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,
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Beirne B. Carter Center for Immunology Research and
Department of Pathology and Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA 22908
Beirne B. Carter Center for Immunology Research and
Department of Pathology and Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4.8% of pulmonary CD8+ T cells at the
peak of the primary response (day 8), whereas matrix 2-specific
CD8+ T cells constituted 50% of the responding
CD8+ T cell population in the lungs. When RSV F-immune mice
undergo a challenge RSV infection, the F-specific CD8+ T
cell response is accelerated and dominates, whereas the primary
response to the matrix 2 epitope in the lungs is reduced by 20-fold.
In addition, we found that activated F-specific effector
CD8+ T cells isolated from the lungs of RSV-infected mice
exhibited a lower than expected frequency of IFN-
-producing
CD8+ T cells and were significantly impaired in ex vivo
cytolytic activity compared with competent F-specific effector
CD8+ T cells generated in vitro. The significance of these
results for the regulation of the CD8+ T cell response to
RSV is discussed. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RSV-specific CD8+ T cell responses are involved in the clearance of the virus and recovery from infection. In BALB/c mice, previous studies have demonstrated that virus-specific CTL are sufficient for effective virus clearance during primary infection (3, 4) and matrix 2 (M2), fusion (F), and N proteins of RSV are major target Ags for MHC class I-restricted CTL activity (5, 6, 7). In humans, RSV-specific CTL develop in response to natural infection (8, 9), and F and N proteins are reported to be primary targets for MHC class I-restricted CTL responses (6, 10, 11). In addition to the role in the clearance of virus, it has been suggested that CD8+ T cells play an important role in the regulation of the differentiation and activation of Th2 CD4+ T cells, which mediate the enhanced lung pathology by the recruitment of eosinophils into the lungs during RSV infection (12, 13). It has also been hypothesized that CD8+ CTL play a role in the pathogenesis of RSV-induced disease. For example, enhanced pulmonary injury was reported in a mouse model of RSV infection in which passive transfer of activated RSV-specific CTL resulted in rapid virus clearance from the lungs but also enhanced pulmonary pathology (3). The immune mechanisms underlying the balance among protection, disease, and recovery from infection is still unclear.
The advent of methods such as MHC class I tetramer and peptide-induced intracellular cytokine staining (ICS) has provided the opportunity to detect and accurately enumerate Ag-specific CTL and to analyze the kinetics of the virus-specific CTL response at the site of infection during the time course of infection. Therefore, the identification of CTL epitopes of RSV and the subsequent application of new technologies may contribute to elucidating the role of CD8+ CTLs in RSV immunity and pathogenesis. Although the CD8+ T cell response to the M2 is directed primarily if not exclusively to a single immunodominant site spanning residues 82–90 of the M2 protein (14), no corresponding site recognized by CD8+ T cells has been identified in the RSV F protein.
In this study, we have identified an immunodominant CTL epitope contained in RSV F protein and have shown that it is Kd-restricted. Using MHC class I tetramer containing this epitope, we visualized and quantitated F-specific CD8+ T cell response during primary and secondary RSV infection of BALB/c mice. We found that the magnitude of F-specific CD8+ T cell response during primary RSV infection is 10-fold lower than the dominant M2-specific response. In contrast, this response hierarchy is reversed in challenge infection of F-primed mice, with a 20-fold suppression of the M2-specific primary CTL response in the lungs. These results established a immunodominance hierarchy between F- and M2-specific CD8+ T cell responses in experimental RSV infection. In addition, functional assays measuring the effector activity of lung CD8+ CTL revealed the defective phenotype of effector functions by these F-specific CD8+ T cells, suggesting that RSV induces immune dysregulation of virus-specific CD8+ T cells. The significance of these results for CD8+ T cell epitope immunodominance and for the host response to RSV infection is discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice (H-2d), 6–8 wk old, were purchased from Taconic Farms (Germantown, NY). Mice were housed in a pathogen-free environment.
