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Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies have examined the cellular immune response of CD40L-deficient (CD40L-/-) mice toward infection with various viruses (14, 15, 16, 17). The results presented by Borrow et al. (14) showing that CD40L-/- mice mount an unimpaired CTL response toward both VSV and LCMV intrigued us, as these two viruses are known to have very different biological characteristics and have previously been found to differ substantially in their requirements for induction of a CTL response (18). LCMV is a natural mouse pathogen that replicates extensively and widely in several organs; following an i.v. infection high virus titers are detectable in the spleen throughout the infection until approximately day 10 postinfection (p.i.) (19). In contrast, VSV is not a natural mouse pathogen, and it replicates poorly in the spleen. Virus can be detected on day 1 p.i., but by approximately day 2 live virus becomes undetectable (20). These differences in biological behavior have been linked to their different requirements in elicitation of efficient CTL responses. Thus, LCMV induces a Th-independent, CD28-independent CTL response, whereas generation of VSV-specific CTLs has been reported to require both Th cells and CD28 (5, 8, 18, 21, 22, 23).
Consequently, the primary aim of this study was to re-evaluate and
compare the immune responses of CD40L-/- mice
following primary infection with these two viruses. Three parameters of
activation were evaluated: cell cycle progression (in vivo
incorporation of 5-bromo-2'-deoxyuridine (BrdU)), phenotypic
conversion, and effector function (cytolytic activity and capacity to
produce IFN-
). Furthermore, based on the assumption that a primary
function of CD40 signaling is to enhance the B7/CD28 interaction
(24), we also wanted to compare the requirements for CD40L
and CD28. This was done by assessing the same parameters in
virus-infected CD28-/- mice. As recent advances
in technology have made it possible to directly visualize Ag-specific T
cells and thus precisely quantitate and compare effector cell
generation (25, 26, 27), we decided to apply one of these
assays (intracellular staining for cytokine (IFN-) following brief
stimulation with specific peptide) to address the above questions.
Because this methodology has not previously been used to analyze the
VSV-induced CD8+ T cell response, initial
experiments were performed to validate the specificity of our
experimental approach and to define the kinetics of the VSV-specific
CD8+ T cells response measured in this
fashion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD40L-/- mice (C57BL/6,
129-Cd40ltm1Imx), CD28-/-
mice (C57BL/6-Cd28tm1Mak), and
IFN--/- mice
(C57BL/6-ifgtm1) were the progeny of breeder
pairs obtained from The Jackson Laboratory (Bar Harbor, ME). MHC class
II-deficient (Aß-/-) mice and their wild-type
littermates, all back-crossed five times onto a C57BL/6 background,
were obtained from Taconic Farms (Germantown, NY). C57BL/6 were
purchased from Bomholtgaard (Ry, Denmark). Mice from outside sources
were always allowed to rest for 1 wk before entering into experiments;
by that time the animals were about 7–8 wk old. Animals were housed
under controlled (specific pathogen-free) conditions that included the
testing of sentinels for unwanted infections according to Federation of
European Laboratory Animal Science Association standards; no such
infections were detected.
Virus infection
LCMV of the Armstrong strain (clone 53b) was provided by M. B. A. Oldstone (Scripps Clinic and Research Foundation, La Jolla, CA) (14). For i.v. infection of mice, 4800 PFU of LCMV in a volume of 0.3 ml was used. Inoculation of immunocompetent mice by this route is followed by transient, immunizing infection (19, 28). VSV of the Indiana strain, originally provided by K. Berg of this institute, was produced, stored, and quantified as described previously (29). For infection with VSV, mice were inoculated with 106 PFU i.v. This virus dose is nonlethal to immunocompetent mice, but is lethal to B cell-deficient mice (29). Titrations of organs for LCMV were performed by intracerebral inoculation of 10-fold dilutions of 10% organ suspensions into young adult Swiss mice, and titers were calculated by the Kärber method and expressed as the 50% lethal dose.
