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Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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50% of the
responding T cell population. Following secondary exposure to SV5, high
avidity CD8+ T cells again are the exclusive cell type
present at early times postinfection (d2). Similarly, high avidity
cells were preferentially elicited at d3 following infection with the
unrelated vaccinia virus. We also made the observation that the
immunodominance profile has not been established at d3 postinfection
with SV5. However, by d5 a clear immunodominance pattern arises and is
permanently maintained. These data indicate that high avidity cells are
the predominant population responding at early times postinfection
following respiratory infection with SV5 or vaccinia virus. However, as
the response progresses, low avidity cells are activated/expanded to a
greater extent compared with high avidity cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have used simian virus 5
(SV5)3 as a model
system to study the immune response following viral infection in the
respiratory tract (3). SV5 has long been considered a
prototypic member of the paramyxoviridae family of viruses whose
members include common respiratory pathogens such as respiratory
syncytial virus, mumps virus, and the human parainfluenza viruses 1–4.
Members of this family of viruses have nonsegmented negative strand RNA
genomes. The 15-kb SV5 RNA genome consists of seven tandemly linked
genes that encode eight different proteins. The small number of viral
proteins expressed by SV5 allows us to easily follow the immune
response elicited by infection with the virus.
Of the many peptides encoded by a complex Ag, such as a viral particle, that can potentially be presented and elicit a CD8+ T cell response, only a limited number do so (for review, see Ref. 4). Within this small fraction often a single epitope elicits the immunodominant response, while the remaining immunogenic epitopes elicit subdominant immune responses (4, 5, 6). Although the mechanisms that contribute to the emergence of immunodominance are not fully understood, a number of factors have been shown to contribute, at least in part, to the establishment of immunodominance, including peptide affinity and the level of presentation at the cell surface (7). It has also been shown that immunodominance is flexible and is relative for a given set of immunogenic epitopes. For example, following loss of an immunodominant epitope, the response to subdominant epitopes is able to compensate for the loss of the immunodominant determinant (8, 9). There is significant interest in elucidating the mechanisms involved in controlling immunodominance, as it has been suggested to have a role in immune escape and delayed clearance as a result of the intense focusing of the immune response toward a single epitope.
Although CD8+ T cells have a well-documented role in the clearance of viral infections, not all Ag-specific T cells are equivalent in their ability to reduce viral load or protect from challenge. CD8+ T cell avidity is one characteristic that influences the efficacy of a given cell. We and others have demonstrated the in vivo importance of high avidity CD8+ T cells (10, 11, 12). In adoptive transfer studies, high avidity cells were much more effective than low avidity cells in reducing viral load. The reports to date have used two models: vaccinia virus delivered i.p. (10) and lymphocytic choriomeningitis virus administered intracerebrally (12). To our knowledge, no studies have evaluated the avidity of responses to viral infections in the respiratory tract, the site of entry for the majority of viruses.
Little is known about the in vivo regulation of high vs low avidity T cells. For example, the parameters promoting the activation and expansion of high and low avidity cells and the kinetics with which they respond to viral infection are not known. In addition, many questions remain in our understanding of the mechanisms by which immunodominance is established and maintained. In an effort to understand the regulation of the antiviral CD8+ T cell response from its initiation to the establishment of memory, immunodominance hierarchies and functional avidity were analyzed in BALB/c mice during the course of a respiratory tract infection with SV5. The findings from these studies have important implications for our understanding of the antiviral response elicited following respiratory tract infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were purchased from the Frederick Cancer Research and Development Center (Frederick, MD). P815 is a DBA/2-derived (H-2d) mastocytoma. All research performed on mice in this study complied with federal and institutional guidelines set forth by the Wake Forest University Animal Care and Use Committee. Recombinant viruses were constructed, as previously described (3), using the W3 SV5 strain or the WR vaccinia virus strain.
Immunizations
Mice were anesthetized with Avertin (2,2,2-tribromoethanol) by i.p. injection. Anesthetized mice were then immunized intranasally (i.n.) with the indicated amount of virus in 50 µl of PBS.
Tissue sampling
Lymphocytes from the lung were isolated from infected mice, as previously described (13). Briefly, lungs were extracted and pooled from two to four mice, finely chopped, and incubated at 37°C for 20 min in 10 ml of digestion mixture. The digestion mixture consisted of RPMI 1640, 10% FCS, 1 mg/ml of collagenase D (Roche), and 20 µg/ml DNase I type IV from bovine pancreas (Sigma-Aldrich, St. Louis, MO). Following digestion, the tissue was pushed through a nylon cell strainer and centrifuged over a Ficoll gradient (Sigma-Aldrich) to isolate mononuclear cells. Mediastinal lymph nodes (MLN) and spleens were isolated and pooled from two to four mice that had been immunized as indicated. Single cell suspensions were made.
