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and IL-4 Responses to Viral Infections: Requirements for IL-21
Departments of
*
Molecular Microbiology and Immunology and
Medicine, Division of Biology and Medicine, Brown University, Providence, RI 02912; and
Emory Vaccine Center, Emory University, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4 as
well as IL-2. Experiments in mice either
ß2-microglobulin-deficient, lacking MHC class I molecules
and CD8+ T cells, or Aßb-deficient, lacking
MHC class II molecules and CD4+ T cells, demonstrated that
mixtures of T cell responses were required for optimal ex vivo cytokine
productions. Intracellular cytokine expression analyses of cells from
immunocompetent and immunodeficient mice showed that CD8+ T
cells were predominant IFN- producers, and that expansion of
CD8+ T cells primed to make IFN- was independent of
CD4+ T cells in vivo. Studies in IL-2-deficient mice
demonstrated that this cytokine promoted IFN- and IL-4 responses,
and ex vivo experiments showed that exogenous IL-2 was required to
maintain high-level IFN- production by in vivo-primed
CD8+ T cells. Conditions associated with cytokine decreases
were accompanied by reduced detectable plasma Ab responses. The results
indicate that, although IL-2-dependent CD8+ T cell
proliferation does not require endogenous CD4+ T cells,
IL-2 production by the CD4+ T cells may promote continued
cytokine release from activated CD8+ T cells. By defining
these critical steps in cellular and cytokine interactions for shaping
endogenous immune responses, the studies advance understanding of the
unique conditions regulating CD8+ T cell responses to viral
challenges. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and type 2 responses by IL-4, IL-5, IL-6, and IL-10
expression (1). Particular cytokines can selectively promote type 1 or
type 2 responses during CD4+ T cell differentiation.
Priming by IL-12 and/or IL-12-induced IFN- preferentially polarizes
to type 1 CD4+ T cell responses. These events can occur in
the host and have been shown to contribute to resistance during
infections with certain intracellular parasites and bacteria (2). In
contrast to nonviral pathogens, endogenous cytokine responses to a
number of acute viral infections have mixtures of type 1 and type 2
cytokines (3, 4, 5, 6, 7). Moreover, during infections with lymphocytic
choriomeningitis virus
(LCMV)3, T cell IFN-
responses can occur in the absence of detectable IL-12 protein
induction and are not inhibited by treatments neutralizing in vivo
IL-12 function (8). Thus, viral infections appear to differ from those
with other pathogens in not eliciting polarized cytokine responses, and
in apparently having IL-12-independent pathways priming and/or
promoting T cell IFN- responses.
Prominent CD8+ T cell responses distinguish viral from many
other infections. Following LCMV inoculations, CD8+ T cell
responses, necessary for viral clearance (9, 10, 11, 12), include proliferation
(13, 14, 15) and acquisition of virus-specific cytotoxic activity (9, 11, 13, 14, 16). In addition, CD8+ as well as CD4+
T cells have been shown to contribute to IFN- expression during a
number of viral infections (4, 6) including LCMV (17). Hence,
virus-induced CD8+ T cell responses involve cell expansion
along with induction of the effector functions resulting from CTL
activation and IFN- production. Although these virus-induced
CD8+ T cell responses are well documented, requirements for
their development are poorly understood. The focus of most studies to
date has been on development of CTL. During acute LCMV infections,
virus-induced CTL responses may be enhanced by, but do not require, the
endogenous presence of CD4+ T cells (11, 16, 18, 19, 20, 21, 22, 23),
IFN- (24, 25), IL-2 (26), or IL-4 (27). In regard to proliferation,
studies from this laboratory have shown that, although CD4+
T cells are major producers of IL-2, these cells are not required (20, 28), whereas IL-2 is a significant factor (29) for LCMV-induced
expansion of CD8+ T cells. Reductions in CD8+ T
cell expansion resulting from IL-2 deficiencies are accompanied by
proportionally inhibited overall CTL development (29). IL-2 deficiency
also results in reduced IFN- production (29), but the mechanism and
affected cell type(s) have not been identified. With the exception of
the suggested lack of role for IL-12 (8), virtually nothing is known
about requirements for in vivo T cell IFN- responses to viral
infections. Moreover, the role for CD4+ T cells in
promoting CD8+ T cell IFN- production has not been
characterized.
The studies presented here were undertaken to ascertain how
CD4+ and CD8+ T cells act to promote
virus-induced T cell cytokine production. Although CD8+ T
cell expansion and CTL development can proceed independently of
CD4+ T cells, both CD4+ and CD8+ T
cell responses were needed in vivo for optimal cytokine production as
evaluated by continued release in culture, with CD8+ T
cells the predominant IFN- producers as evaluated by cytoplasmic
staining of cytokine. IL-2 was required for both IFN- and IL-4
production, and was necessary for continued IFN- production by
activated CD8+ T cells. These results demonstrate that
during acute viral infections, IL-2 contributes to cytokine production
at the levels of priming/expansion and maintenance of cytokine
production by CD8+ T cells. They suggest that although
CD4+ T cells are not always required for CD8+ T
cell expansion and CTL development, this cell type can play critical
roles in supporting sustained CD8+ T cell IFN-
responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free male and female C57BL/6NTacfBR mice, and
male homozygous MHC class II-deficient
C57BL/6TacfBR-[KO]Aßb N5 or wild-type MHC class II
control C57BL/6TacfBR-[KO]Aßb N6 mice, were purchased
from Taconic, Germantown, NY. C57BL/6J-B2
mtm1Unc and C57BL/6J-Il2tm1Hor
mice, obtained from The Jackson Laboratory (Bar Harbor, ME), were
maintained at Brown University, Providence, RI, by breeding through
homozygous and heterozygous matings, respectively. Mice were typed as
+/+, +/-, or -/- for IL-2 gene alleles by PCR amplification.
