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J Virol, August 1998, p. 6637-6645, Vol. 72, No. 8
Department of Microbiology, Mount Sinai
School of Medicine, New York, New York 10029
Received 16 March 1998/Accepted 18 May 1998
During secondary immune responses to influenza virus,
virus-specific T memory cells are a major source of gamma
interferon (IFN- Gamma interferon (IFN- Previous studies revealed protective roles for IFN- Most cases of influenza virus infection throughout the human population
are, in fact, reinfections with drift or shift variants, and only a
minority of them are primary infections. There is ongoing emergence of
new shift variants subsequent to gene reassortment among strains of
different subtypes. Consequently, influenza virus poses a continuous
danger of morbidity and mortality for primed and naive human
populations, since the immune memory is limited to cross-reactive
T-cell epitopes located on the more conserved internal proteins.
Since a major source of IFN- Mice.
Mice bearing nonfunctional IFN- Viruses and immunization.
Influenza viruses A/PR/8/34 (H1N1)
and A/HK/68 (H3N2) were grown in 10-day-old embryonated hen eggs
incubated at 37°C. The allantoic fluid was harvested 48 h later
and stored at
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Protective Role of Gamma Interferon during the
Recall Response to Influenza Virus
and
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
). We assessed the contribution of IFN- to
heterologous protection against the A/WSN/33 (H1N1) virus of wild-type
and IFN-
/ mice previously immunized with the A/HK/68 (H3N2)
virus. The IFN-/ mice displayed significantly reduced survival
rates subsequent to a challenge with various doses of the A/WSN/33
virus. This was associated with an impaired ability of the IFN-/
mice to completely clear the pulmonary virus by day 7 after the challenge, although significant reduction of the virus titers
was noted. However, the IFN-/ mice developed type A influenza
virus cross-reactive cytotoxic T lymphocytes (CTLs) similar to the
wild-type mice, as demonstrated by both cytotoxicity and a
limiting-dilution assay for the estimation of CTL precursor frequency.
The pulmonary recruitment of T cells in IFN-/ mice was not
dramatically affected, and the percentage of CD4+ and
CD8+ T cells was similar to that of wild-type mice. The T
cells from IFN-/ mice did not display a significant switch
toward a Th2 profile. Furthermore, the IFN-/ mice retained the
ability to mount significant titers of WSN and HK virus-specific
hemagglutination-inhibiting antibodies. Together, these results are
consistent with a protective role of IFN- during the
heterologous response against influenza virus independently of the
generation and local recruitment of cross-reactive CTLs.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
) is a
cytokine produced by NK cells, CD4+ Th1 cells, and a subset
of CD8+ T cells, and it is thought to be a major defense
arm during the immune response against intracellular bacteria, certain
parasites, and viruses (2). IFN- exerts two major
effects, directly inhibiting the ability of some microbes to multiply
and stimulating the cellular immune response. The direct effect of
IFN- is mediated by the induction of cellular products that
interfere with the microbial metabolism (19) or promote the
apoptosis of infected cells (8, 17). The indirect effects of
IFN- on the generation and function of specific immune effectors is
very complex and range from upregulation of antigen processing
(11) and presentation in the context of major
histocompatibility complex (MHC) class I (39) and II
(30, 34) molecules to modulation of the priming
(10), recruitment (36), and death of activated T
lymphocytes (25). Furthermore, IFN- exerts stimulatory
effects on the function of macrophages and NK cells (7).
in animal models
of infection with herpes simplex virus (33), cytomegalovirus (16), murine hepatitis virus 3 (26), lymphocytic
choriomeningitis virus (22), and adenovirus (39).
