By
From the * Department of Molecular Microbiology and Immunology, Division of Biology and Medicine,
Brown University, Providence, Rhode Island 02912; the Genetics Institute, Inc., Andover,
Massachusetts 01810; and the § Emory Vaccine Center and Department of Microbiology and
Immunology, Emory University School of Medicine, Atlanta, Georgia 30322
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Abstract |
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Viral infections induce CD8 T cell expansion and interferon (IFN)- production for defense,
but the innate cytokines shaping these responses have not been identified. Although interleukin (IL)-12 has the potential to contribute, IL-12-dependent T cell IFN-
has not been detected
during viral infections. Moreover, certain viruses fail to induce IL-12, and elicit high levels of IFN-
/
to negatively regulate it. The endogenous factors promoting virus-induced T cell
IFN-
production were defined in studies evaluating CD8 T cell responses during lymphocytic
choriomeningitis virus infections of mice. Two divergent supporting pathways were characterized. Under normal conditions of infections, the CD8 T cell IFN-
response was dependent
on endogenous IFN-
/
effects, but was IL-12 independent. In contrast, in the absence of
IFN-
/
functions, an IL-12 response was revealed and substituted an alternative pathway to
IFN-
. IFN-
/
-mediated effects resulted in enhanced, but the alternative pathway also promoted, resistance to infection. These observations define uniquely important IFN-
/
-controlled pathways shaping T cell responses during viral infections, and demonstrate plasticity of
immune responses in accessing divergent innate mechanisms to achieve similar ultimate goals.
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Introduction |
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Immune responses to various classes of infectious agents
have many overlapping, but certain unique or uniquely
dominant, characteristics (1). The elements appear to be in
place to access and/or deliver mechanisms most effective in
defense against the eliciting infectious organism. Innate cytokine responses can have roles in shaping downstream adaptive, as well as other innate, immune responses (2). The paradigm emerging from bacterial and parasite studies has
IL-12 as the pivotal innate cytokine for promoting NK and T helper type 1 (Th1) cell IFN- responses (3). Roles
for this cytokine in immune responses to viruses are less
clear. Biologically active IL-12 is not induced during all
viral infections (10). The cytokine is part of the innate
immune response and stimulates NK cell IFN-
production during murine cytomegalovirus (MCMV)1 (10, 11)
and influenza virus (12), but not lymphocytic choriomeningitis virus (LCMV) (11), infections of mice. Moreover, IL-12-dependent T cell IFN-
responses are not demonstrable in number of viral infections including those that do
or do not induce detectable IL-12 (11, 12, 14).
In contrast to infections with other agents, many viruses
elicit high levels of the innate cytokines, type 1 interferons
(i.e., IFN-/
) and adaptive CD8 T cell responses. During
LCMV infections lacking detectable IL-12, early and dramatic elevations in IFN-
/
concentrations are induced
on days 2 and 3 after infection (10, 11, 13, 15), and T cell
immune responses characterized by profound CD8 T cell
expansion and IFN-
production are elicited on or after
days 7-9 (16). Specificity of the CD8 T cell responses has been proven by direct visualization with binding of
MHC class I tetramer molecules complexed to LCMV
epitopes (23), and by stimulation of IFN-
expression with
LCMV peptides (23). The innate cytokines important
for promoting these T cell responses have not been defined. IFN-
/
cytokines can contribute to a variety of
immunoregulatory effects (26), and are reported to promote T cell IFN-
production under certain culture conditions (27). Thus, they may be a class of innate cytokines uniquely regulating adaptive T cell responses to viral infections.
The studies reported here were undertaken to define
roles for innate cytokines in supporting antiviral T cell responses. The experiments, carried out during LCMV infections of mice, demonstrate for the first time the major endogenous innate pathways to CD8 T cell IFN- during
viral infections. They show that the IFN-
/
cytokines are
dominant in promoting conditions for this T cell IFN-
production. Moreover, they demonstrate that although T
cell IFN-
responses are IL-12 independent in the context
of IFN-
/
induction and function, IL-12 revealed in the
absence of IFN-
/
functions can substitute to promote
IFN-
production. This alternative pathway is beneficial
but not sufficient for induction of optimal protection.
Taken together, the data define unique factors and conditions regulating immune responses to viral infections. Furthermore, they result in the discovery of alternative innate
cytokine pathways for promoting IFN-
responses.
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Materials and Methods |
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Mice.
Mice deficient in IL-12, as a result of targeted disruption of the IL-12p35 gene (IL-12p35 KO), were generated and bred at Genetics Institute, using the p35 SK+ vector (Stratagene Inc.), the embryonic stem cell system in 129/sv mice, and established procedures (30). Mice were backcrossed onto the C57BL/6 genetic background for two to four generations. Homozygous mutant (
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Treatments.
The LCMV infections were established i.p. on day 0 with 2 × 104 PFU of Armstrong strain, clone E350 (11, 13, 35, 36). CD8 T cells were depleted in vivo by treatment i.p. with 0.5 mg of monoclonal anti-CD8 antibody 2.43, prepared from ascites, on day 5 after infection. Control treatment was with partially purified P3NS1 ascites containing 0.5 mg rat IgG (Sigma Chemical Co.). Sheep antimurine IFN-Cytokine Analyses.
Samples for quantitation of IFN-Flow Cytometric Analysis.
As per modification of published techniques (40), cells were stimulated on 24-well cluster plates previously coated overnight with 0.5 ml 10 µg/ml purified hamster anti-mouse CD3Viral Plaque Assays.