IFN-
ELISPOT assay
The ELISPOT assays were performed, as previously described
(3). Briefly, responder cells isolated from the spleen,
MLN, or lungs of mice immunized with either wild-type (WT) rSV5 or
recombinant vaccinia viruses (VV) expressing SV5 polypeptides were
cocultured with P815 stimulators that had been infected with the
indicated virus 18–24 h prior and then UV inactivated. Because P815
cells express MHC class I Ags and not MHC class II Ags, the IFN-
production observed was mediated through class I-restricted T cells. To
enumerate the numbers of high and low avidity cells, titrated numbers
of responders were cultured in the presence or absence of saturating
concentrations (as determined by flow cytometric analyses) of
anti-CD8 Ab (clone 53 6.72) in the form of ascites. Following
36–48 h of coculturing of responder and stimulator cells at 37°C,
the plates were developed, as described previously (3).
The number of spots was determined with the aid of a stereoscope. The
relative avidity of cells was determined by the differential
sensitivity of high vs low avidity CD8+ T cells
to blocking with anti-CD8 Ab. Higher avidity T cells produce
IFN- in the presence of anti-CD8-blocking Ab following
stimulation by APC infected with virus, and T cells of lower avidity
are blocked by anti-CD8 Ab (3, 14, 15). Nonspecific
spot production was assessed by culturing the highest input number of
responder cells in the presence of stimulator cells that were
uninfected or were infected with viruses expressing irrelevant Ags
(i.e., the HIV glycoprotein, gp160). The number of spots in the absence
of infection was negligible.
Peptide ELISPOT assays were performed by incubating P815 stimulators with titrated doses of M285–293 (IPKSAKLFF) peptide at 37°C for 3 h. Stimulator cells were then washed and cultured with T cells from the MLN of immunized mice in an ELISPOT assay. As a negative control, T cells were cocultured with P815 cells that had not been loaded with peptide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In an effort to further understand the response to respiratory
infection with SV5, kinetic experiments were performed to study the
emergence of the SV5-specific response following i.n. immunization.
Responder cells from the MLN, spleen, and lung were isolated at
different time points postinfection and analyzed by ELISPOT assay.
Stimulators for this assay were P815 cells infected with vaccinia
viruses expressing individual SV5 proteins. Importantly, P815 cells do
not express MHC class II, and therefore only class I-restricted
CD8+ T cells are stimulated to produce IFN-.
At day 3 (d3) postinfection, SV5-specific CD8+
effector T cells were detected in the MLN, but not the spleen or lung
(Fig. 1
). The presence of cells in the
MLN, but not the spleen, is expected given i.n. delivery of the virus.
No SV5-specific cells were detected at d2 postinfection (data not
shown). Significant expansion of T cells occurred in the MLN between d3
and d7 with a subsequent decline by d12. The peak of the SV5-specific
response at d7 is similar to that reported for i.n. infection with
influenza virus (16).
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Analysis of the memory response (d40) demonstrated that the majority of memory cells were found to reside in the spleen. This is in agreement with the results reported in other systems (17). Furthermore, SV5-specific cells were detectable in the lung, but as would be expected, the number was much smaller than at d12.
High avidity CD8+ T cells dominate the initial response to SV5
We have previously shown that the immunodominant response to SV5
in BALB/c mice is directed against an epitope in the M protein, whereas
subdominant responses are directed against epitopes in the P, F, and HN
proteins (3). To address the emergence of T cells of high
vs low avidity over time, we performed a kinetic analysis of the
antiviral CD8+ T cell response in BALB/c mice
immunized i.n. with SV5. On the indicated days postinfection, responder
cells from the MLN were isolated and tested in an ELISPOT assay for
CD8+ T cells that recognize the SV5 P, M, F, or
HN proteins. Our assay for T cell avidity is based on the observation
that low avidity cells require CD8 engagement to elicit effector
function, whereas high avidity cells are relatively CD8 independent for
activation (3, 15, 18, 19, 20, 21, 22). As previously described, we
were able to quantify the number of high and low avidity CTL specific
for SV5 epitopes by including anti-CD8-blocking Ab in cocultures of
responding T cells and stimulators (P815 cells infected with VV-P,
VV-M, VV-F, or VV-HN) (3). Only those CTL that are of high
avidity will produce IFN- in the presence of anti-CD8-blocking
Ab (filled bars). Cultures incubated in the absence of Ab detect the
total response to each protein (hatched bars). The number of low
avidity cells (CD8 dependent) can be determined by subtracting the
number of cells detected in the presence of anti-CD8 Ab from the
total response.