IL-2-mutated mice were used at 
9 wk of age. Others were used at 5 to
28 wk. Mice were age, sex, and littermate matched for experiments and
handled in accordance with institutional guidelines for animal care and
use. Infections were initiated i.p. on day 0 with 2 x
104 plaque-forming U of LCMV, Armstrong strain, clone E350.
Plasma samples were collected by retroorbital harvest with heparinized
Natelson blood collecting tubes and centrifugation.
Preparation of cells and conditioned media samples
Leukocytes were obtained, after teasing apart spleens, by passing through nylon mesh and lysing erythrocytes. Viable cell yields were determined by trypan blue exclusion. To enrich CD8+ T cells, murine CD8+ T cell subset column kits (R&D Systems, Minneapolis, MN) were used for negative selection per manufacturer recommendations. Recovered CD8+ T cells ranged from 55 to 85% purity, with <0.3% CD4+ T cells. Conditioned media samples were prepared by incubating 107 splenic leukocytes/ml at 37°C for 24 h in media containing 10% FBS. To quantitate IL-4 or IL-2 production, rat anti-mouse IL-4 receptor (Genzyme, Cambridge, MA) or rat anti-mouse IL-2 receptor p55 (PharMingen, San Diego, CA) mAbs, respectively, were added to block factor consumption. In some cases, up to 2500 U/ml of recombinant human IL-2 (Cetus, Emeryville, CA) were added. Unless otherwise indicated, samples were prepared in the absence of additional stimuli, Ag, or mitogens. Cellfree supernatant fluids were harvested and stored at -20°C until use.
Flow cytometric analysis
Two-color flow cytometric analyses were performed as described
(30, 31), using anti-CD4 R-phycoerythrin (PE)-conjugated rat mAb
RM4-5, anti-CD8
FITC-conjugated rat mAb 53-6.7,
anti-CD8ß.2 FITC-conjugated rat mAb 53-5.8, or control Abs
lacking specificities for murine determinants (all from PharMingen).
Intracellular flow cytometric analyses were conducted using a published
procedure (32) with modifications. Cells were resuspended at
106 cells/ml in 10% FBS RPMI, and brefeldin A (Sigma,
St. Louis, MO), at 1 mg/ml, was added at 0.01 ml per ml of cells for
2 h at 37°C. Where indicated, cells were stimulated with
anti-CD3 for a total of 6 h at 37°C, with brefeldin A added
during the last 2 h of incubation, using cluster plates previously
adsorbed with purified hamster anti-mouse CD3 mAb 145-2C11
(PharMingen). Cells were collected, washed with PBS, incubated at
106 cells per test for 30 min on ice with biotinylated rat
anti-CD4 mAb clone RM4-5 and anti-CD8 FITC-conjugated rat
mAb 53-6.7, washed with staining buffer (0.5% BSA and 0.006%
NaN3 in PBS), incubated for 30 min on ice with
streptavidin-PerCP (Becton Dickinson, Mountain View, CA), washed,
resuspended at 2 x 106 cells/ml in PBS, and fixed
with an equal volume of 4% formaldehyde in PBS. After 20 min of
incubation at room temperature, cells were PBS washed, resuspended at
107 cells/ml in staining buffer, stored overnight at 4°C,
washed again with staining buffer, treated with permeabilization
buffer (1% saponin in staining buffer; Sigma), resuspended in 25
µL of permeabilization buffer containing 300 µg/ml rat Ig (Sigma),
incubated for 10 min at room temperature, and incubated an additional
20 min after addition of anti-mouse-IFN- R-PE-conjugated rat mAb
XMG1.2 or anti-mouse-IL-4 R-PE-conjugated rat mAb 11B11
(PharMingen). To prove cytokine specificity of staining, the Abs were
preincubated with either recombinant mouse IFN- (PharMingen) or
recombinant mouse IL-4 (R & D Systems), at 0.25 µg per test, for 20
min before addition to cells. Cells were washed with permeabilization
buffer followed by staining buffer, and acquired immediately at the
Rhode Island Hospital of Brown University on a FACScan (Becton
Dickinson, San Jose), using the LYSYS II software package, or at Brown
University on a FACSCalibur, using the CELLQUEST version 1.2.2 or 3.0
software package. Argon laser output was 15 mW at 488 nm. More than
20,000 events were collected for intracellular cytokine staining
analyses. Where necessary, flow cytometry standard data files were
converted using CONSORT File Exchange version 1.0 and FACSConvert
version 1.0 software programs, before analysis using CELLQUEST software
packages.