Livers and spleens were frozen at ![]() |
Results |
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To conclusively exclude a role for IL-12 in T cell responses to LCMV
infection, mice were made deficient for the p35 subunit of
IL-12 (IL-12p35 KO) by homologous recombination with
a deleted gene construct, as described in Materials and
Methods and Fig. 1. None of the day 8 splenic T cell responses to LCMV infection were significantly reduced in
the IL-12p35 KO mice. Yield and flow cytometric analyses
demonstrated that overall expansion of CD8 T cells was
similar in both WT and IL-12p35 KO (Fig. 2 A). Furthermore, ELISA studies demonstrated that induction of IFN- expression, both in media conditioned with splenic leukocytes (CM) and in serum, was neither blocked nor significantly inhibited in the absence of IL-12 (Fig. 2 B). Because
CD8 T cells are the predominant IFN-
producers during
LCMV infection, IFN-
expression by CD8 T cells also
was measured by flow cytometric analysis for cytoplasmic
protein and shown to be unaffected by absence of IL-12. Upon stimulation ex vivo with immobilized anti-CD3,
~60-70% of the cells induced to express cytoplasmic IFN-
from both types of mice were CD8 T cells, and the proportions of IFN-
-expressing CD8 T cells were 43.2% (± 8.0) and 43.5% (± 4.7) (means ± SEM) in WT and IL-12p35 KO mice, respectively (Fig. 2 C). Likewise, total
numbers of CD8 T cells expressing IFN-
, calculated
based on CD8 T cell yields, were equivalent with 7.9 (± 2.2)
and 9.6 (± 2.7) × 106 cells (Fig. 2 C). The endogenous responses were sufficient to mediate protection against infection because viral titers were below detectable levels in
both infected WT and IL-12p35 KO mice by day 8 (data
not shown). Thus, T cell proliferation and IFN-
production occur in the complete absence of the biologically active IL-12 heterodimer during infections with this virus,
and the immune responses induced under these conditions
are protective.
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Experiments were carried out to characterize roles of IFN-/
in regulating the T cell responses. Although of lower magnitude on the inbred genetic 129 background of these
mice, inductions of CD8 T cell expansion and IFN-
expression were observed and similar in both WT and IFN-
/
R KO mice (Fig. 2, D and E). IFN-
levels, measured
in samples from the IFN-
/
R KO mice, were equal to
or greater than those from WT mice in CM and serum
(Fig. 2 E). Interestingly, serum IFN-
levels were enhanced significantly by more than threefold in samples obtained from IFN-
/
R KO, as compared with WT, mice.
Studies, in IFN-
/
R KO mice depleted of endogenous
CD8 T cells by antibody treatments, demonstrated that 66%
of the enhanced serum IFN-
was dependent on endogenous CD8 T cells. After anti-CD3 stimulation ex vivo,
50-60% of the IFN-
expressing cell types from both IFN-
/
R KO and WT mice were CD8 T cells (data not shown),
and similar proportions of the CD8 T cell subset from both
types of mice were induced to express the cytokine (Fig. 2
F). As these T cell responses are intact, the data suggest that
IFN-
/
deficiencies by themselves do not result in generalized T cell exhaustion or depletion. However, viral titers
were increased in IFN-
/
R KO relative to WT mice (see
below). Hence, the CD8 T cell expansion and IFN-
production are not IFN-
/
dependent, but the conditions
result in decreased resistance to infection.
Because total expansion and IFN- expression were intact, but levels of virus were increased, antigen specificity
of the responding cells was tested. To enumerate CD8 cells
bearing T cell receptors for LCMV antigens, splenic leukocytes were labeled with anti-CD8 and tetramers of Db
MHC class I molecules containing LCMV peptides immunodominant for CD8 T cell responses (NP396-404 or GP33-41), as described in Materials and Methods. Flow cytometric
analyses showed significant increases in percentages of CD8
T cells binding tetrameric Db NP396-404 or Db GP33-41
with cells from day 8 LCMV-infected, relative to uninfected, WT mice (Fig. 3, A and B). Samples from IFN-
/
R KO
mice also had significant increases in proportions of tetrameric Db NP396-404- and Db GP33-41-binding CD8 T
cells after infection (Fig. 3, A and B). T cell frequencies specific for tetramers with NP396-404 were higher in WT,
whereas those with GP33-41 were higher in IFN-
/
R KO, populations. Total numbers of CD8 T cells binding the
complexed tetramers were significantly increased after infection, relative to uninfected samples, in both WT and
IFN-
/
R KO mice (Fig. 3 C). Moreover, because of increases in average splenic cell yields after infection, the total
numbers of CD8 T cells specific for the tetrameric Db LCMV
peptide complexes were higher in the IFN-
/
R KO than
the WT mice, respectively, averaging 247 and 120 × 104
cells per spleen. As a result of their skewed proportional representation, however, the Db GP33-41 binding cells respectively comprised ~75 and 20% of the populations, and appeared to account for a large proportion of the enhanced
expansion of T cells recognizing viral epitopes in the IFN-
/
R KO mice. Thus, as in WT mice, expanding CD8 T
cells in IFN-
/
R KO mice are specific for the virus. However, in the absence of IFN-
/
function, responses are differentially elicited to particular LCMV epitopes.
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To demonstrate specificity of functional CD8 T cells,
IFN- expression responses to the LCMV NP396-404 and
GP33-41 peptides were evaluated ex vivo. Cells from uninfected WT or IFN-
/
R KO mice were not stimulated
by either of these peptides (data not shown). In contrast, on
day 8 after LCMV infection, significant increases in CD8 T
cells expressing cytoplasmic IFN-
, relative to samples prepared in the absence of exogenous stimulation, were observed after peptide stimulation of either WT or IFN-
/
R
KO splenic leukocytes (Fig. 4). Consistent with the Fig.
