JOURNAL OF VIROLOGY, May 2009, p. 4934–4941
Vol. 83, No. 10
Characterization of Respiratory Syncytial Virus M- and M2-Specific
CD4 T Cells in a Murine Model?
Jie Liu, Tracy J. Ruckwardt, Man Chen, Teresa R. Johnson, and Barney S. Graham*
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Received 10 October 2008/Accepted 24 February 2009
CD4 T cells have been shown to play an important role in the immunity and immunopathogenesis of
respiratory syncytial virus (RSV) infection. We identified two novel CD4 T-cell epitopes in the RSV M and M2
proteins with core sequences M213-223(FKYIKPQSQFI) and M227-37(YFEWPPHALLV). Peptides containing
the epitopes stimulated RSV-specific CD4 T cells to produce gamma interferon (IFN-?), interleukin 2 (IL-2),
and other Th1- and Th2-type cytokines in an I-Ab-restricted pattern. Construction of fluorochrome-conjugated
peptide–I-Abclass II tetramers revealed RSV M- and M2-specific CD4 T-cell responses in RSV-infected mice
in a hierarchical pattern. Peptide-activated CD4 T cells from lungs were more activated and differentiated, and
had greater IFN-? expression, than CD4 T cells from the spleen, which, in contrast, produced greater levels of
IL-2. In addition, M209-223peptide-activated CD4 T cells reduced IFN-? and IL-2 production in M- and
M2-specific CD8 T-cell responses to Db-M187-195and Kd-M282-90peptides more than M225-39peptide-stimu-
lated CD4 T cells. This correlated with the fact that I-Ab–M209-223tetramer-positive cells responding to primary
RSV infection had a much higher frequency of FoxP3 expression than I-Ab–M226-39tetramer-positive CD4 T
cells, suggesting that the M-specific CD4 T-cell response has greater regulatory function. Characterization of
epitope-specific CD4 T cells by novel fluorochrome-conjugated peptide–I-Abtetramers allows detailed analysis
of their roles in RSV pathogenesis and immunity.
CD4 T lymphocytes play an important role in the resolution
of primary viral infections and the prevention of reinfection by
regulating a variety of humoral and cellular immune responses.
CD4 T cells provide cytokines and other molecules to support
the differentiation and expansion of antigen-specific CD8 T
cells, which are major effectors for both virus clearance and
immunopathology during primary infection with respiratory
syncytial virus (RSV) (3, 17, 42, 43). CD4 T-cell help is man-
datory for an effective B-cell response (14), which is necessary
for producing neutralizing antibodies that prevent secondary
RSV infection (12, 18, 21). A concurrent CD4 T-cell response
also promotes the maintenance of CD8 T-cell surveillance and
effector capacity (9). Previous studies have shown that inter-
leukin 2 (IL-2) from CD4 T cells can restore CD8 T-cell
function in lungs (10) and that IL-2 supplementation can in-
crease the production of gamma interferon (IFN-?) by CD8 T
cells upon peptide stimulation in vitro (45).
While CD4 T cells are important for providing support to
host immunity, they have also been associated with immuno-
pathogenesis by playing a key role in the Th2-biased T-cell
response (34, 46), which may be the common mechanism of
enhanced lung pathology and other disease syndromes shown
in murine studies (2, 16, 17, 19, 35). Earlier studies showed the
positive association of formalin-inactivated RSV (FI-RSV) im-
munization-mediated enhanced illness upon subsequent natu-
ral RSV infection with a Th2-biased CD4 T-cell response (19,
44). Th2-orientated CD4 T cells elicit severe pneumonia with
extensive eosinophilic infiltrates in the lungs of FI-RSV-immu-
nized mice (13, 24, 48). Patients with severe RSV disease
showed an elevated Th2/Th1 cytokine ratio in nasal secretions
and peripheral blood mononuclear cells (27, 29, 31, 38). In-
creased disease severity has also been associated with polymor-
phisms in Th2-related cytokine genes, such as the IL-4, IL-4
receptor, and IL-13 genes (11, 23, 36). Th2 cytokines from
CD4 T cells can also diminish the CD8 T-cell response and
delay viral clearance (4, 8).
