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* Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Brown University, Providence, RI 02912; and
Department of Microbiology, Ohio State University, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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responses as they occur in situ
are defined here during lymphocytic choriomeningitis virus (LCMV)
infections, and a unique mechanism for the innate cytokines IFN-
and IL-18 in promoting these responses is defined. Infections of mice
with Armstrong or WE strains of LCMV induced an unexpectedly early day
4 IFN- response detectable in serum samples and spleen and liver
homogenates. Production of IFN- was MHC class I/CD8 dependent, but
did not require IL-12, NK cells, TCR-
T cells, MHC class II, or
CD4 T cells. Peak response required specific Ag recognition, as
administration of antagonist peptide partially impaired day 4 IFN-
induction, and viral peptide stimulation enhanced CD8 T cell IFN-
expression in culture. The IFN- response was associated with IL-18
and IFN- expression. Furthermore, both factors augmented
peptide-driven IFN- production in culture, and mice lacking IL-18 or
IFN- functions had reduced day 4 IFN-. Collectively, these
results demonstrate that during viral infections, there is a dramatic
in vivo CD8 T cell response preceding maximal expansion of these cells,
and that the mechanism supporting this response is dependent on
endogenous innate cytokines. Because stimulation by microbial products
is linked to innate cytokine expression, the studies also suggest a
pathway for precisely limiting T cell functions to times of
need. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production observed under culture
conditions with antigenic or TCR restimulation. Measurement of the
responses using these procedures demonstrates peak frequencies of CD8 T
cells primed for IFN- production, but does not assess timing of in
vivo function. Because reductions in viral burden are observed prior to
peak CD8 T cell expansion and priming for function as measured in
culture, in vivo CD8 T cell effector functions must be activated
relatively early during infections. The actual in vivo response has not
been previously characterized, nor have the early pathways responsible
for induction and promotion of initial CD8 T cell functions been
defined.
Type I interferons (IFN-) are a family of innate cytokines
exerting pleiotropic effects during viral infections. They can mediate
both antiviral and immunoregulatory functions (3, 4, 5, 6, 7).
Infections by LCMV elicit high levels of IFN- by days 2–3, but
not detectable, biologically active IL-12 (8, 9, 10, 11). As a
result, NK cell IFN- responses are low or absent during infections
with this virus (10, 12). IFN- can inhibit
replication of certain LCMV strains, including Armstrong and WE, in
vitro (13). Moreover, IFN- play a modest role in
promoting day 8 CD8 T cell IFN- responses in immunocompetent mice
(14). Interestingly, IFN- have been reported to
synergize with another innate cytokine, IL-18, for enhancing human T
cell IFN- expression in culture (15). Whether such
interactions are important in the mouse and/or during viral infections
in vivo is not known. IL-18 is an IFN--inducing factor (16, 17). After proteolytic processing by caspase-1, mature IL-18 is
biologically active and can induce IFN- production by NK and T
cells, particularly in synergy with IL-12 (18, 19, 20, 21). Thus,
there are conditions in which innate cytokines might interact to
promote and shape downstream adaptive responses. An intriguing
possibility is that virus-elicited innate cytokines may boost antigenic
signals delivered to T cells to cooperatively induce high levels of
IFN- production only during active viral infection and/or at times
when virus-specific subsets are present at low frequencies. The
contributions of IL-18 to T cell responses, and whether IL-18 and
IFN- can synergize and promote early antigen-driven IFN-
responses during LCMV infections, are not currently known.
The studies presented in this report evaluate the in vivo kinetics of
IFN- production after LCMV infections and delineate the roles of
innate cytokines in promoting this response. The results demonstrate an
unexpectedly early day 4 peak of IFN- occurring prior to the
dramatic expansion of CD8 T cells. The response is MHC class I/CD8
dependent and associated with IFN- and IL-18 expression, but is
independent of IL-12 or NK cells, MHC class II/CD4, or TCR-
cells. Antagonist peptide treatment impairs the production of day 4
IFN- in vivo, suggesting a role for viral Ag recognition in the
response. IL-18 and IFN- augment viral peptide-driven IFN-
production in culture, and IL-18-deficient or IFN-R-deficient
mice demonstrate impaired day 4 IFN- responses in vivo. Taken
together, the data reveal a previously unappreciated mechanism for
interaction between innate and adaptive immunity to promote delivery of
specific adaptive immune functions by low frequency CD8 T cell subsets
at times preceding their maximal expansion. They also suggest that this
mechanism might be in place to limit delivery of a T cell function to
times associated with significant pathogen burden.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 and homozygous
B6.129S2-Cd8atm1Mak,
B6.129P2-B2mtm2Unc,
B6.129S2-Cd4tm1Mak,
B6.129P2-Tcrdtm1Mom, and
B6.129-II12btm1Jm were purchased from The
Jackson Laboratory (Bar Harbor, ME). In addition, male C57BL/6 and MHC
class II-deficient B6.169-Abtm1
N5-M with control B6.169-Abtm1
N6-W were purchased from Taconic Laboratory Animals and Services
(Germantown, NY). Mice mutated in the IL-18 (22) or
IFN-R gene (14) were bred and maintained at
Brown University (Providence, RI). These animals were age and sex
matched, respectively, with C57BL/6 and 129/SvEv mice as appropriate.
