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-Secreting T Cells After DNA-Based Immunization1
*
Unité de Recombinaison et Expression Génétique, Institut National de la Santé et de la Recherche Médicale, U163, Institut Pasteur, Paris, France; and
Centre de Recherche, Institut National de la Santé et de la Recherche Médicale, U347, Le Kremlin-Bicètre, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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by the activated T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA-based immunization may contribute to immunotherapeutic strategies
by inducing both cellular and humoral responses. This novel method of
immunization refers to the induction of an immune response to a protein
expressed in vivo from a gene introduced directly in the form of pure
plasmid DNA (2, 3). To date, DNA-based immunization has been found to
result in a broad range of immune responses, including neutralizing Abs
as well as cytotoxic and Th cell responses. It was shown recently that
immunization with plasmid DNA vectors can induce a strong bias toward a
Th1 response in animals (4). Th1-type cytokines such as IL-2 and
IFN- are associated principally with cell-mediated immune responses
and play a crucial role in protection from intracellular pathogens,
including viruses. In HBV infection, it has been suggested that an
imbalance between Th2 and Th1 responses may contribute to chronicity
(5).
As a model for the HBV chronic carrier state, we have used transgenic
(Tg) mice that constitutively express hepatitis B surface Ag (HBsAg)
(6). The transgene in these mice consists of a copy of the HBV genome
with the core gene deleted. The sequences encoding the small, middle,
and large HBV envelope proteins under the control of an endogenous HBV
promoter are expressed principally in the liver from before birth (7).
As observed for chronic HBV carriers, large amounts of subviral
particles are present in the sera. In this model, we have shown
previously that the immune response induced after a single i.m.
injection of DNA results in the complete clearance of circulating HBsAg
and in the long-term control of transgene expression in hepatocytes
(8). This response did not involve a detectable cytopathic effect in
the liver. We now report, using adoptive transfer of fractionated
primed spleen cells from non-Tg DNA-immunized mice, that both
HBs-specific CD4+ and CD8+ T lymphocytes are
involved in the regulation of the HBV mRNA expressed in the livers of
Tg mice. These T cells operate without major cytopathic effects. These
results suggested that both CD4+ and CD8+ T
cells act by a common mechanism. Following in vitro antigenic
stimulation, both subpopulations secreted type 1 cytokines, especially
IFN-. The role of this cytokine was confirmed using HBV Tg mice
carrying the null mutation for the IFN-R gene. Thus, by
inducing a strong bias toward a Th1 immune response, DNA-based
immunization could represent an alternative approach for the treatment
of individuals chronically infected with HBV.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation and characterization of the HBV envelope Tg mouse
lineage E36 has been reported previously (6, 7). These Tg mice were
produced on a C57BL/6 x SJL/J background and were backcrossed
against C57BL/6 (H-2b) 
20 times before use. Only
5–8-wk-old female mice that were heterozygous for the HBV envelope
transgene (ayw subtype) and their non-Tg littermates were
used. The Tg mice sera contain 200–2000 ng/ml of HBsAg; their livers
exhibit no pathology.
The construction of the IFN-R knockout (IFN-R0/0)
mice has been described previously (9). Briefly, these mice are inbred
129/Sv (H-2b) animals for which the IFN-R has been
inactivated by homologous recombination. The mice were mated with the
HBV Tg mouse lineage to yield heterozygous mice
(Tg/IFN-R+/0). These animals were subsequently interbred
with IFN-R0/0 mice to yield Tg mice homozygous for the
null mutation of the IFN-R gene (Tg/IFN-R0/0).
DBA/2 and C57BL/6 mice were purchased from Iffa-Credo (Lyon, France). All experiments involving mice were conducted in accordance with institutional guidelines.
DNA immunization
Mice were injected on a single occasion with 100 µg of recombinant plasmid DNA expressing the S and preS2 domains of the gene encoding the HBV envelope proteins (pCMV-S2.S) (10) or the Escherichia coli LacZ gene encoding ß-galactosidase (pCMV-LacZ) (11). DNA was purified by anion-exchange chromatography (Endofree Plasmid Kit, Qiagen, Hilden, Germany) and was injected directly into regenerating tibialis anterior muscles as described previously (12). Purified DNA contains only negligible amounts of endotoxin (<0.1 endotoxin unit/µg plasmid DNA).