In vivo depletion of CD4+ cells
Depletion of CD4+ cells was obtained by treatment with clarified ascites fluid containing the CD4-specific mAb GK1.5. Mice were injected with 0.1 ml i.p. on days -1, 0 and +2 relative to virus inoculation, and the efficiency of CD4+ depletion was always verified by flow cytometry at the time of harvesting splenocytes for functional analysis (<1% CD4+ cells were detected).
In vivo BrdU labeling
Mice were given BrdU (Sigma, St. Louis, MO) at 0.8 mg/ml in their drinking water for a period of 3 days (30). BrdU-containing water was protected from light and changed daily.
Cell preparations
Single-cell suspensions of spleen cells were obtained by pressing the organ through a fine steel mesh. When used for analysis by flow cytometry (except for intracellular staining), erythrocytes were lysed by 0.83% NH4Cl treatment (Gey’s solution). Peritoneal cells were obtained by lavage with 5 ml of cold HBSS.
Monoclonal Abs
The following mAbs were purchased from PharMingen (San Diego,
CA) as rat anti-mouse Ab: FITC-conjugated anti-CD49d (common
4-chain of LPAM-1 and VLA-4; R1-2), PE- and CyChrome (Cy)-conjugated
anti-CD8a (Ly-2; 53-6.7, PE-conjugated anti-CD4 (L3T4;
H129.19), biotinylated anti-L-selectin (CD62L, LECAM-1, and Ly-22;
MEL-14), and PE-conjugated anti-IFN-. For BrdU staining,
FITC-conjugated anti-BrdU (Becton Dickinson, San Jose, CA) was
used.
Fluorescence staining and flow cytometric analysis
Staining for flow cytometry was performed as described previously (31, 32, 33). Briefly, 1 x 106 cells were incubated for 5 min in FACS medium (PBS containing 10% rat serum, 1% BSA, and 0.1% NaN3). Subsequently, cells were incubated with relevant Abs in the dark for 20 min at 4°C, after which they were washed three times in PBS with 0.1% NaN3 and fixed with 1% paraformaldehyde in PBS. In case of biotin-conjugated Ab, cells were additionally incubated with streptavidin-Tri-color (Caltag, Burlingame, CA) for 20 min before fixation.
For BrdU staining, cells were stained for surface markers as described above, resuspended in PBS and 1% NaN3, transferred to cold 0.15 M NaCl solution, and fixed by adding cold 96% ethanol drop by drop. After a 30-min incubation on ice, cells were washed once with PBS and resuspended in PBS, 0.01% Tween 20, and 1% paraformaldehyde. After a 1-h incubation at room temperature, cells were pelleted and resuspended in PBS, 0.15 M NaCl, and 4.2 mM MgCl2, pH 5, containing 50 Kunitz units/ml of DNase I (Sigma). After incubation for 15 min at 37°C, cells were washed once in PBS before adding the anti-BrdU Ab. After a 30-min incubation at room temperature, cells were washed in PBS and analyzed.
To detect intracellular IFN-, splenocytes were cultured at 37°C in
96-well round-bottomed plates at a concentration of 1 x
106 cells/well in a volume of 0.2 ml of complete
RPMI medium supplemented with 10 U/well murine rIL-2 (R & D Systems
Europe, Abingdon, U.K.) and 3 µM monensin (Sigma), either with or
without peptide. The peptides were used at a concentration of 0.1
µg/ml (LCMV GP33–41 and
NP396–404) or 1 µg/ml (VSV
NP52–59). After 5–6 h of culture, cells were
washed once in FACS medium (PBS containing 1% BSA, 0.1%
NaN3, and 3 µM monensin) and subsequently
incubated with relevant surface Abs in the dark for 20 min at 4°C.