ELISA
For quantitation of IL-2 and IL-4 proteins, sandwich ELISAs were
performed using protocols from PharMingen. Immulon 4 96-well ELISA
plates (Dynatech, Chantilly, VA) were coated with rat anti-mouse
IL-2 mAb clone JES6-1A12 or rat anti-mouse IL-4 mAb clone BVD4-1D11
(PharMingen) in coating buffer (0.1 M NaHCO3, pH 8.2),
PBST washed, incubated with 5% FBS-PBS, washed, and then samples,
recombinant mouse IL-2 (PharMingen) or recombinant mouse IL-4 (Life
Technologies, Gaithersburg, MD) standards were added in duplicate.
After incubation overnight at 4°C, plates were washed, incubated with
biotinylated rat anti-mouse IL-2 mAb clone JES6-5H4 or biotinylated
rat anti-mouse IL-4 mAb clone BVD6-24G2 (PharMingen) for 45 min at
room temperature, washed, incubated for 30 min with avidin-peroxidase,
washed, and ABTS ((2,2'-azino-di-[3-ethylbenzthiazoline
sulfonate(6)]); Kirkegaard & Perry Laboratories, Gaithersburg, MD)
substrate added. Quantitation of IFN- by ELISA was performed as
described (33) using rat anti-mouse IFN- mAb clone XMG1.2 for
capture, polyclonal rabbit anti-mouse IFN- antisera (gift from
Dr. Philip Scott, University of Pennsylvania, Philadelphia, PA) for
detection, peroxidase-conjugated donkey anti-rabbit Ig (Jackson
ImmunoResearch, West Grove, PA), and recombinant mouse IFN-
(PharMingen) standards. At multiple intervals following ABTS addition,
absorbances were detected at 410 nm using a Dynatech MR-4000 plate
reader, or at 405 nm using a SpectraMax 250 (Molecular Devices,
Sunnyvale, CA) with the SOFTmax Pro software for data analysis. Limits
of detection for IL-2, IL-4, and IFN- ELISAs were 5.2 pg/ml (1 U of
IL-2 equivalent to 26.5 pg of IL-2), 4.9 to 9.8 pg/ml, and 9.8 to 19.7
pg/ml, respectively.
Virus-specific Ab titers
LCMV-specific plasma Ab titers were quantitated as described (34, 35). Briefly, viral Ag was used to coat plates and bound LCMV-specific Abs were detected by peroxidase-conjugated Abs to mouse Ig. LCMV-specific Ab titer was defined as reciprocal of the dilution of plasma resulting in an absorbance at least twice the SD as samples from uninfected mice.
Enzyme-linked immunospot (ELISPOT) assay
ELISPOT assays to detect IL-4-producing cells were performed
essentially as previously described for detection of IFN--producing
cells (29), with modifications. Immulon 4 96-well ELISA plates
(Dynatech) were coated with purified rat anti-mouse IL-4 mAb clone
BVD4-1D11 (PharMingen), with or without 30 µL of 10 µg/ml purified
hamster anti-mouse CD3 mAb (PharMingen), in coating buffer (0.1 M
Na2CO3, 0.1 M NaHCO3, pH 8.5).
Plates were washed with PBST and PBS, incubated with 10% FBS-RPMI for
1 h at room temperature, washed, and cell dilutions in 100-µL
vol of 10% FBS-RPMI were added in duplicate wells. After incubation at
37°C for 24 to 30 h, plates were washed, incubated with
biotinylated rat anti-mouse IL-4 mAb clone BVD6-24G2 (PharMingen)
at 37°C for 1 h, washed again, and streptavidin-alkaline
phosphatase (Zymed, S. San Francisco, CA) diluted 1000-fold in 5%
FBS-PBST was added at 100 µL per well and incubated at 37°C for 30
min. Plates were washed, substrate solution containing 1 mg/ml
5-bromo-4-chloro-3-indolyl-phosphate disodium salt (Sigma), 0.6% (w/v)
SeaPlaque agarose (FMC BioProducts, Rockland, ME) in 0.1 M
2-amino-2-methyl-1-propanol alkaline buffer (Sigma) added, incubated at
room temperature overnight, and blue precipitates enumerated under low
power magnification (x10).
Statistical analysis
The two-tailed Mann-Whitney U test was performed where indicated using the statistical software package DATA DESK 5.0 (Ithaca, NY). Unless otherwise indicated, mean ± SE are shown. Results identified as having less than (<) means had sample values below the level of detection for the assay. These were calculated as averages of readings at the level of detection for those below detection with those above detection. The results are reported as less than mean averages.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-4 production in normal mice
The kinetics of T cell cytokine production elicited during acute
LCMV infections of normal C57BL/6 mice were characterized. To evaluate
responses to stimuli received in vivo, splenic leukocytes isolated at
various times after infection were cultured in media without additional
stimulation for only 24 h. The ex vivo-released IL-2, IFN-, and
IL-4 proteins were quantitated by ELISA. Addition of Abs to IL-2 or
IL-4 receptors to block factor consumption was necessary for optimal
detection of IL-2 and IL-4. Consistent with previous work from this
laboratory (20, 28), IL-2 induction was evident after infections (Fig. 1
A). Cells
(107) from day 7-infected mice were induced to produce
47 ± 5 pg of IL-2. Production, however, approached maximal levels
of 900 pg with cells from day 14-infected mice and continued to be
elevated for 21 days after infection (M. Ruzek and C. Biron,
unpublished observations). IFN- and IL-4 levels also were induced,
with peak levels produced by cells from day 11 or 14 infected mice
(Fig. 1, B and C). Cytokine production on
a per spleen basis showed similar trends except that, because of
maximal cell yields on day 11 after infection, maximal levels of both
IFN- and IL-4 production occurred at this time. Thus, LCMV
infections induce production of IL-2, IFN-, and IL-4.