Viruses and infection of mice
The A2 strain of RSV was a gift from P. L. Collins (National Institutes of Health, Bethesda, MD). RSV (A2 strain) stock was grown on HEp-2 cells (American Type Culture Collection, Manassas, VA) and titered for infectivity. Recombinant vaccinia virus expressing the F protein of RSV (vvF) was a gift from J. L. Beeler (National Institutes of Health). Mice were infected with 5 x 106 PFU vvF by scarification at the base of the tail. Three weeks after priming, mice were lightly anesthetized with ether-chloroform (2:1), and inoculated intranasally with 1 x 106 PFU RSV in 50 µl. At various times after infection, infected mice were sacrificed by cervical dislocation.
Peptides
Synthetic peptides were made by standard 9-fluorenylmethoxycarbonyl chemistry with a model AMS422 peptide synthesizer (Gilson, Middleton, WI).
RMA-S stabilization assay
RMA-S expressing Kd or Ld molecules cells were maintained in selective medium (RPMI 1640 supplemented with 10% FBS, 10 U/ml penicillin G, 10 µg/ml streptomycin sulfate, 2 mM L-glutamine, 5 x 10-5 2-ME, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10 mM HEPES, and 200 µg/ml G-418 (Life Technologies, Gaithersburg, MD)). Cells were incubated at 25°C in a 5% CO2 atmosphere for 3 h, pulsed with indicated amounts of peptides, and then warmed to 37°C for an additional 3 h. Cells were then stained with PE-conjugated anti-Kd mAb (clone SF1-1.1; PharMingen, San Diego, CA), or anti-Ld mAb (clone 28.14.8) followed by FITC-conjugated goat anti-mouse IgG (Caltag Laboratories, San Francisco, CA), washed twice, and analyzed by flow cytometry. All staining manipulations were conducted at 4°C.
Preparation of lung-derived lymphocytes
The lungs were perfused with 5 ml PBS containing 10 U/ml heparin (Sigma, St. Louis, MO) through the right ventricle using a syringe fitted with a 25-gauge needle. The lungs were removed and placed into complete IMDM (supplemented with 10% FBS, 10 U/ml penicillin G, 10 µg/ml streptomycin sulfate, 2 mM L-glutamine, 5 x 10-5 2-ME, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 10 mM HEPES). The tissue was processed through a steel screen to obtain a single-cell suspension, and particulate matter was removed by quick centrifugation at 300 x g. Cells were counted and resuspended at the given cell concentrations for the appropriate in vitro assay.
In vitro stimulation of splenocytes
Pooled spleen cells from vvF-immunized mice were resuspended in complete IMDM supplemented with recombinant human IL-2 (20 U/ml), and 2.5 x 107 cells were added to each well of a six-well plate. Syngeneic splenocyte stimulators were prepared from naive mice by irradiation (2000 rad) and pulsing for 0.5 h at room temperature with the peptide at a concentration of 5 x 10-8 M. Thereafter, cells were extensively washed to remove free unbound peptide, and 5 x 106 cells were added to the each well of responder cells.
Flow cytometry and tetramer staining
MHC class I-peptide tetramers were produced as described
previously (15). Plasmid DNA encoding the extracellular
domain of the H-2Kd H chain and human
2-microglobulin were provided by J. Altman
(Emory University, Atlanta, GA) and E. Pamer (Yale University, New
Haven, CT), respectively. Recombinant proteins were expressed in
Escherichia coli, purified from inclusion bodies,
solubilized, and refolded in the presence of corresponding peptides.
