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Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037; and
Division of Clinical Virology, Karolinska Institut, Huddinge, Sweden
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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90%) (7, 8) observed in babies infected
perinatally by their HBeAg-positive mothers (4). In fact, perinatal
infection with an HBeAg-negative variant of the HBV results in acute
rather than chronic infection (9). A similar finding has been reported
in the woodchuck model of chronic infection (10). Therefore,
conservation of secretion of the HBeAg may represent a viral strategy
to guarantee persistence during vertical transmission of HBV, which is
the major source of chronic infection in endemic areas. However, HBeAg secretion is also conserved in the avian hepadnaviruses, in which in utero tolerance mechanisms are not relevant. Furthermore, adult infection with the HBeAg-negative mutant is often associated with a fulminant course of infection rather than the relatively benign acute course that characterizes most adult-onset infections with wild-type HBV (11, 12). In addition, emergence of the HBeAg-negative mutant during chronic active HBV infection can correlate with an exacerbation of liver injury and a worse prognosis (13). These observations suggest that the HBeAg may function to modulate the immune response during chronic HBV infection in the adult in addition to its effects on neonatal tolerance.
One means of examining the effects of continuous exposure of the immune
system to the secreted HBeAg has been to develop HBeAg-expressing Tg
mice. Studies in HBeAg-Tg mice revealed that the level of Th cell
tolerance was dependent upon the MHC background and Th cell site
recognized by the Tg murine strain. A proportion of Th cells of
HBeAg-Tg mice on an H-2b background (residues
129–140-specific) evade tolerance induction and can be activated in
vivo (14). This HBeAg-Tg model has provided the opportunity to examine
the immunoregulatory properties of circulating HBeAg. For example, a
single injection of the HBeAg-derived Th cell site (peptide 129–140)
into HBeAg-Tg (H-2b) mice leads to sufficient anti-HBe
"autoantibody" (autoAb) production to complex with and mask the
detection of serum HBeAg ((14), and Fig. 1
). Subsequent studies revealed that the
HBeAg-specific (i.e., self-reactive) Th cells that evade tolerance and
mediate anti-HBe autoAb production in HBeAg-Tg mice are
significantly "altered" by their coexistence with the circulating
HBeAg. The HBeAg/self-reactive Th cells surviving in HBeAg-Tg
(H-2b) mice exhibit a unique fine specificity that can be
distinguished from the HBeAg-specific Th cell repertoire of non-Tg mice
and are comprised predominantly of Th2-like cells (15). The
preferential survival of HBeAg-specific Th cells of the Th2-type in
HBeAg-Tg mice is of particular interest because of the serologic
evidence that an imbalance in HBe/HBcAg-specific Th1/Th2 cell function
may contribute to the induction and/or maintenance of persistent HBV
infection (16).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/10 (B10) (H-2b) and (B10 x B10.S)F1 mice were obtained from the breeding colony of The Scripps Research Institute. The designated HBeAg-Tg and HBcAg-Tg mice, which express either HBeAg (9–13 ng/ml) in the serum or HBcAg (0.25 ng/mg) intracellularly, respectively, were produced as described previously (5, 24). Briefly, for the HBeAg-Tg lineage the HBV-DNA fragment (subtype ayw, coordinates 1804–2804) containing the complete precore plus core open reading frame was cloned between the mouse metallothionein I promoter (Mtp) (coordinates -700 to +64) and polyadenylation recognition sequences (coordinates 930–1241), such that expression of HBeAg was controlled by the Mtp. The microinjected HBV-DNA fragment used to produce the HBcAg-Tg lineage consisted of the HBV (ayw) core sequences (coordinates 1890–2804) under the control of the mouse Mtp and polyadenylation sequences. B10 MRL-Faslpr (H-2b) mice were obtained from The Scripps Research Institute breeding colony. B6Smn.C3H-Fasgld (H-2b) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice homozygous for the gld or lpr mutation and either homozygous or hemizygous for the HBeAg transgene were produced by first crossing homozygous lpr or gld mice with HBeAg-Tg mice that were homozygous for the HBeAg transgene. The F1 mice produced were then intercrossed to produce F2 mice of the appropriate genotypes. Subsequently, mice of the desired genotype were produced by intercrossing F2 mice. Nontransgenic and Fas-FasL wild-type or heterozygous littermates were used as control mice. All mice designated as lpr or gld were homozygous for the lpr or gld mutations. The mice designated as HBeAg-Tg or HBcAg-Tg were hemizygous for the HBe/HBcAg transgenes with the exception of the HBeAg-Tg mice used for cytokine analysis, which were homozygous. (HBeAg-Tg x lpr/lpr) or (HBeAg-Tg x gld/gld) mice were designated e/lpr or e/gld.