2 H experiments, proportions of total primed CD8 T cells
responding to anti-CD3 stimulation for IFN-
expression
remained similar (Fig. 4 B). However, even though the total proportions of cells specific for binding tetramers with
LCMV peptides (Fig. 3 B), and of cells expressing cytoplasmic IFN-
after anti-CD3 stimulation (Fig. 4 B), were
similar, lower proportions of cells from IFN-
/
R KO as
compared with WT mice were stimulated in response to
peptides (Fig. 4 B). Interestingly, despite dramatic increases
in cells binding tetrameric Db GP33-41 (Fig. 3), the proportions and numbers of cells responding to this peptide for
IFN-
expression were similar or only marginally higher in
IFN-
/
R KO compared with WT populations (Fig. 4).
Thus, the reduced IFN-
responses with cells from IFN-
/
R KO mice appeared to be a consequence of specifically
binding populations failing to detectably respond to GP33-41 stimulation for IFN-
expression. Evaluation of cytokine in CM demonstrated similar levels of population responsiveness to the LCMV peptides (Table I). These studies demonstrate induction of LCMV-specific CD8 T cell IFN-
production responses in the absence of IFN-
/
functions.
Taken together with the tetramer binding results, they provide evidence that a proportion of the CD8 T cells specifically expanding in response to viral epitopes are altered
in their requirements for stimulation and/or magnitude of
functional responses.
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Kinetic studies of viral clearance during the experiments reported here, sampling on various days after LCMV infection, demonstrated that viral burdens in both IL-12p35 KO (data not shown)
and WT mice were below detection by day 8 after infection. Extended studies of WT and IFN-/
R KO mice
showed that WT mice had viral burdens that peaked
around day 4.5 at levels of 6.2 (± 0.2) and 5.0 (± 0.2) log
PFU/g of spleen and liver, respectively, and were below detection by day 7 (Fig. 5). Similar kinetics of splenic and
hepatic viral titers were observed in IFN-
/
R KO mice,
but these peaked at the higher levels of 10.3 (± 0.2) and
10.5 (± 0.0) log PFU/g of respective tissue on day 4.5 after
infection, and declined by day 7 (Fig. 5). Interestingly, decreases in splenic and liver viral loads were observed in the
IFN-
/
R KO mice over time to 4.7 (± 0.2) and 4.2 (± 0.0) log PFU/g of tissue, respectively, on day 14 after infection, and below the limits of detection by day 28 after
infection (Fig. 5). Thus, absence of an endogenous IL-12
response does not increase sensitivity to LCMV infection, and although absence of IFN-
/
functions does, viral
clearance is eventually achieved.
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Because IL-12 expression can be revealed during LCMV
infection by neutralization of endogenous IFN-/
functions (13), a released IL-12 induction may substitute in supporting T cell responses under these conditions. Measurements of IL-12 p70 in serum demonstrated that the factor
was induced to detectable levels on days 1.5 and 3 of LCMV
infection in IFN-
/
R KO, but not WT, mice (Fig. 6). Therefore, IL-12 effects on the T cell responses were examined in IFN-
/
R KO mice by treatments with antibodies neutralizing IL-12. Although anti-IL-12 treatments
did not modify IFN-
production responses as compared
with control antibody treatments in WT mice, they significantly reduced IFN-
production on day 8 in LCMV- infected IFN-
/
R KO mice; relative to cells isolated
from control-treated IFN-
/
R KO mice, 70% decreases in
CM spontaneous production of IFN-
were observed (Fig.
7 A, left). Furthermore, anti-IL-12 treatment of IFN-
/
R
KO mice, resulted in a >85% reduction in CM IFN-
levels, as compared with anti-IL-12-treated WT mice (Fig. 7 A, left, striped bars); i.e., from 66.4 (± 16.0) to 7.8 (± 2.0) pg/106 cells. Consistent with the experiments shown in
Fig. 2, B and E, only low levels of serum IFN-
were detected in WT mice, but were increased by more than
threefold in IFN-
/
R KO mice (Fig. 7 A, right). Given
the contribution of CD8 T cells to serum IFN-
(see above),
the stimulation of CD8 T cell IFN-
expression by LCMV
epitopes (Fig. 4), and the significant increases in viral burdens (Fig. 5, day 8), elevated in vivo stimulation of the
CD8 T cells by LCMV epitopes was likely to have contributed to higher serum IFN-
levels in the IFN-
/
R KO
mice. IL-12 also participated in this enhanced response because neutralization of the factor resulted in a 40% reduction in serum IFN-
levels (Fig. 7 A, right). Thus, in the
absence of endogenous IFN-
/
effects during a viral infection, IL-12 can be induced and substitute to provide
conditions supporting IFN-
responses.
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To confirm and extend these studies, IFN-/
effects
were examined under the reciprocal conditions in mice
lacking endogenous IL-12. For these experiments, WT and
IL-12p35 KO mice were treated with antibodies neutralizing IFN-
/
or control antibodies. Cytokine neutralization
of WT mice resulted in IFN-
responses comparable with
those in IFN-
/
R KO mice; i.e., IFN-
production in
CM was not blocked and was induced to elevated levels in
serum (Fig. 7 B). Relative to control-treated mice, antibody-mediated neutralization of IFN-
/
in IL-12p35 KO mice
significantly inhibited IFN-
levels spontaneously produced
in CM (Fig. 7 B, left). Moreover, in comparison with anti-
IFN-
/
-treated WT mice, serum IFN-
levels were reduced by >90% as a result of IFN-
/
neutralization in the
IL-12p35 KO mice; i.e., whereas the anti-IFN-
/
-
treated WT mice had levels of 1,454.2 (± 171.2) pg/ml in
serum, the anti-IFN-
/
-treated IL-12p35 KO mice had
only 110.9 (± 30.6) (Fig. 7 B, right, striped bars). Thus, in
the absence of endogenous IL-12, IFN-
/
-mediated effects are primarily responsible for endogenous conditions
promoting the IFN-
responses to viral infections.