All mice were of the H-2b haplotype and were used between 6
and 12 wk of age in accordance with institutional guidelines for animal
care and use.
In vivo treatments, sample preparation, and cytokine measurements
Infections were established i.p. on day 0 with 2 x
104 PFU of either LCMV Armstrong strain clone E350 or the
hepatotropic strain WE (12). In NK cell depletion
experiments, either rabbit anti-AGM1 or anti-NK1.1 (PK136) Ab
preparations, or the respective control rabbit IgG (Sigma-Aldrich, St.
Louis, MO) or P3NS1 preparations, were administered i.p. on day
-0.5 and day 3 (10). Efficacy of NK cell
depletions were verified by lack of
NK1.1+CD3
- (after anti-AGM1) or
2B4+CD3- (after anti-NK1.1)
(23) populations by flow cytometry and/or lack of YAC-1
target cell lysis in standard 51Cr release assays
(24). In peptide administration studies, mice received 2
mg i.p. on day 3 postinfection of either control viral peptide
NP396–404 (FQPQNGQFI), an irrelevant OVA peptide
OVA257–264, or an antagonistic synthetic peptide
(SMIENLEYM) (25, 26, 27), purchased from Dr. B. Evavold (Emory
University, Atlanta, GA). At indicated times after infection, mice were
anesthetized with methoxyflurane (Pittman-Moore, Mundelein, IL) or
isoflurane (Abbott Laboratories, North Chicago, IL). For determination
of cytokine levels, serum samples, tissue homogenates from spleens and
livers, and splenic leukocytes for cellular assays were prepared as
described (24). In specific experiments, CD8+
or IFN--secreting cells were enriched by MACS of splenocytes labeled
with MACS CD8a microbeads or a mouse IFN- secretion assay kit,
respectively, according to manufacturer’s directions (Miltenyi Biotec,
Auburn, CA). IFN- and IL-18 levels were measured by sandwich ELISA
(24). Biologic assays for IFN- activity were
measured as protection against vesicular stomatitis virus-induced
cytopathic effects (28). Specificity for IFN- was
verified using neutralizing Abs to the factors.
Flow cytometric analyses and intracellular IFN- staining
Surface and intracellular IFN- protein stainings were done
similar to described methods (14). CD8 T cells were
phenotypically identified as CD8+CD4-
populations after labeling with PerCP-conjugated anti-CD4 (RM4-5)
and FITC-conjugated anti-CD8 (53-6.7) or isotype-matched control
Abs (BD PharMingen, San Diego, CA). In some experiments, cells were
additionally stained with allophycocyanin-conjugated
H-2Db tetramers binding immunodominant LCMV peptides
NP396–404 or gp33–41 (KAVYNFATM) (29).
Analysis of activated CD8 cell subsets was conducted with
FITC-conjugated anti-CD62 ligand (MEL-14),
CyChrome-conjugated CD44 (IM7), and PE-conjugated CD8 (53-6.7). For
surface staining, more than 60,000 events were collected using
CellQuest software and FACSCalibur (BD Biosciences, San Jose, CA) with
argon laser output operating at 15 mW at 488 nm. As per modification of
published methods, intracellular staining for IFN- was done after
5 h of in vitro restimulation at 37°C with brefeldin A
(Sigma-Aldrich) added during the last 3 h of culture
(14). To restimulate, 106 cells were cultured
in 96-well microtiter plates containing 200 µl of 10% FCS-RPMI
1640 or medium supplemented with NP396–404 and
gp33–41 at 100 ng/ml each. After incubation, samples were stained for
surface markers, fixed, permeabilized, and stained with
allophyocyanin-conjugated anti-mouse IFN- (XMG1.2) or isotype
control Ab. At least 150,000 events were collected with a second laser
operating at 635 nm. Specificity of intracellular IFN- staining was
demonstrated by competition with unconjugated XMG1.2 Ab and/or
recombinant murine IFN- (eBioscience, San Diego, CA).