Genotyping by PCR
Faslpr and Fas+ were typed by conventional PCR methodology (D. C. Roopenian, personal communication, The Jackson Laboratory, Bar Harbor, ME). The following oligonucleotides were purchased from Life Technologies (Grand Island, NY) and used to distinguish the Faslpr allele from the wild-type allele of Fas: FAS1, 5'-GAT TCC ATT TGC TGC TGT GT-3'; FAS2, 5'-CTT CAT AAC TGG TGT CGC AA-3'; and FAS3, 5'-CAG GGA AAT GTA GCA AGA TG-3'. Amplification was conducted in 50 µl DNA, 1.5 mM MgCl2, 1 µM each of three oligonucleotides, 100 µM each of four deoxynucleoside triphosphates, 1.25 U Taq polymerase, and cycling conditions were as follows: (94°C x 2'); (94°C x 1', 55°C x 1', 72°C x 2') x 35; and (72°C x 7'). FAS1 + FAS3 yields a 135-bp product for the Fas wild-type allele and FAS2 + FAS3 yields a 271-bp product for the lpr allele. The products were visualized on a 1.5% agarose gel.
FasLgld was distinguished from FasL+ using mutagenically separated PCR technology as described by Rust et al. (25). Primers were purchased from Life Technologies and had the following sequences: GLD1, (forward) 5'-TGC ACT ACT GGA CAG ATA TGG GCC CAC A-3'; GLD2, (normal reverse) 5'-AGA TCA TTT TAA ATT GCT TTT CTT TTT AAA GCT TAT ACA AGC CGA GAA A-3'; and GLD3, (mutant reverse) 5'-TCT TTT AAA GCT TAT ACA AGC CGA AAT G-3'. Amplification was conducted in a PTC 150 MJ Research thermal cycler (Cambridge, MA) using 0.5 ml thin-walled tubes. The 100 µl reaction volume contained 10 µl DNA, 1.5 mM MgCl2, 20 µM each of four deoxynucleoside triphosphates, 0.1 µM each GLD1 and GLD2, 0.2 µM GLD3, 2.5 U Taq-polymerase, and cycling conditions were as follows: (94°C x 1', 60°C x 1', 72°C x 1') x 38; and (72°C x 7'). GLD1 + GLD2 yields a 176-bp product for the normal gene and GLD1 + GLD3 yields a 155-bp product for the mutant form of the gene. The products were visualized on a 3.5% NuSieve GTG Agarose/1% agarose (catalogue no. 50072, FMC Bioproducts, Rockland, ME) composite gel.
Recombinant proteins and synthetic peptides
The HBV core gene encodes two polypeptides. Initiation of translation at the first start codon (AUG) results in a 25-kDa precore protein that is secreted as HBeAg after removal of 19 residues of the leader sequence and 34 COOH-terminal amino acids. Initiation of translation at the second AUG leads to the synthesis of a 183-amino acid 21-kDa protein that assembles to form particles that comprise the virion nucleocapsid (HBcAg). Although HBeAg and HBcAg are serologically distinct, these Ags are cross-reactive at the level of Th cell recognition because they are colinear throughout most of their primary sequence. rHBcAg of the ayw subtype was produced in Escherichia coli and purified as described previously (26). An rHBeAg corresponding in sequence to serum-derived HBeAg encompassing the 10 precore amino acids remaining after cleavage of the precursor and residues 1 to 149 of HBcAg was produced as described previously (27). The presence of the 10 precore amino acids prevents particle assembly, and HBeAg is recognized efficiently by HBeAg-specific mAbs but displays little HBc antigenicity (27). An aliquot of truncated HBcAg was reduced and denatured by boiling in SDS/2-ME (1.0%) and then alkylated. The SDS was removed by dialysis, and the material was resolubilized in 3 M urea. This preparation consisted predominantly of monomers (16 kDa) with some dimer formation upon nonreducing PAGE and was designated P16. P16 does not bind HBcAg- or HBeAg-specific mAbs.