To evaluate the specificity of CD8 T cell responses for
LCMV, under the conditions of anti-IL-12 treatment in
IFN-/
R KO mice, experiments were carried out examining peptide or anti-CD3 stimulation for cytoplasmic IFN-
expression, specific stimulation by peptides for IFN-
production in CM, and surface binding of tetramers complexed with peptides immunodominant for CD8 T cells. Overall, both the control and anti-IL-12 antibody treatments modestly blunted magnitudes of the ex vivo-
detected specific responses. As a result, intracytoplasmic labeling was not sensitive enough to identify changes in the
low proportions of CD8 T cells specifically responding to
peptides with cytoplasmic IFN-
expression (see Fig. 4).
However, it was possible to demonstrate decreases, resulting from the blocking IL-12 function in IFN-
/
R KO
mice, in the proportion of CD8 T cells primed for anti-
CD3 stimulation of IFN-
expression; i.e., 18.3% (± 2.2)
to 12.8% (± 0.2). The anti-IL-12 treatments also resulted
in significant 46 and 53% inhibitions of NP396-404- and
GP33-41-stimulated IFN-
production, respectively, in
CM (Fig. 8). In contrast to the effects on IFN-
responses,
blocking of both cytokine pathways did not inhibit LCMV-induced CD8 T cell expansion; increased proportions and
numbers of CD8 T cells specific for Db NP396-404 and Db
GP33-41 were observed with or without IL-12 neutralization (data not shown). These studies demonstrate that the
conditions of a revealed IL-12 pathway, in the absence of
IFN-
/
-mediated functions, result in IFN-
production
promoted in a virus-specific manner by epitopes immunodominant for CD8 T cell responses. In contrast, the results indicate that specific CD8 T cell proliferation can occur in the absence of both IL-12 and IFN-
/
-mediated
effects.
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To assess the
contribution of IL-12 to virus clearance in the absence of
IFN-/
function, viral titers were measured in LCMV-infected mice having had both factors blocked. Anti-IL-12
treatment of IFN-
/
R KO mice resulted in statistically
significant (P < 0.01) increases in viral titers of almost 1 log
by day 14 after infection; spleens and livers from anti-IL-12-treated IFN-
/
R KO had 5.8 (± 0.0) and 5.3 (± 0.1)
log PFU/g, respectively, as compared with the 5.1 (± 0.1)
and 4.4 (± 0.1) log PFU/g of tissue observed in control-treated IFN-
/
R KO mice (means of three mice per
group ± SEM). These results show that the endogenous
IL-12 response in IFN-
/
R KO mice promotes the antiviral state of the host, but cannot substitute for endogenous
IFN-
/
in clearing virus expediently.
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Discussion |
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These studies have characterized divergent innate pathways for promoting IFN- responses during viral infections, with induction of high level IFN-
/
resulting in
conditions supporting one and acting to limit a potential alternative IL-12 pathway. The experiments demonstrate (a)
an IFN-
/
pathway for IFN-
induction, and (b) IL-12
independence of the CD8 T cell responses of expansion
and IFN-
production, during LCMV infections. Moreover, they show that in the absence of endogenous IFN-
/
-
mediated functions, an IL-12 response is revealed and sufficient to support induction of an IFN-
response, but not
peak protection. Despite delayed clearance of viral burdens,
the CD8 T cell expansion and IFN-
responses elicited in
the presence of the alternative IL-12 pathway are LCMV
specific because the expanded cells bind MHC tetramer molecules complexed with the LCMV epitopes NP396-404
or GP33-41, and are stimulated to express IFN-
by viral
peptides. Although the substituted IL-12 acts to promote
IFN-
production, it does not appear to be required for expansion of virus-specific CD8 T cells. It is biologically significant, however, as viral titers increase if both pathways are
blocked. Thus, major contributions to adaptive CD8 T cell
responses are made by innate cytokines, predominantly IFN-
/
, but alternatively IL-12, during viral infection.
These innate cytokine immunoregulatory pathways can
be contrasted to those characterized in response to nonviral
intracellular pathogens. Similar to the Th1 responses defined under conditions of bacterial or parasitic stimuli (1,
3), LCMV infections induce IL-2 and IFN- production. However, in the other microbial infections, IL-12 is
the pivotal cytokine promoting Th1 responses (4, 8, 42).
The studies presented here conclusively demonstrate that,
in the complete absence of endogenous biologically active IL-12 resulting from genetic mutation of the IL-12p35
subunit, LCMV-induced CD8 T cell expansion and IFN-
expression proceed normally (Fig. 2, A-C). The lack of a
role for IL-12 in induction of T cell IFN-
expression confirms and extends an earlier report from this laboratory
demonstrating that neutralization of endogenous IL-12
function by treatment with antibodies directed against the p40 chain does not inhibit LCMV induction of the T cell
responses in IFN-
/
competent mice (11). Moreover, it
is in agreement with the recent reports of lack of IL-12
effect on T cell IFN-
responses under the conditions of
treatments with antibody directed against the p40 chain
during influenza virus infections (12) and genetic mutation
of either the p35 or both the p35 and p40 molecules during
mouse hepatitis virus infections (14). Thus, there are indications in a variety of viral infections that T cell IFN-
responses are IL-12 independent.
In addition to showing the lack of importance for IL-12
in the presence of IFN-/
functions, however, the studies
demonstrate an IFN-
/
role in supporting T cell IFN-
production during viral infections. They define the existence of this pathway for the first time and characterize the
in vivo conditions under which it is important to the host.