Surprisingly, in the MLN at d3 postinfection, the response to each of
the SV5 proteins was almost exclusively high avidity (Fig. 2A; compare hatched bars with
filled bars). However, by d5 postinfection, low avidity cells were
readily detected and comprised 50% of the total responding
CD8+ T cells. In agreement with our previous work
(3), the P-specific response remained exclusively high
avidity.
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1000-fold more than that required by cells at
d3 (Fig. 2
B). These data confirm the observation that at d3,
high avidity cells dominate the response, but at later time points, low
avidity cells emerge.
At d3, the M-specific response was equivalent in size to the other
SV5-specific responses (Fig. 2A). However, by d5, a clear
immunodominance pattern was present, with the M-specific response
clearly established as immunodominant. The same avidity and
immunodominance phenotypes detected at d5 continued into the memory
population (d40) (Fig. 2A).
In contrast to the MLN, in the spleen the M-specific response was the
largest response at every time point tested, even d5, which was the
earliest time at which virus-specific cells could be detected (Fig. 3). Although the responses at d5 were
small in three separate experiments performed, the M-specific response
was the largest response in each experiment and was
statistically different in two of the three experiments
(p 
0.05). In addition, the d5 population
contained both high and low avidity cells (Fig. 3). The immunodominance
and avidity patterns, as well as the kinetic delay, suggest that the
CD8+ T cells detected in the spleen were
activated in the MLN, entered circulation, and subsequently entered the
spleen.
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High avidity CD8+ T cells are highly underrepresented in the lung compared with other tissues
Although the presence of T cells in the lung following infection
with a number of viruses has been reported, the avidity of the cells
present was not assessed (16, 23, 24). Given the critical
nature of avidity in determining clearance efficacy in other viral
models (10, 11, 12), it was important to determine the avidity
of SV5-specific CD8+ T cells present at the site
of infection. At various time points postinfection with SV5, cells were
isolated from the lung and analyzed directly ex vivo by ELISPOT assay.
As previously noted, the emergence of CD8+ T
cells in the lung is delayed compared with the MLN (d3) with only very
low numbers of detectable Ag-specific CD8+
effector T cells before d7 (Fig. 1). When the functional avidity was
assessed, a striking observation was made. When compared with the
response in the MLN, there was a major skewing toward low avidity cells
in the lung at d9-d40 (Fig. 4).
Interestingly, the responses to the subdominant P and HN epitopes were
also underrepresented in the lung compared with the MLN.
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SV5 is known to replicate in the lungs of infected mice
(25). In addition, infection of cells in vitro shows that
SV5 can replicate to high titers without producing apparent cytopathic
effects. Conversely, vaccinia virus infection is associated with
cytopathic effects and has been shown to spread systemically following
i.n. infection in mice (26). To determine whether the
early emergence (d3) of high avidity T cells was specific to SV5
infection, BALB/c mice were immunized i.n. with either a recombinant
vaccinia virus expressing the full-length P protein (rVV-P) or M
protein (rVV-M) from SV5. At d3 postinfection, responder cells from
immunized mice were harvested and analyzed by ELISPOT. Similar to SV5
infection, both rVV-P and rVV-M infection elicited almost exclusively
high avidity CD8+ T cells in the MLN (Fig. 5). In contrast to SV5 infection,
however, the responses in the spleen of vaccinia virus-immunized mice
were also exclusively high avidity at d3 (Fig. 5). This finding is most
likely the result of the systemic spread of the virus, resulting in the
activation of T cells in the spleen. Similar to SV5, at d5 postvaccinia
virus infection, low avidity M-specific CD8+ T
cells began to emerge in both the MLN and the spleen, although there
was a lower percentage of M-specific low avidity cells detected at d5
as a result of vaccinia virus infection compared with SV5 infection
(64.2 ± 4.2% with SV5 infection compared with 30.5 ± 6.4%
with vaccinia virus infection). Consistent with our previous results
(3), the P-specific response remained exclusively high
avidity (Fig. 5). The percentage of M-specific cells that were low
avidity continued to increase through d7. Interestingly, there was a
larger proportion of low avidity M-specific CD8+
T cells detected in the spleen compared with the MLN, suggesting that
there may be a skewing toward the elicitation of high avidity cells in
the draining lymph node following vaccinia virus infection. In summary,
respiratory infection with either SV5 or vaccinia virus resulted in an
initial population of cells (d3) that was almost exclusively high
avidity. Whether this is the case for other virus infections is
currently under investigation. In addition, these findings demonstrate
that the high avidity response elicited at early times postinfection is
not exclusive to the draining lymph node, but may also occur in other
lymphoid tissues, such as the spleen.