Peptides were synthesized by the simultaneous multiple peptide synthesis method (28) and were kindly provided by Richard Houghten (Torrey Pines Institute for Molecular Studies, La Jolla, CA.). The following HBc/HBeAg-derived synthetic peptides representing Th cell recognition sites were used and designated by amino acid position from the N terminus of HBcAg: 129–140, PPAYRPPNAPIL; and 120–140, VSFGVWIRTPPAYRPPNAPIL.
Serology
HBeAg was measured in diluted Tg mouse sera by a commercial ELISA (HBe enzyme immunoassay; Abbott Laboratories, Chicago, IL), and rHBeAg was used as a standard. Anti-HBc and anti-HBe IgG Ab was measured in murine sera by an indirect solid-phase ELISA using rHBcAg or rHBeAg as the solid-phase ligands as described previously (29). The data are expressed as Ab titers representing the reciprocal of the highest dilution of sera required to yield an OD492 reading three times that of preimmunization sera. IgG isotype-specific ELISAs were performed using IgG1-, IG2a-, IgG2b-, and IgG3-specific second Abs (Southern Biotechnology, Birmingham, AL).
Adoptive transfer of autoAb production in HBeAg- and HBcAg-Tg mice
Non-Tg donor mice were immunized in the hind footpads either with 100 µg of the peptide T cell site 129–140 emulsified in CFA or with CFA alone, and draining popliteal lymph node (PLN) cells were harvested after 10 days and cultured with peptide 120–140 (0.2 µg/ml) or purified protein derivative for an additional 3 days. In vitro-activated PLN cells were then transferred into sublethally irradiated (500 R) HBeAg- or HBcAg-expressing Tg recipient mice. Sera were collected before and at various times after adoptive transfer and analyzed for anti-HBe and anti-HBc Abs by ELISA. In selected experiments, T cells were enriched from the PLN preparation before adoptive transfer, producing similar results. For convenience, and because donor mice were primed with and PLN cells were cultured with peptides that represent T cell recognition sites but not B cell sites (30), unselected PLN cells were routinely used to transfer HBc/HBe-specific T cell help in adoptive transfer experiments.
Determination of Ag-specific cytokine production
Groups of four HBeAg-Tg/Fas-FasL-expressing (e/+) or
HBeAg-Tg/Fas-deficient (e/lpr) mice were immunized
i.p. with rHBeAg (10 µg) emulsified in IFA, and 10 to 14 days later
spleen cells were harvested, pooled, and cultured (8 x
106/ml) with various concentrations of a series of Ags.
Culture supernatants (SN) were harvested at 24 h for IL-2
determination and at 48 h for IL-4 and IFN-
determinations.
IL-2 was measured by the ability of SN to stimulate proliferation of
the IL-2- and IL-4-sensitive NK-A cell line in the presence of mAb
11B11 specific for IL-4 (31). IL-4 was measured by the ability of SN to
stimulate proliferation of the IL-4-sensitive CT.4S cell line
(generously provided by William Paul, National Institutes of Health,
Bethesda, MD) (32). IFN- was measured by two-site ELISA using mAb
HB170 and a polyclonal goat anti-mouse IFN- (Genzyme
Corp., Boston, MA).
Tg autoAb model
Because HBeAg-expressing transgenic mice on a B10
(H-2b) background are not completely T cell tolerant,
injection of the synthetic Th cell site, 129–140, results in
anti-HBe or autoAb production (14). This Tg model is useful for
screening immunomodulatory drugs or therapies. Groups of four to five
e/+, e/lpr, or HBeAg-Tg/FasL-deficient
(e/gld) mice were injected with the peptide Th cell
site 129–140 (50 µg in IFA). The HBeAg-Tg mice were bled before
injection and at 2 wk intervals for the determination of total IgG
anti-HBe as well as isotype-specific anti-HBe Ab levels by
ELISA. Because mouse to mouse variation was minimal (
2-fold) in this
system, sera were pooled before analysis.