The evaluation of CD4 T-cell responses in viral infection is
particularly relevant in the RSV model because of the associ-
ation of RSV and allergic inflammation, which is largely me-
diated by CD4 T cells. Understanding the influence of CD4 T
cells on CD8 T-cell responses and other immunological effec-
tor mechanisms is central to understanding RSV pathogenesis
and developing preventive vaccine strategies for RSV. Our lab
and others have demonstrated that CD8 T cells target RSV
M and M2 proteins with cytolytic effector activities (28, 30, 39).
In this study, we found that both RSV M and M2 proteins also
contain CD4 T-cell epitopes. These epitopes have 11-mer
amino acid core sequences and are associated with the major
histocompatibility complex (MHC) class II molecule I-Ab. Flu-
complexes can identify RSV M- and M2-specific CD4 T cells
from CB6F1 mice following RSV infection in a hierarchical
pattern. Peptides containing the epitopes can stimulate CD4 T
cells from RSV M or M2 DNA-immunized and virus-chal-
lenged mice and can lead to the production of IFN-?, IL-2, and
other Th1- and Th2-type cytokines that can modulate the CD8
T-cell response to RSV M and M2. We also found that CD4 T
cells from the lungs and spleens of immunized mice have
different phenotype and cytokine profiles upon in vitro stimu-
lation. These observations suggest a regulatory role for CD4 T
cells in the host response to RSV infection. The development
* Corresponding author. Mailing address: Vaccine Research Center,
NIAID, National Institutes of Health, 40 Convent Dr., Bethesda, MD
20892-3017. Phone: (301) 594-8468. Fax: (301) 480-2771. E-mail:
?Published ahead of print on 4 March 2009.
of novel MHC class II tetramer reagents allows the character-
ization of epitope-specific CD4 T-cell responses to RSV and
will enable the investigation of basic mechanisms by which
CD4 T cells affect pathogenesis and immunity to viral infec-
MATERIALS AND METHODS
Mice. Pathogen-free BALB/c, C57BL/6, and CB6F1 female mice between the
ages of 8 and 10 weeks were from Charles River Laboratories (Raleigh, NC) and
Jackson Laboratories (Bar Harbor, ME) and were cared for in accordance with
the Guide for the Care and Use of Laboratory Animals, as described previously
(20). The NIH Animal Care and Use Committee approved all animal experiment
protocols in this study. Experimental groups were age matched.
Design and construction of the M/M2 fusion gene. With dominant CD8
epitopes of the RSV M and M2 proteins identified in BALB/c and C57BL/6
mice, respectively (28, 39), a model antigen incorporating both of these target
proteins was designed as follows. The M2 protein (NCBI sequence number
AAB86660) was added to the end of the M protein (NCBI sequence number
AAB86677). The reverse fusion protein was designed by adding the M protein to
the end of M2. These two fusion proteins were then pasted together. This
combination of the two separate fusion proteins was then codon optimized, using
GeneOptimizer technology by GeneArt (Regensburg, Germany), for expression
in mammalian cells. The use of this design results in a single codon-optimized
gene sequence, which facilitates the subsequent cloning of either the M/M2 or
M2/M fusion protein or the single M or M2 protein into a variety of vectors due
to the unique use of flanking enzyme restriction sites. The M/M2 gene cassette
was cloned into the p8400 plasmid, which utilizes a CMV/R promoter, as pre-
viously described (5). Expression was verified by Western blotting. HEK 293 cells
were transfected with p8400 containing the M/M2 gene according to the Invitro-
gen (Carlsbad, CA) Lipofectamine 2000 protocol. Cells were lysed with Invitro-
gen (Carlsbad, CA) NuPAGE lithium dodecyl sulfate sample buffer, and the
lysates were run on 4-to-12% Bis-Tris gels. Gels were stained with a polyclonal
anti-RSV antibody (Maine Biotechnology, Portland, ME) for the primary anti-
body and with a peroxidase-conjugated AffiniPure rabbit anti-goat immunoglob-
ulin G (H?L) antibody (Jackson ImmunoResearch Labs, West Grove, PA) for
the secondary antibody.