The results are consistent with reported enhancing effects of
IFN-
/
for T cell IFN-
production under certain specific conditions in culture (27, 43). However, the culture
studies have been limited to examining the IFN-
roles for
modest effects in association with IL-12 (28, 43), CD4 T
cell subset IFN-
responses (27, 43), and/or dramatic effects in association with the IFN-
-inducing factor (IGIF), sometimes called IL-18 (29). Ongoing studies in our laboratory
are evaluating a potential accessory role for IGIF under the
conditions of viral infections. The results presented here
contribute to defining the complete system by demonstrating that during viral infections IFN-
/
cytokines are dominant for T cell IFN-
responses, mediate these effects in
the absence of IL-12, and act on CD8 T cell subsets.
The experiments also identify a secondary IL-12 response
absent in IFN-/
R-competent but revealed in IFN-
/
R-
deficient mice (Figs. 6-8). The lack of IL-12 appearance in
the presence of IFN-
/
functions is consistent with the
known negative regulation of IL-12 by the cytokines (13).
The alternative IL-12 response is beneficial to the host because it can substitute in promoting IFN-
production and
facilitates clearance of virus. However, it is apparently sub-optimal because the conditions may fail to access the direct
antiviral effects of IFN-
/
and result in delayed viral
clearance. Thus, the responses elicited in the context of the
IFN-
/
-mediated effects are clearly better for defense
against this particular infectious agent, but the host can activate substitute defense mechanisms. Although the two divergent in vivo pathways to T cell IFN-
are clearly established, it is not known if the IFN-
/
and IL-12 effects are
mediated directly or indirectly, or at the priming as compared with the production phases. Experiments are underway examining these.
Remarkably, LCMV elicits particularly high circulating
levels of IFN-/
, and under these conditions does not induce IL-12 (10, 11, 13, 15). Immune responses to this infection may represent those on one end of a spectrum ranging
from exclusive dependence on IFN-
/
, to codominant
regulation by IFN-
/
and IL-12, to exclusive dependence
on IL-12 for promoting IFN-
production. Our hypothesis
is that relative contributions would depend on presence or
absence and magnitude of induction levels. MCMV and influenza infections induce more mixed responses with detectable IL-12 (10, 44), and certain bacterial and parasitic infections also may elicit both IL-12 and IFN-
/
expression
(6, 45, 46). Other intracellular bacteria may preferentially
elicit IL-12 responses (4). Direct comparison of infections
indicates that LCMV induces up to threefold higher levels
and sustains longer production periods of serum IFN-
/
relative to MCMV (Cousens and Biron, unpublished results). The levels achieved during LCMV infection are sufficient to mediate significant negative regulation of IL-12
(13). Thus, the picture emerging is that the immune system
is equipped to induce both IFN-
/
and IL-12 simultaneously; either will lead to conditions promoting IFN-
production, but conditions of high IFN-
/
expression
make these cytokines dominant because they also are inhibiting the IL-12 response. As stated above, such conditions appear to be beneficial because they access direct antiviral functions and are particularly conducive for induction
of protective responses. Moreover, as the dramatic T cell
responses to LCMV render the host more sensitive to
IL-12 toxicities (35, 36), they may additionally act to protect from detrimental immune responses.
Although effects of IFN-/
on IFN-
production are
demonstrated, the results also indicate that total and virus-specific CD8 T cell expansions are IFN-
/
independent
(Figs. 3 and 4), and that they occur even if both IFN-
/
and IL-12 functions are blocked (data not shown). Thus,
other factors must be promoting CD8 T cell expansion
during LCMV infections. At least one adaptive cytokine,
IL-2, is apparently available to carry out this function. CM
levels of IL-2 are similar with cells from IFN-
/
R KO,
IL-12p35 KO, and WT mice, and only reduced by about
half with cells from mice blocked in both IFN-
/
and IL-12 functions (data not shown). This cytokine is critical for
CD8 T cell expansion, and as a result of supporting T cell
proliferation, for peak T cell IFN-
responses (17, 21).
Thus, the innate cytokine IFN-
/
and/or IL-12 responses and the downstream consequences of these responses do not appear to be as important as IL-2 for T cell
expansion. In this regard, it has been suggested that IFN-
/
may stimulate IL-15 production to promote proliferation of memory CD8 T cells during early viral infections
(47, 48). Our results suggest that an IFN-
/
induction of
IL-15 is not essential for virus-specific CD8 T cell proliferation during acute infections. However, it is interesting to
note that specificities of the expanded T cells are somewhat
skewed in the absence of IFN-
/
(Figs. 3 and 4). Thus,
there are additional unidentified IFN-
/
effects contributing to selection of the T cell repertoire activated against infection.
The skewing of responses is observed at the level of relative proportions of CD8 T cells specifically binding tetrameric molecules complexed with NP396-404 or GP33-41
(Fig. 3). However, as the total numbers of cells binding one
or the other are similar in WT and IFN-/
R KO mice,
the magnitude of the CD8 T cell proliferative responses is
IFN-
/
function independent. Nevertheless, overall responses to peptide stimulation for CD8 T cell expression of cytoplasmic IFN-
expression (Fig. 4), and peptide stimulation for IFN-
production (Table I) are reduced. Moreover, the total proportions of CD8 T cells primed to specifically respond by expressing cytoplasmic IFN-
after ex
vivo stimulation with the LCMV peptides NP396-404 or
GP33-41 account for 90-95% of those sensitized to anti-
CD3 stimulation during infections of WT mice, but represent only 25-60% of those sensitized during infections of
IFN-
/
R KO mice (Fig. 4). Thus, anti-CD3 reveals cells
primed for T cell functions but failing to respond to the
NP396-404 or GP33-41 peptides. The populations stimulated by anti-CD3, but not the tested LCMV peptides,
could represent T cells having receptors (a) recognizing other LCMV epitopes, (b) nonspecifically activated, and/or
(c) altered in magnitudes of functions and/or requirements
for stimulation. There is evidence for the first two of these
under other conditions. Although NP396-404 and GP33-41 represent the major immunodominant LCMV epitopes
detected in MHC H-2b mice (23, 39, 49, 50), other
minor epitopes have been identified (39). Despite prominence of the virus-specific T cell responses, "bystander" activation of memory phenotype T cells has been reported
during LCMV infections (47). However, the last possibility
seems most likely because CD8 T cells binding tetrameric
Db GP33-41 are dramatically expanded. Further experiments are needed to conclusively distinguish between these
possibilities. Nevertheless, the studies clearly document expansion of a large proportion of cells specific for LCMV
epitopes and responding with IFN-
production.