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Results |
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Although the MRL-lpr/lpr and
MRL-gld/gld strains are most severely affected,
other strains carrying the lpr and gld mutations
also develop lymphadenopathy and are prone to developing autoimmunity
with differing kinetics (33). Therefore, the simplistic prediction was
that HBeAg-Tg/lpr or gld mice would be more
susceptible to induced anti-HBe autoAb production as compared with
HBeAg-Tg/+ mice. In fact, quite the opposite occurred. As shown in
Figure 1
, e/lpr mice produced significantly less
anti-HBe autoAb (i.e., 16- to 32-fold less) as compared with e/+
mice during the first 12 wk after the HBeAg Th cell peptide 129–140
was injected. Thereafter, the anti-HBe autoAb titers of the e/+ and
e/lpr mice began to merge. It should be noted that by wk 32,
lymphadenopathy was beginning to become evident in e/lpr
mice. The diminished early anti-HBe autoAb production demonstrated
by e/lpr mice cannot be attributed to a generalized
hyporesponsiveness conferred by the lpr mutation, since in a
control experiment littermate non-Tg wild-type (+/+) and lpr
(+/lpr) mice differed by only 2- to 4-fold in their
anti-HBe Ab responses to immunization with recombinant HBeAg (Fig. 1, inset). It was also notable that the IgG isotype
distribution of anti-HBe Abs produced by +/+ vs +/lpr
mice was not significantly different (data not shown). This result
indicates that the lpr mutation has no general effect on IgG
isotype (see next section).
Similar experiments were performed comparing induced anti-HBe
autoAb production in HBeAg-Tg/+ mice with HBeAg-Tg/gld mice.
Groups of five e/+ or e/gld mice were injected with the
HBeAg-specific Th cell site 129–140 and anti-HBe autoAb titers
were measured by ELISA thereafter. The e/gld mice produced
from 8- to 64-fold less anti-HBe autoAb during the first 20 wk of
observation as compared with e/+ mice (Fig. 2). In view of the fact that Fas-FasL
interactions do not play a role in clonal deletion in the thymus, the
results of these two experiments in Fas- and FasL-defective HBeAg-Tg
mice suggest that Fas-FasL interactions, and by inference Th cell
apoptosis, play an important role in regulating the peripheral immune
response to the secreted HBeAg. The interpretation of the role played
by Fas in HBeAg-Tg mice is somewhat complicated by the observation that
in the absence of Fas-mediated clonal deletion of HBeAg (self)-reactive
Th cells (i.e., in e/lpr mice), anti-HBe autoAb
production decreases rather than increases. This issue is addressed in
the next section.
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One explanation for why the absence of Th cell clonal deletion in
the periphery in Fas-FasL-defective HBeAg-Tg mice results in diminished
as opposed to increased anti-HBe autoAb production would be a
preferential depletion of the Th1 subset of HBeAg-specific Th
cells by Fas-mediated apoptosis. To examine this possibility, the IgG
isotype patterns of anti-HBe autoAb produced by HBeAg-Tg/+ and
HBeAg-Tg/lpr or gld mice were determined. As
illustrated in Figure 3, the anti-HBe
autoAb response in e/+ mice induced by injection of the Th cell site
129–140 is exclusively comprised of the IgG1 isotype. Only the peak
response at 6 wk is shown; however, the predominance of the IgG1
isotype is evident from wk 2 throughout the observation period. In
marked contrast, the induced anti-HBe autoAb response in
e/lpr mice is characterized by a 16-fold reduction in the
IgG1 isotype as compared with e/+ mice and also by the production of
significant quantities of IgG2a and IgG2b anti-HBe Abs (Fig. 3).
Injection of peptide 129–140 into +/+ or +/lpr control mice
did not elicit anti-HBe autoAb production due to the absence of
endogenous HBeAg.