In vitro stimulations
Spontaneous production of IFN- in culture was determined by
plating 106 cells/well in 96-well microtiter plates with
200 µl of 10% FCS-RPMI 1640. Anti-CD3-elicited IFN- production
was similarly done using plates coated overnight with 150 µl of 10
µg/ml anti-CD3 (145-2C11) (BD PharMingen). To evaluate the
ability of exogenous cytokines to enhance Ag-driven IFN- production
in culture, cells from LCMV-infected mice were plated at
106 cells/well (bulk splenocytes) or 2 x
105 cells/well (CD8 enriched). Indicated concentrations of
NP396–404 were added with or without recombinant murine
IL-18 (R&D Systems, Minneapolis, MN) and/or universal IFN- A/D (PBL
Biomedical Laboratories, New Brunswick, NJ) to a final volume of 200
µl/well. NP396–404 was chosen because this is the
immunodominant epitope in H-2b hosts (30, 31, 32, 33, 34).
Universal IFN- A/D is a hybrid factor constructed from recombinant
human IFN-A and IFN-D and exerts bioactivity on mouse cells.
Cultures were incubated for 24 h at 37°C prior to harvesting of
supernatants for determination of IFN- production by ELISA.
Statistical analyses
Data were analyzed using statistical functions and the two-tailed homoscedastic Student’s t test function from Microsoft Excel 98 (Microsoft, Redmond, WA). Unless otherwise indicated, results are given as means ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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responses
Acute LCMV infections are known to induce a profound expansion of
CD8 T cells with activation of CTL activity and priming for ex vivo
IFN- production that are apparent at high levels by day 8
postinfection (29, 35, 36, 37, 38, 39). Consistent with these
observations, flow cytometric analyses using H-2Db
tetramers binding immunodominant LCMV peptides NP396–404
or gp33–41 demonstrated that splenic proportions of virus-specific CD8
T cells were not readily apparent on day 4, but were dramatically
elevated by day 8 in C57BL/6 mice infected with either LCMV Armstrong
or WE (Fig. 1
A).
Concomitantly, high levels of ex vivo production of IFN- in
spontaneous and anti-CD3-elicited splenocyte-conditioned media were
observed by day 8, but not prior to days 4–6 (Fig. 1B).
Armstrong infections typically induced higher magnitude splenic CD8 T
cell responses than infections with WE. However, it is not clear
whether these ex vivo assays demonstrating CD8 T cell priming for
IFN- production accurately represent their functions in vivo,
particularly since clearance of LCMV is known to be underway prior to
day 8 in the spleen (1, 14, 40, 41, 42).
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during LCMV infections were done. Levels of IFN- were measured in
serum samples and organ homogenates to assess in vivo expression of
cytokines. These experiments demonstrated an unexpectedly early peak of
IFN- production on day 4 in response to LCMV infections (Fig. 2). With Armstrong, levels reached
0.19 ± 0.03 ng/ml in serum and 2.8 ± 0.6 and 0.4 ±
0.1 ng/g tissue in spleen and liver, respectively; with WE, IFN-
levels reached 0.65 ± 0.11 ng/ml in serum and 7.1 ± 0.7 and
0.6 ± 0.1 ng/g tissue in spleen and liver, respectively. Taken
together, the results reveal a dichotomy between when peak responses
occur for in vitro measurements of CD8 T cell priming of IFN-
production and when IFN- is endogenously expressed in vivo during
acute infections.
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responses
To determine the requirements for the early IFN- response,
different immune components were eliminated through the use of
genetically manipulated or Ab-treated mice. Because spleen IFN-
levels were higher than in the liver, subsequent experiments focused on
systemic and splenic responses. Disruption of particular arms of innate
immunity did not impair day 4 IFN- expression in either NK
cell-depleted mice or IL-12p40-deficient mice (Table I), nor was it impaired in
IL-12p35-deficient mice (data not shown). The IFN- response was also
intact in mice lacking TCR- cells, CD4 T cells, or MHC class II
(Table I). Thus, IFN- production did not require NK cells or CD4 or
TCR--bearing T cells. However, mice lacking MHC class I and/or
CD8 T cells, as a result of genetic disruptions in
2-microglobulin or CD8, demonstrated significant
reductions of 75–95% in systemic and splenic IFN- responses to day
4 Armstrong and WE infections (Table I). Thus, both strains elicited a
MHC class I/CD8-dependent IFN- response during acute infections.