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, and IL-4 production in the absence of
CD8+ or CD4+ T cells
Mice genetically lacking functional histocompatibility
molecules were used to evaluate consequences of CD8+
and CD4+ T cell deficiencies for cytokine responses.
Effects of CD8+ T cells were examined in
C57BL/6-ß2-microglobulin
(ß2m)-/- mice rendered CD8+ T
cell deficient as a result of MHC class I expression deficiency. At day
7 after LCMV infection, splenic leukocyte populations from
ß2m-/- mice were induced to produce IL-2
levels comparable to those of cells from immunocompetent C57BL/6 mice,
i.e., 80 ± 18 pg per 107 cells (Fig. 1
D). However, IL-2 production by cells from
ß2m-/- mice fell to baseline values on days
11 or 14 after infection. Likewise, IFN- production by cells from
LCMV-infected ß2m-/- mice was normal on
days 5 through 9, but dramatically reduced by day 11 (Fig. 1E). IL-4 production remained undetectable using
cells isolated from ß2m-/- mice at any of
the infection times examined (Fig. 1E). Despite
decreases in cell yields following infection in
ß2m-/- mice, cytokine production on a per
spleen basis showed similar kinetic trends. To examine effects of
CD4+ T cells, responses were evaluated in
Aßb-/- mice rendered CD4+ T cell deficient
as a result of MHC class II expression deficiency. Splenic leukocytes
from Aßb-/- mice produced no detectable IL-2 and
IFN-, and reduced levels of IL-4 (Table I). Splenic cell yield increases were
induced during infections of Aßb-/- mice, and cytokine
production on a per spleen basis showed similar kinetic trends. These
results indicate that although detectable IL-4 can be induced without
CD4+ T cells and detectable IL-2 and IFN- can be induced
without CD8+ T cells, optimal induction of all three
cytokine responses to viral infections, as measured by ex vivo
production, requires in vivo presence of both responses.
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As IL-2, IFN-, and IL-4 contribute to induction and modulation
of B cell and Ab responses, virus-specific Ab responses were evaluated.
Plasma anti-LCMV Ab titers were induced in normal C57BL/6 mice by
day 7, continued to increase on day 11, and remained elevated on day 21
after infection (Fig. 2A). In contrast,
anti-LCMV Ab titers in CD8+ T cell-deficient
ß2m-/- mice were not apparent on day 7, and
were induced to approximately 2-log lower levels on days 11 and 21.
CD4+ T cell-deficient Aßb-/- mice also
demonstrated impaired induction of anti-LCMV Ab titers on both days
7 and 11 (Fig. 2B). These results show the expected
CD4+ T cell requirement for optimal T-dependent Ab
production. In addition, they indicate that during infections, the in
vivo presence of CD8+ T cells associated with optimal
induction of cytokines promotes conditions leading to high titer
detectable Ab production.
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Earlier in situ hybridization and ex vivo production studies from
this laboratory have shown that although IL-2 mRNA transcripts can be
detected in both CD4+ and CD8+ T cell
subsets from normal LCMV-infected mice, CD4+ T cells
account for virtually all detected IL-2 released in culture (20, 28).
Intracellular flow cytometric analyses for cytokine expression were
conducted to ascertain whether CD8+ and CD4+ T
cells contributed directly or indirectly to optimal cytokine
production, and to extend characterization of cell subset cytokine
expression to IFN- and IL-4. Preliminary experiments using published
techniques with brefeldin A (32), to allow accumulation of cytokine
within synthesizing cells, demonstrated marginally detectable IFN-,
but not IL-2 or IL-4, proteins in cells from infected but not
uninfected mice. To enhance analyses and quantitate numbers of cells in
vivo primed to express cytokine, expression was facilitated by
short-term cell incubation with immobilized anti-CD3. This step
enhanced detection of IFN- and allowed modest detection of IL-4, but
not IL-2, protein. Thus, studies of intracellular cytokine expression
were limited to IFN- and IL-4. Specificity of staining was
demonstrated by cold competition with soluble unlabeled cytokine.