The generation of mice lacking functional IFN- genes (7)
allowed the assessment of the role of this cytokine during primary
infection with influenza virus (12). Surprisingly, mice
lacking IFN- did not display a reduced ability to recover from
primary infection with the A/JAP/57 (H2N2) strain of influenza virus
and mounted cytotoxic T-lymphocyte (CTL) activity comparable to that of
their wild-type counterparts. Furthermore, CTL clones obtained from
IFN-/ mice, adoptively transferred into wild-type recipients
previously challenged with influenza virus, mediated effective recovery
from infection (12). However, no data were available
regarding a protective role of IFN- during the secondary response to
influenza virus.
during the immune response to shift
variants of influenza virus are the memory T cells specific for
cross-reactive epitopes, we studied the secondary response to the
A/WSN/33 (H1N1) virus strain of IFN-/ mice previously immunized
with the A/HK/68 (H3N2) virus strain. Besides the fact that the WSN
virus is of a different subtype, it bears certain mutations in
neuraminidase that are responsible for its increased replication
ability and virulence (23). In the present report, we show
that IFN- plays a protective role during the memory response to a
virulent strain of influenza virus of a subtype that is different from
that of the strain used for priming. However, this role is independent
of the generation and local recruitment of effector T cells specific
for epitopes conserved among influenza viruses of different subtypes.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
genes
(IFN-/), obtained by gene targeting (7) and bred into
a BALB/c genetic background, were purchased from The Jackson Laboratory
(Bar Harbor, Maine). The BALB/c genotype and potential genetic
contamination were routinely monitored during breeding at The Jackson
Laboratory by assessing phenotypic, biochemical, and serological
markers. As a control, we used haplotype- and age-matched BALB/c
animals. Mice were housed in the Mount Sinai Animal Facility and
immunized at 8 to 12 weeks of age.
80°C. Influenza virus A/WSN/33 (H1N1) was grown on
MDBK cells in Dulbecco modified Eagle medium (DMEM) supplemented with
1% bovine serum albumin (BSA) at 37°C in a humidified atmosphere in
the presence of 7% CO2. The supernatant was harvested 2 days later. The virus concentration was measured on MDCK cells, and the
titers were expressed as 50% tissue culture-infective doses
(TCID50) per milliliter.
/ mice were immunized with the live HK virus
by intraperitoneal inoculation of 106 TCID50 in
200 µl of saline. Similar groups of mice were immunized intramuscularly three times at 3-week intervals with 30 µg of a
plasmid (NPV1) expressing the nucleoprotein (NP) of the A/PR/8/34 virus, which was previously shown to induce cross-reactive CTLs against
type A influenza viruses (38).
Infection and measurement of pulmonary virus titers.
Mice
were challenged via the aerosol route with 100% lethal doses of the
WSN or PR8 virus in the form of supernatant or allantoic fluid diluted
in 10 ml of saline. The infection was carried out for 30 min in an
aerosol chamber to which a nebulizer (Ace Glass Inc., Long Island,
N.Y.) was attached, connected to a vacuum-pressure system pump. The
nebulizer was loaded with 1.5 × 107, 2.25 × 107, or 3 × 107 TCID50 of the
WSN virus or 1.5 × 105 TCID50 of the PR8
virus. Control and IFN-/ mice were simultaneously infected. Mice
were observed daily after infection for up to 20 days, and their
clinical status, namely, respiratory pattern and weight loss, was
assessed. At least seven mice per group were infected for survival rate
analysis. The surviving mice were sacrificed, and their pulmonary virus
titers were measured.
Isolation of lymphocytes from spleens and lungs. The mice were sacrificed by injection with anesthetics (ketamine and xylazine), followed by bleeding of the axillar arteries. Spleens were aseptically removed, and after fine mincing, the fragments were passed through cell strainers. The erythrocytes were lysed by hypotonic shock. Lung tissue fragments were pretreated with 8 U of collagenase per ml in RPMI medium-1% BSA for 90 min at 37°C, in accordance with a previously described method (35). The fragments were passes through cell strainers, and the erythrocytes were lysed by hypotonic shock. The resulting cells were incubated in petri dishes at 37°C for 30 min to remove the plastic-adherent cells. The nonadherent cells were further used in functional assays or for staining. Bronchoalveolar lavage (BAL) lymphocytes were obtained by tracheal cannulation and three consecutive washings with 1 ml of saline, in accordance with a previously reported method (1). The BAL lymphocytes from three mice were pooled and used for functional assays.
Cytotoxicity assay and estimation of pCTL frequency.
