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Beirne B. Carter Center for Immunology Research and
Departments of Microbiology and Pathology, 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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genes and
predominantly express V14 TCR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Respiratory syncytial virus (RSV)3 is a ubiquitous human respiratory pathogen. Clinically apparent infection most frequently occurs in young children. It is estimated that 60% of infants are infected within their first year of life (4). RSV is the leading cause of viral lower respiratory tract infection worldwide, accounting for >90,000 hospitalizations in the United States each year (5). In addition to children, RSV infection is also an important cause of severe respiratory illness in elderly and immunocompromised individuals (6, 7, 8, 9, 10). Despite the significant health and economic burden produced by RSV, currently there is no safe and effective licensed RSV vaccine. Early RSV vaccine trials in human children using formalin-inactivated (FI)-RSV had to be halted because children receiving the FI-RSV vaccine displayed greater morbidity and mortality with subsequent natural RSV infection than control unvaccinated children (11, 12, 13, 14, 15). Histological examination of the lungs of the most severely affected children at autopsy revealed peribronchiolar infiltration and increased levels of eosinophils in the lungs and blood (12). This was one of the first demonstrations of immune-mediated injury as the result of vaccination in humans. Thus, before a new vaccine can be developed, a better understanding of the pathogenesis of the RSV vaccine-enhanced illness is critical.
The enhanced pathology exhibited by the children that received the FI-RSV vaccine can be simulated by priming BALB/c mice either with FI-RSV (16, 17, 18) or with a recombinant vaccinia virus (vv) expressing the G glycoprotein of RSV (vvG) (19) followed by intranasal inoculation with infectious RSV. In this latter model the vvG-primed mice develop pulmonary eosinophilia following intranasal RSV infection (19). This influx of eosinophils into the lung resembles the pathology found in the children from the 1960’s vaccine trials discussed above. Studies using the BALB/c model have suggested that the induction of memory CD4+ T cells producing Th2 cytokines as the result of vvG vaccination mediates enhanced disease (19, 20, 21). Transfer of RSV G-specific Th2 CD4+ T cell lines into naive recipients has been shown to induce lung eosinophilia following intranasal RSV infection (20). In addition, analysis of several RSV G protein frameshift mutants has revealed that a particular region spanning aa 193–205 of the G protein is required to induce pulmonary eosinophilia, a process that is dependent on CD4+ T cell recognition of G (22). Furthermore, a separate study established that immunization of BALB/c mice with a peptide derived from aa 184–198 of the G protein led to the development of pulmonary eosinophilia after intranasal infection with RSV (23). Finally, recent studies from our laboratory have demonstrated that both Th1 and Th2 CD4+ T cells are elicited to a single RSV G-derived peptide encompassing aa 183–197 (24). Since lung and blood eosinophilia were also seen in human FI-RSV vaccinees with augmented disease, it is likely that lung pathology induced by FI-RSV or vvG immunization results from priming of Th2 memory CD4+ T cells. Thus, these observations implicate RSV G-specific memory CD4+ T cells as likely mediators of the immunopathology observed in FI-RSV-vaccinated individuals following natural RSV infection.
Despite the importance of RSV G-specific memory
CD4+ T cells in mediating experimental RSV
vaccine-enhanced disease, very little quantitative information
currently exists concerning this population at the site of RSV
infection, the lung. In addition, the fine specificity of the RSV
G-specific memory CD4+ T cell population is
presently undefined. To address both of these important issues, we
sought to better characterize the RSV G protein-specific memory
CD4+ T cell response in terms of both its
magnitude and its fine specificity. Intracellular cytokine analysis of
lung-derived memory effector CD4+ T cells from
vvG-sensitized mice infected with RSV revealed that >40% of the
CD4+ T cells produced IFN-
or TNF-
following stimulation with a peptide spanning the immunodominant region
of the G protein of RSV. Using a series of analogue peptides truncated
at either the amino- or carboxyl-terminal end of the immunodominant
region of RSV G183–197, we demonstrate that
there is a single I-Ed-restricted
CD4+ T cell epitope with a core 9-aa sequence of
185–193 that is recognized by both Th1 and Th2 memory effector
CD4+ T cells. Strikingly, we also report that the
RSV G-specific memory effector CD4+ T cells that
have entered the lung following pulmonary RSV infection predominantly
express V14 TCR. These results demonstrate for the first time that
RSV G-specific effector CD4+ T cells are directed
to a single immunodominant I-Ed-restricted G
epitope and employ TCR with a highly conserved TCR V-chain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/cAnNTac (H-2d) mice were purchased from Taconic Farms (Germantown, NY) and used at 8–12 wk of age for all experiments. Mice were housed in a pathogen-free environment.
Viruses and infection of mice
Recombinant vv expressing the attachment (vvG) glycoprotein of
RSV was a gift from J. L. Beeler (Food and Drug Administration,
National Institutes of Health, Bethesda, MD). The generation and
characterization of this virus have been described previously
(25). Recombinant vv expressing -galactosidase
(vvB-gal) was used as a negative control. The A2 strain of RSV was a
gift from P. L. Collins (National Institute of Allergy and
Infectious Diseases, National Institutes of Health). RSV was grown in
HEp-2 cells (American Type Culture Collection, Manassas, VA). Groups of
four mice were infected with 3 x 106 PFU of
vvG or vvB-gal in a 10-µl volume by scarification with a 25-gauge
needle at the base of the tail. Three weeks after priming, mice were
lightly anesthetized with metaphane (Malinckrodt Veterinary, Mundelein,
IL) and intranasally inoculated with 2 x
106 PFU of RSV in 50 µl. At various times
postinfection (p.i.), control and infected mice were sacrificed by
cervical dislocation.