High levels of cytoplasmic IFN- protein were detected on day 7 after
LCMV infection (Fig. 3A). Both single
positive CD8+ and CD4+ T cells were shown to
express IFN-; averages from multiple samples demonstrated that these
populations accounted for 80 to 90% of cells expressing the factor
(Fig. 3B). Proportionally more CD8+ T
cells synthesized IFN-; in the experiment shown with
n = 3, CD8+ T cells (Fig. 3C) averaged 54% (±6) IFN- positive (Fig. 3D), whereas the CD4+ T cells (Fig. 3C) averaged only 27% (±5) positive (Fig. 3E). In multiple experiments after LCMV infection,
the percentages of CD8+ T cells that were IFN- positive
ranged from 45 to 84% on day 7, and 39 to 78% on day 11 after
infection, whereas those for CD4+ T cells ranged from 16 to
52% for day 7, and 12 to 56% for day 11. Because of preferential
subset expansion, CD8+ T cells accounted for approximately
sevenfold more of the IFN--expressing cells in the spleen than did
CD4+ T cells (Fig. 4,
A and B). Although cytoplasmic IL-4
protein expression was detectable under these conditions (Fig. 3, F and G), both proportions of expressing
cells and intensity of staining remained low. Thus, conditions and
reagents available for use were not sensitive enough for reliable
quantitation of IL-4-expressing cells. This was consistent with
detection of IFN- but not IL-4 production in media without blocking
of cytokine consumption using Abs to the receptor, and greater than
fourfold excess peak production of IFN- compared with IL-4 (Fig. 1C). Analyses with cells isolated on day 11 after
infection yielded similar results (Fig. 4, A and
B, and data for IL-4 not shown). Thus, although these
experiments provided limited information about cellular sources of
IL-4, they conclusively demonstrate that high proportions and numbers
of CD8+ T cells are activated and are the predominant cell
type induced to directly synthesize IFN- during the infection.
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production by separated CD8+ T cells
The aforementioned experiments, examining cytoplasmic expression
with cells from immunocompetent mice and ex vivo cytokine production
without additional stimulation with cells from both immunocompetent
mice and T cell subset-deficient mice, did not distinguish between ex
vivo roles in facilitating reciprocal subset responses and in vivo
roles for promoting and/or expanding cytokine-expressing T cells. To
evaluate the contribution of other cell populations in facilitating ex
vivo cytokine production, highly purified cells activated in an
immunocompetent context were prepared and examined for ex vivo cytokine
production in the absence of additional stimuli. Under these
conditions, CD8+ T cell production of IFN- was low.
In one representative experiment, total cell populations from day 11
LCMV-infected C57BL/6 mice produced 1952 pg, but purified day 11
CD8+ T cell populations produced only 138 pg of IFN- per
107 cells. Thus, populations of cells with only 20 to 30%
CD8+ T cells made >10-fold higher levels of IFN- than
did populations of cells that were 
90% CD8+ T cells.
These results mirror the reduced IFN- production by cells from
CD4+ T cell-deficient mice (Table I) and indicate that,
although CD8+ T cells are the predominant IFN- source,
they require additional signals for sustained IFN- production.
To examine cellular roles for promoting and/or expanding
cytokine-expressing T cells in vivo, cytoplasmic IFN- expression by
populations activated in the absence of either CD8+ or
CD4+ T cell responses was examined. Averages of multiple
experiments demonstrated that infection induced changes in proportions
and numbers of CD4+ T cells in the CD8+ T
cell-deficient ß2m-/- mice and of
CD8+ T cells in the CD4+ T cell-deficient
Aßb-/- mice similar to those changes during infections
of immunocompetent mice (Table II). On
day 7 after infection, CD4+ T cells in immunocompetent and
ß2m-/- mice were, respectively, 9 and 12%
with similar total cell numbers per spleen, and CD8+ T
cells in immunocompetent and Aßb-/- mice were
significantly increased to, respectively, 28 and 38%, with about
fourfold rises in total cell numbers. Cytoplasmic IFN- staining
(Fig. 5) demonstrated that priming and
expansion for cytokine expression in the present T cell subsets was
within the range of observed responses of both subsets in
immunocompetent mice (Fig. 5). Low proportions of IFN--expressing
cells were induced in ß2m-/- mice (Fig. 5A), and these were predominantly CD4+ T
cells (Fig. 5, B and C), whereas high
proportions of IFN--expressing cells were induced in
Aßb-/- mice (Fig. 5D), and these were
primarily CD8+ T cells (Fig. 5, E-G). Remarkably, averages of
IFN--positive CD4+ T cells were 12% (±0.2) in
ß2m-/- (Fig. 5C) and 9%
(±1) in immunocompetent mice, and those of IFN--positive
CD8+ T cells mice were 18% (±3) (Fig. 5E) in Aßb-/- compared with 27%
(±2) in immunocompetent mice, used in the same experiments. Moreover,
total numbers of virus-induced IFN--expressing CD4+ T
cells in the CD8 cell-deficient ß2m-/- mice
and IFN--expressing CD8+ T cells in the CD4
cell-deficient Aßb-/- mice were similar to those in
immunocompetent mice (Fig. 6). Thus,
inductions and expansions of T cell subsets primed to make IFN- were
independent of the presence or absence of the reciprocal subset in
vivo. Taken together with the ex vivo production studies, these results
demonstrate that CD4+ T cells are not required for in vivo
responses, but do promote continued IFN- production by
CD8+ T cells in culture.
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and IL-4 production in the absence of IL-2
CD4+ T cells are major, but do not appear to be
the only, producers of IL-2 during LCMV infection (20, 28, 29). IL-2
may contribute to optimal IFN- production by supporting the in vivo
expansion and priming phases of producing cells and/or by facilitating
the maintenance of cytokine production from activated cells. To
determine whether IL-2 was required for IFN- and IL-4 production
during viral infection, mice rendered IL-2 deficient by genetic
mutation were LCMV infected, and cytokine production was measured in ex
vivo cell-conditioned media. In contrast to cells from control
IL-2+/+ or IL-2+/- mice, those from
IL-2-/- mice produced little to no detectable IFN-
(Fig. 7A) or IL-4 (Fig. 7B). IFN- and IL-4 production on a per spleen
basis showed the same trends. The concurrent deficiencies in cytokine
production were accompanied by impaired virus-specific Ab induction,
with >2-log less plasma anti-LCMV Ab titers in
IL-2-/- mice by day 9 after infection (Fig. 7C).
