood mononuclear cells showed a significant number of IFN- -producing cells in response to HBcAg in the same experiment. Kinetics of HBcAg induced IFN- -producing cells in acute and chronic hepatitis B infection HBcAg-specific T cell activation was shown to be associated with viral clearance (Jung et al., 1995). However, the cytokine profile of unselected PBMC in the course of resolving hepatitis has not yet been described. On the other hand, chronic hepatitis B has been suggested to be associated with an absence of a Th1- or the predominance of a Th2-like lymphokine profile. To exclude the possibility that lymphokine determinations at single time points did not adequately reflect the spectrum of lymphokines produced during the entire course of the disease, we prospectively and serially at various time points determined the lymphokine profile of patients during infection. Representative results for acute infection are shown in Fig. 3. In acute self-limiting hepatitis, a significant number of IFN- -producing cells are readily detectable during the acute phase of infection and this number declines after viral elimination. A significant number of IL-4-producing cells were not detectable. The ELISPOT assay more frequently gave significant positive results than the T cell proliferation assay (Fig. 3). Long-term observation of cytokine-producing cells and of T cell proliferation in response to HBcAg in chronic hepatitis B infection has been done in patients undergoing antiviral treatment with famciclovir and lamivudine (Fig. 4). A response was characterized by an increase of IFN- -producing cells when the HBV DNA level declined or became negative. Again, the detection of interferon- -producing cells was more frequent than T cell proliferation. The cytokine profile of chronically infected patients was not dominated by Th2 lymphokines as reflected by the absence of IL-4-producing cells (data not shown). Lymphokine response to HBcAg/HBeAg peptides: Epitope dependency of the lymphokine response Since it has been suggested that the pattern of lymphokine response is epitope dependent (Milich et al., 1995), we did ELISPOT assays using overlapping HBcAg/ HBeAg derived peptides covering the entire HBc protein in PBMC from 46 patients with acute hepatitis and from 28 patients with chronic hepatitis. We determined the kind of lymphokines produced as well as the distribution of the inducing epitopes within the HBcAg/HBeAg. Ten patients with acute hepatitis and 16 patients with chronic hepatitis did not respond to either peptide, which confirms the specificity of the response (data not shown). In contrast, 38 patients with acute hepatitis and 12 patients with chronic hepatitis showed a positive reaction to at least one of the peptides and to nucleocapsid protein as a positive control. Stimulation of PBMC with HBcAg derived peptides led exclusively to IFN- production. In contrast to the data obtained in mice (Milich et al., 1995) IL-4 production could not be induced by any epitope (data not shown). As shown in Fig. 5 IFN- was inducible in both patient groups by almost all peptides used. Epitope mapping with the ELISPOT assay in acute hepatitis showed that amino acids (aa) most frequently induced IFN- production (44.12% of the patients). Other immunogenic sequences could be identified in the amino-terminal part of the protein, aa 1 25 (33.33%) and aa 21 45, (13.88%) and in the carboxy-terminal part, aa (36.11%) and aa (19.44%). All peptides were immunogenic for PBMC of both acutely and chronically infected patients.

4 168 JUNG ET AL. FIG. 2. Number of IL-4-producing cells in three patients with acute and chronic hepatitis B in response to tetanus toxoid (TT) and the nucleocapsid protein of HBV (HBcAg). The mean of the crude data is presented. Except for aa and a part of aa all epitopes described are shared by HBcAg and HBeAg. Comparison of the results in proliferation and ELISPOT assays To answer the question whether proliferation and ELISPOT assays record similar cellular activities, we compared the results of the two assays in 51 patients with acute hepatitis B and 56 patients with chronic hepatitis. Using the Pearson correlation method and a linear regression analysis, no correlation between the results obtained in the two assays could be demonstrated (R in acute hepatitis, R in chronic hepatitis B) (Fig. 6). FIG. 3. Determination of IFN- -producing cells and T cell proliferation (SI) upon stimulation with HBcAg (1 g/ml) during the course of acute hepatitis B infection in three patients. Black columns represent significant values. The results have been corrected by subtracting the background spots and represent the mean of duplicates. In patient 1 HBsAg disappeared in November 1995, patient 2 lost HBsAg at the beginning of December 1994, and patient 3 lost HBsAg at the end of August 1995.