Folded complexes consisting of H-2Kd,
2-microglobulin, and antigenic peptide were
purified by gel filtration over a Sephacryl S-300HR column (Amersham
Pharmacia Biotech, Piscataway, NJ) and enzymatically biotinylated with
the biotin-protein ligase BirA (Avidity, Denver, CO). The biotinylated
monomer complexes were further purified over a Mono-Q ion exchange
column (Amersham Pharmacia Biotech) and tetramerized with PE-labeled
streptavidin (Molecular Probes, Eugene, OR). Tetramers were
stored at 5 mg/ml at 4°C in PBS (pH 8.0) containing
0.02% sodium azide, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 0.5
mM EDTA. Freshly explanted lung lymphocytes were purified by
density gradient centrifugation and stained in PBS, 3% (w/v) FCS,
0.09% (w/v) NaN3 using
fluorochrome-conjugated Abs and MHC class I tetramers. The Abs used
were anti-CD8
(clone 53-6.7), anti-CD11a (clone 2D7),
anti-CD25 (clone 7D4), anti-CD43 (clone 1B11), and
anti-CD62L (clone MEL-14). All Abs were purchased from
PharMingen. After staining, cells were fixed in PBS, 2% (w/v)
paraformaldehyde, and events acquired using a FACSCalibur flow
cytometer (BD Biosciences, San Jose, CA). Dead cells were excluded on
the basis of forward and side light scatter. Data were analyzed using
CellQuest software (BD Biosciences).
Intracellular cytokine staining
To enumerate the number of IFN--producing cells,
intracellular cytokine staining was performed as previously described
(16). In brief, 106 freshly
explanted lung lymphocytes were cultured in a culture tube. Cells were
left untreated or stimulated with F85–93 peptide
(1 µM) and were incubated for 5 h at 37°C in 7%
CO2. Brefeldin A (10 µg/ml; Sigma) was added
for the duration of the culture period to facilitate intracellular
cytokine accumulation. After this period, cell surface staining was
performed as above, followed by intracellular cytokine staining using
the staining buffer containing 0.5% (w/v) saponin (Sigma). The Abs
used were anti-IFN- (clone XMG1.2) or its control isotype Ab
(rat IgG1).
CTL assay
Standard chromium release assay was performed as described (13). Briefly, 5 x 103 sodium [51Cr]chromate-labeled P815 cells were added as target cells per well of U-bottom 96-well plates (Costa, Cambridge, MA) and tested for lysis by effector cells in the presence of relevant or irrelevant peptide. Effectors from lung-derived lymphocytes or in vitro-cultured splenocytes were prepared by density gradient centrifugation and CD8+ T cells were subsequently enriched by positive selection using MACS (Miltenyi Biotec, Auburn, CA). Typical purification results in 80–90% CD8+ cell purity. Percent specific lysis was calculated as ((51Cr release with effector cells - spontaneous 51Cr release)/(total 51Cr release with 1% Triton X-100 - spontaneous 51Cr release)) x 100. Spontaneous 51Cr release from target cells in all assays was <5% of the detergent lysis. The percent specific lysis values represent the mean values of quadruplicate wells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify potential CTL epitopes within F protein, we synthesized an overlapping panel of 15-mer synthetic peptides spanning the 574-aa F protein of the RSV A2 strain and tested these peptides for a target cell sensitization in CTL assays. Effector cells used in the screening were derived from the spleens of BALB/c mice that had been primed with vvF, stimulated in vitro with RSV-infected APC, and tested on P815 target cells pulsed with overlapping peptides in standard 51Cr release assay. We have previously shown that this priming and in vitro stimulation strategy leads to robust in vitro secondary CTL activity by immune splenocytes in response to RSV-infected APC (13). In this initial screening, two peptides were identified that exhibited lytic activity above background killing with irrelevant peptide (data not shown). One peptide included residues 85–93 of the F glycoprotein, which contains a canonical H-2Kd-binding motif (KYKNAVTEL; critical anchor residues are in bold) and the other peptide included F residues 352–360, which contain an H-2Ld-binding motif, i.e., FPQAETCKV. We then synthesized the two nonamer peptides, F85–93 and F352–360, conforming to the predicted motifs and containing the putative CTL target sequences, and tested the ability of these peptides to sensitize target cells for recognition by RSV F-specific CTL generated in vitro. Target cells sensitized with F85–93 or F352–360 peptide were lysed by immune CD8+ T cell effectors generated in vitro from the spleen of vvF-immunized but not vvM2-immune mice (data not shown).