LCMV is a relatively noncytopathic virus. In the absence
of T cell responses and/or under specific conditions of diminishing CTL responses, sometimes called "T cell exhaustion," persistent LCMV infections can be established.
Although detectable CTL function is not induced during
LCMV infection in the absence of IFN-/
(33; data not
shown), our results indicate that the conditions are sufficient for resistance and eventual viral clearance (Fig. 8).
Thus, they are in contrast to the suggestion of others that, in the absence of IFN-
/
-mediated regulation of viral
replication, LCMV-induced T cell exhaustion results from
an overwhelming viral burden (33, 34). Those investigators
have based their hypothesis on the lack of CTL activity
without enumerating CD8 T cell numbers. In our studies,
the two are dissociated; i.e., CD8 T cell expansion and
IFN-
production occur in the absence of apparent virus-specific CTL function (data not shown). However, different isolates of LCMV vary for spontaneous induction, in
immunocompetent mice, of T cell exhaustion as characterized by lack of CTL (51, 52) and persistent infection (51-
53), and it has been demonstrated that at least one of these
conditions also results in the lack of virus-specific CD8 T
cell expansion (54). Clearly, this is not the case under the
conditions of infection in our studies. However, it is interesting to note that during chronic LCMV infections, the specificity of CD8 T cell responses can vary such that
NP396-404-specific cells are deleted and functionally unresponsive GP33-41-specific cells are maintained (55), and
that the skewing of specific T cells during infections of
IFN
/
R KO mice is in this direction; i.e., reduced
NP396-404 and increased GP33-41 specific cells (Fig. 3).
Thus, parameters in addition to the absence of IFN-
/
functions must be required to extinguish defense and establish viral persistence, but protection mediated in the absence
of IFN-
/
functions may be shifting in dependence towards T cell subsets sustained for longer periods of time
during infections and extended antigen stimulation.
In summary, data presented here define unique divergent
regulatory pathways promoting IFN- responses to viral
infection, controlled by IFN-
/
or IL-12. They demonstrate that the strong and protective CD8 T cell responses
of expansion and IFN-
production are induced though
IL-12-independent pathways during infections of immunocompetent hosts. Moreover, the studies show that in the absence of endogenous IFN-
/
, an IL-12 response can be
revealed and substitute conditions to promote IFN-
production. Although not resulting in induction of the most
effective antiviral immune responses, the IL-12 substitution
is beneficial. Thus, the results define uniquely IFN-
/
-
controlled pathways for promoting peak defense during viral infections inducing these cytokines, and the plasticity of
immune responses in accessing an alternative pathway to
reach certain of the same goals.
![]() |
Footnotes |
---|
Address correspondence to Christine A. Biron, Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Box G-B629, Brown University, Providence, RI 02912. Phone: 401-863-2921; Fax: 401-863-9045; E-mail: christine_biron{at}brown.edu
Received for publication 29 December 1998 and in revised form 22 February 1999.
The authors thank Drs. Ion Gresser, Phillip Scott, and Giorgio Trinchieri for their generous gifts of antibodies and hybridomas, Dr. Stan Wolf for helpful discussions, and Dr. Kaja Murali-Krishna for technical advice.
This work was supported in part by National Institutes of Health grants CA41268, AI42373, and NS21496. L.P. Cousens was supported in part by National Institutes of Health Environmental Science Training Grant T32-ES07272.
Abbreviations used in this paper CM, conditioned media; LCMV, lymphocytic choriomeningitis virus; MCMV, murine cytomegalovirus; WT, wild-type.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Biron, C.A., and R.T. Gazzinelli. 1995. Effects of IL-12 on immune responses to microbial infections: a key mediator in regulating disease outcome. Curr. Opin. Immunol. 7: 485-496 [Medline]. |
2. | Medzhitov, R., and C.A. Janeway. 1997. Innate immunity: impact on the adaptive immune response. Curr. Opin. Immunol. 9: 4-9 [Medline]. |
3. | Heinzel, F.P., R.M. Rerko, P. Ling, J. Hakimi, and D.S. Schoenhaut. 1994. Interleukin 12 is produced in vivo during endotoxemia and stimulates synthesis of gamma interferon. Infect. Immunol. 62: 4244-4249 [Abstract]. |
4. | Hsieh, C.-S., S.E. Macetionia, C.S. Tripp, S.F. Wolf, A. O'Garra, and K.M. Murphy. 1993. Development of Th1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science. 260: 547-549 [Medline]. |
5. | Manetti, R., P. Parronchi, M.G. Giudizi, M.P. Piccinni, E. Maggi, G. Trinchieri, and S. Romagnani. 1993. Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells. J. Exp. Med. 177: 1199-1204 [Abstract]. |
6. |
Scharton-Kersten, T.,
L.C.C. Afonso,
M. Wysocka,
G. Trinchieri, and
P. Scott.
1995.
IL-12 is required for natural
killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis.