Immunization and challenge. We used VRC plasmid p8400 as a vector for
delivery of the RSV M/M2 DNA. Mice were immunized with 50 ?g of RSV
M/M2 DNA intramuscularly via the quadriceps muscle 30 days before RSV
challenge. The challenge stock was derived from the A2 strain of RSV by
sonicating HEp-2 cell monolayers as previously described (20). Mice were anes-
thetized intramuscularly with ketamine (40 ?g/g of body weight) and xylazine (6
?g/g of body weight) and were inoculated intranasally with 107PFU of live RSV
in 100 ?l of Eagle’s minimal essential medium with 10% fetal bovine serum.
Synthetic peptides. Fifteen-mer peptides, overlapping by 11 amino acids, span-
ning the entire M and M2 proteins of RSV strain A2 were obtained from New
England Peptide, Inc. (Gardner, MA). All peptides had a purity of ?80% by
analytical high-performance liquid chromatography. All truncated peptides of
RSV M and M2 proteins were from Biosynthesis, Inc. (Lewisville, TX), with
?95% purity confirmed by analytical high-performance liquid chromatography
by Jan Lukszo at the NIAID peptide core facility (Bethesda, MD). Peptide
OVA323-339(ISQAVHAAHAEINEAGR) was from AnaSpec (San Jose, CA).
All peptides were dissolved into dimethyl sulfoxide and adjusted to the concen-
trations indicated in experiments with RPMI 1640 culture medium.
Cell culture and antibody reagents. The cell culture medium was RPMI 1640
from HyClone (Logan, UT), supplemented with 10% heat inactivated fetal
bovine serum, 2 mM glutamine, and 10 U/ml penicillin and 10 ?g/ml strepto-
mycin. Fluorochrome-conjugated antibodies used to study the T-cell phenotype
and cytokine production with flow cytometry were anti-CD3–cyanine 7 (Cy7)
allophycocyanin (APC), anti-IFN-?–APC, anti-IL-2–phycoerythrin (PE), and
anti-CD45RB–fluorescein isothiocyanate, which were obtained from BD Bio-
sciences (San Jose, CA). Unconjugated antibodies to CD4, CD62L, and CD8
were from BD Biosciences and were conjugated with Cy5.5-PE, Alexa Fluor 488
and Alexa Fluor 688 from Invitrogen (Carlsbad, CA), and Cy7-PE, respectively.
Cy7 and Cy55 were obtained from Amersham Life Sciences (Pittsburgh, PA),
and PE was obtained from ProZyme (San Leandro, CA). Protocols for fluoro-
chrome-antibody conjugation are available at http://drmr.com/abcon/index.html.
Conjugates were validated by comparison with commercial conjugates. Ethidium
monoazide bromide (EMA; Invitrogen) and propidium iodide (PI; Sigma
-Aldrich, St. Louis, MO) were used as viability markers to exclude dead cells
from the analysis (32). Antibodies used to study MHC class II haplotype
restriction of peptide presentation were anti-I-Ab(clone KH74), anti-I-Ad(clone
39-10-8), anti-I-Ak(clone 11-5.2), and anti-I-A&E (clone M5/114.15.2), all from
BD Bioscience, and anti-I-E (clone 14-4-4S) from eBioscience, Inc. (San Diego,
CA).The expression of FoxP3 was assessed with a fluorescein isothiocyanate
-conjugated anti-FoxP3 antibody and a FoxP3 fixation/permeabilization kit by
following the manufacturer’s instructions (eBioscience, Inc.).