Peptides
The following peptides were used: hen egg lysozyme
(HEL)108–119 (WVAWRNRCKGTD) (26)
and mouse hepatitis virus N 267–275 (ILNKPRQKR) (27)
in addition to the RSV G protein-derived peptides shown in Tables II
and III. All peptides were synthesized by the University of Virginia
Biomolecular Research Facility using standard techniques of F-moc
chemistry.
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The lung vascular bed was flushed via the right ventricle with 4–5 ml of PBS containing 10 U/ml heparin (Sigma, St. Louis, MO) to remove any blood. The lungs were removed aseptically; carefully dissected away from the heart, the thymus, and the bronchial lymph nodes; and placed into RPMI medium (Life Technologies Laboratories, Grand Island, NY) supplemented with 10% FCS (Atlanta Biologicals, Norcross, GA), 10 U/ml penicillin G, 10 µg/ml streptomycin sulfate, 2 mM L-glutamine, 5 x 10-5 2-ME, 1 mM sodium pyruvate (Life Technologies), 0.1 mM nonessential amino acids (Life Technologies), and 10 mM HEPES (Life Technologies). The tissue was finely minced and pressed through a wire screen. Particulate matter was removed by quick centrifugation at 1000 rpm. Lung mononuclear cells were obtained by preparing single-cell suspensions from a pool of four mice per group. Cells were counted and resuspended at the given cell concentrations for the appropriate in vitro assay.
Flow cytometric analysis and intracellular cytokine staining
For multicolor FACS analysis, approximately 2 x 106 cells were stained with PE-conjugated anti-CD4 (clone RM4-5) and one of the following FITC-conjugated mAb: anti-LFA-1 (clone 2D7), anti-CD25 (clone 7D4), anti-CD44 (clone IM7), anti-CD62L (clone MEL-14), anti-CD69 (clone H1.2F3), rat IgM (clone R4-22), rat IgG2a (clone R35-95), rat IgG2b (clone A95-1), or hamster IgG (clone A19-3) in PBS supplemented with 2% FCS and 0.02% NaN3 (staining buffer). All mAb used in FACS staining were purchased from PharMingen (San Diego, CA). Stained cells were fixed and E lysed with FACS lysing solution (Becton Dickinson, San Jose, CA) washed, resuspended in staining buffer, and analyzed in two-color mode using a Becton Dickinson FACScalibur flow cytometer (Mountain View, CA).
For intracellular cytokine staining approximately 2 x
106 cells were stimulated for 5 h in the
presence of 1 µg/ml of brefeldin A (Sigma) in the presence (1 µM)
or the absence of one of the RSV G protein-derived peptides (or control
I-Ed-restricted peptide derived from HEL and/or
mouse hepatitis virus). In some experiments, an
anti-I-Ad (clone 34-5-3) or
anti-I-Ad/I-Ed (clone
M5/114.15.2) mAb was added to the culture for the duration of the
stimulation. The cells were subsequently washed in staining buffer,
blocked with purified anti-FcRII/III mAb (clone 2.4G2;
PharMingen), and stained with CD4-allophycocyanin. After fixation
with FACS lysing solution, the cells were washed in permeabilization
buffer (staining buffer containing 0.5% saponin; Sigma) and stained
with PE-conjugated anti-cytokine mAb or an isotype-matched control
mAb (all from PharMingen). The following mAb were used:
anti-IL-2 (clone JES6-5H4), anti-IL-3 (clone MP2-8F8),
anti-IL-4, (clone BVD4-1D11), anti-IL-5 (clone TRFK5),
anti-IL-6 (clone MP5-20F3), anti-IL-10 (clone JES5-16E3),
anti-GM-CSF (clone MP1-22E9), anti-IFN- (clone XMG1.2),
anti-TNF-, rat IgG1 (clone R3-34), rat IgG2a (clone R35-95), and
rat IgG2b (clone A95-1). Control cells known to express the cytokine of
interest were used in all experiments as positive controls (MiCK-1,
MiCK-2, and MiCK-3; PharMingen). Background staining with the
appropriate isotype-matched control mAb was subtracted from each
individual. Single-color controls were used in all multiparameter FACS
analyses for electronic compensation, and between 60,000 and 80,000
events were acquired from each preparation. Lymphocyte populations were
first gated based on forward scatter and 90° side scatter and then
analyzed using CellQuest software (Becton Dickinson).
Restimulation in vitro and cytokine ELISA
Cultures of 2 x 106 lung mononuclear
cells pooled from four mice per group were incubated in the presence or
the absence of one of the RSV G-derived peptides and incubated at
37°C in 1-ml total volume of RPMI-10% FCS supplemented as described
above in 24-well plates (Costar, Cambridge, MA). Supernatants were
harvested at 24, 48, and 72 h and stored at -80°C before being
assayed for cytokines by ELISA according to the manufacturer’s
instructions. Briefly, Immulon 2 plates (Dynatech, Chantilly, VA) were
coated overnight at 4°C with anti-cytokine mAb, washed the next
day with PBS-0.05% Tween-20 (Sigma), and blocked with PBS-10% FCS.