Cells were washed twice in PBS with 3 µM monensin and resuspended in
100 µl of PBS/monensin, and 100 µl 2% paraformaldehyde in PBS was
added. After 30 min of incubation in the dark at 4°C, cells were
washed in FACS medium and resuspended in PBS with 0.05% saponin. After
10 min of incubation in the dark at 20°C, cells were pelleted and
resuspended in PBS with 0.05% saponin and relevant Abs. After
incubation for 20 min at 4°C, cells were washed twice in PBS/saponin
and analyzed.
Cell samples were analyzed using a FACSCalibur (Becton Dickinson), and at least 104 mononuclear cells were gated using a combination of forward angle and side scatter. Data analysis was conducted by use of the CellQuest program (Becton Dickinson), and results are presented as dot plots.
Cytotoxicity assays
The activity of CTLs was assessed in 51Cr release assays (26, 33). Targets for evaluation of LCMV-specific cytotoxicity were MC57G cells infected with LCMV for 48 h; uninfected MC57G cells served as control targets. VSV-specific cytotoxicity was evaluated using either VSV-infected or VSV-NP52–59-pulsed EL-4 cells as specific targets; unpulsed EL-4 cells served as control targets. The assay time was 5 h, and the percentage of specific lysis was calculated as previously described (26).
Assay for IFN- release
Cell culture supernatants of splenocytes (1 x 106 cells/0.2 ml/well) incubated for 9 h with or without relevant peptides were assayed using kits purchased from Endogen (Cambridge, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, techniques to directly enumerate Ag-specific
CD8+ T cells have been developed, e.g., staining
with tetrameric peptide-MHC complexes and detection of intracellular
IFN- following brief in vitro stimulation with relevant peptides
(25, 26, 27). Several reports describing these methods
primarily in the context of the LCMV infection have recently been
published, but to our knowledge the T cell response of VSV-infected
animals has not previously been analyzed by applying any of these
techniques. In the present study staining for intracellular IFN- was
selected as the primary means to quantitate CD8+
responses in virus-infected mice. Therefore, the aim of our initial
experiments was to validate the use of this methodology under our
experimental conditions. Groups of C57BL/6 mice were infected with
either VSV or LCMV, and on day 6 (VSV) or 8 (LCMV) p.i., freshly
explanted spleen cells were cultured in vitro without stimulation (no
peptide) or with LCMV GP33–41 or VSV
NP52–59. These time points were chosen based on
previous analysis of maximal primary CTL responses to these two viruses
(34, 35, 36). Flow cytometry was used to assess the
frequencies of peptide-specific IFN-+
CD8+ T cells (Fig. 1
). Spleen cells from virus-infected
animals made no IFN- in the absence of peptide stimulation;
background frequencies were always <1%.
IFN-+CD8+ cells were
only detectable when splenocytes were incubated with the peptide
related to the virus with which the mice had been primed, thereby
confirming the specificity of the assay and in addition demonstrating
that no cross-reactivity exists at the CD8+
level. Spleen cells from uninfected mice did not produce any IFN-
after stimulation with any of the viral peptides, and peptide
stimulation did not induce
IL-5+CD8+ in any group
(data not shown), demonstrating that both viral infections were
associated with the generation of Tc1 cells (37, 38).
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-/- mice were
infected with 106 PFU of VSV, and on day 6
p.i. spleen cells were harvested and stimulated with
VSV-NP52–59. As expected, no
IFN-+ cells could be detected in VSV-infected
IFN--/- mice; this was not due to lack of
activation of CD8+ T cells, as about 50%
expressed the activation marker VLA-4 (data not shown). About 15% of
wild-type CD8+ T cells reacted with the
immunodominant VSV-NP52–59 peptide (Fig. 1
), and
detailed phenotypic analysis of the induced
CD8+IFN-+ cells
characterized these as being VLA-4high,
LFA-1high, CD44high, and
mostly Mac-1+ (data not shown), the expected
phenotype of recently activated CD8+ cells
(31). Kinetics of CD8+ T cell proliferation and differentiation in VSV-infected C57BL/6 mice
To study the kinetics of the clonal expansion of
CD8+ T cells during a VSV infection, mice were
infected with VSV on day 0 and given BrdU in their drinking water for a
period of 3 days before analysis; on days 3, 6, 9, and 72 p.i.,
spleen cells were harvested, and
CD8+BrdU+ cells were
quantified. As shown in Fig. 2, splenic
CD8+ T cells were observed to proliferate above
background during days 0–3 and days 6–9 p.i., but peak proliferation
was observed between days 3–6 p.i., during which period about 50% of
the CD8+ T cells had incorporated BrdU. The level
of CD8+ T cell proliferation in uninfected mice
is depicted on day 0. Proliferation of CD8+ T
cells later after infection is identical to that observed in
uninfected mice.