Peptide–MHC class II molecule tetrameric complexes. I-Ab–M209-223–APC
and I-Ab–M226-39–PE tetramers were prepared by the MHC tetramer core fa-
cility of NIAID (Atlanta, GA). I-Ab–hCLIP conjugated with APC or PE was
used as a control.
In vitro stimulation and study of cytokine expression. The isolation of lym-
phocytes from mouse spleens and lungs has been described previously (40).
Isolated lymphocytes were cultured with an appropriate peptide at 2 ?g/ml for
1 h, together with 1 ?g/ml of costimulatory antibodies to CD28 and CD49d (BD
Bioscience). Brefeldin A (BD Bioscience) at 1 ?g/ml was added to the culture for
another 4 h of culturing. Cultured cells were collected and stained with fluoro-
chrome-conjugated antibodies to cell phenotype markers and with EMA for 10
min at room temperature, followed by 10 min of exposure to light for photoco-
valent binding of EMA to DNA. After treatment with a Cytofix/Cytoperm kit
(BD Bioscience) according to the manufacturer’s instructions, cells were stained
with fluorochrome-conjugated antibodies to cytokine molecules for 10 min at
room temperature, washed with staining medium, and fixed with 0.25% parafor-
maldehyde. Data from stained cell samples were acquired by flow cytometry on
an LSR-II system (BD Bioscience).
For MHC class II restriction studies, lymphocytes were precultured with an-
tibodies to specific MHC class II molecules for 1 h before the addition of a
peptide for stimulation.
For quantitative assessment of cytokines in culture supernatants of stimulated
CD4 T cells, lymphocytes were cultured with peptides and costimulatory anti-
bodies without brefeldin A for 24 h. Supernatants were harvested and stored at
?70?C until they were tested by protein arrays at the SearchLight Sample
Testing Service of Pierce (Rockford, IL).
Flow cytometric analysis. Sample data were analyzed with FlowJo (version 6.3;
Tree Star, San Carlos, CA). Cell doublets were excluded using forward scatter
(FSC) area versus FSC height parameters. A lymphocyte gate was created using
FSC and side scatter properties. EMA- or PI-stained cells were excluded so as to
minimize background staining caused by nonspecific binding to dead cells. Non-T
cells were excluded by gating on CD3?cells. CD4 T cells were selected by gating
out CD8?cells and gating on CD4?cells. Cytokine-producing CD4 T cells were
plotted by gating on anti-IFN-? and anti-IL-2 antibody-stained cells. A two-tailed
Student t test was used for statistical analysis.
CD4 T-cell epitopes of RSV M and M2 proteins. We tested
15-mer peptides from the RSV M and M2 proteins for the
stimulation of RSV-specific CD4 T cells. For initial screening,
we grouped these 108 overlapping peptides into 18 pools con-
figured with the aid of DeconvoluteThis! version 1.2 (37).
When cultured with spleen lymphocytes from RSV M/M2
DNA-immunized and RSV-challenged CB6F1 mice, peptides
in pools 3, 4, 11, 12, 17, and 18 specifically stimulated CD4 T cells
to produce IFN-? and IL-2 (Fig. 1A and B). DeconvoluteThis!
analysis suggested individual peptides 41, 42, 46, 50, 51, 53, 54, 58,
68, 69, 71, and 72 for further testing. Among these, peptides 53,
54, and 69 potently stimulated CD4 T cells to produce IFN-? and
to these peptides (data not shown).
Peptides 53 (M209-223[NKGAFKYIKPQSQFI]) and 54
(M213-227[FKYIKPQSQFIVDLG]) are from the RSV M pro-
tein and share an 11-mer sequence. We tested a series of
trimmed peptides (Table 1) for the stimulation of specific CD4
T cells. Comparing the responses to trimmed peptides with
the CD4 T-cell responses to the original full-length peptides
M209-223and M231-227, we identified an 11-mer peptide, M213-223
(FKYIKPQSQFI), that retained more than 60% potency for
stimulating CD4 T cells. Depleting a single amino acid from
VOL. 83, 2009 RSV M- AND M2-SPECIFIC CD4 T CELLS4935
either end dramatically affected the potency of stimulation.