Flow cytometric analyses of CD8 cells demonstrated that, whereas only
7–8% expressed CD44highCD62Llow in uninfected
mice, by day 4 postinfection 
20% of CD8 cells acquired this
activated phenotype. These results demonstrate that a subset of CD8 T
cells is indeed activated as early as day 4. Furthermore, the data
support a role for CD8 T cells in contributing to in vivo IFN-
production at this time.
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responses
Because MHC class I was critical for early CD8-dependent IFN-
responses, the contributions of Ag signaling were investigated by
interfering with Ag presentation on MHC class I to CD8 T cells. To
antagonize in vivo H-2Db-restricted interactions, a
synthetic blocking peptide (SMIENLEYM) known to block LCMV-specific CTL
activity was used (25, 26, 27). Two different control peptides
were tested in separate experiments, using an immunodominant viral
peptide (NP396–404) and an irrelevant OVA peptide
(OVA257–264). Since administered peptides were expected to
have short half-lives in vivo, control and blocking peptides were
injected 1 day before peak IFN- responses on day 4 after infection.
The studies demonstrated that, as compared to control peptide
treatment, administration of blocking peptide reduced systemic and
splenic IFN- responses by 25–50% in C57BL/6 mice (Table II). Control peptide treatments yielded
similar day 4 IFN- responses as compared to infected, but untreated
mice. The differences between control and antagonist peptide treatments
were statistically significant in most, but not all, experiments. It
was not possible to ascertain whether blocking peptide treatments were
sufficient to interfere with all H-2Db-TCR interactions
involving viral Ag in vivo. Nevertheless, the decreases were
reproducibly observed in additional independent studies, demonstrating
a contribution of Ag signaling during the 24-h period preceding the day
4 IFN- response to LCMV infections. The results therefore support an
Ag-specific stimulation of CD8 T cells as a factor contributing to the
day 4 IFN- response.
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. Spontaneous expression of IFN-
was difficult to detect ex vivo, thus cells were briefly restimulated
with H-2b immunodominant viral peptides
NP396–404 and gp33–41. Under these conditions, <0.1% of
CD8 T cells from uninfected mice expressed intracellular IFN-.
However, after 4 days of LCMV infection, 0.7% of CD8 T cells were
induced to express IFN- compared to 
0.2% of CD4 T cells (Fig. 3A). Thus, consistent with
data from genetically manipulated mice (Table I
), CD8 T cells were
found to be the predominant source of IFN-. Because these
frequencies of IFN--expressing CD8 T cells were low, bulk
splenocytes were enriched for CD8+ cells prior to
restimulation. These experiments confirmed that 0.4% of enriched
CD8 T cells were induced to express intracellular IFN- after
infections (Fig. 3B). Populations from LCMV Armstrong-, as
compared to WE, infected mice typically exhibited a greater magnitude
ex vivo IFN- responses. To delineate the contributions of CD8 T
cells to the IFN- response under these conditions, bulk splenocytes
from Armstrong-infected mice were briefly restimulated with viral
peptides and magnetically sorted for IFN--secreting cells.
Phenotypic identification of the IFN--secreting populations revealed
that CD8 T cells comprised 47% of the isolated cells (Fig. 3C). Although some nonspecific signal from
IFN-+CD8- populations was detected in both
uninfected and day 4 Armstrong-infected mice, the data nevertheless
established that virus-specific CD8 T cell subsets were primed for
IFN- production by day 4 and were the predominant source of this
cytokine. The collective studies thus indicate that MHC class I-TCR
interactions are important for the in vivo CD8-dependent IFN-
response and that likely this response is primarily from Ag-specific
CD8 T cell subsets.