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, the number of VSV-NP-specific
CD8+ T cells was determined at different time
points after VSV infection. No
CD8+IFN-+ T cells could
be detected in naive or day 3 infected mice. On day 6 p.i., when
the response peaks, 10–15% of the CD8+ T cells
were specific for the immunodominant VSV-NP52–59
peptide. The number of IFN-+ T cells then
decreased with time, but a small percentage (2–3%) of
NP52–59-specific CD8+
cells was still detectable in the spleen after 2 mo. Notably, maximal T
cell proliferation and differentiation were observed several days after
infectious virus had disappeared from the spleen (approximately day
2 p.i.). We did not detect a substantial number of
NP52–59-specific T cells in the peripheral lymph
nodes (<1%). However, in both acutely infected and immune mice a
relatively high percentage of VSV-specific cells was detected in the
peritoneum (Fig. 3). This is in keeping
with the expected circulation pattern of primed cells favoring
patrolling of nonlymphoid organs.
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In the above experiments we have validated our methodology and defined the optimal time point to evaluate the CD8+ T cell response to VSV regarding both proliferation and differentiation. We subsequently applied this approach to investigate the immune response of CD40L-/- mice.
CD40L-/- and wild-type mice were infected with
106 PFU of VSV and given BrdU in their drinking
water from days 3–6. On day 6 p.i., cell proliferation,
expression of surface markers, and effector cell differentiation were
evaluated. Naive C57BL/6 mice served as controls and provided cut-off
values in the flow cytometric analyses. As shown in Fig. 4, the response of
CD40L-/- mice was significantly impaired for
all four parameters investigated. Proliferation of both
CD4+ and CD8+ T cells was
reduced >2-fold in CD40L-/- mice compared with
that in wild-type mice. Almost half the CD8+ T
cell population in wild-type mice had acquired an activated phenotype
by day 6 p.i., whereas only a minor subset of
CD8+ cells in CD40L-/-
mice shifted from a naive phenotype
(VLA-4lowL-selectinhigh)
to an activated phenotype
(VLA-4highL-selectinlow).
Regarding effector cell differentiation, evaluated through detection of
intracellular IFN-, the response in CD40L-/-
mice was also significantly reduced (
3-fold).
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Unimpaired immune response in LCMV-infected CD40L-/- mice
It has previously been reported that the primary CTL response of CD40L-/- mice toward LCMV is unimpaired (14, 15, 16, 17). Consequently, we examined the response of LCMV-infected CD40L-/- mice to confirm that the impaired response observed with VSV-infected CD40L-/- mice was virus related and not due to general immune impairment of our CD40L-/- mice.
Proliferation of CD4+ and
CD8+ cells and the frequency of
CD8+IFN-+ cells were
evaluated. CD40L-/-,
CD28-/-, and wild-type mice were infected with
4800 PFU of LCMV and given BrdU in their drinking water from days 5–8,
and on day 8 p.i. splenocytes were analyzed by flow cytometry. As
shown in Fig. 5, proliferation of both
cell subsets in LCMV-infected CD40L-/- mice was
comparable to the proliferative response observed in wild-type mice.
Furthermore, no impairment in the production of intracellular IFN-
in CD40L-/- mice was observed. Essentially
similar results were obtained in LCMV-infected
CD28-/- mice, except that
CD4+ T cell proliferation was significantly
impaired in CD28-/- mice (Fig. 5).