To determine whether these peptides corresponded to valid
Kd- and Ld-restricted F
epitopes, we first examined the ability of in vitro generated CTL from
vvF-primed mice to recognize L929 (H-2k) cells
stably expressing the transfected Kd,
Ld or Dd MHC class I genes,
respectively. Because L cells are not productively infected with RSV,
the targets were infected with vvF to express the endogenously
processed F glycoprotein. Control target cells were infected with
vaccinia virus expressing -galactosidase. As Fig. 1
A demonstrates, F-specific
CTL lysed vvF-infected L cells expressing the Kd
molecule but not the Ld or
Dd transfectants.
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B
demonstrates, F85–93 efficiently bound to and
stabilized Kd over a range of peptide
concentrations. In contrast, F352–360 stabilized
Ld poorly when compared with a known
Ld-binding CD8+ T cell
epitope from murine CMV pp89 protein (17). However,
F352–360 did stabilize Kd
molecules despite lacking a Kd binding motif in
its primary sequence but did so only at the higher peptide
concentrations
(10-6–10-7 M). This
difference in peptide dose dependence of MHC class I binding was
likewise observed at the level of target cells sensitization.
F-specific CTL generated in vitro in response to infectious RSV lysed
F85–93-pulsed target cells over a range of
peptide concentrations (Fig. 1C).
F352–360-pulsed targets were also lysed by
F-specific CTL, but efficient lysis was observed only at the highest
peptide concentrations. The above findings suggested that the
F85–93 peptide likely defined a bona fide
CD8+ CTL epitope generated in response to RSV
infection. The inability of RSV F-specific CTL to recognize
Ld-expressing target cells as well as the
poor response of the Ld motif-containing
F352–360 peptide in the RMA-S stabilization
assay suggested that this peptide did not define an
Ld-restricted epitope in F protein. Rather,
F352–360 was likely to be either a mimotope
(18) of the dominant Kd-restricted
F85–93 epitope or a weak subdominant
Kd-restricted epitope.
To assess the contribution of these two F epitopes in the
CD8+ T cell response to RSV during natural
infection, we harvested lung-infiltrating lymphocytes from vvF-primed
mice undergoing challenge RSV infection at the peak of the response in
the lungs (days 5 and 6). CD8+ T cells in the
lungs responding to F85–93 or
F352–360 were enumerated by ICS/IFN- after
short term in vitro peptide stimulation. As Fig. 2A shows, 30% of the
CD8+ T cells infiltrating the lungs responded
with IFN- production after stimulation with either peptide at the
highest peptide concentration used (1 µM). However, whereas the
F85–93 peptide elicited a vigorous IFN-
response from lung-infiltrating CD8+ T cells over
a broad range of stimulating peptide concentrations, the IFN-
response elicited by F352–360 rapidly declined
as the peptide dose decreased. This finding favors the view that the
F352–360 peptide defines a mimotope possibly of
the dominant F85–93 epitope.