Samples were added at 50 µl/well, and a standard curve was
constructed for each plate by using eight 2-fold dilutions of
recombinant cytokine. Following the addition of the samples, the plates
were incubated overnight at 4°C before the addition of biotinylated
anti-cytokine mAb. Avidin-peroxidase (Sigma) followed by the
peroxidase substrate, 3,3',5,5'-tetramethyl-benzidine dihydrochloride
(Sigma) were used to develop the ELISA. The reaction was stopped with 2
N H2SO4. Plates were read
at 450 nm using an EL 340 plate reader and analyzed using DeltaSoft 3
software (both from Bio-Tek Instruments, Winooski, VT). The following
pairs of mAb were used: anti-IL-2, JES6-1A12 and biotinylated
JES6-5H4; anti-IL-4, 11B11 and biotinylated BVD6-24G2;
anti-IL-5, TRFK5 and biotinylated TRFK4; anti-IL-6, MP5-20F3
and biotinylated MP5-32C11; anti-IL-10, JES5-2A5 and biotinylated
SXC-1; anti-IFN-, R4-6A2 and biotinylated XMG1.2 (all from
PharMingen). Purified and biotinylated anti-IL-13 mAb were
purchased from R&D Systems (Minneapolis, MN). Recombinant murine IL-2,
IL-4, IL-5, IL-6, IL-10, IFN- (PharMingen), and IL-13 (R&D Systems)
were used as standards. The limit of detection was 31 pg/ml for
IL-2, 125 pg/ml for IL-4, 63 pg/ml for IL-5, 63 pg/ml for IL-6, 156
pg/ml for IL-10, 125 pg/ml for IL-13, and 312 pg/ml for IFN-.
Enzyme-linked immunospot assays
Nitrocellulose-based 96-well microtiter plates (Millititer HA,
Millipore, Bedford, MA) were coated overnight at 4°C with 5 µg/ml
of either anti-IL-5 (clone TRFK5, PharMingen) or anti-IFN-
(clone R4-6A2, PharMingen) mAb diluted in PBS, washed the next day
with PBS, and blocked with RPMI-10% FCS. After washing with PBS, lung
mononuclear cells pooled from four mice per group were added to the
wells (1 x 105 to 7.8 x
102 cells/well as responders with 2 x
106 peptide-pulsed irradiated (2000 rad)
splenocytes as stimulators) in RPMI 1640–10% FCS in a total volume of
200 µl/well. Following the addition of the samples, the plates were
incubated overnight at 37°C for 48 h, washed with PBS-0.05%
Tween-20 (Sigma) before the addition of biotinylated anti-cytokine
mAb (anti-IL-5 clone TRFK4 and anti-IFN- clone XMG1.2, both
from PharMingen), followed by avidin-peroxidase (1/400 dilution;
Sigma). Spots were visualized by developing with the substrate
3-amino-9-ethylcarbazole (Sigma). All assays were performed in
triplicate, and mean number of cytokine-secreting cells was calculated
from the triplicate assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Examination of activation marker expression on
CD4+ T cells infiltrating the lungs of vvG-primed
mice challenged with RSV revealed that most of the
CD4+ T cells in the lungs express a
LFA-1highCD44highCD62Llow
activated phenotype by days 5–6 p.i. (Fig. 1). Expression of the early activation
markers CD25 and CD69 peaked earlier on days 3–4 p.i. In contrast,
mice that had been vvG-primed but not infected with RSV did not have
CD4+ T cells expressing a highly activated
phenotype. Moreover, CD4+ T cells from control
mice (i.e., primed with vvB-gal) undergoing a primary RSV infection
exhibited a slightly delayed development of the activated phenotype on
day 5 p.i. compared with the accelerated memory response that
occurred by day 3 p.i. in vvG-primed mice. Thus, as expected, the
majority of the CD4+ T cells accumulating in the
lungs of vvG-primed mice in response to RSV infection express an
activated/memory cell phenotype.
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demonstrates that a high frequency of
cytokine-producing CD4+ T cells are elicited to
this single peptide. By day 5 p.i., >40% of the
CD4+ T cells made IFN- or TNF- following
peptide stimulation. The peak in the percentage of
CD4+IFN-+ cells was on
day 5 p.i. This was also the peak in the total number of
CD4+ IFN-+ cells in the
lung. The mean numbers of total
CD4+IFN-+ cells from
four mice per group were as follows: day 3, 2.1 x
105; day 4, 1.5 x
106; day 5, 7.4 x
106; day 6, 5.1 x
106; day 7, 3.5 x
106; and day 9, 1.9 x
106. We also observed a peak in the percentage of
CD4+ T cells capable of producing the cytokines
IL-3 and GM-CSF on day 5 p.i. In addition, by day 6 p.i.,
>20% of the lung CD4+ T cells produced another
Th1-associated cytokine, IL-2 following peptide stimulation. We were
unable to detect CD4+ T cells secreting the
Th2-associated cytokines IL-4 and IL-5 in response to the
G183–197 peptide by intracellular cytokine
staining (data not shown). However, we were able to detect a slight
(<1.5%) increase in the percentage of IL-10-producing
CD4+ T cells. Interestingly, we did observe a
more significant 10–15% increase in the percentage of IL-6-producing
CD4+ T cells that was not dependent on peptide
stimulation (data not shown). The percentage of cytokine-secreting
CD4+ T cells in response to peptide was low
(<3%) in vvG-primed mice without a challenge infection with RSV or in
mice undergoing a primary RSV infection (vvB-gal-primed, RSV day 5
p.i.). Thus, these results demonstrate that RSV infection of vvG-primed
mice results in a substantial influx of memory effector
CD4+ T cells into the lung that predominantly
express an activated cell phenotype and that as many as 40% are
capable of producing the effector cytokines IFN- and/or TNF-
following stimulation with the G183–197
peptide.