Peptide 69 (M225-39[HNYFEWPPHALLVRQ]) is from the
RSV M2 protein. Neither of its adjacent peptides (peptides 68
and 70) stimulated a CD4 T-cell response, suggesting that the
amino acids at both ends are essential for epitope recognition.
We trimmed the sequence starting from either end and tested
the trimmed peptides as we did with the RSV M peptides. By
so doing, we identified an 11-mer peptide, M227-37(YFEWPP
HALLV), that retained more than 60% potency to stimulate
CD4 T cells (Table 2).
FIG. 1. Identification of peptides stimulating specific CD4 T-cell cytokine production. Spleen lymphocytes from RSV M/M2 DNA-immunized and
RSV-challenged CB6F1 mice were cultured with peptide pools or individual peptides at final concentrations of 2 ?g/ml. Cells were harvested and stained
with fluorochrome-conjugated antibodies specific to CD3, CD4, CD8, and EMA extracellularly, as well as with antibodies to IFN-? and IL-2
intracellularly. (A) CD4 T cells were gated from the CD3?, EMA?, and CD8?lymphocyte populations. The frequency of cytokine-producing cells
was assessed with fluorochrome-conjugated anti-IFN-? and anti-IL-2 staining. (B and C) Cells were cultured with peptide pools 1 to 18 (B) or with
selected individual peptides and with peptide pools 17 and 18 as positive references and peptide pools 7 and 9 as negative references (C). The
percentages of IFN-?- and IL-2 producing CD4 T cells are shown; they are averages for spleen cell samples from four mice, with the background
subtracted (as determined by CD4 T-cell responses to an irrelevant OVA323-339peptide).
TABLE 1. Defining the minimal CD4 T-cell epitope of RSV M protein
Frequencyaof CD4 T cells producing:
Relative responsebof CD4
T cells producing:
0.47 ? 0.16
0.43 ? 0.12
0.38 ? 0.12
0.39 ? 0.13
0.33 ? 0.12
0.16 ? 0.05
0.04 ? 0.02
0.05 ? 0.02
0.69 ? 0.21
0.63 ? 0.18
0.51 ? 0.15
0.49 ? 0.16
0.42 ? 0.13
0.22 ? 0.09
0.05 ? 0.03
0.03 ? 0.02
0.03 ? 0.01 0.02 ? 0.0263
aPercentage of CD4 T cells that were stained intracellularly with fluorochrome-conjugated antibodies specific to cytokines. Data were from four mice and are
expressed as means ? standard deviations.
bThe average frequency of IFN-?- or IL-2 producing CD4 T cells responding to M209-223or M213-227was defined as 100%. The responsiveness of CD4 T cells to
other peptides was evaluated and reported as a percentage of the response to full-length peptides.
cOVA323-339was used as a negative control to assess background cell response.
4936 LIU ET AL.J. VIROL.
Characteristics of RSV M- and M2-specific CD4 epitopes.
We titrated the M209-223and M225-39peptides and found that
spleen CD4 T cells could respond to the peptides at concen-
trations as low as 0.03 ?g/ml. At a peptide concentration of 2
?g/ml, the cytokine production of CD4 T cells reached its peak
in response to both peptides within 5 h in culture (Fig. 2A). We
used both peptides at 2 ?g/ml for subsequent experiments. We
found that the frequency of cytokine-producing CD4 T cells
increased in spleens (Fig. 2B) and lungs (data not shown) on
days 4 through day 10 post-RSV challenge.