Primary
CTL assays were carried out by incubating various numbers of freshly
harvested effector cells with 5 × 103
51Cr-labelled P815 cells in 96-well U-bottom plates. The
target cells were previously infected with influenza viruses
(106 TCID50/105 cells) for 1 h
at 37°C in FCS-free medium supplemented with 1% BSA. In parallel,
target cells were coated with an NP147-155 synthetic peptide, which
corresponds to the major CTL epitope of the PR8 virus, presented in the
context of Kd molecules. As a negative control,
we used a Db-restricted peptide derived from NP
of the PR8 virus. After 4 h of incubation at 37°C in 5%
CO2, the plates were centrifuged and the supernatants were
harvested and counted in a gamma counter (Automatic/Wallac-Finland).
For secondary CTL assays, mixed lymphocyte cultures were prepared and
incubated for 3 to 5 days in RPMI medium supplemented with 10% FCS, 50 mM 2-mercaptoethanol, 1% nonessential amino acids (Gibco BRL), and 2%
HEPES buffer (Gibco BRL) at a density of 4 × 106
cells/ml with a responder-to-stimulator cell ratio of 1. Before incubation, the stimulator cells were irradiated and infected with the
A/PR/8/34 or A/HK/68 virus, both of which share the dominant class
I-restricted epitopes with the A/WSN/33 virus. The PR8 virus was used
instead of the WSN virus, since the latter productively infects and
kills a broad range of cells, including lymphocytes (23). In
vitro stimulation was performed by using stimulator cells from naive
animals of a similar strain, i.e., IFN-/ for responder cells
harvested from IFN-/ mice. Before incubation with target cells,
effector cells were separated on Histopaque-1083 (Sigma, St. Louis,
Mo.). The results were expressed as the mean percent specific lysis of
triplicates ± the standard deviation (SD) after subtraction of
the background, that is, the percent lysis of noninfected, noncoated
P815 target cells.
Measurement of cytokine production.
Lymphocytes (2 × 105) were incubated with the same number of stimulator
cells that had previously been irradiated and pulsed for 30 min with
the sucrose-purified live PR8 or HK virus (50 µg of virus/200 µl of
DMEM-1% BSA/106 stimulator cells harvested from the same
mouse strain as the responder cells). In parallel, 2 × 105 responder cells were coincubated with 1-µg/ml
concanavalin A (ConA). After 3 days of incubation in RPMI medium
supplemented with 10% FCS, the supernatants were harvested and the
concentrations of interleukin 2 (IL-2), IL-4, and IFN- were measured
by sandwich enzyme-linked immunosorbent assay (ELISA) with anti-mouse
cytokine specific reagents (Biosource International, Camarillo,
Calif.). Samples with known concentrations were included in order to
plot standard curves. The concentration of the samples was interpolated from the standard curve. The results were expressed as the mean of
triplicates ± SD (picograms per milliliter). Values lower than the background plus 3 SDs were considered to be below the limit of
detection and assigned a value of 0. Generally, the sensitivity of the
method was below 5 pg/ml.
FACS analysis. Lymphocytes from three mice in each group were isolated by collagenase treatment of lung tissue and analyzed in three-color mode on an EPICS-Coulter fluorescence-activated cell sorter (FACS; Coulter Corporation, Hialeah, Fla.) equipped with a 488-nm argon laser. The cells were washed with cold PBS-2% BSA and stained for 30 min on ice with the following antibodies: 29B anti-mouse CD3 (Quantum Red), H129.19 anti-mouse CD4 (phycoerythrin), and 53-6.7 anti-mouse CD8 (fluorescein isothiocyanate) purchased from Sigma. The cell samples were run after fixation in PBS-1% paraformaldehyde supplemented with 0.1% sodium azide. The number of T cells in each subset was individually estimated, taking into consideration the total number of cells separated from the lung tissue by collagenase digestion. The results were expressed as the mean cell number in a particular subset ± the standard error of the mean (SEM).