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The results previously published regarding the responses of
CD40L-/- mice to VSV and LCMV have been
obtained primarily by analyzing the virus-specific CTL response
(14). To enable a comparison between our results obtained
by use of flow cytometry with those from the literature obtained by CTL
assays, we also evaluated the cytotoxic response of
CD40L-/- mice upon infection with the two
different viruses. CD40L-/- mice and wild-type
mice were infected with 106 PFU of VSV or 4800
PFU of LCMV, and splenocytes were analyzed on day 6 (VSV) or day 8
(LCMV) p.i. Both total virus-specific and VSV-NP-specific
cytotoxicities of VSV-infected animals were examined. The results
presented in Fig. 6 support the results
obtained by flow cytometry (Figs. 4 and 5); LCMV-infected
CD40L-/- mice showed no impairment in cytotoxic
activity, whereas a significant 3-fold reduction in cytotoxicity was
noted in VSV-infected, CD40L-/- mice. Notably,
similar results were obtained whether total or NP-specific CTL activity
was assayed, demonstrating that the impaired CD8+
response did not involve only one particular epitope.
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According to recent theories regarding induction of
CD8+ T cell responses, some viruses activate APCs
directly, thereby bypassing the need for Th help that is normally
mediated through CD40/CD40L interaction (9, 13). This
prediction appeared to hold true regarding LCMV, as the immune response
of CD40L-/- mice to this virus was
indistinguishable from that of wild-type mice. Consequently, we wanted
to examine whether coinfection of VSV-infected
CD40L-/- mice with LCMV would normalize the
response to VSV. This was performed by coinfecting
CD40L-/- and wild-type mice with VSV and LCMV
and enumerating VSV-NP-specific
IFN-+CD8+ T cells on day
6 p.i. As depicted in Fig. 7, coinfection with LCMV augmented
the response of both CD40L-/- and wild-type
mice. In wild-type mice the effect was primarily seen as a higher
production of IFN- on a per cell basis, as evidenced by higher mean
fluorescence intensity. However, in
CD40L-/- mice a significant, 2- to 3-fold
increase in the frequency of VSV-specific
IFN-+ T cells was observed (Figs. 7 and 8).
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, it may be concluded that the
augmentation of the VSV response following coinfection with LCMV is not
due to cross-reactivity at the CD8+ level.
However, cross-reactivity at the CD4+ level could
not be ruled out. Therefore, to test whether CD4+
T cells were critically involved in the augmented response of
coinfected mice, we evaluated the response of CD4-depleted and
coinfected CD40L-/- mice. Efficient depletion
of CD4+ cells did not impair the help provided by
coinfection with LCMV (Fig. 8), demonstrating that cross-reactivity on
the CD4+ level was not the mechanism underlying
the augmented VSV-NP response seen in VSV-infected
CD40L-/- mice coinfected with LCMV.
To test whether the B7/CD28 interaction was a prerequisite for LCMV to
augment the VSV-specific response,
CD28-/- mice were
infected with both viruses, and the VSV-NP specific response in
coinfected mice was compared with that in
CD28-/- mice infected with VSV only. In
contrast to CD40L, the requirement for which could partially be
bypassed by coinfection with LCMV, no significant augmentation of the
VSV-specific response was observed in coinfected
CD28-/- mice (Fig. 7).
Generation of VSV-NP-specific CD8+ T cells does not require CD4+ help
The above results clearly demonstrate that the
CD8+ T cell response of VSV-infected animals is
impaired when CD40L is absent (Figs. 4 and 6–7). This could lead to
the conclusion that VSV-infected mice require
CD4+ help to mount a CD8+ T
cell response, and mice made deficient of CD4+ T
cells would therefore be expected to resemble
CD40L-/- mice. To examine whether this was the
case, both CD4-depleted wild-type C57BL/6 mice and MHC class
II-/- mice were infected with
106 PFU of VSV, and their
CD8+ T cell responses were compared with that of
normal mice. As shown in Fig. 9,
deficiency of CD4+ T cells, regardless of whether
it is acute (mAb depletion) or chronic (MHC class
II-/-), was associated with little or no
reduction in the CD8+ response to VSV.