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The above experiments demonstrate the presence of memory effector
CD4+ T cells at a high frequency in the lungs of
vvG-primed mice after intranasal RSV challenge using intracellular
cytokine staining to detect IFN- production following stimulation
with a peptide representing aa 183–197 of the G glycoprotein of RSV.
Therefore, we chose to use this sensitive single-cell assay to more
finely map the CD4+ T cell epitope(s) contained
within the 183–197 region of RSV G. To verify that we were using an
optimal peptide concentration to stimulate cytokine production by
lung-derived effector CD4+ T cells in the
intracellular IFN- assay, we performed a peptide dose-response curve
using the RSV G183–197 peptide. Fig. 2 shows that the maximal percentage of
IFN--producing CD4+ T cells is elicited at a
peptide concentration of 1 µM and higher. In contrast, no increase in
the percentage of IFN--producing CD4+ T cells
was observed following stimulation with a control
I-Ed binding peptide derived from HEL.
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), and tested for their capacity to
stimulate the production of IFN- by lung-derived memory effector
CD4+ T cells from vvG-primed mice 5 days after
intranasal challenge infection with RSV. Fig. 3 demonstrates that peptides with
successive aa deletions from the N-terminus are stimulatory until the
isoleucine at aa position 185 is deleted. Consecutive deletions from
the C-terminus revealed that loss of the lysine at position 193 ablated
the ability to stimulate IFN- production by these memory effector
CD4+ T cells. Fig. 3 also demonstrates that T
cell responsiveness was not critically dependent on peptide length,
since both the nonstimulatory peptide 183–192 and the stimulatory
184–193 peptide are 10 aa residues in length. These results suggest
that there is a single CD4+ T cell epitope within
RSV G requiring a minimal 9-aa core sequence spanning residues 185–193
(see underlined amino acids in Table II). Furthermore, to obtain a
significant response, at least two additional amino acids at the N- and
C-termini of the core 9 aa sequence are needed (Fig. 3, see peptide
183–195). However, since we could not detect Th2 cytokine production
using the intracellular cytokine stain following peptide stimulation of
lung effector CD4+ T cells ex vivo, we used the
enzyme-linked immunospot assay to determine the relative frequency of
IFN-- and IL-5-secreting cells specific to the RSV
G183–195 peptide. The mean numbers of
cytokine-secreting cells per million lung mononuclear cells from a
representative experiment with a pool of four mice per group were as
follows: IFN-, 9280; and IL-5, 590. This suggests that there is
approximately a 15-fold higher frequency of cells capable of making
IFN- than IL-5 in response to the RSV
G183–195 peptide.
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assay, cell culture supernatants of
peptide-stimulated lung mononuclear cells isolated 5 days after
intranasal RSV challenge of vvG-primed mice were assayed for cytokines
by ELISA. As Fig. 4 demonstrates, using
the same series of truncated peptides as that used in the intracellular
cytokine assay discussed above, that production of the Th1-associated
cytokine IFN- maps to the same 9-aa core epitope defined by aa
185–193. Moreover, production of the Th2-associated cytokines IL-5 and
IL-6 also maps to peptides containing this same core sequence (Fig. 4).
Interestingly, another Th2 cytokine, IL-13, appears to map to this same
epitope, but in vitro production of IL-13 was more dependent on peptide
length. Similar results were obtained with IL-2 and IL-10 as well (data
not shown). No IL-4 production was detected following short term
stimulation of lung effector T cells on day 5 post-RSV infection (data
not shown). Taken together, these results demonstrate that both Th1 and
Th2 RSV-specific memory effector CD4+ T cells
recognize a single immunodominant epitope in the G protein of RSV with
a 9-aa core sequence of 185–193. To insure that the single epitope
defined above was indeed restricted to I-Ed, we
incubated CD4+ T cells with the 183–197 peptide
in the presence of mAb specific either for I-Ad
or for both I-Ad/I-Ed. Fig. 5 shows that IFN- production by
CD4+ T cells is inhibited by mAb that recognize
both I-Ad and I-Ed, but not
I-Ad alone, demonstrating that
I-Ed is the restricting MHC molecule for memory
effector CD4+ T cells responding to the G epitope
containing the 185–193 aa core sequence.
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The above results suggest that there is a single
I-Ed-restricted CD4+ T cell
epitope within RSV G183–197 with a 9-aa core
sequence of 185–193. To further analyze the specificity of the
RSV-specific memory effector CD4+ T cells, 12
variant peptides were synthesized in which individual residues within
the optimal 183–195 sequence (see Fig. 3) were replaced by alanine to
determine the amino acids critical for T cell stimulation (Table III). These alanine-substituted peptides
were used to stimulate IFN- production by RSV G-specific memory
effector lung CD4+ T cells using the
intracellular IFN- assay. Fig. 6 shows
that the substitution of alanine for isoleucine at position 185 or
arginine at position 188 greatly abolished T cell recognition and
IFN- production, consistent with the predicted roles of these two aa
as I-Ed anchor residues. Alanine substitution at
the two other predicted I-Ed anchor residues
(i.e., aa 190 and 193) also results in significant inhibition of
IFN- production. However, of particular note was the finding that
alanine substitution at position 187, 189, 191, or 192 dramatically
diminished T cell recognition and IFN- production. This result was
striking, since these residues probably represent TCR contact residues.