Measurement of virus-specific HI antibodies. The hemagglutination inhibition (HI) assay was carried out after treatment of sera with receptor-destroying enzyme (RDE/neuraminidase; Sigma) overnight at 37°C to remove the nonspecific activity due to serum sialoproteins. The RDE was inactivated by incubation with 2.5% sodium citrate at 56°C for 30 min. Twofold serial dilutions of RDE-treated sera were incubated with a 0.5% human erythrocyte saline suspension in the presence of agglutinating titers of the WSN or HK virus. After 45 min of incubation in 96-well round-bottom flexible plates (Falcon) at room temperature, the results were read and expressed as the log2 of the last inhibitory dilution. Negative controls (blank sera) and positive controls (hemagglutinin [HA]-specific monoclonal antibodies) were included in the experiment. The results were expressed as geometric mean of individual HI titers ± the standard error (SE).
Statistical analysis. We performed statistical analysis of samples obeying binomial distribution, namely, the survival rates. The P values were computed by Fisher's exact test. For estimation of pCTL frequency by limiting-dilution analysis, we used linear interpolation by the least-squares method.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Survival of immunized IFN-/ mice subsequent to heterologous
and homologous infections.
We have studied the protection from
heterologous challenge conferred by live-virus immunization in the
absence of IFN-. IFN-/ and wild-type BALB/c mice were
immunized intraperitoneally with the live HK (H3N2) virus and
challenged 1 month later with various 100% lethal doses of the WSN
(H1N1) virus. In all cases, the IFN-/ mice displayed lower
survival rates than their wild-type counterparts subsequent to the
heterologous challenge (Fig. 1A to C).
The difference in the survival rates was statistically significant in
the case of a challenge with a dose of 1.5 × 107 or
2.25 × 107 TCID50 (P values of
0.038 and 0.009, respectively). In contrast, both IFN-/ and
BALB/c WSN-immunized mice challenged with the homologous virus
displayed complete protection in terms of survival rates (Fig. 1D).
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, most of the
mice immunized with the HK virus and challenged with the WSN virus
rapidly lost approximately one-third of their weight and died
at around day 7 after challenge (Fig.
2A). In contrast, most of the
wild-type mice and a few of the IFN-/ mice immunized with the HK
virus and challenged with the WSN virus exhibited a smaller decrease in
body weight and total or partial recovery of their initial weight (Fig.
2).
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plays an important
protective role during recall responses to shift variants of influenza
virus, although in the absence of IFN-, mice can recover from lethal
heterologous infections. In contrast, the absence of IFN- did not
impair a protective memory response to a homologous challenge.
Clearance of pulmonary virus in the absence of IFN-.
To
address the question of whether the decreased survival of HK-immunized
IFN-/ mice challenged with the WSN virus was due to defective
clearance of the pulmonary virus, we measured virus titers in the lungs
at 3 and 7 days after infection. Wild-type and IFN-/ naive mice
displayed significant pulmonary virus titers at days 3 and 7 after
infection (Table 1). Wild-type mice immunized with the live HK virus cleared the pulmonary virus by day 7 after the heterologous challenge. In contrast, HK-immunized IFN-/ mice did not clear the virus, although they displayed significantly decreased titers at day 7 compared to naive wild-type and
IFN-/ mice challenged with the WSN virus (Table 1). Furthermore, at day 3 after the heterologous challenge, the HK-immunized
IFN-/ mice displayed significantly higher titers of virus than
their wild-type counterparts.
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/ mice against a heterologous challenge was associated with
an impaired ability to completely clear the pulmonary virus. However,
in the absence of IFN-, the immune mechanisms mediated a significant
reduction of pulmonary virus titers by day 7 after the heterologous
challenge.
Induction of cross-reactive CTLs against type A influenza viruses
in the absence of IFN-.
We took advantage of the fact that the
WSN strain of influenza virus, due to a mutation in neuraminidase
(23), exhibits high-level and promiscuous replication
associated with the ability to induce enhanced levels of virus-specific
cytotoxicity that are detectable in primary CTL assays without
secondary in vitro stimulation. Thus, we have studied the in vivo
priming of WSN-specific CTL responses in BALB/c and IFN-/ mice
infected with the WSN virus. Freshly harvested splenocytes from
wild-type and IFN-/ mice infected with the WSN virus 7 days
previously displayed significant CTL activity against virus-infected
target cells (Fig. 3). The IFN-/
mice exhibited slightly lower CTL activities that were not
significantly different from those of their wild-type counterparts. Thus, the ability to mount primary cytotoxicity was not impaired in the
absence of IFN-.