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There is evidence that the responding CD8+ T
cell population following initial exposure to Ag is often different
from the cells that are elicited following subsequent exposures to the
same Ag. For example, it has been shown that following secondary
exposure there is a decrease in the diversity of TCR V
segments used
in the responding CD8+ T cell population
(27). In addition, following secondary influenza virus
infection, the relative dominance of some T cell epitopes is diminished
when compared with the primary response (28, 29). Thus, it
was of interest to determine whether the observed pattern of
immunodominance and, more importantly, the early emergence of high
avidity cells were specific to the primary immune response. Mice were
initially immunized i.n. with 1 x 106 PFU
of WT SV5. At d40 postinfection, mice were challenged i.n. with 1
x 107 PFU of the same virus. Responders were
harvested and analyzed by ELISPOT d2 after secondary immunization. Day
two was the earliest time at which Ag-specific expansion could be
detected (data not shown). As a control, mice that were initially
infected with SV5 were challenged with PBS and analyzed at d2. There
was a significant expansion (20-fold) of Ag-specific
CD8+ T cells in the MLN d2 after challenge with
SV5 compared with the negative control (Fig. 6A). As was observed in the
primary response, the earliest detectable responding SV5-specific cells
were exclusively high avidity. Interestingly, immunodominance was
apparent in the secondary response even at d2, as evidenced by the
larger response to the M epitope compared with P, F, and HN (Fig. 6A). Thus, once established following primary SV5 exposure,
immunodominance is a constant feature of the SV5-specific response. The
response in the spleen was not significantly different from the
PBS-challenged mice, demonstrating a lag in the response in the spleen
analogous to that observed during the primary response. Together, these
data demonstrate that the early appearance of high avidity cells in the
MLN is not specific to the primary response to virus, but rather is a
general characteristic of the antiviral CD8+ T
cell response to SV5.
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B). There was little change in the splenic
responding population at this time point. As the response in the MLN
begins to decline by d7, the response in the spleen had increased
significantly (Fig. 6C). However, there was no change in the
immunodominance hierarchies or the avidity of the SV5-specific
responses compared with the response following primary exposure to SV5
(Fig. 2). Taken together, these results indicate that after the initial
response to virus, subsequent exposures to the SV5 do not change the
relative avidity of the responding population. In addition, once
established in the primary response, immunodominance is present even at
the earliest time at which virus-induced expansion of the
CD8+ T cells can be detected following secondary
exposure to SV5. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The most interesting observation in our studies was that high avidity
CD8+ T cells were selectively activated and/or
expanded at early times following infection, as indicated by the
response in the MLN at d3 (Fig. 2A), which was almost
exclusively high avidity cells. This was followed by the later
emergence of low avidity cells as the response progressed. In contrast,
the response in the spleen consisted of relatively low numbers of cells
until d7 postinfection, at which time a mixture of high and low avidity
CD8+ T cells was detected (Fig. 3). As i.n.
infection with SV5 results in an infection in the respiratory tract
(25), it is likely the cells that are detected in the
spleen at d5 are cells that were initially activated in the MLN and
then migrated to the spleen. This interpretation is supported by
the results of a recent study that analyzed the response to
cutaneous infection with HSV 1 (32). This immune response
was initiated in the draining lymph node by d2 postinfection. However,
CD8+ T cells were not detected in the spleen
until d4 postinfection.
Our data with vaccinia virus infection provide further evidence for the
selective activation and/or expansion of high avidity
CD8+ T cells at the initiation of the response.