5 VIRUS-SPECIFIC CYTOKINE INDUCTION IN HEPATITIS B INFECTION 169 FIG. 5. Production of IFN- in patients with acute (n 36) and chronic (n 12) hepatitis B infection in response to HBcAg derived peptides. The figure shows the percentage of patients responding significantly to stimulation with HBcAg derived peptides. FIG. 4. Determination of IFN- -producing cells and T cell proliferation (SI) upon stimulation with HBcAg (1 g/ml) during the course of chronic hepatitis B infection in one patient undergoing famciclovir and lamivudine treatment. Dark columns represent significant values. The results have been corrected by subtracting the background spots and are presented as means. Comparison of significant positive test results showed that in acute hepatitis B the ELISPOT assays gave significant results in 60.78% of the patients, whereas the proliferation assay was positive in only 29.41%. In chronic hepatitis B infection a significant antigen-specific IFN- production was detectable in 25.41% of the patients and a significant specific cell proliferation in 17.86%. These data show that the ELISPOT assay is more sensitive in detecting antiviral cellular activity. the ELISPOT assay allow one to obtain a cytokine snapshot of a given pathological sample. This is likely to minimize antagonistic or synergistic cytokine interactions that occur during the prolonged cultures that ELISA determinations require. Third, the ELISPOT assay allows determination of the frequency of cytokine-producing cells. Finally, this method allows the detection of the protein released into the extracellular environment and therefore adds important information when compared with techniques measuring cytokine mrna levels. DISCUSSION There are several lines of evidence showing that cytokines that are secreted as a response to viral antigens control immune reactions determining the outcome of infection. In the present study, peripheral blood mononuclear cells from patients with acute or chronic hepatitis B infection were stimulated with viral antigen and tested for T cell proliferation and for cytokine production (IFN-, IL-4) in an ELISPOT assay. We have chosen to use ELISPOT assays to study, at the protein level, local cytokine production rather than bulk-release detection methods for several reasons. First, relatively few cells are necessary to perform the ELISPOT assay, and in situations where few producing cells were present, this assay allows the detection of cells secreting a given cytokine. In contrast, ELISA assays are not sufficiently sensitive for the detection of the cytokine in the corresponding cell culture supernatant. Second, the short incubation times necessary to perform FIG. 6. Analysis of interferon- -producing cells and T cell proliferation in acute (n 51 patients) and chronic hepatitis B (n 56 patients) using the Pearson correlation method. Results are demonstrated in a linear regression analysis. No correlation could be demonstrated (R in acute hepatitis B, R in chronic hepatitis B).