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production
Although IL-12 is not required for CD8 T cell responses to LCMV
infections, several other innate cytokines may promote and/or augment T
cell synthesis of IFN-. In particular, IFN- and IL-18 have
been reported to synergize for human T cell IFN- in culture
(15). To determine whether IFN- and/or IL-18 were
induced during LCMV infections, the kinetics of expression for these
innate cytokines was assayed. Consistent with other reports
(8), IFN- was induced to high systemic levels
following LCMV infections and peaked on days 2–3 (Fig. 4). In addition, in vivo production of
IFN- in spleen homogenates was demonstrated, and was found to
have similar kinetics of induction as in serum (Fig. 4). Likewise,
IL-18 was induced early in serum samples and spleen homogenates (Fig. 4). Systemic levels of IL-18 were induced from undetectable levels in
uninfected mice to 0.11 ± 0.01 and 0.14 ± 0.02 ng/ml by day
6 after Armstrong and WE infections, respectively. Splenic levels of
IL-18 peaked earlier on day 3, reaching 339 ± 30 and 342 ±
51 ng/g tissue after Armstrong and WE infections, respectively (Fig. 4). Splenic expression of the 18-kDa biologically active form of IL-18
was verified by Western blotting (data not shown). Taken together,
these experiments demonstrate that IFN- and IL-18 are induced
early after infections, and thus are available for potentially
supporting day 4 IFN- responses.
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and/or IL-18 could indeed promote
Ag-driven IFN- responses, splenocytes from day 4 and day 8
LCMV-infected mice were cultured with varying concentrations of the
immunodominant LCMV peptide NP396–404 in combination with
IFN- and/or IL-18. Day 4 IFN- responses were modest under these
culture conditions (Fig. 5), presumably
due to the low frequencies of CD8 T cells specific for this viral
epitope on day 4 (Fig. 1). Nevertheless, the addition of either IFN-
or IL-18 tended to enhance the modest Ag-driven IFN- response on day
4. Splenocytes from either day 8 Armstrong- or WE-infected mice
produced high levels of IFN- in response to viral Ag, and production
also could be augmented in the presence of either IFN- or IL-18
(Fig. 5). Consistent with the greater accumulation of responding CD8 T
cells in the spleens of Armstrong-, as compared to WE-, infected mice,
measurements of in vitro production by isolated cell populations
resulted in higher levels of IFN- from Armstrong rather than WE
infections, particularly on day 8 (Figs. 5 and 6). At suboptimal concentrations, the
combination of IL-18 and IFN- synergistically enhanced viral
peptide-driven IFN- production following either day 4 or day 8 LCMV
infections (Fig. 6). These innate cytokines also synergized with Ag for
driving IFN- production from CD8-enriched cell populations. Enriched
CD8 populations pooled from day 8 Armstrong-infected mice produced 1.3
ng/ml IFN- after stimulation with 50 ng/ml NP396–404
peptide. Addition of either 1000 U/ml IFN- or 0.4 ng/ml IL-18
enhanced IFN- production to 4.6 and 3.0 ng/ml, respectively.
Combining all three stimuli augmented production of IFN- by enriched
CD8 T cells to 10.2 ng/ml, suggesting that these factors could directly
act upon CD8 T cells. Collectively, these studies demonstrate that
innate cytokines act in concert with Ag to augment CD8 T cell IFN-
production in culture.
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in
promoting IFN- responses, mice lacking IL-18 or IFN- functions
were infected with LCMV to determine their IFN- responses in vitro
and in vivo. These animals were genetically mutated in the genes
encoding either IL-18 or the IFN- receptor. Splenic leukocytes
from Armstrong-infected, IL-18-deficient mice demonstrated reduced
IFN- production when cultured with viral Ag (Fig. 7A). Control C57BL/6 mice
produced 306.3 ± 16.4 pg/ml IFN- at the highest concentration
of NP396 peptide. In contrast, IL-18-deficient populations
only secreted 67.0 ± 7.7 pg/ml IFN-, but production was
augmented to 561.5 ± 136.7 pg/ml by supplementing exogenous
IL-18. Similarly, control 129 mice produced 1277.4 ± 37.1 pg/ml
IFN-, whereas only 64.3 ± 9.8 pg/ml were detected from
IFN-R-deficient populations (Fig. 7B). Since the
genetic defect in these mice is for the cytokine receptor, cytokine
reconstitution studies were not attempted. However, in either IL-18- or
IFN-R-deficient mice, viral Ag was able to stimulate IFN-
production in a dose-dependent manner, albeit weakly. This suggested
that virus-specific CD8 T cells were present but impaired. The
reconstitution of a vigorous IFN- response by exogenous IL-18
further indicated that these cells were functional once the limiting
defect was corrected. Moreover, the day 8 expansion of total and
virus-specific CD8 T cells was similar between respective control mice
and IL-18- or IFN-R-deficient mice (Table III). Experiments were also done to
investigate endogenous in vivo responses in these immunodeficient mice.