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response of lung
CD8+ T cells stimulated by the individual F
peptides with the IFN- response of lung
CD8+ T cells exposed to a equimolar mixture of
the two peptides at 1 µM concentration. As shown in Fig. 2B, the frequency of CD8+ T cells
responding to the peptide mixture did not exceed the frequency of
IFN--secreting cells responding to F85–93 peptide
alone. This result strongly suggests that F85–93 is a
real immunodominant epitope of RSV F protein and F352–360
is a mimotope that can stimulate an IFN- response by
F85–93-specific cells only at nonphysiological
concentrations. Magnitude of F85–93-specific primary and secondary CD8+ T cell response in the lungs
Infection of BALB/c mice with RSV results in the recruitment of
H-2d-restricted CD8+ CTL
responses to M2 and F viral proteins to the lungs that can be detected
by ex vivo pulmonary CTL-mediated lysis (19). However,
the actual magnitude of F-specific CD8+ T cell
response has not been quantitated in the lungs during RSV infection. To
gain a precise measure of the pulmonary CD8+ T
cell response, we used tetramer technology to quantitate the numbers of
CD8+ T cells responding to RSV
F85–93 epitope and the previously identified
immunodominant M282–90 epitope present in the
RSV M2 protein during primary and challenge RSV infection. Naive
(primary) and vvF-primed (secondary) mice were infected intranasally
with RSV, and the frequencies of RSV-specific
CD8+ T cells infiltrating the lungs in the course
of infection were analyzed using Kd MHC I
tetramers containing the F85–93 or the
M282–90 peptide (designated as
KdF85–93Tet or
KdM282–90Tet, respectively). Fig. 3A demonstrates that, after
primary infection, the pulmonary F85–93-specific
CD8+ T cell response reaches detectable levels by
day 5 postinfection and peaks on day 8. When compared with M2-specific
CD8+ T cell response, the magnitude of
F85–93-specific CD8+
response in the lungs was relatively weak, reaching a maximal frequency
of 4.8% at day 8. By contrast, the response to the dominant,
Kd-restricted M2 epitope was 10-fold higher,
reaching 50% of lung CD8+ T cells at the same
time point (Fig. 3A). In companion
studies,5 the kinetics
of M2-specific CD8+ T cell accumulation in the
lungs during primary infection and the rate of decline of M2-specific
CD8+ T cells at later times in infection directly
paralleled the pattern observed in the response to the
F85–93 epitope. These data clearly demonstrate
that during natural infection the frequencies of primary
CD8+ T cells responding to different RSV proteins
vary significantly for the immunodominant sites within these two
CD8+ T cell target proteins. Although the
absolute numbers of responding CD8+ T cells vary
among individual animals within an experiment and between experiments
(within a 2-fold range over a series of experiments), the hierarchy of
responsiveness for these two determinants was consistent with
M2-specific CD8+ T cells, demonstrating a 10-fold
higher frequency. Total CD8+ T cell numbers
increased in the lungs at early times during primary infection (days 4
and 5) before RSV-specific CD8+ T cells were
detected. This presumably reflects the nonspecific recruitment of
CD8+ T cells to the lungs in response to
pulmonary inflammation. We estimate that at the peak of the primary
response to infection in the lungs (days 8–10) at least 50–60% of
the total CD8+ T cells present in the lungs are
RSV-specific with the majority of the RSV-specific
CD8+ T cells (90%) likely representing
M2-specific T cells.
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B). As expected, after challenge infection of F-primed
mice, there was a significant expansion and the accumulation of
F85–93-specific CD8+ T
cells in the lungs, which peaked earlier (at day 6) and exhibited
10-fold higher frequency than peak primary response (56% of
total lung CD8+ T cells vs 4.8% in the
primary). Thus, the memory response to RSV F in the lungs was dominated
by CD8+ T cells directed to
F85–93 epitope. Interestingly, the strong
response to the M2 epitope in the primary response was not observed
during the recall response to the F protein. M2-specific
CD8+ T cells, which accounted for 50% of the
CD8+ T cells responding to RSV at the peak
primary response (days 8–10), now accounted for <3% of the
CD8+ T cells accumulating in the lungs at day 10
postinfection (Fig. 3B). We did routinely observe a
slight increase in the overall numbers of
CD8+ T cells accumulating in the lungs during
challenge infection as compared with primary infection (<50%). This
increase in CD8+ T cell numbers can be
accounted for by the expanded population of F-specific
CD8+ T cells infiltrating the lungs during
challenge infection. Although not reported here, the reduced frequency
of primary M2-specific CD8+ T cells accumulating