Serology
Blood was collected from anesthetized mice by retrobulbar puncture using heparinized glass pipettes; HBsAg was measured in the plasma using a commercial ELISA kit (Monolisa AgHBs; Diagnostics Pasteur, Marnes la Coquette, France). Quantitation of mouse anti-hepatitis B surface Ab (HBsAb) was performed by ELISA (13). The serum alanine aminotransferase activity in the plasma was measured using a commercial kit (Enzyline, BioMérieux, Lyon, France).
Histologic procedures
Tissues were fixed in alcoholic Bouin’s fixative. Sections of paraffin-embedded tissues were cut at 5-µm thickness and stained with hematoxylin-eosin and trichrome.
Northern Blot analysis
The total RNA in the liver was extracted from mechanically pulverized frozen tissue by RNA-Plus (Bioprobe, Montreuil-sous-Bois, France). The RNA (40 µg) was fractionated on 1% formaldehyde-agarose gels and blotted onto nylon membranes; next, membranes were hybridized with 32P-labeled DNA probes synthesized from HBV DNA fragment or from a 0.2-kb PstI cDNA fragment of the murine 18S ribosomal RNA gene (Valbiotech, Paris, France) using the Rediprime DNA labeling system (Amersham, Les Ulis, France).
Adoptive transfer of spleen cells
Splenocytes obtained from individual naive or primed female mice
were prepared for adoptive transfer by lysis of RBCs in a single-cell
suspension (incubation with Tris-buffered ammonium chloride for 5 min
at 4°C). After four washes with RPMI 1640 medium (Life
Technologies, Cergy Pontoise, France), the remaining white blood cells
were counted and resuspended in 200 µl of PBS. Each recipient mouse
was injected with cells obtained from a single spleen. For the transfer
of subpopulations, 
3–6 x 106 cells were injected
into the retroorbital cavity of recipient mice that had been
sublethally irradiated (5 Gy) at 2 h before cell transfer.
Lymphocyte subset fractionation
T cell subpopulations were isolated from the total spleen cell population using CD4/CD8 subset column kits (R&D Systems, Abingdon, U.K.). The purity of the CD4+ and CD8+ lymphocytes was confirmed by cytofluorometry analysis using a FACScan (Becton Dickinson, Le Pont de Claix, France). The percentages of undesired cell subsets in the enriched cell population are <4% for CD4+ T cells, <1% for CD8+ T cells, and <6% for B cells.
CTL generation in mice
Groups of mice were immunized with pCMV-S2.S DNA vector. Spleens
were removed from immunized mice at 8–12 wk postinjection, and
single-cell suspensions were prepared. Cells (107
cells/well) were suspended in 2 ml of 
-minimum essential
medium tissue culture medium supplemented with 10 mM HEPES
buffer, 1 mM sodium pyruvate, nonessential amino acids, 5 x
10-5 M ß-mercaptoethanol, antibiotics, and 10% FCS
(Life Technologies) in 24-well plates. Responder spleen cells from
C57BL/6 or DBA/2 mice were stimulated with 106 irradiated
RBL5/S or P815/S transfectants, respectively, expressing the small
envelope protein (14). After 5 days in culture, one-half of the medium
was replaced with fresh medium; cells were used as effectors in a
standard chromium release assay that was performed 2 or 3 days later.
Targets were RBL5 or P815 cells infected with recombinant vaccinia
virus expressing the HBV envelope and labeled with 51Cr
(3.7 MBq/106 cells, reference number
CJS4, Amersham). After a 4-h incubation at 37°C, 50 µl of
the supernatant was removed from each well and counted on a beta
counter as described previously (15). The percentage of specific
release was calculated as follows [(experimental release -
spontaneous release)]/[(total release - spontaneous release)]
x 100. Total release was measured by resuspending target cells
in lysis buffer. Spontaneous release was obtained from targets
incubated with medium alone and is usually <15% of the total release.
T cell proliferation assay
Spleen cell suspensions from pCMV-S2.S-immunized mice were cultured in triplicate using 96-well round-bottom plates at 5 x 106 cells/ml in 200 µl of RPMI 1640 medium (Life Technologies) containing 2% mouse sera with different concentrations of either HBsAg particles (3, 1, 0.3, and 0.1 µg/ml), preS2 synthetic peptides (10, 3, 1, and 0.3 µg/ml), or medium alone. A set of peptides covering the entire preS2 domain of HBV (ayw subtype) was used to detect minimal epitopes recognized by activated T cells. Spleen cells were stimulated for 96 h, and [3H]thymidine (reference number TRK 120, specific activity 25 Ci/mmol; Amersham) was added (1 µCi/well). Cells were incubated for an additional 18 h, and the [3H]thymidine incorporation into DNA was measured after harvesting.