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by measuring the cytotoxicity of freshly harvested splenocytes
from wild-type and IFN-/ mice infected with the WSN (H1N1) virus
that had been immunized 1 month previously with the live HK (H3N2)
virus. Both the IFN-/ mice and their wild-type counterparts
developed significant secondary CTL activities against the WSN virus,
as well as the dominant NP-specific CTL epitope that is
Kd restricted and conserved among the subtypes
of type A influenza virus, namely, NP147-155 (Fig.
4). In contrast, we measured no significant CTL activity against another NP peptide that is recognized by CTLs from C57BL/6 mice. Surprisingly, wild-type mice immunized with
the HK virus and challenged with the WSN virus displayed slightly lower
levels of cytotoxicity than IFN-/ mice. To address this point in
a quantitative manner, we measured the frequency of virus-specific
pCTLs in spleens by limiting-dilution analysis. As shown in Fig.
5, the frequency of the pCTLs induced by
HK immunization followed by WSN challenge was slightly higher in
IFN-/ mice than their wild-type counterparts (1 in 12 × 103 versus 1 in 15.5 × 103). Thus, both
the primary and secondary CTL responses were not decreased in the
absence of IFN-.
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Local recruitment of virus-specific effector cells in mice that
lack IFN-.
The impaired clearance of pulmonary virus by
IFN-/ mice immunized with the HK virus and challenged with the
WSN virus may have been due to a reduction in the local recruitment of
virus-specific T cells. Previous studies showed a correlation between
the rapid recruitment of virus-specific CTLs into the lungs of infected mice and complete clearance of the pulmonary virus by day 7 after challenge (6). We studied the local recruitment of the
effector cells by measuring the virus-specific CTL activity of
nonadherent cells harvested from collagenase-digested lungs 3 days
after a challenge with the WSN virus. After in vitro expansion with
virus-infected splenocytes, effector cells derived from both
IFN-/ and wild-type mice displayed significant and comparable
CTL activities (Fig. 6). Thus, the
absence of IFN- did not impair the early recruitment of
virus-specific pCTLs, which are thought to play the major protective role during the immune response to a shift variant.
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/ and
wild-type mice immunized with the HK virus and challenged with the WSN
virus. After in vitro stimulation in the presence of ConA or
antigen-presenting cells (APC) pulsed with live virus, the T cells from
IFN-/ mice and their wild-type counterparts produced similar
amounts of IL-2 (Table 2). Interestingly,
besides the lack of IFN-, the knockout mouse T cells isolated from
lungs produced significantly smaller amounts of IL-4 after in vitro stimulation with either ConA or live viruses. However, 7 days after
infection, the T cells isolated from lungs of IFN-/ mice displayed enhanced production of IL-4 following in vitro stimulation with ConA (data not shown).
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/ and wild-type mice at day 3 after the heterologous infection (Table
3). Similarly, no significant differences
in the percentage of the major T-cell subsets were noted in the cell
populations harvested by collagenase treatment or BAL from IFN-/
and wild-type mice at day 3 after the heterologous infection (data not
shown).
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, the
recruitment of functional virus-specific T cells in the lungs of
influenza virus-infected mice is not impaired.
Induction of influenza virus-neutralizing antibodies in the absence
of IFN-.
Previous studies suggested that T-dependent
virus-neutralizing antibodies may participate in the clearance of
influenza virus (28, 32). We tested the ability of
IFN-/ mice to mount protective HI antibodies, although the
design of the experiment limited their eventual protective role, since
the mice were immunized with an H3N2 strain and subsequently infected
with an H1N1 strain of influenza virus. IFN-/ and wild-type mice
immunized with the live HK virus mounted significant and comparable
titers of HI antibodies specific for the homologous strain and lacking
cross-reactivity for the heterologous strain, namely the WSN virus
(Table 4). Mice immunized with the HK
virus and challenged with the WSN virus developed similar primary
responses in terms of HI antibodies specific for the WSN virus,
independently of the presence of a functional IFN- gene. No boost
effect was noted regarding the HK-specific HI antibodies subsequent to
infection with the WSN virus (Table 4). Thus, neither the primary nor
the secondary humoral response, in terms of virus-neutralizing
antibodies, was significantly impaired in the absence of IFN-.