While substitution at one of several different TCR contact residues
might be expected to affect the recognition of a single
CD4+ T cell clone and its progeny, a
heterogeneous population of effector CD4+ T cells
derived from different clones would be expected to employ a diverse
array of TCR with differing sensitivities to substitution at TCR
contact residues and therefore might not be expected to be similarly
affected by substitution in individual TCR contact residues. For this
reason it is noteworthy that substitution at positions outside the
minimal core sequence (i.e., position 183, 194, or 195) did not
significantly alter recognition by the memory effector
CD4+ T cells, although in some instances the
composition of a residue flanking the core epitope has been reported to
affect CD4+ T cell recognition
(29, 30, 31). Similar results were observed using ELISA of
cell culture supernatants for the Th2 cytokines IL-5, IL-6, IL-10, and
IL-13 following in vitro stimulation with the same panel of
alanine-substituted peptides (data not shown).
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repertoire of RSV G peptide-specific memory effector
CD4+ T cells
Although residues 187, 189, 191, and 192 could represent secondary
MHC anchors contributing to the stability of the peptide/MHC complex,
the finding that a high proportion of the memory effector
CD4+ T cells was equally sensitive to alanine
substitutions at G185–193 core residues other
than the four predicted I-Ed anchor sites
suggested that these substitutions may be disrupting potential TCR
contact residues necessary for the response of an oligoclonal T cell
population. To examine this possibility, we challenged vvG-primed mice
with RSV and stained lung-derived effector CD4+ T
cells following stimulation with the RSV
G183–197 peptide for intracellular IFN- and
TCR V usage using a panel of V-specific mAb. The TCR V
repertoires of the IFN-+ and
IFN-- CD4+ T cell
populations are shown in Fig. 7.
Surprisingly, among the activated CD4+ T cells
infiltrating the lungs of vvG-primed, RSV-infected mice, both the
IFN-+ and IFN--
subsets of CD4+ T cells are highly enriched for
V14+ cells. Since we have demonstrated above
that both Th1 and Th2 effector CD4+ T cells are
elicited to the same I-Ed-restricted epitope, it
is likely that the
IFN--V14+ population
reflects Th2 CD4+ T cells that we are unable to
detect in the intracellular cytokine assay. Thus, RSV infection of
vvG-primed BALB/c mice results in the generation and recruitment of a
RSV-specific CD4+ T cell population that
predominantly expresses a V14 TCR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Second, both Th1 and Th2 memory effector CD4+ T
cells are directed against the same immunodominant RSV G epitope.
Third, the RSV G-specific memory effector CD4+ T
cells express a highly restricted V TCR repertoire.
The study of virus-specific T cell populations has been greatly aided by recent technological advances, including the development of tetrameric complexes of MHC glycoprotein loaded with peptides and the staining for intracellular cytokines in cells stimulated with peptides (34, 35), both of which have allowed the direct visualization and quantitation of Ag-specific T cell populations (36). For the most part, these techniques have been used to analyze MHC class I-restricted CD8+ T cells in several different viral and tumor model systems (34, 35, 37, 38, 39, 40, 41, 42, 43, 44). These studies have all shown that the magnitude of the Ag-specific CD8+ T cell response is greater than previously recognized. Interestingly, recent results from our laboratory using the influenza model suggest that the use of tetramers to quantitate the frequency of virus-specific CD8+ T cell populations may still result in an underestimate of the total virus-specific CD8+ T cell frequency (45). Nevertheless, the advent of the tetramer technology and the intracellular cytokine stain have led to a better understanding of the kinetics, phenotype, and frequency of virus-specific CD8+ T cell populations.
In contrast to the great number of studies examining
CD8+ T cell responses using these new techniques,
much less is known about virus-specific CD4+ T
cells. For technical reasons, it is far easier to create stable
peptide-loaded MHC class I tetramers than stable MHC class II
tetramers. However, in most of the systems studied to date, there is a
good correlation between the results obtained with tetramers and
intracellular IFN- staining (35, 36). Thus, the
intracellular cytokine assay probably provides a viable alternative for
quantitating virus-specific CD4+ T cell
responses. As was the case with virus-specific
CD8+ T cells (35, 46), recent work
performed in the lymphocytic choriomeningitis virus system has
demonstrated that the virus-specific CD4+ T cell
frequency is much greater than previously thought (47). In
the murine RSV model, there is little information on the frequency of
cytokine-secreting virus-specific CD4+ T cells
during either an acute RSV infection or a memory response in RSV
Ag-primed mice undergoing challenge infection with RSV. One study
examined the frequency of MHC class II-restricted CTL by limiting
dilution analysis following acute RSV infection, reporting frequencies
of <1% for virus-specific T cells (48). Here we show
that by 5 days after RSV infection of vvG-primed BALB/c mice, >40% of
the CD4+ T cells in the lung are specific to a
single immunodominant RSV G epitope. This frequency is probably a
significant underestimate of the total frequency of RSV G
protein-specific CD4+ T cells due to our
inability to currently detect peptide-specific
CD4+ T cells secreting Th2 cytokines such as IL-4
and IL-5 using the intracellular cytokine assay. Interestingly, others
have recently failed to detect IL-4 and/or IL-5 in LCMV
peptide-specific CD4+ T cells
(49, 50, 51, 52). It is currently unclear why no IL-4- or
IL-5-secreting CD4+ T cells can be detected using
the intracellular cytokine stain, since during murine RSV infection the
production of these cytokines can be readily detected in cell culture
supernatants following in vitro stimulation with RSV-derived peptides
(21) (Fig. 4). It may be that the production of the Th2
cytokines is dependent on the cell cycle status of the
CD4+ T cells as has been suggested by several
recent studies (53, 54). Future studies comparing the
frequency of RSV G peptide-specific CD4+ T cells
using the intracellular IFN- assay and MHC class II tetramers loaded
with the RSV G immunodominant peptide defined in this report should
help resolve this important issue.