in the lungs of F-primed mice after challenge RSV infection was evident
at day 8 (data not shown) and day 10 (Fig. 3B) after
infection. Defective effector functions of F85–93-specific CD8+ T cells in the lungs
We recently reported that pulmonary RSV infection results in the
defective expression of effector activity by pulmonary M2-specific
CD8+ T cells.5 Therefore,
it was of interest to investigate whether the functions of F-specific
CD8+ T cells accumulating in the lungs after RSV
infection are likewise defective in the expression of effector
activity. We tested the effector activity of F-specific
CD8+ T cells by examining the IFN- synthesis
by
KdF85–93Tet+CD8+
T cells and by direct ex vivo CTL assay of lung-infiltrating
lymphocytes. As shown in Fig. 3B and Table I, at the peak of the memory response to
challenge infection (day 6), 56% of the CD8+
T cells infiltrating the lungs stained with the
KdF85–93Tet, whereas only
27% of the CD8+ cells produced IFN- in
response to cognate peptide (i.e., 49% of the expected percentage
of F85–93-specific cells based on tetramer
staining). This lower than expected frequency of IFN--secreting
F-specific CD8+ T cells was observed whether the
lung CD8+ T cells were stimulated with
peptide-pulsed resident APC present in the lung cell suspension or
peptide-pulsed P815 tumor cells were used to stimulate cytokine
synthesis. We calculated the percentage of
IFN-+KdF85–93Tet+
cells by dividing the fraction of
IFN-+CD8+ T cells by the
fraction of the
KdF85–93Tet+CD8+
T cells because stimulation of activated effector
CD8+ T cells by Ag leads to the rapid
down-regulation of TCR levels and decreased efficiency of tetramer
staining (Ref. 20) and data not shown). A simultaneous
analysis of the expression of activation markers on lung-infiltrating
cells revealed that >90% and >80% of the
KdF85–93Tet+CD8+
T cells infiltrating the lungs during infection exhibited
CD11ahighCD62Llow and
CD25high activated phenotypes, respectively
(Table I). Noticeably, 60% of
KdF85–93Tet+CD8+
T cells displayed the CD43(1B11)high phenotype,
suggesting that these F-specific CD8+ T cells may
not fully differentiate into terminal effectors. The 1B11 Ab is known
to detect an activation-associated isoform of CD43 reported to be
displayed at high levels on fully differentiated cytolytic
CD8+ effector T cells (21). For the
primary response, the frequency of IFN--secreting
KdF85–93Tet+CD8+
cells were too close to the background value to be reliably used to
calculate the exact proportions of competent effectors (data not
shown).
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assay. In this instance we observed a
one to one correspondence between tetramer staining and cytokine
synthesis in the frequency of Ag-specific CD8+ T
cells present in the lung at the peak of infection (data not shown).
Thus, the diminished frequency of IFN--producing cells observed
during RSV infection appears not to be a general property of
CD8+ T cells accumulating in the lungs during
experimental virus infection.
We next investigated the direct ex vivo lytic activity of the
activated, F85–93-specific
CD8+ T cells infiltrating the lungs of RSV
F-immune mice undergoing a challenge RSV infection in parallel with
effector cells generated by in vitro stimulation of splenocytes from
F-immune mice with F85–93 peptide. Viable
CD8+ T cells were purified from each source and
assayed with F85–93 peptide-pulsed
51Cr-labeled P815 target cells over a range of
E:T ratios (Fig. 4). Activated
F85–93-specific CD8+ T
cells generated by in vitro stimulation of F-immune precursors
displayed potent in vitro cytolytic activity. In contrast, pulmonary
effector CD8+ T cells exhibited significantly
reduced lytic activity that was approximately one-third of the activity
observed with the in vitro generated effectors when normalized to the
actual numbers of F85–93-specific cells as
determined by tetramer staining. Taken together, these results suggest
that pulmonary RSV infection may inhibit the expression of effector
activity by RSV F-specific CD8+ T cells recruited
to the lungs during viral infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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assay, we visualized
the expansion of F85–93-specific CD8+ T
cells in the lungs of infected mice during primary and secondary
infection. In contrast to the response to the immunodominant M2 epitope
(which represents 50% of total CD8+ T cells
in the lungs responding to primary RSV infection), the
F85–93-specific response represented only 4.8%
of pulmonary CD8+ T cells infiltrating the lungs
during primary infection. However, during challenge RSV infection of
F-primed mice, the recall response to F85–93 now
vastly exceeded the primary response to the
M282–90 epitope in the lung compartment (53%
vs 2.4%).