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production in IL-2-/- mice is due in part, but
not entirely, to decreased frequencies of IFN--expressing cells as
quantitated by ELISPOT (29). The decreased IL-4 production seen in
IL-2-/- mice at late times after infection could also be
attributed to lower frequencies of IL-4-expressing cells. Numbers of
IL-4-expressing cells by day 11 after infection reached 19,575 ±
2,987 (n = 7) in IL-2+/+ mice and
20,783 ± 1,052 (n = 5) in
IL-2+/- mice but were significantly less at 7,049 ±
1,300 (n = 4) in IL-2-/- mice
(p < 0.05). These results indicate that IL-2
functioned in vivo during viral infections to promote expansion of not
only IFN--expressing cells, but also IL-4-expressing cells.
Characterization of the IL-2 requirement for CD8+
T cell IFN- production
Earlier studies from this group have demonstrated the importance
of IL-2 for in vivo CD8+ T cell expansion (29). To
determine whether IL-2 also acts on primed CD8+ T cells to
promote continued production of IFN-, the effects of IL-2 addition
were examined during the cytokine production phase in culture. The
addition of 2500 U/ml of IL-2 did induce IFN- produced into
conditioned media by CD8+ T cells prepared from uninfected
immunocompetent mice (Fig. 8A). The value,
however, was lower than that produced by total day 11 populations. In
contrast, although highly purified CD8+ T cells activated
in the immunocompetent mice failed to produce detectable IFN- in
culture, the addition of 2500 U/ml of IL-2 to these cells restored
their ability to produce IFN- and resulted in enhanced production to
approximately 1300 pg/107 cells (Fig. 8A). Interestingly, although total cells and purified
CD8+ T cells from day 11 CD4+ T cell-deficient
Aßb-/- mice did not produce IFN- on their own in
culture, the addition of 2500 U/ml of IL-2 to the purified
CD8+ T cells resulted in greatly enhanced IFN-
production to 6000 pg/107 cells (Fig. 8B). IL-2 did not appear to act by improving cell
viabilities, as recoveries remained approximately 50% after a 24-h
incubation in the presence or absence of added factor. Taken together
with the earlier studies, these results indicate that IL-2 acts both to
promote the in vivo expansion and priming of IFN--expressing
CD8+ T cells and to facilitate continued CD8+ T
cell IFN- production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production (11, 13, 16, 17). In
the case of LCMV infections, the proliferative and cytotoxic responses
are CD4+ T cell independent (18, 19, 20, 21, 22) but IL-2 dependent
(29), and IL-2 contributes to overall IFN- responses (29). The
studies presented here characterized roles for T cell subsets and IL-2
in IFN- and IL-4 responses. LCMV infection induced T cell IL-2,
IFN-, and IL-4 responses detected by ex vivo production into
conditioned media. Peak induction levels of these cytokine responses
were dependent upon the presence of CD8+ T cell,
CD4+ T cell, and IL-2 responses. As evaluated by priming
for intracellular IFN- expression, CD8+ T cells were
primarily responsible for the IFN- response. The endogenous presence
of IL-2, but not CD4+ T cell, responses were required for
induction and expansion of CD8+ T cells primed to make
IFN-. However, both CD4+ T cells and IL-2 facilitated
continued production of IFN- by activated CD8+ T cells.
The results delineate an in vivo role of IL-2 in promoting T cell
cytokine responses to viral infections by supporting T cell expansion
for both IFN- and IL-4 responses, and by promoting continued IFN-
production from activated CD8+ T cells. As CD4+
T cells are potent producers of IL-2, they also suggest that, although
the cells are not required for the IL-2-dependent CD8+ T
cell expansion in vivo, CD4+ T cells may play a role after
CD8+ T cell activation in providing IL-2 for continued
IFN- production.
By precisely defining roles for IL-2 in promoting T cell cytokine
responses including CD8+ T cell IFN- production, these
studies significantly advance understanding of the regulation of immune
responses to viral infections. The results raise important issues
concerning in vivo requirements for T cell cytokine responses. It has
been proposed that CD8+ T cells, like CD4+ T
cells, are polarized to synthesize either the type 1 or type 2
cytokines (36, 37). However, in contrast to characterization of
conditions contributing to polarization of CD4+ T cell
responses (1, 2, 32), those for CD8+ T cell response
polarization have been less thoroughly examined (38). There are
indications that in vivo conditions necessary for promoting
CD8+ T cell IFN- production differ from those promoting
CD4+ T cell IFN- production. In particular, under the
conditions of LCMV infection used for the studies presented in this
report (8) and in contrast to studies of T cell IFN- responses to
other types of infections (2, 39), it is not possible to detect IL-12
protein, and T cell IFN- production is not significantly modified by
treatments with Abs neutralizing this factor (8) or blocked in
IL-12-deficient mice (L. P. Cousens, R. Peterson, S. Hau, A.