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Having found nearly parallel requirements for CD40L and CD28
during the induction phase of the antiviral CD8+
response (Fig. 5), we wanted to test whether this was also the case
during the late immune phase in LCMV-infected mice. Using
CD40L-/- mice we and others have recently
demonstrated a requirement for CD40L in maintenance of virus-specific T
cell memory and long term immune surveillance (14, 16). To
evaluate whether a similar requirement existed for CD28, and a number
of reports suggest that this could be the case (39, 40, 41, 42),
CD28-/-, CD40L-/-, and
wild-type mice were infected with LCMV, and 2 and 4 mo later
virus-specific CD8+ cells were enumerated. In
addition, spleens and lungs were assayed for persistent infection at 4
mo (Fig. 10). As previously reported,
virus could be detected in the organs of all
CD40L-/- mice (n = 6), whereas
little or no virus was detected in CD28-/- and
wild-type mice (n = 4–6). As an underlying mechanism
for this failure to permanently control the infection in
CD40L-/- mice, we found that although similar
numbers of splenic LCMV-specific CD8+ cells could
be found in CD40L-/- and wild-type mice at 4 mo
p.i., LCMV-specific CD8+ T cells from
CD40L-/- mice suffered a functional impairment,
as evidenced by a lesser capacity to synthesize IFN-. A similar
trend was observed after 2 mo p.i. (data not shown), at which time
CD40L-/- mice still controled the infection
(16); this is consistent with the assumption that
impairment of CD8+ T cell function is the cause,
and not an effect, of deficient virus control. As confirmation that the
observed reduction in mean fluorescence intensity was
functionally relevant, Ag-induced secretion of IFN- in vitro was
also evaluated. Following 9 h of stimulation of splenocytes from
CD40L-/- and wild-type mice with LCMV
GP33–41 and NP396–404, we
found that wild-type cells produced about 10-fold more IFN- than did
cells from CD40L-/- mice. In contrast to the
situation in CD40L-/- mice, slightly lower
frequencies of virus-specific CD8+ T cells were
detected in CD28-/- mice, but the cells were
qualitative normal. Thus, CD40L, but not CD28 plays a role in
maintaining CD8+ effector capacity in
LCMV-immune mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production at the single-cell level clearly
demonstrates that about one-third of the activated
CD8+ T cells are specific for a single
immunodominant epitope (NP52–59), and memory
cells with this specificity can be detected in low numbers even at 2 mo
after infection. The effector cells generated are of the Tc1 subtype
(IFN-+IL-5-) (35, 40, 43) and are phenotypically identical to the effector cells
induced by LCMV infection (44). However, despite these
similarities, the requirements for costimulation are significantly
different. Many viral infections are known to be capable of eliciting a strong CD8+ T cell response in the absence of CD4+ T cells, thus somehow bypassing the need for CD4-mediated activation of APCs (5, 6, 7, 8). This activation is generally assumed to be mediated through CD40/CD40L interaction. Recently published data from Borrow et al. (14 and Bachmann and coworkers (45) indicate that the immune response of CD40L-/- mice toward LCMV as well as VSV is unimpaired. However, it is known that these two viruses differ significantly with respect to their capacity to spread in the host, and evidence exists suggesting that these viruses differ markedly in their requirements for costimulation (18). Thus, LCMV induces a Th-independent, CD40L- and CD28-independent CTL response, whereas generation of a VSV-specific CTL response has been reported to depend on Th help and be CD28 dependent (18, 45). Consequently, it would be expected that the requirement for CD40/CD40L interaction would also differ.