J. Immunol.
154:
5320-5330
|
7. | Seder, R.A., and W.E. Paul. 1994. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu. Rev. Immunol. 12: 635-673 [Medline]. |
8. | Sypek, J.P., C.L. Chung, S.E.H. Mayor, J.M. Subramanyam, S.J. Goldman, D.S. Sieburth, S.F. Wolf, and R.G. Schaub. 1993. Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response. J. Exp. Med. 177: 1797-1802 [Abstract]. |
9. |
Wysocka, M.,
M. Kubin,
L.Q. Vieira,
L. Ozmen,
G. Garotta,
P. Scott, and
G. Trinchieri.
1995.
Interleukin-12 is required for interferon-![]() |
10. |
Orange, J.S., and
C.A. Biron.
1996.
Characterization of early
IL-12, IFN-![]() ![]() |
11. |
Orange, J.S., and
C.A. Biron.
1996.
An absolute and restricted requirement for IL-12 in natural killer cell IFN-![]() |
12. |
Monteiro, J.M.,
C. Harvey, and
G. Trinchieri.
1998.
Role of
interleukin-12 in primary influenza virus infection.
J. Virol.
72:
4825-4831
|
13. |
Cousens, L.P.,
J.S. Orange,
H.C. Su, and
C.A. Biron.
1997.
Interferon-![]() ![]() ![]() |
14. |
Schijns, V.E.C.J.,
B.L. Haagmans,
C.M.H. Wierda,
B. Kruithof,
I.A.F.M. Heijnen,
G. Alber, and
M.C. Hornizek.
1998.
Mice lacking IL-12 develop polarized Th1 cells during
viral infection.
J. Immunol.
160:
3958-3964
|
15. |
Welsh, R.M..
1978.
Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice. I. Characterization of natural killer cell induction.
J. Exp. Med.
148:
163-181
|
16. | Biron, C.A.. 1994. Cytokines in the generation of immune responses to, and resolution of, virus infection. Curr. Opin. Immunol. 6: 530-538 [Medline]. |
17. |
Cousens, L.P.,
J.S. Orange, and
C.A. Biron.
1995.
Endogenous IL-2 contributes to T cell expansion and IFN-![]() |
18. |
Gessner, A.,
D. Moskophidis, and
F. Lehmann-Grube.
1989.
Enumeration of single IFN-![]() |
19. |
Kasaian, M.T., and
C.A. Biron.
1989.
The activation of IL-2
transcription in L3T4+ and Lyt-2+ lymphocytes during virus
infection in vivo.
J. Immunol.
142:
1287-1292
|
20. | Moskophidis, D., S.P. Cobbold, H. Waldmann, and F. Lehmann-Grube. 1987. Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response. J. Virol. 61: 1867-1874 [Medline]. |
21. |
Su, H.C.,
L.P. Cousens,
L.D. Fast,
M.K. Slifka,
R.D. Bunjiro,
R. Ahmed, and
C.A. Biron.
1998.
CD4+ and CD8+ T
cell interactions in IFN-![]() |
22. | Zinkernagel, R.M., and P.C. Doherty. 1997. The discovery of MHC restriction. Immunol. Today. 18: 14-17 [Medline]. |
23. | Murali-Krishna, K., J.D. Altman, M. Suresh, D.J.D. Sourdive, A.J. Zajac, J.D. Miller, J. Slansky, and R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity. 8: 177-187 [Medline]. |
24. | Butz, E.A., and M.J. Bevan. 1998. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity. 8: 167-175 [Medline]. |
25. | van der Most, R.G., K. Murali-Krishna, J.L. Whitton, C. Oseroff, J. Alexander, S. Southwood, J. Sidney, R.W. Chesnut, A. Sette, and R. Ahmed. 1998. Identification of Db- and Kb-restricted subdominant cytotoxic T-cell responses in lymphocytic choriomeningitis virus-infected mice. Virology. 240: 158-167 [Medline]. |
26. | Ahmed, R., and C.A. Biron. 1998. Immunity to viruses. In Fundamental Immunology. 4th ed. W.E. Paul, editor. Lippincott-Raven Publishers, New York. 1295-1334. |
27. |
Brinkman, V.,
T. Geiger,
S. Alkan, and
C.H. Heusser.
1993.
Interferon ![]() ![]() |
28. |
Manetti, R.,
F. Annunziato,
L. Tomasevic,
V. Gianno,
P. Parronchi,
S. Romagnani, and
E. Maggi.
1995.
Polyinosinic
acid: polycytidylic acid promotes T helper type 1-specific
immune responses by stimulating macrophage production of
interferon-![]() |
29. |
Saraneva, T.,
S. Matikainen,
M. Kurimoto, and
I. Julkunen.
1998.
Influenza A virus-induced IFN-![]() ![]() ![]() |
30. | Gossler, A., T. Doetschman, R. Korn, E. Serfling, and R. Kemmler. 1986. Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc. Natl. Acad. Sci. USA. 83: 9065-9069 [Abstract]. |
31. | Lufkin, T., A. Dierich, M. Le Meur, M. Mark, and P. Chambon. 1991. Disruption of the Hox-1.6 homeobox gene results in defects in a region corresponding to its rostal domain of expression. Cell. 68: 1105-1119 . |
32. | Mansour, S.L., K.R. Thomas, and M.R. Capecchi. 1988. Disruption of the proto-oncogene int-2 in mouse embryo- derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 336: 348-352 [Medline]. |
33. | Muller, U., U. Steinhoff, L.F.L. Reis, S. Hemmi, J. Pavlovic, R.M. Zinkernagel, and M. Aguet. 1994. Functional role of type I and type II interferons in antiviral defense. Science. 264: 1918-1921 [Medline]. |
34. | van den Broek, M.F., U. Muller, S. Huang, M. Aguet, and R.M. Zinkernagel. 1995. Antiviral defense in mice lacking both alpha/beta and gamma interferon receptors. J. Virol. 69: 4792-4796 [Abstract]. |
35. |
Orange, J.S.,
S.F. Wolf, and
C.A. Biron.