Depletion of CD4+ or CD8+ cells of murine spleen cells was achieved by high-gradient magnetic cell sorting using a MidiMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) (16). Briefly, CD4+ or CD8+ cells were magnetically labeled with CD4 (L3T4) or CD8a (Ly-2) microbeads and passed through a separation column that had been placed in the magnetic field of a magnetic-advanced cell sorting separator. The effluent fraction was collected as the depleted fraction. The percentage of undesired T cells was <2%.
Cytokine assay
In vivo-primed spleen cells were cocultured with Ags in parallel
with the proliferation assays. Culture supernatants were collected each
day, and concentrations of IL-2, IL-4, IFN-, and TNF- were
determined by ELISA using commercial kits (Genzyme, Cambridge, MA). For
these assays, the limit of detection was 5 pg/ml of IL-2, IL-4, or
IFN- and 15 pg/ml of TNF-.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both CD4+ and CD8+ T cells control transgene expression
Fully immunocompetent C57BL/6 non-Tg mice were immunized once with
100 µg of pCMV-S2.S DNA vector. Total T cells and CD4+ or
CD8+ T cells were purified from spleens at 8–12 wk after
DNA injection. Tg recipient mice were immunosuppressed by sublethal
total body irradiation to confine the analysis to mechanisms expressed
by the donor cells alone. Injection of the primed CD8+ T
cell suspensions obtained from pCMV-S2.S-immunized non-Tg donor mice
into Tg littermate recipients resulted in a rapid clearance of
circulating HBsAg in the absence of Ab production. The HBsAg became
undetectable in the sera from 12–33 days posttransfer, depending upon
the initial HBsAg concentration in the serum (Fig. 1
, left panel). This
regulation required 106 CD8+ T cells to
obtain the clearance of the circulating HBsAg (data not shown).
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, right panel). For the recipient Tg mice having the
highest HBsAg concentration in the sera before transfer, complete
clearance was slightly delayed. In mice displaying faster Ag clearance,
FACS analysis of the transferred cells showed that contaminant
CD8+ T cells did not exceed 3 x 104. This
finding corresponds to a number of CD8+ T cells that we
have shown by dilution experiments not to be sufficient to clear the
HBsAg (see above) and indicates that the transferred CD4+ T
cells by themselves are responsible for the observed decrease in HBsAg.
We subsequently investigated the effect of the purified T cell
subpopulations on HBV mRNA expression in the livers of Tg mice.
As we have reported previously, adoptive transfer of
HBsAg- but not ß-galactosidase-primed spleen cells
was able to induce a complete disappearance of HBV mRNA in livers taken
from 17 days posttransfer (Fig. 2,
lanes 2 and 3, respectively) up to 5 mo (8). This
effect is mediated by pCMV-S2.S-primed T cells, because the transfer of
these cells in the absence of B cells is sufficient to completely
abolish the detection of HBV mRNA in the livers of recipient Tg mice
(Fig. 2, lane 4). Most importantly, both purified
HBsAg-primed CD8+ and CD4+ T cells were able to
reduce HBV mRNA in the livers of Tg mice to an undetectable level by 33
days after adoptive transfer (Fig. 2, lanes 6–8 and
10–12, respectively). Since we could not correlate the
observed decrease of HBV mRNA in the liver to a hypermethylation of the
transgene and to a subsequent switch-off of gene expression (data
not shown), this indicates that each of the T cell subpopulations
was responsible for the down-regulation of the transgene
expression.
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C57BL/6 mice have been classified as a nonresponder haplotype to
HBsAg at the CTL level in response to immunization with different
exogenous or virally expressed HBsAg preparations. Nevertheless, this
nonresponse can be circumvented after in vivo priming using DNA-based
immunization (17). Spleens from C57BL/6 non-Tg mice immunized by a
single injection of pCMV-S2.S were removed at 8 wk postinjection;
primed T cells were tested for their lytic activity in vitro.
Spleen cells restimulated in vitro with HBsAg-expressing transfected
cells (RBL5/S) were able to specifically lyse autologous target cells
(RBL5) infected with recombinant vaccinia virus expressing the HBV
envelope proteins but not target cells infected with a wild-type
vaccinia virus (Fig. 3A,
left).
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50 days posttransfer. Histologic
examination of livers showed few necrotic foci only on some sections
taken 10–17 days posttransfer (Fig. 3A, right).