Dorner, and C. A. Biron, manuscript in preparation). In contrast,
this report definitively establishes IL-2 as a significant factor in
the induction of IFN--producing CD8+ T cells during
viral infections.
Because of the approaches we used, we were able to distinguish the
effects on in vivo activation and expansion from those on continued
production. For analysis of the in vivo effects, cells were subjected
to short-term stimulation with anti-CD3 and incubated with
brefeldin A to block protein secretion. This approach demonstrated
profound in vivo expansion and priming of IFN--expressing
CD8+ T cells, and the role for IL-2 in supporting this
response. For analysis of ex vivo cytokine production, media were
conditioned for 24 h in the absence of further stimulation. This
approach demonstrated that mixtures of total cells were substantially
better producers of cytokines than were separated or purified T cell
subsets, and that the addition of IL-2 could substitute for T cell
mixtures in supporting continued IFN- production by purified in
vivo-activated CD8+ T cells. Although purification of
CD8+ T cell subsets also may have resulted in the loss of
APCs previously loaded with viral peptides in vivo, this did not
account for their reduced ability to produce IFN- (Fig. 8), because
CD8+ T cells in mixed, but CD4+ T
cell-deficient, populations from Aßb-/- mice also were
impaired in their ability to make IFN- (Table I and Fig. 8). Thus,
the presence of in vivo-activated CD4+ T cells and/or
exogenously added IL-2 was required to support continued peak
CD8+ T cell IFN- production. This may explain the
requirement for CD4+ T cells in addition to
IFN--expressing CD8+ T cells during adoptive transfer to
clear persistent viral infections (40).
CD8+ T cells are responsible for almost all of the control
of LCMV during acute conditions of infection (11, 12, 18, 19, 20, 21, 23, 29).
As a result, the ß2m (30) and IL-2 (29)-deficient mice
used in these experiments have higher viral burdens than either
immunocompetent or Aßb-deficient (23) mice. Our studies
began with observations in immunodeficient mice, but also used
responses either in, or with cells from, immunocompetent mice. As
examples, identification of the CD8+ T cell subset
contribution to IFN- expression is based on studies of production
with total cells from immunocompetent and immunodeficient mice (Fig. 1
and Table I), and by cytoplasmic expression in cell subsets from
immunocompetent and immunodeficient mice (Figs. 3 through 6).
Characterization of the role for IL-2 in IFN- production by
CD8+ T cells is based both on responses in IL-2-deficient
mice and on ex vivo production by in vivo-activated T cells from
immunocompetent and Aßb-deficient mice (Fig. 8). Thus,
the conclusions are not confounded or limited by differences in
endogenous viral burdens.
Our observations are consistent with results from in vitro stimulation
studies establishing that 1) IL-2 expression precedes T cell production
of cytokines such as IFN- or IL-4 (41, 42, 43); 2) IL-2 can facilitate T
cell mitogen- or Ag-induced IFN- production from a variety of cells,
including CD8+ T cells (44, 45, 46, 47, 48, 49, 50, 51); and 3) IL-2 promotes
expansion of, as well as influences cytokine production by, T cell
populations, including CD8+ T cells (38, 42, 47, 49, 52, 53). To our knowledge, the studies reported here are the first to
identify viral infections as in vivo conditions under which IL-2
functions to facilitate IFN- production by simultaneously promoting
expansion and maintaining cytokine production of CD8+ T
cells.
The frequencies of IL-4-producing cells induced during infection of IL-2-containing compared with IL-2-deficient mice are consistent with work from in vitro systems indicating that IL-2 is required for development of IL-4-producing cells (42, 54, 55). However, although certain of those studies suggest that IL-2 can maintain IL-4 production in the absence of proliferation (54), supplementation with IL-2 failed to restore IL-4 production into media conditioned with cells from day 11 LCMV-infected IL-2-deficient mice in our experiments (data not shown). Despite apparent differences in requirements for supporting continued IL-4 production, both the in vitro and in vivo studies demonstrate that IL-2 promotes IL-4 responses as a result of supporting expansion of cells to become IL-4 producers. Given that proliferation of low frequency Ag-specific T cells is required during any primary challenge of immunocompetent hosts, this may not be surprising. However, the results underscore the limitations of using approaches with high frequency Ag-specific T cells, such as mice or cells from mice transgenic for a single TCR, to dissect cytokine requirements for T cell cytokine responses.
It is interesting to note that although CD4+ T cells or
IL-2 appeared to be interchangeable for supporting continued IFN-
production by CD8+ T cells in culture, CD4+ T
cell deficiencies (Figs. 5, 6, and 8) were not equivalent to IL-2
deficiency (Fig. 7) during the in vivo expansion and priming phase,
i.e., the in vivo events were IL-2 dependent but CD4+ T
cell independent. This observation is consistent with the previously
demonstrated dependence of CD8+ T cell proliferation and
overall CTL induction on IL-2 (29) but not CD4+ T cells
(20). Our laboratory has shown that CD8+ T cells are
induced to express IL-2 as detected by in situ hybridization (20, 28),
and has suggested that this is the source for IL-2 in supporting
CD8+ T cell proliferation in vivo. In contrast to the high
level protein production by CD4+ T cells, however, it has
been difficult to demonstrate IL-2 production by CD8+ T
cells (20). In experiments conducted during the LCMV infection studies
presented here, it was possible to see low levels of IL-2 in media
conditioned with purified CD8+ T cells, but this was again
lower than that detected in purified CD4+ T
cell-conditioned media (data not shown). Thus, it appears as though
either CD8+ T cells make sufficient IL-2 to support their
proliferation and priming in vivo but not to support their continued
IFN- production, and/or conditions exist in vivo that are not
duplicated in culture to enhance IL-2 production by CD8+ T
cells.