In contrast to previous reports (14, 45) we find that the
generation of VSV-specific TC1 cells is impaired,
although not entirely absent, in CD40L-/- mice.
A possible explanation for the discrepancy between our results and
those published could have been that previous reports solely evaluated
CTL activity. For this reason, we have investigated several parameters
of activation (cell proliferation, activation markers, and IFN-
production) in addition to cytotoxic capacity and found both clonal
expansion and differentiation of VSV-specific
CD8+ T cells in CD40L-/-
mice to be impaired. In agreement with results from other groups
(14, 15, 17), we found the response of
CD40L-/- mice to LCMV to be indistinguishable
from that of wild-type mice, demonstrating that there was no general
immune impairment of these mice. Thus, we are without any obvious
explanation for the discrepancy observed. However, the difference in
CTL activity is on the order of 3-fold, which may easily be overlooked
if not backed by more precise assays. Furthermore, in one study
conclusions on CTL responsiveness were based on comparison with +/-
mice, not +/+ mice, and with a molecule such as CD40L, a gene-dose
effect is very likely. Indeed, a reassessment of the latter report
reveals that +/- mice had a CTL response less than half that in
wild-type mice.
Notably, the dependence upon CD40L observed in VSV-infected animals did not reflect an absolute requirement for CD4+ T cells. This finding is at variance with the results of a recent study (5). However, differences in the environment that surrounded the infected mice may provide an explanation to this apparent discrepancy. Thus, it has previously been observed that while MHC class II-deficient mice respond optimally to influenza virus when bred under specific pathogen-free conditions, the CTL response in these mice is impaired under conventional conditions (46). Most important, under our experimental conditions CD4+ T cells are not essential, since neither acute depletion of CD4+ T cells nor a chronic deficiency such as that found in MHC class II-/- mice had a substantial impact on the response. Therefore, the results indicate that CD8+ T cells may suffice to deliver the necessary signals. Given that we found a significant role for CD40L in the context of the VSV-specific CD8+ response, this provides indirect evidence that CD8+ cells may generate their own help through this ligand. Apparently this may be critical during VSV, but not LCMV, infection. Perhaps the underlying reason for this difference is to be found in the extremely transient nature of viral replication following i.v. inoculation of VSV.
The observation that neither Th cells nor CD40/CD40L interaction are required for induction of CD8+ responses to some viruses indicate that either 1) CD8+ T cells activate APCs also through CD40L-independent mechanisms; or 2) APCs may be activated directly by viral infection. Recent data from Ruedl et al. (45) show that naive CD8+ T cells following antigenic stimulation are capable of activating dendritic cells directly in the absence of CD40 and CD4+ T cells. Apparently this activation does not require direct interaction between CD8+ T cells and dendritic cells (DCs), but nevertheless leads to up-regulation of costimulatory molecules on the DCs. However, these results were obtained using TCR transgenic mice, in which the percentage of virus-specific CD8+ T cells is very much higher than that under normal physiologic conditions. Whether this CD8-mediated activation of DCs is relevant during the initiation of an immune response in the natural setting, i.e., when the frequency of virus-specific CD8+ T cells is low, has not been proven. More likely this mechanism could play a role later in the infection, when virus-specific CD8+ T cells have undergone clonal expansion and have become frequent.
It appears more reasonable to focus on the APCs, especially the DCs. It
is known that viral infection in vitro may activate DCs directly
(9), thereby up-regulating costimulatory molecules such as
B7.1, B7.2, and CD40. Recently, it has been found that VSV and LCMV
infections lead to very different levels of activation of DCs in vivo,
as LCMV induces a much stronger up-regulation of costimulatory
molecules than VSV (45). This further stresses the
differences previously observed between the two viruses and may be a
reflection of the difference in their capacity to replicate in the
murine host. Although other types of APCs may participate in activation
of virus-specific CD8+ T cells (21, 47), there is every reason to assume that virus-induced
activation should function in a parallel manner for all major subtypes
of APCs. Hence, the observation that infection with LCMV more
efficiently induces the up-regulation of costimulatory molecules on DCs
readily explains our results regarding coinfection with LCMV and VSV.