As seen with SV5, i.n. infection of BALB/c mice with recombinant
vaccinia viruses (rVV-P or rVV-M) resulted in exclusively high avidity
P- and M-specific CD8+ effector T cells at d3
postinfection in the MLN and the spleen (Fig. 5). Intranasal infection
of BALB/c mice with the WR strain of vaccinia causes a systemic
infection with detectable levels of virus in the blood and the spleen
by d3 postinfection (26). Thus, it is likely that naive
cells are activated initially in the spleen as well as the MLN. An
exclusively high avidity early antiviral response was also detected
after a secondary challenge with SV5 (d2 postchallenge) (Fig. 6A). In total, these results demonstrate that the high
avidity nature of the initial responding CD8+ T
cells is a property of both the primary and secondary antiviral
response following respiratory tract infection with SV5 or vaccinia
virus. These results differ from a study that showed a time-dependent
increase in the avidity of cells specific for an epitope from
lymphocytic choriomeningitis virus (15). The reason for
this discrepancy in results is not known, but could reflect a
difference in the route of infection or the virus used. In striking
contrast to the responses detected in the spleen and the MLN, the
effector population in the lung following infection with SV5 showed a
dramatic skewing toward low avidity cells (Fig. 4). The mechanism
responsible for the underrepresentation of high avidity cells is
currently under investigation.
The mechanism responsible for the emergence of the disparate populations present at d3 vs d5 is not known. Certainly, the environment present at d1-d2, which is directing the populations detected at d3, could be significantly different from that present at later times. One factor that may change over the course of infection and that could influence the populations present is the level of peptide presentation.
Peptide/MHC density has been shown to play a crucial role in vitro in the generation of CTL of a defined avidity (10, 12, 30, 33). In these studies, low concentrations of peptide were found to preferentially expand high avidity CD8+ T cells, while high concentrations of peptide stimulated proliferation of low avidity CTL. It is currently not known whether peptide/MHC density exerts a similar pressure on the activation of naive precursors in vivo. However, based on the in vitro studies, it is attractive to hypothesize that high avidity precursors require less Ag than low avidity precursors to initially become activated. This model would predict that at early times postinfection, virus peptide/MHC determinant densities are low, and thus only sufficient to activate high avidity cells. However, over time, determinant densities may have reached sufficient levels to also activate low avidity cells, thus accounting for the lag in detection of low avidity cells in our experiments. Our data support this model and indicate that by d5 there is a >30-fold increase in the number of SV5-specific low avidity cells detected in the MLN compared with d3 postinfection. However, there is only a 5-fold increase in the number of high avidity cells. The mechanism by which peptide could be limited early on postinfection is unknown, but factors such as cytokine environment could explain enhanced levels of peptide at later times postinfection. For example, it is well established that IFN mediates up-regulation of proteins that facilitate Ag processing and presentation as well as immunoproteasome formation (34). It is possible that at early times postinfection, the composition of the proteosomes is such that only low levels of immunogenic epitopes are presented, promoting preferential activation of high avidity cells. However, during the course of a viral infection, infected cells are triggered to synthesize cytokines that create an inflammatory environment. These soluble mediators of inflammation have been shown to activate APCs, which allows for more efficient priming of Ag-specific T cells (35). Therefore, it is possible that once a potent inflammatory environment is established, low avidity cells are able to overcome any lack of stimulatory agents (i.e., limiting Ag dose and cytokines) that are not present at sufficient levels during the beginning of the infection, and thus are activated at later time points.
In summary, the study presented in this work provides new and important insights into the emergence and maturation of the antiviral response following respiratory tract infection with two different viruses. The results show that at d3 postinfection, there is no evidence of immunodominance. However, by d5, immunodominance hierarchies are permanently established. Furthermore, the initial CD8+ T cells present in these antiviral populations in the MLN are exclusively high avidity. The high avidity response at early times postinfection is independent of subsequent exposures to Ag and of the virus administered. Elucidation of the mechanisms responsible for the control of the selective activation and expansion of high avidity T cells and the emergence of immunodominance may allow for the design of vaccines that elicit high avidity cells to multiple epitopes, which should provide optimal protection against pathogens.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martha A. Alexander-Miller, Department of Microbiology and Immunology, Room 5108, Gray Building, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail address: marthaam{at}wfubmc.edu
3 Abbreviations used in this paper: SV5, simian virus 5; d, day; i.n., intranasal; MLN, mediastinal lymph node; VV, vaccinia virus; WT, wild type.
Received for publication May 28, 2002. Accepted for publication October 23, 2002.
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References |
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), and lung (
) were isolated for analysis by
ELISPOT. Total SV5-specific spots at each time point were calculated by
subtracting the background out of each SV5 protein-specific response
and adding them together. Each time point represents the average of
three or four separate experiments.