Although the studies with ß2m-/- mice (Fig. 1) definitively established that peak cytokine responses during LCMV
infections are dependent upon the endogenous presence of
CD8+ T cells, they did not define mechanisms responsible
for the lack of detectable IL-4, as well as the initiation but aberrant
premature ending of IL-2 and IFN-, responses in these mice. The
ß2m-/- mice have a number of reported
endogenous and LCMV-induced changes that are not apparent in
immunocompetent mice (56, 57, 58, 59), including lack of NK1+ T
cells (56) and LCMV-induced and IL-2-dependent increases in NK
(T-) cells (Refs. 31 and 57). Experiments were conducted
to ascertain the role of these in the modified cytokine responses.
Analysis of mRNA by the highly sensitive technique of competitive
reverse transcriptase-PCR did not reveal induction of early IL-4 during
LCMV infection (data not shown). Moreover, NK1+ T cells,
which are potent IL-4 producers missing in the
ß2m-/- mice (56), did not appear to be the
major producers of LCMV-induced IL-4 because 1) highly purified
NK1-CD4+ and NK1-CD8+
T cell populations from LCMV-infected immunocompetent mice both made
IL-4 as measured in ELISPOT assays (data not shown), and 2) SJL mice,
also reported to lack the NK1+ T cell subset (60), have
readily detectable LCMV-induced T cell IL-4 responses (data not shown).
The expanded NK (T-) cells (31, 57) did not appear to be
responsible for the altered ß2m-/- IFN-
response because NK cell depletions did not deplete IFN- production
in culture. It was not possible to demonstrate high level cytoplasmic
IFN- expression in these cells at late times after infection of
either immunocompetent or ß2m-/- mice (data
not shown). Although it is not yet clear how the aberrant cytokine
profile in ß2m-/- mice is regulated,
contributing mechanisms are currently under investigation in this
laboratory and are likely to be distinct from those identified with the
IL-2 or Aßb-/- mice in this report.
The plasma virus-specific Ab levels suggest that consequences of
impaired cytokine production as a result of either CD4+ T
cell, CD8+ T cell, or IL-2 deficiency include secondary
effects on B cells and/or B cell Ab production. Increased clearance of
IgG may have contributed to the differences in
ß2m-/- mice (61, 62), but not the
CD4+ T cell- or IL-2-deficient mice. However, although our
studies did not carry out in depth analyses of Ab isotypes, time points
associated with both IgM and IgG responses were examined. In addition,
as IL-2-deficient (29) and CD8+ T cell-deficient (22, 31, 63), but not CD4+ T cell-deficient (23) mice have impaired
ability to clear acute LCMV infection, virion-Ab complexes may have
interfered with the ability to detect total Ab production in these
mice. This possibility is suggested by the findings of decreased
LCMV-specific Ab titers in ß2m-deficient mice without a
corresponding decrease in frequencies of Ab-forming cells (63). The
decreased virus-specific Ab titers in
Aßb-deficient mice are consistent with
reports demonstrating CD4+ T cell requirements for Ab
responses but not viral clearance (11, 16, 18, 22, 23). However, in all
three cases, i.e., lower IL-4, IFN-, and IL-2, the reduced
virus-specific Abs could be a direct result of impaired cytokine
production because 1) decreased virus-specific Ab responses also are
observed in ß2m-/- mice following challenge
with viruses less dependent upon CD8+ T cell responses for
clearance (64, 65), and 2) decreased Ab responses are induced in the
absence of virion-Ab complexes after immunization with the
nonreplicating Ag, 2,4,6-trinitrophenyl-conjugated keyhole limpet
hemocyanin (65). As IL-2-deficient mice have impaired vesicular
stomatitis virus-specific IgG responses (26), and IL-4-deficient mice
have impaired parasite-specific Ab responses (66, 67), our observations
suggest that reduced cytokine responses explain and/or contribute to
the decreased Ab titers in ß2m-/- and
Aßb-/- mice during LCMV infection. Whatever the
mechanism, the data demonstrate that there are biologic consequences
associated with the three deficiencies.
In summary, these studies show that LCMV infection induces the
simultaneous expression of IL-2, IFN-, and IL-4. Moreover, they
demonstrate that viral infection not only induces CD8+ T
cells to proliferate and acquire cytotoxic activity, but also to become
the major producers of large quantities of IFN-, and that IL-2 is a
required cytokine for this T cell IFN- response. They suggest that
viral infections induce distinct regulators to uniquely shape
endogenous T cell cytokine responses.
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Acknowledgments |
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antisera. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Christine A. Biron, Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Box G-B629, Brown University, Providence, RI 02912. E-mail address:
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; ß2m, ß2-microglobulin; PE, phycoerythrin; ELISPOT, enzyme-linked immunospot.
Received for publication October 30, 1997. Accepted for publication January 14, 1998.
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C57BL/6 controls; * p <
0.05, ** p < 0.005.