Thus, we obtained evidence indicating that in
CD40L-/- mice, LCMV was able to provide help
for the response toward VSV in an Ag-nonspecific, CD4-independent
manner. The stronger, and probably prolonged, activation of APCs
associated with LCMV infection (perhaps due to a more potent and
prolonged IFN-ß response (48, 49)) would cause DCs
and other APCs to become more efficient in activating VSV-specific
CD8+ T cells. In contrast, coinfection did not
bypass the requirement for CD28. The latter finding is consistent with
previous reports indicating that CD28 is directly involved in T cell
activation, and that the importance of this molecule is inversely
related to the strength of the TCR-dependent signal 1
(23). Because coinfection does not change the basic
behavior of VSV, the transient replication of this virus may limit Ag
presentation and thus explain the stringent requirement for
costimulation in this case.
The finding that certain viral infections may provide nonspecific help, thus bypassing control mechanisms normally perceived to be involved in preventing autoaggression, supports the theory put forward by Ruedl et al. (45) concerning autoimmune diseases involving self-reactive T cells not cross-reactive with viral epitopes. They propose that certain self-Ags are present at low amounts at all times, but due to an immature state of DCs are ignored by the T cells. However, during a viral infection DCs may become nonspecifically activated and up-regulate costimulatory molecules, resulting in activation of self-reactive T cells, thus shifting the balance from tolerance to autoimmunity.
Notably, despite the fact that CD28 and CD40L appear to play almost parallel roles during initiation of the CD8+ T cell response, consistent with the idea of the interrelationship of these signaling pathways, the importance of these molecular interactions is very different during the memory phase. Thus, LCMV-infected CD40L-/- mice show evidence of impaired CD8+ T cell function and do not permanently control the infection. Notably, this is observed even following infection with an LCMV strain (Armstrong) that has limited potential for causing persistent infection in wild-type mice. In contrast, no evidence for failure of immune control was found in CD28-/- mice, even though the frequency of virus-specific CD8+ cells tended to be slightly lower. This may indicate that CD28/B7 interactions play little role once a significant population of primed T cells has been established (40). In contrast, CD40/CD40L interaction may be needed to enable APCs to maintain the activated state of the memory cell population required for continued immune surveillance (50); during this phase of the infection viral replication is too limited to contribute significantly to conditioning of APCs.
In conclusion, the results demonstrate that the requirement for CD40/CD40L interaction to elicit a CD8+ T cell response varies considerably between viruses and over time. CD40L is critical for generation of VSV-specific TC1 cells regarding clonal expansion as well as differentiation, whereas the response to LCMV initially is CD40L independent. In the long term, CD40L seems to be required to maintain the functional capacity of primed CD8+ T cells even when the virus load is very low (low virus dose, virus strain with little capacity to persist (51)). Interestingly, coinfection with a CD40L-independent virus may augment the response to a CD40L-dependent virus/Ag. The latter finding may have implications for understanding the relationship between viral infections and autoimmune disease.
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2 Address correspondence and reprint requests to Dr. Allan Randrup Thomsen, Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, 3C Blegdamsvej, DK-2200, Copenhagen N, Denmark. E-mail address:
3 Abbreviations used in this paper: CD40L, CD40 ligand; CD40L-/-, CD40L deficient; VSV, vesicular stomatitis virus; LCMV, lymphocytic choriomeningitis virus; p.i., postinfection; BrdU, 5-bromo-2'-deoxyuridine; CD28-/-, CD28 deficient; IFN--/-, IFN- deficient; Cy, CyChrome; GP, glycoprotein; NP, nucleoprotein; VLA-4, very late Ag-4; DC, dendritic cell.
Received for publication October 4, 1999. Accepted for publication January 14, 2000.
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T cell subsets in viral immunity. Annu. Rev. Immunol. 1:123.
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