IL-18-deficient C57BL/6 mice exhibited significant reductions of
40–60% in serum and splenic IFN- production after infections by
either Armstrong or WE (Fig. 8A). More dramatically, in
both compartments the day 4 IFN- response to WE infections in 129
IFN-R-deficient mice was significantly reduced by >95% (Fig. 8B). Collectively, the results demonstrated that lack of
IL-18 or IFN- functions impairs the ability of Ag to stimulate
virus-specific IFN- responses in culture. The results therefore
define critical requirements for endogenous innate cytokines in
promoting day 4 IFN- responses in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production. We show for the first time
the induction and expression of IL-18 during LCMV infections and
delineate its role, in conjunction with IFN-, in promoting an
unexpectedly early in vivo day 4 IFN- response. This response occurs
with either LCMV Armstrong or WE infections, is NK cell independent but
CD8 dependent, and precedes the dramatic expansion of CD8 T cells seen
after infection. Antagonist peptide studies and experiments
restimulating with LCMV peptides demonstrate that day 4 IFN- is
driven by Ag-specific responses. Either IFN- or IL-18 can
significantly augment IFN- responses in culture, and both factors
synergize for this effect. Correspondingly, day 4 IFN- responses
require IFN- and, to a lesser extent, IL-18 for expression. Our
studies delineate the induction of endogenous innate cytokines and the
promotion by these factors of a heretofore unappreciated CD8 T
cell-mediated IFN- response by low-frequency subsets early after
LCMV infection. Thus, they define a unique mechanism by which innate
cytokines promote T cell responses.
Although the frequencies of virus-specific CD8 T cells for any single
immunodominant epitope were below the limits of effective detection,
our data indicate that on day 4 postinfection 0.3 to 0.7% of CD8 T
cells were primed for intracellular IFN- expression following ex
vivo restimulation with immunodominant viral peptides (Fig. 3). This is
commensurate with early estimates of naive LCMV-specific CTL precursor
frequencies of 1 in 560,000 (43) and with more recent
approximations of 1 in 10,000 (44). Taking into account a
6- to 24-h division time for activated T cells, our measurements of day
4 IFN--producing CD8 T cells are in close agreement with expected
virus-specific precursor frequencies after about 3–4 days of in vivo
stimulation. It is not entirely surprising to us that a low-frequency
subset of cells could produce the levels of IFN- observed on day 4.
NK cells, which typically only comprise about 2% of splenocyte
populations, produce 10-fold greater levels of IFN- at their peak
response to murine cytomegalovirus infection (24).
However, in vivo contributions to IFN- on day 4 of LCMV infection
may be drawn from a broader range of CD8 T cell subsets than those
quantitated with the LCMV peptides tested here. Although the day 4
IFN- response is at least partially driven by viral Ag presented on
H-2Db (Table II), it is possible that additional
H-2Kb interactions, subdominant peptides, and/or emerging
low-affinity CD8 T cells also contribute to IFN- production
(34). We cannot exclude the possibility that other cell
populations may be contributing to CD8-independent IFN- responses.
Others have demonstrated that in the absence of a normal high-affinity
CTL response, lower affinity CTL and/or alternative-epitope CTL can be
generated against LCMV (45, 46). Interestingly, in this
hierarchical context IFN- is required by the resultant low-affinity
CTL response for clearance of virus (47). Although there
are a number of pathways by which this effect could be mediated,
IFN- might enhance CTL function by promoting the expression of MHC
class I molecules and the resulting Ag presentation by these molecules
to CD8 T cells (48). Thus, prior to the expansion of CD8 T
cells specific for high-affinity immunodominant viral epitopes, the
early IFN- response may be a composite derived from more diverse CD8
T cell subsets driven by LCMV Ags and augmented by IFN- and
IL-18, and the cytokine response might act to help promote defense
through direct antiviral and/or immunoregulatory effects.