Because the lytic activity of H-2b-restricted
CD8+ T cells is not very strong in vitro, we decided to use
T cells derived from H-2d mice, which are described as good
responders at the CTL level after DNA-based immunization (17). Using a
single pCMV-S2.S DNA injection, HBs-specific CTL precursors were
efficiently primed in DBA/2 mice, since in vitro-restimulated spleen
cells efficiently lysed autologous target cells (p815) infected with
recombinant vaccinia virus expressing the HBV envelope protein (Fig. 3B, left). The CTL populations generated in
H-2d mice expressed a higher specific cytolytic reactivity
than the CTL populations from H-2b mice immunized with the
same vector (compare Fig. 3, A and B, left
panel).
Injection of HBs-primed precursor T cells derived from the spleens of DBA/2 mice immunized with pCMV-S2.S and displaying in vitro cytolytic activity were injected directly into F1 hybrid Tg mice that had been backcrossed one generation against DBA-2 mice before transfer (H-2bxd mice). This resulted in a mild and transient increase in the serum glutamic-pyruvic transaminase activity observed only 1 day after transfer (n = 3, pretransfer 29 ± 9 U/L; posttransfer 79 ± 15 U/L).
To further increase the number of specific lytic T cells, spleen cells
derived from DBA-2 immunized mice were restimulated in vitro with
HBsAg-expressing transfectant cells before transfer. Bulk CTL lines
(9 x 106) restimulated in vitro two times with p815/S
transfectant (>70% CD8+ cells and 13% CD4+
cells) were injected i.v. into H-2bxd Tg mice;
transaminase activity was monitored in the sera at different timepoints
posttransfer. Serum glutamic pyruvic transaminase activity was slightly
elevated after transfer (n = 3, 207 ± 45 U/L at
day 1; 99 ± 21 U/L at day 2) to above normal values (22 ± 3
U/L), but this activity returned to basal level after 3 days.
Histologic examination of thin liver sections taken sequentially from
Tg recipients showed only few small foci of mononuclear cells and
acidophilic bodies at 2 days posttransfer (Fig. 3B,
right). This observation correlates well with the mild
elevation in transaminase levels. These results indicate that in our
model, although the transferred CD8+ T cells exhibit
cytolytic activity in vitro, their major effector function in vivo
seems to be the control of transgene expression rather than the
killing of hepatocytes.
T cells primed by pCMV-S2.S immunization have a Th1 phenotype
Because the transfer of purified HBs-primed CD4+
T cells also induced the clearance of HBsAg and the control of HBV mRNA
in the livers of the transferred Tg mice, it was important to
characterize the function and specificity of the HBs-primed
CD4+ T cells in vitro. Spleen cells that were harvested
from C57BL/6 non-Tg mice at 3–5 wk after pCMV-S2.S immunization and
cultured in the presence of HBsAg particles or peptides derived from
the preS2 domain of the HBV envelope middle protein are specifically
stimulated in vitro (Fig. 4). The fine
specificity of the Th cells was determined using a panel of synthetic
peptides (Fig. 4A). The three preS2-overlapping peptides
(109–134, 124–148, and 139–163) used to cover the entire preS2
domain are recognized by T cells. A shorter peptide (126–138) was also
recognized by T cells to the same extent and defined a Th epitope
generated by DNA-based immunization in C57BL/6 mice. This peptide is
included in a larger sequence that has been shown previously to induce
T cell proliferation in C57BL/6 mice (18). Depletion experiments showed
that only CD4+ T cells but not CD8+ T cells are
induced to proliferate after in vitro stimulation with these Ags (Fig. 4B).
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and TNF- in the absence of IL-4 was observed. This pattern
was not affected by the depletion of CD8+ T cells (Table I). Similarly,
CD4+-depleted primed splenocytes displaying lytic activity
in vitro (data not shown) also produced Th1 cytokines after stimulation
with irradiated RBL5/S cells presenting HBV envelope peptides (Table I). These results are in agreement with the isotype profile of secreted
IgG present in the sera of pCMV-S2.S-immunized C57BL/6 mice (8, 19).