6 170 JUNG ET AL. When peripheral blood mononuclear cells of patients with acute or chronic hepatitis B infection were stimulated with PHA IFN- and IL-4 production could be easily detected although the number of IL-4-secreting cells was lower than the number of IFN- -producing cells. In response to HBcAg, IFN- but not (number of cells was not significant) IL-4 was produced, indicative of a Th1-type response and in agreement with what has been published by others using HBcAg-specific T cell lines and clones (Penna et al., 1997). Although the studies were done with unselected polyclonal cells, we saw a clearer picture in terms of lymphokine profile than Penna et al. (1997), who showed IFN- production in combination with IL-4 and/or IL-5 production in 5/15 polyclonal T cell lines. These differences might be explained by the different techniques used (ELISA versus ELISPOT). However, our failure to detect IL-4-secreting cells in acutely and chronically infected patients upon stimulation with HBcAg is not likely to be explained by experimental conditions (i.e., sensitivity of the test) because IL-4-producing cells were detectable upon stimulation with PHA and with TT (Fig. 3). The kind of lymphokine profile detected in chronic hepatitis B infection did not differ from acute hepatitis B infection. The preferential activation of IFN- -producing cells was not HBcAg epitope dependent as could be shown in this study by using HBcAg derived peptides in an ELISPOT assay. As in acute self-limited hepatitis, IFN- is also the lymphokine produced in response to HBcAg in chronic hepatitis. This cytokine pattern has been confirmed by the data of Bertoletti et al. (1997), who analyzed 291 clones derived from six patients with chronic hepatitis B and demonstrated a Th1 profile. However, the data published so far allow only a comparison between intrahepatic clones of chronically infected patients and peripheral T cell clones and lines of acutely infected individuals. In contrast, in the study presented here, we were able to directly compare the same cell compartment within the two patient groups. The cytokine response in chronic hepatitis differs from that in acute hepatitis in that the response in acute hepatitis is significantly more vigorous and more frequently detectable. Patients with acute hepatitis typically mount a significant IFN- response during the entire course of infection and lymphokine production declines after viral elimination. The typical lymphokine pattern in chronic hepatitis B is characterized by a weak or absent antigen-specific IFN- production. In contrast, chronically infected patients responding to antiviral treatment with reduction of HBV DNA (interferon- lamivudine, famciclovir) show a transient increase of IFN- -producing cells. Although it is believed that liver damage reflected by elevated transaminases is mediated by functionally active virus-specific lymphocytes no correlation was found between significant results in the ELISPOT assay and transaminases. This may be explained by the fact that peripheral lymphocytes do not necessarily simultaneously reflect T cell activity in the liver and detection of virus-specific lymphocytes in the periphery possibly indicates virus-specific cells coming from or going into the liver. The ELISPOT assay provides a powerful means to detect and enumerate cells on the basis of their cytokine production. This was a prerequisite to detect the few IFN- -producing cells typical for many patients with chronic hepatitis and to compare the precursor cell frequencies between the two patient groups. In addition to the determination of the frequency of cytokine-producing cells and the kind of lymphokine produced, the ELISPOT assay seems to be an important tool to assess antiviral immune responses. Thus far measurement of T helper cell activity has been confined to T cell proliferation assays. Comparison between proliferation and ELISPOT cytokine production shows no correlation between the results of the two test systems. A significant number of acutely infected patients responded to viral antigen with a significant IFN- production, but with no T cell proliferation. On the other hand only a few patients demonstrated a significant T cell proliferation without cytokine production. These data may indicate that the ELISPOT assay measures cytokine production by several different cell populations responding to viral antigens, whereas the T cell proliferation assay reflects predominantly the activity of CD4 T cells. Alternatively, the ELISPOT assay may assess a quality of CD4 T lymphocytes that is not reflected by T cell proliferation. The quantitation of lymphokine-producing cells is likely to more precisely and sensitively reflect the biological situation during an antiviral immune response in acute and chronic hepatitis B infection and thus