The factors that potentially contribute to immunodominance hierarchy
include the binding of peptides to MHC class I molecules, the
efficiency of Ag processing, the precursor frequency of T cells capable
of responding to a given epitope, and antigenic competition in which
immune response to one determinant are suppressed by
simultaneous exposure to more potent determinants (24, 25). Using a peptide binding prediction algorithm
(http://bimas.dcrt.nih.gov/molbio/hla_bind/), the estimated scores
for half-time of dissociation from H-2Kd molecule
were 5760 for M282–90 sequence and 3456 for
F85–93 sequence, respectively. However, we found
that F85–93 peptide binds to
Kd molecule more efficiently than
M282–90 peptide in the
RMA-S-Kd stabilization assay (Fig. 1B). Therefore, it is unlikely that the relatively weak
CD8+ T cell response to
F85–93 in the lungs during primary response is
due to the differences in binding affinity with MHC class I molecules
between the F85–93 epitope and the
immunodominant M282–90 epitope.
A number of mechanisms could account for the relatively weak response
to F85–93 during primary infection including
lower abundance of F protein relative to M2 protein in virus infected
cells, inefficient processing (fragmentation) of the epitope from the
native protein, and/or inefficient transport by the TAP transporter
complex. It is unlikely that the weak response to
F85–93 detected during primary infection is due
to the failure of specific CTL precursors to expand in vivo because
on challenge infection of F-primed mice we observed a robust response
to the F85–93 epitope in the infected lungs. The
reduced primary response to the dominant M282–90
epitope during challenge infection (2.4% vs 50% in naive
animals) likely reflects competition between the expanded population of
F85–93-specific memory CTL precursors and the
M2-specific CD8+ primary T cell precursors for
the virus-infected APC. This phenomenon of antigenic competition
between epitopes has been well documented (26, 27). In the
current instance, the reduced primary M2-specific
CD8+ T cell response in F-primed animals
undergoing challenge infection is not at the level of processing and
presentation of the F85–93 and
M282–90 epitopes but most likely due to the
rapid elimination of virus-infected APC by the F-specific memory
effector CD8+ T cells. Because priming with the
RSV F glycoprotein also leads to the production of circulating
F-specific neutralizing Abs, the generation of virus-infected APC
necessary for the activation of memory and primary
CD8+ T cells may be limited by the effect of
neutralizing Abs as well as the early generation of F-specific memory
effectors. Although the mechanistic basis for the relatively weak
response to the RSV F protein by CD8+ T cells
remains to be determined, the ability to simultaneously detect several
distinct Ag-specific T cell populations during the course of RSV
infection in the lungs using tetramer staining should provide a
powerful tool for the investigation of the hierarchical relationships
established in vivo.
We also identified a mimotope, F352–360, that is
recognized by IFN--secreting CD8+ T cells
generated during the course of the in vivo response to the
F85–93 epitope. Although this mimotope contains
a well-conserved motif for H-2Ld binding, we were
unable to demonstrate Ld-restricted CTL
responding to infectious RSV, and the F352–360
epitope was unable to stabilize the expression of surface
Ld molecules. In support of the view that
F352–360 is a mimotope, this peptide exhibited a
low binding affinity to H-2Kd molecules in the
RMA-S stabilization assay with stabilization occurring only at high
nonphysiological concentrations. This requirement for high peptide
concentrations in Kd stabilization was paralleled
by the result obtained with peptide stimulation of cytokine synthesis
by lung-infiltrating CD8+ T cells in the
ICS/IFN- assay. To date, there have been few
H-2Kd-restricted epitopes that do not display the
characteristic Kd binding motif (reviewed in Ref.