1994.
Effects of IL-12
on the response and susceptibility to experimental viral infections.
J. Immunol.
152:
1253-1264
|
36. | Orange, J.S., T.P. Salazar-Mather, S.M. Opal, R.L. Spencer, A.H. Miller, B.S. McEwen, and C.A. Biron. 1995. Mechanism of interleukin 12-mediated toxicities during experimental viral infections: role of tumor necrosis factor and glucocorticoids. J. Exp. Med. 181: 901-914 [Abstract]. |
37. | Gresser, I., M.G. Tovey, C. Maury, and M.-T. Bandu. 1976. Role of interferon in the pathogenesis of virus diseases in mice as demonstrated by the use of anti-interferon serum. J. Exp. Med. 144: 1316-1323 [Abstract]. |
38. | Moskophidis, D., M. Battegay, M.-A. Bruendler, E. Laine, I. Gresser, and R.M. Zinkernagel. 1994. Resistance of lymphocytic choriomeningitis virus to alpha/beta interferon and to gamma interferon. J. Virol. 68: 1951-1955 [Abstract]. |
39. | van der Most, R.G., A. Sette, C. Oseroff, J. Alexander, K. Murali-Krishna, L.L. Lau, S. Southwood, J. Sidney, R.W. Chesnut, M. Matloubian, and R. Ahmed. 1996. Analysis of cytotoxic T cell responses to dominant and subdominant epitopes during acute and chronic lymphocytic choriomeningitis virus infection. J. Immunol. 157: 5543-5554 [Abstract]. |
40. | O'Garra, A., and K.M. Murphy. 1996. Role of cytokines in determining T cell function (mouse). In Weir's Handbook of Experimental Immunology. 5th ed. D.M. Weir, editor. Blackwell Science, Cambridge, MA. 226.1-226.10. |
41. |
Altman, J.D.,
P.A.H. Moss,
P.J.R. Goulder,
D.H. Barouch,
M.G. McHeyzer-Williams,
J.I. Bell,
A.J. McMichael, and
M.M. Davis.
1996.
Phenotypic analysis of antigen-specific T
lymphocytes.
Science.
274:
94-96
|
42. | Mattner, F., J. Magram, J. Ferrante, P. Launois, K. Di Padova, R. Behin, M. Gately, J.A. Louis, and G. Alber. 1996. Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response. Eur. J. Immunol. 26: 1553-1559 [Medline]. |
43. |
Wenner, C.A.,
M.L. Guler,
S.E. Macatonia,
A. O'Garra, and
K.M. Murphy.
1996.
Roles of IFN-![]() ![]() |
44. | Grundy, J.E., J. Trapman, J.E. Allan, G.R. Shellam, and C.J.M. Melief. 1982. Evidence for a protective role of interferon in resistance to murine cytomegalovirus and its control by non-H-2-linked genes. Infect. Immun. 37: 143-150 [Medline]. |
45. |
Diefenbach, A.,
H. Schindler,
N. Donhauser,
E. Lorenz,
T. Laskay,
J. MacMicking,
M. Röllinghoff,
I. Gresser, and
C. Bogdan.
1998.
Type 1 interferon (IFN-![]() ![]() |
46. |
Yaegashi, Y.,
P. Nelson,
A. Sing,
C. Galanos, and
M.A. Freudenberg.
1995.
Interferon ![]() ![]() |
47. | Tough, D.F., P. Burrow, and J. Sprent. 1996. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science. 272: 1947-1950 [Abstract]. |
48. | Zhang, X., S. Sun, I. Hwang, D.F. Tough, and J. Sprent. 1998. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity. 8: 591-599 [Medline]. |
49. | Gairin, J.E., H. Mazarguil, D. Hudrisier, and M.B.A. Oldstone. 1995. Optimal lymphocytic choriomeningitis virus sequences restricted by H-2Db major histocompatibility complex class I molecules and presented to cytotoxic T lymphocytes. J. Virol. 69: 2297-2305 [Abstract]. |
50. | Yanagi, Y., A. Tishon, H. Lewicki, B.A. Cubitt, and M.B.A. Oldstone. 1992. Diversity of T-cell receptors in virus-specific cytotoxic T lymphocytes recognizing three distinct epitopes restricted by a single major histocompatibility complex molecule. J. Virol. 66: 2527-2531 [Abstract]. |
51. | Ahmed, R., R.S. Simon, M. Matloubian, S.R. Kolhekar, P.J. Southern, and D.M. Freedman. 1988. Genetic analysis of in vivo-selected viral variants causing chronic infection: importance of mutation in the L RNA segment of lymphocytic choriomeningitis virus. J. Virol. 62: 3301-3308 [Medline]. |
52. | Moskophidis, D., F. Lechner, H. Pircher, and R.M. Zinkernagel. 1993. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral effector T cells. Nature. 362: 758-761 [Medline]. |
53. | Pfau, C.J., J.K. Valenti, D.C. Pevear, and K.D. Hunt. 1982. Lymphocytic choriomeningitis virus killer T cells are lethal only in weakly disseminated murine infections. J. Exp. Med. 156: 79-89 [Abstract]. |
54. |
Gallimore, A.,
A. Glithero,
A. Godkin,
A.C. Tissot,
A. Pluckthun,
T. Elliot,
H. Hengartner, and
R. Zinkernagel.
1998.
Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class
I-peptide complexes.
J. Exp. Med.
187:
1383-1393
|
55. |
Zajac, A.J.,
J.N. Blattman,
K. Murali-Krishna,
D.J.D. Sourdive,
M. Suresh,
J.D. Altman, and
R. Ahmed.
1998.
Viral
immune evasion due to persistence of activated T cells without effector function.
J. Exp. Med.
188:
2205-2213
|