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Protection conferred by DNA immunization of IFN-/ mice.
Whereas the memory CTLs specific for epitopes on internal proteins are
thought to play the major protective role during the response to a
heterologous challenge, T cells that are specific for HA epitopes that
are conserved among certain variants of different subtypes can play a
role as well. We addressed the role of IFN- in the absence of T
memory cells specific for HA. It was previously shown that DNA
immunization of BALB/c mice with a plasmid (NPV1) expressing NP of
influenza virus PR8 elicited cross-reactive CTL immunity that was
protective against a lethal challenge (4, 38).
/ and wild-type mice with NPV1 and challenged
them at 1 month after the completion of immunization with lethal doses
of the PR8 virus. As shown in Table 5,
the wild-type mice completely cleared the virus by day 7 after the
challenge. In contrast, the IFN-/ mice did not completely clear
the virus, although they displayed 100 times lower titers than did
naive BALB/c mice infected with similar doses of the PR8 virus (4, 6). Again, wild-type BALB/c and IFN-/ mice mounted
comparable CTL responses (Table 5). Furthermore, except for the lack of IFN- secretion, T cells obtained by BAL and those harvested from the
spleens of IFN-/ mice produced similar amounts of IL-2 and IL-4
compared with those obtained from wild-type mice (Table 5). Thus, in
the absence of IFN-, mice immunized with NPV1 did not completely
clear the pulmonary virus after a challenge with the PR8 virus.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this investigation, we have studied the requirement for IFN-
during the secondary immune response of influenza virus-immunized mice
against a strain of a different subtype. We found that both survival
and the ability to completely clear the pulmonary virus were impaired
in the absence of IFN-. However, the immunized mice lacking
functional IFN- genes displayed significantly decreased pulmonary virus titers after a heterologous challenge, compared to
their nonimmunized counterparts. The generation and pulmonary recruitment of virus-specific CTLs and the induction of antibody responses were not impaired in the absence of IFN-. Besides
the lack of IFN- production, the T cells isolated from various
compartments of IFN-/ mice did not display dramatic differences
in the ability to produce IL-2 or IL-4. Finally, in the absence of
IFN-, the clearance of pulmonary virus in mice previously immunized
with a plasmid expressing NP was also impaired.
The T-cell immunity against class I- and class II-restricted epitopes that are conserved among drift and shift variants of influenza virus plays an important role in the recovery from lung infection, since the B-cell epitopes exhibit a high natural genetic variation. The role of memory T cells during the recall response to influenza virus was previously suggested by two types of data: first, adoptive transfer experiments with T-cell lines or clones demonstrated the ability of virus-specific T cells to mediate clearance of pulmonary virus (24, 40). Secondly, experiments carried out with B-cell-deficient mice showed that in the absence of antibodies, the cellular immunity conferred significant protection during secondary responses to influenza virus (3). However, the antibodies were essential for the recovery from primary infection (14) and greatly enhanced the protection during the secondary response against homologous strains (3).
The role of T-cell immunity in protection against a heterologous
challenge with strains of a different subtype is strongly supported by
our data regarding the survival (Fig. 1) and virus clearance (Table 1)
of wild-type mice immunized with the HK virus and challenged with
lethal doses of the WSN virus (Fig. 1). The mechanisms by which
CD8+ and CD4+ T cells specific for influenza
virus epitopes contribute to the clearance of pulmonary virus are
thought to be different. Whereas CD8+ T cells are endowed
with ability to directly lyse infected cells and are thought to play
the major role in the clearance of influenza virus (1),
CD4+ T cells exert their protective effects in a
pleiotropic manner. Previous reports showed that the generation of
neutralizing antibodies is a process that depends on T help
(32), and a recent study demonstrated the requirement for B
cells in protection conferred by adoptive transfer of virus-specific
CD4+ T cells (14). It has been previously shown
that MHC class II-restricted T cells, in certain transgenic models, are
endowed with the ability to directly lyse virus-infected cells
(20). However, the in vivo protective role of such class
II-restricted cytotoxicity has been challenged (5, 37). The
virus-specific CD4+ T cells were shown to up-regulate the
induction and local recruitment of CD8+ T cells during
primary infection in transgenic mice expressing a T-cell receptor
specific for an HA peptide (5). The role of proinflammatory
cytokines produced by CD4+ and CD8+ T cells
during infection with influenza virus is not clear. A previous study
showed that Th1, but not Th2, clones mediated effective clearance of
influenza virus from infected lungs, indirectly suggesting a beneficial
role for IFN- (13). The study of mice lacking functional
IFN- genes did not reveal a protective role for IFN- during the
primary response to the A/JAP/57 strain of influenza virus
(12).