Given that the induction of innate cytokine responses can be linked to
stimulation with microbial products, their requirement for enhancing an
Ag-specific response may serve as a mechanism for ensuring the
appropriateness of expressing T cell effector functions. Our results
complement the observations, during infections with the intracellular
pathogens Toxoplasma gondii (49) and
Leishmania major (50), that continuous IL-12
exposure is required for maintaining T cell IFN- production and host
resistance. The IL-12 requirement exists despite continued antigenic
stimulation, either in culture with exogenous Ag or in vivo after
infection reactivated due to loss of resistance. Using innate cytokines
to promote Ag stimulation of T cells may limit induction of T cell
functions to times when they are needed. On the other hand, there are
examples where Ag is critical for priming of T cell functions, but once
activated, the responses can occur independently of further antigenic
stimulation in the presence of cytokines (39). Following
transient exposure to Listeria monocytogenes Ag in culture,
for example, peptide-specific CD8 T cells are capable of undergoing
limited replication, after Ag deprivation, promoted by exogenous IL-2
(50). Similarly, IL-12 and IL-18 costimulation induces
polarized CD4 TH1 cells to secrete IFN- in the absence
of exogenous specific Ag (51, 52). Thus, once primed with
Ag, T cells may be stimulated by cytokines to produce IFN- without
further TCR-mediated signaling. Such mechanisms may make it possible to
call memory CD8 T cells into early IFN- production by exposure to
innate cytokines and might provide a mechanism for nonspecific
activation during viral infections. Under the day 4 infection
conditions studied here, however, the CD8 T cell IFN- responses
occur in the context of antigenic stimulation and are partially
dependent upon TCR signaling (Table II). Nevertheless, it is possible
that the residual IFN- response, not inhibited by antagonistic
peptide, is due either to other cell types being recruited to
participate in the response or to virus-specific CD8 T cells, primed
before day 3, but no longer requiring Ag to respond to
cytokine-mediated signals for IFN- production. Taken together, our
data and others demonstrate that cytokines may be a second signal
required for accessing Ag-primed T cell responses. By requiring a
second signal from innate cytokines induced during an acute infection,
a safeguard is created by which T cell functions are only driven when
licensed to do so in vivo.
The intracellular signals inducing T cell IFN- expression can arise
from TCR and/or cytokine stimulation via different pathways. In
TH1 CD4 T cells, TCR-induced IFN- is likely initiated by
the NFAT (53, 54, 55), whereas IL-18-induced IFN- requires
GADD45 (56). It is unclear whether CD8 T cells have a
similar dichotomy in receptor-mediated pathways, but differences
clearly exist between CD4 and CD8 T cells in their requirements for
IFN- induction by TCR stimulation (57). Our results
show that day 4 IFN- responses to LCMV infections are primarily
derived from CD8 T cells rather than CD4 T cells. Both Ag and IL-18
play important roles in driving this response, which also requires
IFN- (Table II and Fig. 7). Although we have not defined the
intracellular pathways for synergism in this report, the interplay
between IFN- and IL-18 can occur at several mechanistic levels.
IFN- can enhance IL-18-mediated signaling by inducing caspase
gene expression, up-regulating IL-18R components (58),
and/or increasing expression of the IL-18R signaling adaptor molecule
myeloid differentiation factor 88 (59). Indeed,
after LCMV infections, CD8 T cells up-regulated expression of the
IL-18R component, IL-1R-related protein, indicating that these
populations are likely primed to respond to this factor (data not
shown). Thus, during in vivo infections a combination of TCR and
cytokine signals contribute to CD8 T cell IFN- responses,
particularly at early times when viral titers and Ag load are high but
virus-specific CD8 T cell frequencies are still relatively low. At
these low in vivo E:T cell ratios, dichotomous but convergent TCR and
cytokine signaling pathways are likely to contribute to the synergism
observed in promoting CD8 T cell IFN- responses on day 4
postinfection.
IFN- cytokines exert pleiotropic effects during viral infections,
and it is becoming clear that during LCMV infections, these innate
factors possess complex immunoregulatory roles. The type I IFNs are a
family of structurally related factors encoded by a single IFN- and
multiple IFN- genes. These molecules all utilize the same
heterodimeric receptor complex. Although certain subtle differences
have been suggested, the various IFN- members generally have
overlapping biological effects (60). Our laboratory has
recently demonstrated that IFN- and multiple IFN- gene products
are induced during LCMV infection (61). In the present
study, we demonstrate that a universal recombinant type I IFN supports
Ag-driven T cell IFN- production in vitro and that LCMV-induced
IFN- endogenously promote T cell IFN- responses in
vivo.
These experiments define a unique mechanism for innate cytokine
promotion of CD8 T cell IFN- production. IFN- has been shown to
be essential for defense against a variety of infections (9, 10, 24, 62, 63, 64). During LCMV infections, its role has been more
controversial, with IFN- reported to modestly inhibit viral
replication in vitro (13) and to enhance viral clearance
(65, 66, 67). We and others have shown that IFN- are
important for control of LCMV (13, 14, 68). In contrast,
IL-18 was dispensable for clearance of virus (data not shown), possibly
due to incomplete abrogation of the IFN- response. This is
consistent with the greater importance of IFN-, as compared to
IL-18, for promotion of IFN- (Fig. 8). However, it remains to be
determined whether IFN- plays a role in optimal host defense at
specific times prior to maximal expansion of virus-specific
CTL.