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). The
overall anti-HBe autoAb response in e/gld mice is
reduced and the IgG isotype pattern is broadened to include IgG2a,
IgG2b, and IgG3 as compared with the IgG1 predominance observed in e/+
mice. In the mouse, IgG1 switching is influenced by IL-4, a cytokine
predominantly produced by Th2 cells, and IgG2a and IgG3 switching are
mediated by IFN-, a cytokine produced by Th1 cells and NK cells (34, 35). Therefore, the anti-HBe IgG isotype data indicate that a mixed
population of HBeAg-specific Th1 and Th2 cells coexist in the periphery
of e/lpr and e/gld mice. In e/+ mice, the induced
anti-HBe autoAb response is almost exclusively dominated by IgG1
production, suggesting that HBeAg-specific Th1 cells are depleted by
Fas-mediated mechanisms, and HBeAg-specific Th2 cells are
spared.
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To determine whether Fas-mediated mechanisms affected
HBeAg-specific cytokine production in HBeAg-Tg mice, e/+ and
e/lpr mice were immunized with 10 µg of rHBeAg and
Ag-specific in vitro cytokine production by splenic Th cells was
measured. A panel of HBc/HBe-derived Ags was used (i.e., HBcAg; HBeAg;
P16, the structural subunit polypeptide; and peptide 120–140) to
recall Th cell cytokine production in vitro. Splenic Th cells of
HBeAg-primed e/+ mice produced IL-4 significantly more efficiently than
e/lpr HBeAg-primed Th cells recalled in vitro with HBeAg or
peptide 120–140 (Fig. 5, upper
panel). However, the comparative differences in in vitro
cytokine production between e/+ and e/lpr mice were not as
dramatic as the differences in in vivo anti-HBe autoAb production.
Due to assay variability, many of the differences observed between e/+
and e/lpr mice did not attain statistical significance;
however, these differences were reproducibly observed. For example,
when differences in splenic IFN- production were observed between
e/+ and e/lpr mice, it was always more efficient in
e/lpr mice as compared with e/+ mice (Fig. 5). No
differences were observed in Ag-specific IL-2 production between e/+
and e/lpr mice. As expected from the IgG isotype analysis,
especially in e/lpr mice, these cytokine profiles clearly
indicate the presence of mixed Th1/Th2 cell populations in the in vitro
Ag cultures. Nevertheless, the trend is consistent with the in vivo Ab
data suggesting a relative Th1-like bias in HBe/HBcAg-specific
e/lpr Th cells and a relative Th2-like bias in
HBe/HBcAg-specific e/+ Th cells. When comparing e/+ and
e/lpr mice, the greater differences observed for in vivo
anti-HBe production vs in vitro cytokine production may also
reflect the fact that for the cytokine studies exogenous HBeAg was used
for immunization. In the other studies (i.e., anti-HBe autoAb
production and adoptive transfers) endogenous/transgenic HBeAg or HBcAg
served as the source of Ag in vivo. Endogenous Ags may better mimic Ag
exposure during an HBV infection and reduce possible artifacts due to
immunization and in vitro culture.
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Because the experiments performed in e/lpr and
e/gld mice suggested that HBeAg in the serum preferentially
deleted HBeAg-specific Th1 cells, and because previous studies
indicated that HBcAg preferentially elicits Th1-like cells (36), it was
of interest to determine whether the Th1-mediated HBcAg-specific immune
response would be influenced by the presence of HBeAg in the serum. For
this purpose, peptide 129–140-primed Th cells were adoptively
transferred into either HBeAg-Tg, HBcAg-Tg, or dual HBeAg/HBcAg-Tg
recipients, and the IgG isotype profile of the anti-HBe and
anti-HBc Abs produced were determined. As depicted in Figure 6, adoptive transfer of 129–140-primed
Th cells into e/+ mice resulted in anti-HBe Ab production dominated
by the IgG1 isotype (i.e., Th2-like pattern) and no anti-HBc Abs
were produced (not shown). In contrast, adoptive transfer of the same
129–140-primed Th cells into HBcAg-Tg (+/c) mice resulted in the
production of IgG2a and IgG2b anti-HBc Abs and no IgG1 anti-HBc
Abs (i.e., Th1-like pattern). Adoptive transfer of 129–140-primed Th