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R are not susceptible to HBV mRNA
down-regulation by T cells
Since one of the major cytokines produced by pCMV-S2.S-primed T
cells is IFN-, we decided to investigate the possible role of this
cytokine in the down-regulation of HBV mRNA. For that purpose,
we used mice with a deficient IFN-R (IFN-R0/0), which
provides an excellent in vivo model to study the regulatory function of
endogenous IFN- (9). The HBV Tg mouse lineage was mated with
IFN-R0/0 mice to yield Tg mice that were heterozygous
(Tg/IFN-R+/0) or homozygous for the null mutation of the
IFN-R gene (Tg/IFN-R0/0). The HBsAg in the sera from
most of the Tg/IFN-R0/0 mice was not eliminated after
immunization with pCMV-S2.S DNA (Fig. 5A). Nevertheless, a decrease
was observed in half of the mice at 4 wk postimmunization; 2 of 10 mice
almost completely cleared HBsAg in 12 wk. In contrast, in five of five
heterozygous mice (Tg/IFN-R+/0), a single injection of
this plasmid DNA was sufficient to decrease HBsAg titers in 2 wk and to
clear the Ag in 4–8 wk (Fig. 5A). Since the clearance of
HBsAg correlates in the HBsAg Tg mice with the appearance of Abs (Fig. 5B, 8 , we checked the ability of
IFN-R0/0 mice to mount an Ab response. Following
pCMV-S2.S DNA immunization, anti-HBsAbs were induced in
non-Tg/IFN-R0/0 mice at a level and with a kinetic that
were comparable with those induced in C57BL/6 mice (8), whereas only
low levels of anti-HBsAbs were detectable in the sera of the
Tg/IFN-R0/0 mice (Fig. 5B). The lower Ab
titers in the Tg/IFN-R0/0 mice as compared with the
non-Tg/IFN-R0/0 mice are due to the persistence of Ag in
the serum and to the formation of Ag/Ab complexes. This finding
indicates that, despite the efficient production of hepatitis B
surface-specific Abs by IFN-R0/0 mice that could clear
the Ag, the presence of a functional receptor is required for the
long-term elimination of HBsAg. At 12 wk after pCMV-S2.S immunization,
Northern blot analysis of total RNA from the livers of
Tg/IFN-R0/0 immunized mice showed that HBV mRNA,
although variable from animal to animal, was clearly detectable (Fig. 6, lanes 7 and 9)
and was decreased in the livers of mice that partially cleared (Fig. 6, lane 8) the envelope Ag from their serum. In contrast, HBV
mRNA remained undetectable in the livers taken at the same time from
pCMV-S2.S-immunized Tg/IFN-R+/0 mice (Fig. 6, lanes 5 and 6). This observation indicates that
in these mice, the control of HBV mRNA in the liver operates primarily
via signaling through a functional IFN-R, whereas the decrease of
the HBsAg and HBV mRNA observed over time in some of the knockout mice
may involve other TH1 cytokines such as TNF- that could also play a
role in this down-regulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The
lytic activity of CD8+ T cells played little or no role. A single i.m. injection of the plasmid pCMV-S2.S encoding the HBV small and middle envelope proteins was shown previously to be sufficient to break B and T cell unresponsiveness to these antigenic proteins in Tg mice, which express the same envelope coding sequences in their livers. In the experiments reported here, we used animals that had a complete immune response as a source of donor cells to see which populations were capable of regulating transgene expression. In the non-Tg mice after pCMV-S2.S DNA immunization, the HBs-specific CD8+ T cells displayed lytic function in vitro on autologous cells expressing endogenously synthesized HBsAg, whereas the HBs-specific CD4+ T cells specifically recognized HBsAg particles as well as specific peptides derived from the preS2 part of the HBV middle protein. When assessed in vivo for their effector functions in Tg mice, these two subpopulations of T cells efficiently cleared the HBsAg from the sera and controlled hepatic transgene expression via a noncytopathic mechanism. Adoptive transfer experiments of each of these subpopulations resulted in the elimination of the transgene expression product from the sera of the Tg recipient mice; this elimination was first detected at 12 days posttransfer. The rate of clearance could be accelerated to appear by 7 days by cotransfer with pCMV-S2.S-primed B cells (8), because of the synergistic effect of the anti-HBsAbs. Northern blot analysis of total HBV mRNAs from the livers of the recipient Tg mice revealed that HBV mRNA levels were reduced to undetectable levels by 1 mo after transfer of either CD4+ or CD8+ HBs-specific T cells. Nevertheless, HBV mRNAs were still detectable by RT-PCR amplification (data not shown), suggesting that transgene expression is not extinguished but rather controlled. We cannot rule out that all aspects of the immune response are not identical in Tg mice compared with non-Tg mice, or that all parts of the immune response are not activated in Tg mice postimmunization. Nevertheless, passive transfer in recipient Tg mice or direct immunization resulted in loss of HBsAg and mRNA to a similar degree.