Crystal structures of peptide/MHC complexes, elution of peptides
associated with MHC class II molecules, and peptide binding assays have
revealed that MHC anchor residues are usually located at positions 1,
4, 6, and 9 of the core 9 aa that often define
CD4+ T cell epitopes (55, 56, 57, 58, 59, 60). By
analyzing the sequences of peptides known to bind
I-Ed molecules, Rammensee et al. were able to
define an I-Ed peptide motif with the following
preferred aa: W, Y, F, I, L, or V at anchor position 1; K, R, or I at
anchor position 4; I, L, V, or G at anchor position 6; and K or R at
anchor position 9 (28). Examination of the aa sequence of
RSV G183–197 revealed three potential
CD4+ T cell epitopes (bold indicates the
positions of putative I-Ed anchor residues):
184–192 (AICKRIPNK),
185–193 (ICKRIPNKK),
and 189–197 (IPNKKPGKK),
all of which closely fit the MHC class II I-Ed
binding motif. Since these potential epitopes overlap each other, we
chose to use the sensitive intracellular cytokine assay to define the
N- and C-terminal residues important for recognition of this region by
memory effector CD4+ T cells obtained from the
lung following a natural RSV infection. Functional screening of a panel
of truncated synthetic peptides identified the N- and C-terminal
boundaries as aa 185–193, indicating that a single epitope was
recognized by RSV G-specific CD4+ T cells capable
of secreting Th1 cytokines. We confirmed that Th2 memory effector
CD4+ T cells present in the lungs were directed
to this single 9-aa core epitope by ELISA analysis of Th2 cytokines
secreted into the supernatant of lung memory effector
CD4+ T cells stimulated in short term culture
with the same series of peptides as that used above in the
intracellular IFN- assays. To our knowledge, this is the first
report using intracellular cytokine staining to define an
immunodominant CD4+ T cell epitope recognized by
effector CD4+ T cells taken directly from a site
of infection. Thus, our results demonstrate the utility of this
approach in such epitope-mapping studies.
A series of independent observations by several groups has suggested
that there are one or more CD4+ T cell epitopes
present in the RSV G glycoprotein located somewhere between residues
183 and 205. Using frameshift mutant G proteins expressed in
recombinant vv to sensitize mice, Sparer et al. showed that the region
of the G protein between residues 193 and 203 was necessary to induce
eosinophilia and weight loss in RSV-infected mice (22).
Since eosinophilia is characteristic of a Th2 response (20, 21, 61), these results suggested that a Th2 epitope exists within
this region. In contrast, the enhanced weight loss that occurs in
vvG-primed mice challenged with RSV is most likely mediated by TNF-
produced by Th1 cells. Thus, the results of Sparer et al. suggest that
both a Th1 and a Th2 CD4+ T cell epitope were
disrupted by their frameshift mutant analyses. A separate study
performed by Tebbey et al. demonstrated that mice primed with a peptide
derived from aa 184–198 of RSV G in adjuvant develop eosinophilia
following RSV infection (23). More recently, our
laboratory has shown that both Th1 and Th2 CD4+ T
cells are directed against a single region spanning aa 183–197 of the
RSV G glycoprotein (24). Taken together, all these
observations pointed to one or more immunodominant
CD4+ T cell epitope(s) within aa 183–197 of RSV
G. The 9-aa core epitope we have defined here agrees with these
previous studies, since this epitope falls within the 184–198 peptide
used by Tebbey et al. (23) as well as being within the
region of the G protein (i.e., 193–205) disrupted by the studies of
Sparer et al. (22). Our results directly demonstrate that
a single region of the RSV G protein containing the 185–193 core
sequence accounts for the Th1 and Th2 cytokine response in vvG-primed
mice undergoing challenge RSV infection.
In the present study we demonstrate that most of the RSV G
peptide-specific CD4+ T cells that can be
detected using the intracellular IFN- assay express V14 TCR. In
addition, we show that approximately 30% of the activated
CD4+ T cells that fail to make IFN- following
peptide stimulation also express V14 TCR. Many of these
IFN-- cells may represent virus-specific Th2
CD4+ T cells that are not easily detected using
the intracellular cytokine stain. This seems likely, since we have
shown here that Th2 cytokines are produced in short term in vitro
cultures using the CD4+ epitope we have defined
in this report (see Fig. 4). In addition, in vitro activation of
lung-derived CD4+ T cells isolated from
vvG-primed mice with a V14 TCR-specific mAb induces the production
of both Th1 and Th2 cytokines (S. M. Varga and T. J.
Braciale, unpublished observations). Thus, it is possible that
CD4+ T cells expressing a V14 TCR directed
against the immunodominant epitope we have defined in this study may
differentiate into either Th1 or Th2 CD4+ T cells
in vivo. A previous study examining CD4+ T cells
specific to an I-Ed-restricted nucleoprotein
epitope of mouse hepatitis virus demonstrated that a single epitope
could elicit both Th1 and Th2 CD4+ T cells
(27). However, the mouse hepatitis virus
nucleoprotein-specific Th1 and Th2 CD4+ T cells
expressed TCR with distinctly different V gene usage
(27). At present we do not know whether the restricted
V usage of the G185–193 specific
CD4+ T cells reflects the selection of
V14+ T cells with a unique TCR -chain,
i.e., a single unique complementarity-determining region 3 or TCR
-chains with limited complementarity-determining region 3 length and
sequence diversity. Also, since TCR V usage by the memory effector
CD4+ T cells has not been analyzed as yet, we
cannot determine whether these responding lung effector
CD4+ T cells represent a single clone or a panel
of memory CD4+ T cell clones with limited TCR
diversity. Experiments are in progress to address each of these
issues.