Our results suggest that IFN- exerts protective roles during a
secondary response to influenza virus strains of different subtypes.
Decreased survival rates of IFN-/ mice previously immunized with
the HK virus after a challenge with three different doses of the WSN
virus (Fig. 1) were associated with impaired clearance of pulmonary
virus (Table 1). This result indicates that the lack of IFN- does
not merely exacerbate the viral pneumonia but primarily prevents
complete viral clearance by day 7 after challenge. However, even in the
absence of IFN-, there was a significant reduction of virus lung
titers in the immunized versus nonimmunized animals (Table 1), as well
as clinical recovery in some mice (Fig. 1 and 2). Since the antibody
response plays an unlikely role in the protection against a
heterologous challenge (Table 4), these results suggest that T-cell
immune mechanisms are operational during secondary responses to
influenza virus in the absence of IFN-. Indeed, IFN-/ mice
mounted not only primary CTL responses, as shown by the presence of
effector cells among freshly harvested splenocytes (Fig. 3), but
secondary CTL responses as well against a cross-reactive dominant
epitope that is presented in the context of Kd
class I molecules (Fig. 4). Furthermore, data depicted in Fig. 4 and an
estimation of the frequency of virus-specific pCTLs (Fig. 5) indicate a
slightly enhanced CTL response in the absence of IFN-. This can be
due to the prolonged presence of virus or, alternatively, to a
previously speculated role of IFN- in down-regulating T-cell
responses (25). Although our results are concordant with a
previous report showing increased in vitro generation of alloreactive cytotoxicity in the absence of IFN- (7), we did not
address these slight differences by performing subsequent experiments.
The inability of HK-immunized IFN-/ mice to completely clear the
pulmonary virus upon a heterologous challenge with the WSN virus was
not due to defective recruitment of virus-specific CTLs into the
infected lungs (Fig. 6). This result correlated with the comparable
presence of CD8+ together with CD4+ T cells in
the lungs of IFN-/ and wild-type mice challenged with the WSN
virus (Table 3). Similar results were obtained by staining the T cells
from BAL, although slightly reduced numbers of CD4+ T cells
with an activated phenotype were noted in IFN-/ compared to
wild-type mice (data not shown). However, our data cannot not rule out
the possibility that the access of CTLs to certain compartments or
cells of lung tissue is impaired in the absence of IFN-.
Interestingly, the absence of IFN- did not lead to increased IL-4
production by locally recruited T cells (Table 2). This result, which
is concordant with a previous study (31) but discordant with
another report (12), indicates that the inability of
IFN-/ mice to completely clear the virus and recover from the
infection was not due to exacerbated Th2 activity. However, we cannot
exclude the possibility of slight increases in the frequency of
IL-4-producing Th cells, which are difficult to assess by measuring
IL-4 in cell culture supernatants. Furthermore, the absence of IFN-
did not significantly impair a humoral response in terms of
neutralizing antibodies (Table 4). This is further substantiated by the
complete protection of immunized IFN-/ mice during a challenge
with a homologous strain (Fig. 1).
The protective role of IFN- was further assessed in a model lacking
the involvement of Th memory cells against the dominant class
II-restricted epitopes on HA. The results shown in Table 5 indicate the
inability of CTLs primed by a plasmid expressing NP to completely clear
the pulmonary virus in the absence of IFN-. Our results are not
concordant with a previous report showing that influenza virus-specific
CTL clones from IFN-/ mice could clear the pulmonary virus
following adoptive cell transfer (12). A possible cause for
this discrepancy is the fact that the recipient mice used in that
particular protocol were wild-type mice bearing functional IFN-
genes (12).