Our group has previously shown that IFN- function to inhibit
endogenous IL-12 expression during LCMV infection (11). In
addition, 2 days after viral infections, IFN- induce a refractory
state in which NK cell IFN- responses to IL-12 and
anti-CD3-elicited IFN- production from splenocytes are inhibited
(69). The refractory state is temporary and responsiveness
to both stimuli is restored by 4 days postinfection. Along with the
studies presented here, the data demonstrate a biphasic role for
IFN- in the regulation of IFN- responses during LCMV
infections. Initially, IFN- function to inhibit IL-12 and day 2
IFN- expression. However, after the refractory period expires,
IFN- clearly enhance Ag-driven IFN- production in vitro (Figs. 5 and 6) and promote the endogenous day 4 IFN- response (Fig. 7).
This duality of purpose likely exists to temporally coordinate host
immune responses for optimal efficacy. Since both IFN- and
IFN- can signal through STAT1, initial refractoriness to TCR
stimulation may prevent inappropriate IFN- expression and
competition for signal transducers at times when IFN- exert
critical antiviral and/or other immunoregulatory effects. Additionally,
deferred activation of T cell functions may avoid premature
IFN--induced cell death or exhaustion prior to expansion of
immunodominant virus-specific CD8 T cell subsets, and/or before the
delivery of antiviral functions (70, 71). Indeed,
administration of high-dose IL-12 during LCMV infection drives IFN-
levels to greatly elevated levels, but is detrimental to the expansion
of CD8 T cells and hinders control of viral replication
(72). Thus, IFN- are an important coordinator of
host immune responses that span both innate and adaptive immunity.
The mechanisms by which IFN- might act to promote IFN-
expression in the human as compared to the mouse are controversial. The
issue stems from the observation that one pathway to IFN- induction,
STAT2-dependent activation of STAT4, can be elicited in response to
IFN- in human but not mouse cells (73). It should be
noted that the studies reported here demonstrate a mechanism by which
IFN- promote IFN- responses in the mouse by acting with IL-18.
Because these effects have also been reported in human culture systems
(15), at least this pathway may be operational in both
species. Particular biochemical differences between the species may
have less significance in the context of the mixed cytokine and
cellular responses elicited during pathogen challenge. Interestingly,
Ab depletion of NK cells resulted in higher in vivo IFN- levels as
compared to control-treated animals (Table I). Although the mechanism
for this is unclear, it would be consistent with previous work from our
laboratory demonstrating that NK cells can regulate the magnitude of
CD4 and CD8 T cell responses to LCMV infection (74). Thus,
cellular as well as cytokine cross-talk are likely to exist between
innate and adaptive immunity.
In summary, our data demonstrate endogenous roles for innate cytokines
in promoting early adaptive immune responses. Since we have captured
the functioning of CD8 T cells in vivo, our results question the in
vivo relevance of ex vivo measurements of T cell functions, as peak Ag-
or anti-CD3-elicited IFN- responses in vitro occur between days
7 and 9. We show that the endogenous in vivo peak is much earlier.
Access to CD8 T cell effector functions, and hence control of viral
replication by adaptive immunity, can thus begin as early as 4 days
after infection. These conditions may promote early antiviral defense
at times when virus-specific cells are at low frequencies. Furthermore,
the pathways defined in these studies are a mechanism for ensuring the
appropriateness of accessing Ag-specific CD8 T cell functions as they
require a second signal from innate cytokines induced during the acute
infection.
|
Acknowledgments |
|---|
, Drs. Kiyoshi Takeda and Shizuo Akira
(Osaka University, Osaka, Japan) for providing breeding pairs of
IL-18-deficient mice, and the National Institute of Allergy and
Infectious Diseases Tetramer Facility at Emory University
(Atlanta, GA) for the LCMV tetramers. We are also grateful to Drs. Marc
Dalod and Thais Salazar-Mather, along with Rachelle Salomon, Casey
Lewis, and Wanda Montas for their insightful discussions and expert
assistance. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Christine A. Biron, Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Brown University, Box G-B6, Providence, RI 02912. E-mail address: christine_biron{at}brown.edu
3 Abbreviation used in this paper: LCMV, lymphocytic choriomeningitis virus.
Received for publication March 27, 2002. Accepted for publication September 16, 2002.
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) or presence (•) of
increasing size, respectively) of increasing doses of IFN-