A great deal of evidence indicates that MHC class II-restricted
CD4+ T cells play an important, yet often
unappreciated role in antiviral immunity (2, 62, 63, 64, 65, 66, 67, 68, 69).
During RSV infection, CD4+ T cells can contribute
to the antiviral immune response by providing help for B cells to make
antiviral Abs as well as secreting cytokines such as IFN-. However,
during a RSV memory response, G protein-specific
CD4+ T cell responses can also be detrimental to
the host by mediating immunopathology and enhancing disease (32, 33, 70). In this report we show that G protein-specific memory
CD4+ T cells are directed against a single
I-Ed-restricted immunodominant epitope. This
epitope elicits a memory response yielding both Th1 and Th2 effector
CD4+ T cells. While we cannot formally exclude
the possibility that the same effector CD4+ T
cell in the lungs is producing both Th1 and Th2 cytokines, our evidence
to date supports the view that Th1 and Th2 cytokines are produced by
different effector CD4+ T cells. Both these
effector cell types may contribute to the enhanced pathology observed
in immune individuals. In addition, we have demonstrated for the first
time that the majority of RSV G protein-specific
CD4+ T cells express a restricted V14 TCR.
Such a focused T cell response eliciting both Th1 and Th2 effector
cells and capable of mediating enhanced disease could be related to the
finding that only a subset of the children who received the FI-RSV
vaccine in the 1960s went on to exhibit enhanced disease upon RSV
infection (11, 12, 13, 14, 15). Our future studies in the murine RSV
model will examine whether the CD4+ T cells that
exclusively express V14 TCR are capable of mediating RSV
vaccine-enhanced disease. Our results suggest that a comparable
analysis of CD4+ T cell specificity, effector
activity, and TCR usage may be warranted in the human.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas J. Braciale, Beirne B. Carter Center for Immunology Research, MR-4 Building, 400 Lane Road, Room 4012, University of Virginia, Charlottesville, VA 22908.
3 Abbreviations used in this paper: RSV, respiratory syncytial virus; FI, formalin-inactivated; vv, vaccinia virus; vvG, vv expressing the G glycoprotein of RSV; vvB-gal, recombinant vv expressing -galactosidase; p.i., postinfection; HEL, hen egg lysozyme.
Received for publication June 26, 2000. Accepted for publication September 6, 2000.
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and IL-4 responses to viral infections: requirements for IL-2. J. Immunol. 160:5007. is essential for destruction of cells and development of insulin-dependent diabetes mellitus. J. Exp. Med. 185:531.This article has been cited by other articles:
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|
|
|
|
 |
|
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|
|
|
|
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|
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|
|
|
|
|
|
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|
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|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
E. L. Wissinger, W. W. Stevens, S. M. Varga, and T. J. Braciale Proliferative Expansion and Acquisition of Effector Activity by Memory CD4+ T Cells in the Lungs following Pulmonary Virus Infection J. Immunol., March 1, 2008; 180(5): 2957 - 2966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Castilow, D. K. Meyerholz, and S. M. Varga IL-13 Is Required for Eosinophil Entry into the Lung during Respiratory Syncytial Virus Vaccine-Enhanced Disease J. Immunol., February 15, 2008; 180(4): 2376 - 2384. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Olson and S. M. Varga CD8 T Cells Inhibit Respiratory Syncytial Virus (RSV) Vaccine-Enhanced Disease J. Immunol., October 15, 2007; 179(8): 5415 - 5424. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Monick, L. S. Powers, I. Hassan, D. Groskreutz, T. O. Yarovinsky, C. W. Barrett, E. M. Castilow, D. Tifrea, S. M. Varga, and G. W. Hunninghake Respiratory Syncytial Virus Synergizes with Th2 Cytokines to Induce Optimal Levels of TARC/CCL17 J. Immunol., August 1, 2007; 179(3): 1648 - 1658. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Lee, S. A. Miller, D. W. Wright, M. T. Rock, and J. E. Crowe Jr. Tissue-Specific Regulation of CD8+ T-Lymphocyte Immunodominance in Respiratory Syncytial Virus Infection J. Virol., March 1, 2007; 81(5): 2349 - 2358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Alderson, P. McGowan, J. R. Baldridge, and P. Probst TLR4 agonists as immunomodulatory agents Innate Immunity, October 1, 2006; 12(5): 313 - 319. [Abstract] [PDF]
|
|
|
|
 |
|