18), although there have been reports of epitopes for
other class I molecules, including Kb
(29) and Kk (30), the
sequences of which do not conform to their corresponding described
motifs. The results reported here strongly suggest that
F352–360 is a mimotope and does not represent a
true CD8+ T cell epitope generated during RSV
infection.
Using a combination of tetramer staining and functional assays, we
demonstrated that F85–93-specific
CD8+ T cells infiltrating the lung were defective
in the expression of effector functions such as IFN- production and
cytolytic activity. This result is consistent with our recent
observation that RSV inhibits the expression of effector activity by
activated M2-specific CD8+ T cells infiltrating
the lungs during pulmonary RSV infection by interfering with
TCR-mediated signaling.5 As demonstrated here,
the suppressive effect of RSV on the development of
CD8+ T cell effector activity is observed only
with RSV-specific CD8+ T cells responding in the
lungs to RSV infection. Taken together, these results strongly suggest
that RSV has evolved a novel mechanism of suppression of the host
CD8+ T cell response during natural infection,
resulting in a selective defect in the terminal differentiation and/or
the expression of effector activity by virus-specific
CD8+ CTL at the site of infection. However, this
interpretation must be tempered by the fact that difference in both the
extent of virus replication in the lungs and the magnitude of the
inflammatory response to infection between RSV and type A influenza
could contribute to the apparent deficiency in the expression of
effector activity exhibited by RSV-specific CD8+
T cells.
In summary, we have used synthetic peptide screening and tetramer technology to identify and characterize the CD8+ T cell response to RSV F protein in BALB/c mice and to quantitate the magnitude of F-specific CD8+ T cell response during primary and challenge RSV infection. Although the primary CD8+ T cell response in the infected lungs to the F85–93 epitope is less than the response to the immunodominant M282–90 epitope derived from the RSV M2 protein, nonetheless the response to this F epitope is readily detectable during primary infection. The memory response to F85-93 substantially exceeds the primary response to the immunodominant M2 epitope and appears to inhibit the development of the primary response to the M282–90 epitope in the lungs. Although antigenic competition likely accounts for the relatively weak response to the M282–90 epitope during the accelerated and robust recall response to F85–93 in F-primed animals, the basis for the difference in the magnitude of the CD8+ T cell response to these two CTL epitopes in the lungs during primary RSV infection remains to be determined. We also found that like the CD8+ T cells responding to the immunodominant M282–90 epitope, F85–93-specific CD8+ T cells responding in the lungs to RSV infection exhibited a selective defect in the expression of ex vivo cytolytic activity and cytokine synthesis. A detailed characterization and analysis of CD8+ T cell epitopes and responsiveness both in experimental RSV infection and in the human should provide insight not only into the hierarchy of RSV-specific CD8+ T cell responses in vivo but also into the role of cellular immunity in controlling infection and in pathogenesis of RSV-induced pulmonary disease.
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2 Current address: Division of Pulmonary Medicine and Allergy, The Children’s Hospital Research Foundation, Room 4048, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039.
3 Address correspondence and reprint requests to Dr. Thomas J. Braciale, Beirne B. Carter Center for Immunology Research, Box 4012, MR4, University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail address: tjb2r{at}virginia.edu
4 Abbreviations used in this paper: RSV, respiratory syncytial virus; F, fusion; M2, matrix 2; ICS/IFN-, intracellular cytokine staining for IFN-; vv, vaccinia virus.
5 J. Chang and T. J. Braciale. Respiratory syncytial virus infection suppresses lung CD8+ T cell effector activity and peripheral CD8+ T cell memory in the respiratory tract. Submitted for publication.
Received for publication June 7, 2001. Accepted for publication August 14, 2001.
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