Together, our results suggest that IFN- exerts its protective effect
during the secondary response against heterologous strains of influenza
virus in a manner that is independent of the generation and local
recruitment of virus-specific effector T cells. Furthermore, our data
do not support the model in which IFN- mediates the up-regulation of
cytotoxic activity by T helper cells, despite the fact that a
previous study associated the activation of CD4+ T cells
with the availability of IFN- (31). There are at least two potential ways for IFN- to mediate its protective effects: (i)
by up-regulating the presentation of viral peptides in the context of
class I molecules of infected cells, thus allowing virus clearance by
CTLs, and (ii) by inhibiting virus replication in infected cells. A few
previous studies carried out in other experimental models support the
first mechanism. Thus, it was shown that IFN- contributed to the
clearance of recombinant adenovirus constructs by up-regulating the
expression of peptide-class I complexes on infected hepatocytes
(39). Another report showed that IFN- was required for
optimal processing and presentation of foreign peptides in the context
of class I molecules on nonprofessional APC (11). A very
recent study demonstrated the production of IFN- by neurons and
attributed the up-regulation of MHC class I expression on these cells
to an autocrine regulatory loop involving IFN- (27). The
WSN virus, which is a neurovirulent strain endowed with the ability to
replicate in cells that do not express furin (23), may
continue to multiply in certain cells without being detected by CTLs in
the absence of IFN-. Thus, IFN- may facilitate the recognition by
influenza virus-specific CTLs of certain permissive cells that are, in
the absence of IFN-, endowed with a low ability to process and
present foreign peptides in the context of class I molecules.
Another possibility is that IFN- acts in a manner independent of the
lysis of infected cells by CD8+ CTLs. A previous study
showed that 
2-microglobulin-deficient mice that lack
CD8+ T cells, when treated with anti-IFN- antibodies,
displayed delayed clearance of influenza virus (31).
Numerous reports support the concept that IFN- may act through other
mechanisms that lead to inhibition of virus replication or death of
infected cells (reviewed in reference 2). Thus,
IFN- up-regulates the expression of ICAM-1 (9) and
FcRI (15, 29), possibly facilitating the lysis of
infected cells by CTLs in a manner dependent or not dependent on
virus-specific antibodies. Other reports described the apoptosis of
bystander cells mediated by nitric oxide produced subsequent to
IFN--dependent activation of macrophages (17). Some
studies suggested that IFN- up-regulates the production of certain
Mx-like factors (i.e., Mg-21), although their function in directly
inhibiting the replication of influenza virus is still uncertain
(21).
Since our model employs transgenic mice, it is theoretically possible
that genetic contamination from strains that were originally used to
construct the knockout might contribute to the susceptibility of
IFN-/ mice to secondary infection with influenza virus. However,
this possibility seems remote since both C57BL/6 and 129/Sv mice mount
effective secondary immune responses against influenza virus, even in
the absence of B cells (3). Furthermore, C57BL/6 mice mount
protective responses against primary infection with influenza virus,
even in the absence of IFN- (12).
Thus, multiple mechanisms may be responsible for the protective effect
exerted by IFN- during recall responses to influenza viruses of
different subtypes. Further work is required to pinpoint the most
important ones, depending on the particular virus strains and the
genetic background of the animals used.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by grant 5PO1AI24671 from the National Institutes of Health.
The HK virus and NPV1 plasmid were kindly donated by Margaret A. Liu (Chiron Vaccines, Emeryville, Calif.). The WSN virus was kindly donated by Peter Palese (Mount Sinai School of Medicine, New York, N.Y.). We are thankful to Sofia Casares and George Italas (Mount Sinai School of Medicine, New York, N.Y.) for technical help regarding FACS analysis.
| |
FOOTNOTES |
|---|
* Corresponding author. Present address: Alliance Pharmaceutical Corp., 3030 Science Park Rd., San Diego, CA 92121. Phone: (619) 558-4300. Fax: (619) 678-4178. E-mail: ABot{at}allp.com.
Present address: Alliance Pharmaceutical Corp., San Diego, CA
92121.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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