 N. W. Lukacs, M. L. Moore, B. D. Rudd, A. A. Berlin, R. D. Collins, S. J. Olson, S. B. Ho, and R. S. Peebles Jr Differential Immune Responses and Pulmonary Pathophysiology Are Induced by Two Different Strains of Respiratory Syncytial Virus Am. J. Pathol., September 1, 2006; 169(3): 977 - 986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Anghelina, L. Pewe, and S. Perlman Pathogenic Role for Virus-Specific CD4 T Cells in Mice with Coronavirus-Induced Acute Encephalitis Am. J. Pathol., July 1, 2006; 169(1): 209 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bukreyev, M. E. Serra, F. R. Laham, G. A. Melendi, S. R. Kleeberger, P. L. Collins, and F. P. Polack The Cysteine-Rich Region and Secreted Form of the Attachment G Glycoprotein of Respiratory Syncytial Virus Enhance the Cytotoxic T-Lymphocyte Response despite Lacking Major Histocompatibility Complex Class I-Restricted Epitopes. J. Virol., June 1, 2006; 80(12): 5854 - 5861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Martinez-Sobrido, N. Gitiban, A. Fernandez-Sesma, J. Cros, S. E. Mertz, N. A. Jewell, S. Hammond, E. Flano, R. K. Durbin, A. Garcia-Sastre, et al. Protection against Respiratory Syncytial Virus by a Recombinant Newcastle Disease Virus Vector J. Virol., February 1, 2006; 80(3): 1130 - 1139. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Harcourt, R. Alvarez, L. P. Jones, C. Henderson, L. J. Anderson, and R. A. Tripp Respiratory Syncytial Virus G Protein and G Protein CX3C Motif Adversely Affect CX3CR1+ T Cell Responses J. Immunol., February 1, 2006; 176(3): 1600 - 1608. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Haring, V. P. Badovinac, M. R. Olson, S. M. Varga, and J. T. Harty In Vivo Generation of Pathogen-Specific Th1 Cells in the Absence of the IFN-{gamma} Receptor J. Immunol., September 1, 2005; 175(5): 3117 - 3122. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. J. Braciale Respiratory Syncytial Virus and T Cells: Interplay between the Virus and the Host Adaptive Immune System Proceedings of the ATS, August 1, 2005; 2(2): 141 - 146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Huang and R. Anderson Modulation of Protective Immunity, Eosinophilia, and Cytokine Responses by Selective Mutagenesis of a Recombinant G Protein Vaccine against Respiratory Syncytial Virus J. Virol., April 1, 2005; 79(7): 4527 - 4532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Dakhama, J.-W. Park, C. Taube, K. Chayama, A. Balhorn, A. Joetham, X.-D. Wei, R.-H. Fan, C. Swasey, N. Miyahara, et al. The Role of Virus-specific Immunoglobulin E in Airway Hyperresponsiveness Am. J. Respir. Crit. Care Med., November 1, 2004; 170(9): 952 - 959. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Elliott, K. S. Pryharski, Q. Yu, L. A. Boutilier, N. Campeol, K. Melville, T. S. Laughlin, C. K. Gupta, R. A. Lerch, V. B. Randolph, et al. Characterization of Recombinant Respiratory Syncytial Viruses with the Region Responsible for Type 2 T-Cell Responses and Pulmonary Eosinophilia Deleted from the Attachment (G) Protein J. Virol., August 15, 2004; 78(16): 8446 - 8454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Johnson, S. M. Varga, T. J. Braciale, and B. S. Graham V{beta}14+ T Cells Mediate the Vaccine-Enhanced Disease Induced by Immunization with Respiratory Syncytial Virus (RSV) G Glycoprotein but Not with Formalin-Inactivated RSV J. Virol., August 15, 2004; 78(16): 8753 - 8760. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Elliott, K. S. Pryharski, Q. Yu, C. L. Parks, T. S. Laughlin, C. K. Gupta, R. A. Lerch, V. B. Randolph, N. A. LaPierre, K. M. H. Dack, et al. Recombinant Respiratory Syncytial Viruses Lacking the C-Terminal Third of the Attachment (G) Protein Are Immunogenic and Attenuated In Vivo and In Vitro J. Virol., June 1, 2004; 78(11): 5773 - 5783. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Johnson, M. N. Teng, P. L. Collins, and B. S. Graham Respiratory Syncytial Virus (RSV) G Glycoprotein Is Not Necessary for Vaccine-Enhanced Disease Induced by Immunization with Formalin-Inactivated RSV J. Virol., June 1, 2004; 78(11): 6024 - 6032. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. de Waal, S. Yuksel, A. H. Brandenburg, J. P. M. Langedijk, K. Sintnicolaas, G. M. G. M. Verjans, A. D. M. E. Osterhaus, and R. L. de Swart Identification of a Common HLA-DP4-Restricted T-Cell Epitope in the Conserved Region of the Respiratory Syncytial Virus G Protein J. Virol., February 15, 2004; 78(4): 1775 - 1781. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Johnson, R. A. Parker, J. E. Johnson, and B. S. Graham IL-13 Is Sufficient for Respiratory Syncytial Virus G Glycoprotein-Induced Eosinophilia After Respiratory Syncytial Virus Challenge J. Immunol., February 15, 2003; 170(4): 2037 - 2045. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. van Bleek, M. C. Poelen, R. van der Most, H. F. Brugghe, H. A. M. Timmermans, C. J. Boog, P. Hoogerhout, H. G. Otten, and C. A. C. M. van Els Identification of Immunodominant Epitopes Derived from the Respiratory Syncytial Virus Fusion Protein That Are Recognized by Human CD4 T Cells J. Virol., December 20, 2002; 77(2): 980 - 988. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Plotnicky-Gilquin, D. Cyblat-Chanal, J.-P. Aubry, T. Champion, A. Beck, T. Nguyen, J.-Y. Bonnefoy, and N. Corvaia Gamma Interferon-Dependent Protection of the Mouse Upper Respiratory Tract following Parenteral Immunization with a Respiratory Syncytial Virus G Protein Fragment J. Virol., September 11, 2002; 76(20): 10203 - 10210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Durbin, T. R. Johnson, R. K. Durbin, S. E. Mertz, R. A. Morotti, R. S. Peebles, and B. S. Graham The Role of IFN in Respiratory Syncytial Virus Pathogenesis J. Immunol., March 15, 2002; 168(6): 2944 - 2952. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 T. R. Johnson, J. E. Fischer, and B. S. Graham Construction and characterization of recombinant vaccinia viruses co-expressing a respiratory syncytial virus protein and a cytokine J. Gen. Virol., September 1, 2001; 82(9): 2107 - 2116. [Abstract] [Full Text] [PDF]
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