cells into dual HBeAg/HBcAg-Tg (e/c) mice resulted in relatively normal
anti-HBe Ab production (i.e., IgG1-dominated). However, the most
significant observation was the dramatic effect circulating HBeAg had
on the anti-HBc response in dual Tg e/c mice. Anti-HBc production
in e/c Tg mice was almost totally ablated, and the low titer
anti-HBc Ab that was produced was composed entirely of the IgG1
isotype, which was not produced in +/c Tg recipients. These results
indicate that HBeAg circulating in the serum of e/c Tg mice inhibited
the production of the Th1 cell-dependent anti-HBc IgG isotypes and
promoted the production of Th2 cell-dependent IgG1 anti-HBc Abs. At
least two mechanisms may explain the effect of serum HBeAg on the
anti-HBc response. First, serum HBeAg preferentially depletes
HBeAg-specific Th1 cells in the periphery of HBeAg-Tg mice (Figs. 3 and 4), and it is these same HBe/HBcAg-specific Th1 cells that mediate
IgG2a anti-HBc Ab production in HBcAg-Tg mice. Second, the
preponderance of HBe/HBcAg-specific Th2-like cells surviving in the
periphery of e/+ or e/c Tg mice may down-regulate HBe/HBcAg-specific
Th1 cells by virtue of the production of cross-regulatory Th2-type
cytokines (i.e., IL-4, IL-10). It is important to note that the effects
of serum HBeAg on the Th cell repertoire are not limited to Th cells
"shaped" in an HBeAg-Tg setting. Adoptively transferred Th cells
from a non-Tg donor were efficiently skewed toward the Th2 phenotype by
circulating HBeAg in e/c Tg recipients.
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Discussion |
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(36), both of which
enhance Th1 cell differentiation. We suggest that circulating HBeAg in HBeAg-Tg mice preferentially depletes HBeAg-specific Th1-like cells in the periphery via Fas-mediated apoptosis, and that HBeAg-specific Th2-like cells survive this process to a greater degree. Reciprocally, HBeAg-specific Th1-like cells can survive in e/lpr and e/gld mice, and the resulting Th1/Th2-mixed population of HBeAg-specific Th cells mediates lower Ab production with a broader IgG isotype profile. It appears that HBeAg-specific Th1 cells can down-regulate HBeAg-specific Th2 cells in e/lpr and e/gld mice, in which HBeAg-specific Th1 cells have not been fully depleted. Therefore, much like self-reactive Th2 cells that have been proposed to be regulatory for pathogenic self-reactive Th1 cells (43, 44), it appears that autoreactive Th1 cells can regulate autoAb-inducing Th2 cells specific for a secreted self Ag.
Although the HBeAg and the HBcAg are cross-reactive at the level of Th cell recognition, these two structural forms have quite different properties. The HBeAg is a nonparticulate secreted protein and the HBcAg is a particulate intracellular protein. The HBcAg can function as a Th cell-independent Ag, whereas the HBeAg is strictly a Th cell-dependent Ag (45). Recent studies indicate that HBeAg and HBcAg use different Ag presentation pathways as well (3). These different characteristics may explain why the same Th cell population (129–140-specific) adoptively transferred into HBeAg-Tg recipients results in IgG1 anti-HBe Ab production and, to the contrary, IgG2a/IgG2b anti-HBc Ab production in HBcAg-Tg recipients. Because the immune response to HBcAg is biased toward Th1-like cells (36) and the secreted HBeAg appears to preferentially deplete Th1-like cells in the periphery, it was of interest to determine whether the presence of HBeAg in the serum would alter the immune response to the intracellular HBcAg. The results of transferring 129–140-specific Th cells into dual HBeAg/HBcAg-expressing Tg recipients were quite revealing. The presence of HBeAg in the serum ablated the characteristic Th1-like anti-HBc Ab response and promoted a Th2-like anti-HBc response (i.e., IgG1 anti-HBc), which did not occur in Tg mice expressing only HBcAg. In addition to independently confirming the ability of HBeAg to deplete Th1-like cells in the periphery, this result has important implications for understanding mechanisms of viral persistence and immunopathogenesis in chronic HBV infection.