The finding that the kinetics of Ag clearance in mice receiving either
CD4+ or CD8+ T cells were similar, and that
these populations appeared equally effective in transferring the
mechanisms of viral RNA elimination, suggested that a function common
to both T cell subsets may be involved. It was demonstrated previously
in other Tg lineages that HBV gene expression was extremely sensitive
to Th1 cytokines (20). In our model, the main cytokines produced by the
activated T cells after pCMV-S2.S immunization were IFN- and
TNF-. This result is in agreement with previous reports showing that
DNA-based immunization is a powerful means of inducing a strong Th1
response in mice (4). IFN- has the potential to influence viral
infections by a number of mechanisms (reviewed in 21 . IFN-
affects different aspects of the specific immune response through the
up-regulation of class I and class II MHC expression (22) and enhances
the expression of the MHC-linked proteins involved in intracellular
peptide Ag processing for presentation by the class I Ag presentation
pathway (23). In our model, evidence to support an IFN-mediated
mechanism was derived from experiments performed in mice with a
disrupted IFN-R gene. The persistence of HBsAg in the sera and the
sustained expression of the transgene in the livers of most of the
pCMV-S2.S-immunized Tg/IFN-R0/0 mice confirmed that this
cytokine plays an important role in the efferent phase of the immune
response to HBV.
No pathogenic effector functions of the HBV-specific T cells primed
after DNA-based immunization were found after adoptive transfer of T
cell subsets in our Tg lineage. This is in contradiction with another
report (24) in which the transfer of a CD4+ T cell clone
displaying a Th1 phenotype and cytolytic activity resulted in
necroinflammatory liver disease in two different transferred HBV Tg
lineages. Several differences could account for these discrepancies.
The number of transferred HBV-specific T cells and consequently the
amount of secreted cytokines (i.e., IFN- and TNF-) following Ag
stimulation could account for the recruitment of T and non-T cells
observed in the liver. In our model, the number of transferred
HBV-specific T cells is much lower and is equivalent to the number
present in a single spleen postimmunization. Even if DNA-based
immunization is powerful enough to induce strong and sustained class I-
and class II-restricted T cell responses, this effect cannot be
compared with the transfer of a clonal population. In addition,
we detected very rare infiltrates in the liver after adoptive transfer,
indicating that the T cells did not need to reach the liver to be
activated. The HBsAg present in large amounts in the sera of the Tg
mice is sufficient to activate the transferred T cells in the lymphoid
organs and to induce these cells to secrete cytokines. Another
difference between the two models include the genetic background of the
Tg lineage. Finally, the level and the site of Ag expression could
greatly influence the sensitivity to IFN-, because considerable
differences in the severity of IFN--induced liver disease were
observed in the two lineages used in this study (24).
At present, little is known about the balance between the limitation of viral infection and the destruction of liver tissue in viral hepatitis. Th1 responses have been reported to be associated with the capacity of the host to resolve or control viral infections (25, 26, 27). Because Th1 cells and their cytokines are involved in delayed hypersensitivity, which is a primary defense against intracellular pathogens, the prevalent Th1 pattern of secreted cytokines can be regarded not only as a mechanism contributing to inflammation but also as an appropriate response of the immune system to hamper viral expression and eventually lead to viral elimination.
|
Acknowledgments |
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receptor knockout
mice, Dr. M. E. Major for p815/S transfectants, and Dr.
J. Reimann for RBL5 and RBL5/S cells. We also thank Yves
Rivière and Katia Schlienger for helpful discussions and critical
reading of the manuscript. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Marie-Louise Michel, Unité de Recombinaison et Expression Génétique, Institut National de la Santé et de la Recherche Médicale U163 Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris cedex 15, France. E-mail address:
3 Abbreviations used in this paper: HBV, hepatitis B virus; Tg, transgenic; HBsAg, hepatitis B surface Ag; HBsAb, hepatitis B surface Ab.
Received for publication April 20, 1998. Accepted for publication July 13, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor. Science 259:1742./ß interferon down-regulate hepatitis B virus gene expression in vivo by tumor necrosis factor-dependent and -independent pathways. J. Virol. 68:1265.: implication for antigen processing. Proc. Natl. Acad. Sci. USA 89:4928.. Virology 202:76.[Medline]
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).
), 5 Tg/IFN-
) were
immunized once by i.m. injection of 100 µg of pCMV-S2.S. A group of
five Tg/IFN-