complete the data obtained with the conventional [H 3 ]thymidine uptake assay. MATERIALS AND METHODS Patients. Fifty-one patients with acute hepatitis and 59 patients with chronic hepatitis B were studied. Acute hepatitis was diagnosed on the basis of elevated aminotransferases, positive hepatitis B surface antigen (HBsAg), HBV DNA, and anti-hbc immunoglobulin M (IgM) as determined by kits obtained from Abbott (Wiesbaden, Germany). All patients with chronic hepatitis B were positive for HBsAg and HBV DNA for more than 6 months and the diagnosis was proven histologically. Isolation of PBMC. Human peripheral blood mononuclear cells were isolated from heparinized blood obtained from patients with AHB and CHB by Ficoll-paque density gradient centrifugation as described elsewhere (Perlmann et al., 1976). The PBMC were washed four times in phosphate-buffered saline (PBS) and resuspended in tissue culture medium (RPMI 1640 medium, obtained from Gibco, Grand Island, NY) containing 2 mm

7 VIRUS-SPECIFIC CYTOKINE INDUCTION IN HEPATITIS B INFECTION 171 L-glutamine, 1 mm sodium pyruvate, 100 U of penicillin per milliliter 100 g of streptomycin per milliliter and 10% human AB serum. Recombinant proteins, synthetic peptides, tetanus toxoid, PPD, and mitogen. Recombinant HBcAg obtained from bacterial extracts of Escherichia coli K-12 strain HB101 was purchased from Biogen (Geneva, Switzerland) (Pasek et al., 1979). Nine overlapping 25-mer peptides covering the entire HBV core sequence (aa 1 25, 21 45, 41 65, 61 85, , , , , ) were synthesized by Multiple Peptide Systems (San Diego, CA). All peptides were purified to more than 95% by high-pressure liquid chromatography. Tetanus toxoid and PPD were used at a concentration of 10 g/ml and obtained from Behring-Werke (Marburg, Germany). PHA (HA 16; Murex Diagnostics, Dartford, United Kingdom) was used at a concentration of 5 g/ml. ELISPOT assay. The ELISPOT assay was performed as described elsewhere (El Ghazali et al., 1993). Briefly, Millititer HA nitrocellulose-bottom 96-well plates (Millipore Co., Bedford, MA) were coated with 100 l ofthe appropriate mab (anti-ifn-, anti-il-4) at a concentration of 15 g/ml in PBS and incubated overnight at 4 C. All antibodies were purchased from Mabtech AB (Stockholm, Sweden). Unbound antibodies were removed by four successive washings with PBS containing 0.05% Tween. The coated wells were filled in duplicate with 100 l of RPMI medium containing 200,000 freshly isolated PBMC together with antigen [HBcAg (2 g/ml), HBcAgderived peptides (10 g/ml, tetanus toxoid (10 g/ml), PPD (10 g/ml)] or PHA (5 g/ml) as positive control and incubated undisturbed for 48 h at 37 C in a humidified atmosphere with 5% CO 2. After incubation the cells were removed by washing the plates four times with PBS. One hundred microliters of the biotin-conjugated mab (Mabtech) was added to each well at a dilution of 1 g/ml and incubated for 4 h at room temperature. Then the plates were rinsed four times by immersion in PBS and exposed to 100 l streptavidin alkaline phosphatase (Mabtech AB) for 1 h. Unbound conjugate was removed by washing thoroughly with PBS and finally 100 l of BCIP/NBT substrate solution (Bio-Rad Laboratories, Richmond, CA) was added and the reactions were stopped. The color reaction was stopped after 60 min by extensive washing under running water and after drying, the number of blue spots was scored using a dissection microscope. Stimulation with PHA was used as a positive control. PBMC proliferation assay. PBMC ( per well) were incubated in 96-well U-bottom plates from Costar (Cambridge, MA) for 5 days in the presence of HBcAg (1 g/ml), peptides (10 g/ml), TT (10 g/ml), or pha (5 g/ml) in 150 of RPMI 1640 medium. The cell cultures were labeled by incubation for 16 h with 2 Ci of [ 3 H]thymidine (sp act 80 mci/mmol; Amersham). The cells were collected and washed on filters from Dunn (Asbach, Germany) with a cell harvester (Skatron, Sterling, VA) and the amount of radioactivity incorporated into DNA was determined using a beta counter (LKB/Pharmacia, Uppsala, Sweden). Triplicate cultures were assayed routinely, and the results were expressed in mean counts per minute. The stimulation index (SI) was calculated as the ratio between the counts per minute obtained in the presence of antigen and those obtained without antigen. An SI of 3 was considered significant. By separation experiments and fluorescence-activated cell sorter (FACS) analysis, it could be demonstrated that proliferation was confined to the CD4 T cell subset (Jung et al., 1991). Statistical analysis. For the purpose of statistical analysis the results are shown as specific spots (number of spots in the presence of HBcAg or HBcAg-derived peptides minus the number of spots without the specific protein). 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