Earlier studies suggested that exposure to the HBeAg in utero may
establish Th cell tolerance to the HBe/HBcAgs and promote chronicity
upon perinatal infection by an HBeAg-positive mother (4, 5). This
current study suggests an additional mechanism by which the HBeAg may
maintain or induce chronicity even during an adult infection. The fact
that HBeAg is secreted and widely disseminated coupled with its ability
to deplete HBe/HBcAg-specific Th1-like cells and spare Th2-like cells
make it a likely candidate to promote viral persistence. HBeAg-specific
Th2-like cells produce anti-inflammatory cytokines (i.e., IL-4,
IL-10) that would be expected to inhibit the expansion of
HBe/HBcAg-specific CTL and Th1 effector cells necessary for the
clearance of this noncytolytic virus. Recent serologic evidence
suggests that a Th1/Th2 subset imbalance in favor of HBe/HBcAg-specific
Th2 cells may play a role in promoting chronic HBV infection (16).
Furthermore, recent studies of cytokine production in liver-derived Th
cells indicated that Th0-like cells were predominant in chronic HBV
patients, Th1-like cytokines were associated with increasing hepatitis
activity, and Th2-like cytokines were associated with decreasing liver
disease during chronic HBV infection (46, 47). Because both HBcAg and
HBeAg are produced during a wild-type HBV infection and because
HBc/HBeAg-specific Th1 and Th2 cells can be cross-regulatory (Fig. 6),
a dynamic balance between these Th cell subsets may exist during an HBV
infection. Alterations in the HBc/HBeAg-specific Th1/Th2 cell balance
would be expected to influence the course of the infection both in
terms of liver injury and viral persistence. Interestingly, emergence
of an HBV mutant that does not produce the HBeAg during chronic active
HBV infection often results in an exacerbation of liver injury and a
worse prognosis (13). Similarly, adult infection with the
HBeAg-negative HBV mutant can be associated with severe fulminant
hepatitis (11, 12). Therefore, the loss or absence of serum HBeAg can
be associated with increased inflammation and liver injury. Both of
these clinical observations are consistent with the hypothesis that the
secreted HBeAg may preferentially deplete inflammatory
HBc/HBeAg-specific Th1 cells and bias the Th1/Th2 balance toward the
anti-inflammatory Th2 phenotype as suggested by this Tg model.
Interestingly, human HBeAg-specific CD4+ Th1 clones, but
not Th0 clones, can be anergized in vitro by stimulation with specific
peptides (48). Secretion of the HBeAg may represent a viral strategy to
promote or maintain a long term infection without eliciting an overly
destructive immune response that would eliminate the virus and/or kill
the infected host.
Several forms of Th1/Th2 cell cross-regulation were observed in this Tg model system. 1) In the absence of Fas activity (i.e., in e/lpr and e/gld mice), HBeAg-specific Th1 cells down-regulate HBeAg-specific Th2 cells resulting in lower anti-HBe Ab production and induction of the IgG2a, IgG2b, and IgG3 isotypes of anti-HBe. 2) In the presence of Fas activity (i.e., in e/+ mice), HBeAg-specific Th2 cells preferentially survive Fas-mediated depletion in the periphery and predominate over HBeAg-specific Th1 cells. 3) As demonstrated by the adoptive transfer of 129–140-specific Th cells into dual HBeAg/HBcAg-Tg recipients, the circulating HBeAg preferentially depletes or alters the function of the Th1 cells that would otherwise mediate IgG2a and IgG2b anti-HBc Ab production. Therefore, at the HBeAg concentration in the serum and the level of HBcAg expression in the liver of the dual HBeAg/HBcAg-Tg mice in this study, HBeAg-specific Th2 cells predominated over HBcAg-specific Th1 cells. Of course, this balance may shift depending on the relative concentrations of the two Ags. These results indicate how dynamic the balance between HBc/HBeAg-specific Th1 and Th2 cells can be even in this noninfectious Tg system. Similar variables and perhaps many more that are unique to an infectious system may influence the HBc/HBeAg-specific Th1/Th2 cell balance during an HBV infection.
Although most of the conditions examined in this study favored
the predominance of HBeAg-specific Th2-like cells, the HBeAg also has
the potential to elicit Th1-like cells (36, 42) and serve as a CTL
target (49, 50). The immune function of HBeAg cannot be viewed only in
the context of in utero tolerance and perinatal infection. For example,
during an adult acute HBV infection, in which in utero T cell tolerance
is not relevant, production of IL-12 and the family of IFNs may be
sufficient to bias the HBc/HBeAg-specific immune response toward the
Th1 phenotype. However, even in the case of adult infection, it appears
that secretory HBeAg may still modulate the vigor of the inflammatory
response to some degree in view of the observation that adult onset
infection with the HBeAg-negative mutant often results in fulminant
hepatitis (11, 12). During a chronic HBV infection, HBeAg-specific Th
cell tolerance may wane with age (5), allowing the emergence of
Th1-like cells. Even in the setting of HBeAg-Tg mice, in vivo treatment
with either IL-12 or IFN- can shift the HBeAg-specific Ab response
from Th2 to Th1 cell predominance (36, 42). Therefore, the context of
the exposure to HBeAg may be important in determining the effect
secretory HBeAg will have on the outcome of an acute or chronic HBV
infection (see Fig. 7). During the
so-called "tolerance phase" of chronic HBV infection, secretory
HBeAg would be expected to bias the HBc/HBeAg immune response toward
the Th2 phenotype and help to maintain viral persistence. However, in
the injury phase of chronic HBV infection, when by definition
HBeAg-specific tolerance has been broken, the HBeAg-specific Th1/Th2
cell balance may shift toward the Th1 phenotype and the HBeAg may serve
as an additional target for CTL recognition as well. A number of
factors may contribute to a time-related shift in HBeAg-specific Th
cells from a Th2-like to a Th1-like phenotype during chronic HBV
infection including: the waning of HBeAg-specific Th1 cell tolerance
with age; neutralization of circulating HBeAg by anti-HBe resulting
in less efficient Fas-mediated Th1 cell depletion in the periphery;
release of HBcAg from hepatocytes, thereby increasing the role of
HBcAg-specific Th cells that tend to be of the Th1 phenotype; and
increased production of IL-12 and the family of IFNs that promote Th1
cell differentiation.
|
). This view is
consistent with the accumulation of multiple mutations within the
precore and core gene regions in chronic HBV patients with liver injury
and the relative absence of precore and core region mutations in
chronic HBV patients without liver injury (13, 51, 52, 53, 54). This hypothesis
also explains the apparent paradox of how an HBeAg-negative mutant can
become predominant during a chronic HBV infection if circulating HBeAg
promotes viral persistence. Serum HBeAg only promotes viral persistence
in the context of a predominant Th2-like HBeAg-specific Th cell
repertoire. In summary, this HBeAg-Tg model illustrates that the secretion of a viral protein (i.e., HBeAg), which may preferentially deplete inflammatory Th1 cells, may represent a viral strategy to promote persistence. Furthermore, skewing of the Th1/Th2 cell balance specific for one viral Ag (i.e., HBeAg) can effect the immune response to another viral Ag (i.e., HBcAg) via cross-regulatory mechanisms. Infections with HBV as well as HIV are characterized by the secretion of excess envelope proteins (i.e., hepatitis B surface Ag and gp120, respectively). It is anticipated that secretory HBsAg may also exert important immunomodulatory effects on Th1/Th2 cell subset distribution during chronic HBV infection as has been proposed for gp120 during chronic HIV infection (37).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David R. Milich, Department of Molecular Biology, CAL-2, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: HBV, hepatitis B virus; HBcAg, hepatitis B core Ag; HBeAg, hepatitis B precore Ag; Tg, transgenic; AIA, activation-induced apoptosis; SN, supernatant; P16, the HBc/HBeAg subunit polypeptide; FasL, Fas ligand; Mtp, metallothionein I promoter; PLN, popliteal lymph node; autoAb, autoantibody; C57BL/10, B10; e/+, HBeAg-Tg/Fas-FasL-expressing; e/lpr, HBeAg-Tg/Fas-deficient; e/gld, HBeAg-Tg/FasL-deficient; +/+, non-Tg wild-type (non-Tg/Fas-expressing); +/lpr, non-Tg lpr (non-Tg/Fas-defective); +/c, HBcAg-Tg; e/c, dual HBeAg/HBcAg-Tg.
Received for publication April 30, 1997. Accepted for publication October 27, 1997.
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