To study the MHC restriction of the CD4 T-cell recognition
of these two peptides, we compared the cytokine production by
CD4 T cells from CB6F1 (I-Ad/b), BALB/c (I-Ad), and
C57BL/6 (I-Ab) mice. We found that CD4 T cells from
C57BL/6 and CB6F1 mice, but not those from BALB/c mice,
responded to these two peptides by producing IFN-? and IL-2
(Fig. 3A). Pretreatment with anti-I-A&E or anti-I-Ab, but not
with anti-I-Ad, anti-I-Ak, or anti-I-E, decreased cytokine pro-
duction by CD4 T cells from CB6F1 mice in a dose-dependent
manner (Fig. 3B; data for anti-I-E not shown).
Quantitative assessment of cytokines in the supernatants of
spleen lymphocytes after stimulation with peptide M209-223or
M225-39revealed significant amounts of IL-1?, IL-4, IL-5, IL-6,
IL-10, IL-13, IL-17, and RANTES (data not shown), in addi-
tion to IFN-? and IL-2, which were detected by intracellular
staining with flow cytometry. The CD4 T cells showed similar
cytokine profiles in response to the M209-223and M225-39pep-
Enumeration of RSV M- and M2-specific CD4 T cells. Using
fluorochrome-conjugated tetramers to assess RSV M- and M2-
specific CD4 T cells in spleen lymphocytes from CB6F1 mice
immunized with RSV M/M2 DNA, we identified 0.47% I-Ab–
M209-223- and 1.55% I-Ab–M225-39-positive cells in the CD4
T-cell population from mice at 7 days after RSV challenge.
There were ?0.05% CD8 T cells stained with either of the
tetramers in the same culture, and I-Ab–hCLIP–APC and –PE
stained 0.08% and 0.04% of CD4 T cells from the same mice,
respectively. The proportion of tetramer-stained CD4 T cells
in naïve mice was ?0.05% (Fig. 4).
TABLE 2. Defining the minimal CD4 T-cell epitope of RSV M2 protein
Frequencyaof CD4 T cells producing:
Relative responsebof CD4
T cells producing:
0.70 ? 0.22
0.71 ? 0.19
0.57 ? 0.20
0.05 ? 0.02
0.80 ? 0.25
0.71 ? 0.20
0.37 ? 0.15
0.75 ? 0.21
0.67 ? 0.18
0.50 ? 0.17
0.55 ? 0.15
0.03 ? 0.04
0.46 ? 0.11
0.02 ? 0.03
0.05 ? 0.04
0.78 ? 0.27
0.86 ? 0.29
0.67 ? 0.21
0.01 ? 0.03
0.99 ? 0.25
0.83 ? 0.26
0.43 ? 0.20
0.87 ? 0.22
0.78 ? 0.25
0.50 ? 0.12
0.70 ? 0.23
0.04 ? 0.02
0.48 ? 0.11
0.02 ? 0.03
0.03 ? 0.04
0.03 ? 0.01 0.02 ? 0.0243
aPercentage of CD4 T cells that were stained intracellularly with fluorochrome-conjugated antibodies specific to cytokines. Data were from four mice and are
expressed as means ? standard deviations.
bThe frequency of IFN-?- or IL-2-producing CD4 T cells responding to M225-39was defined as 100%. The responsiveness of CD4 T cells to other peptides was
evaluated and reported as a percentage of the response to full-length peptides.
cOVA323-339was used as a negative control to assess the background cell response.
FIG. 2. Kinetics of production of cytokines by CD4 T cells in re-
sponse to peptides M209-223and M225-39. (A) Spleen lymphocytes iso-
lated from mice at 7 days postchallenge were cultured with peptide
M209-223(‚), M225-39(?), or OVA323-339(F) at the concentrations
indicated. (B) Spleen lymphocytes isolated from mice at 4, 7, and 10
days postchallenge were cultured with peptide M209-223, M225-39, or
OVA323-339at 2 ?g/ml. Cells were harvested and stained with fluoro-
chrome-conjugated antibodies for cytokine assessment. CD4 T-cell
responses are shown as means ? standard deviations for five mice from
one experiment representative of three.
VOL. 83, 2009RSV M- AND M2-SPECIFIC CD4 T CELLS 4937
Cytokine profile and phenotype of CD4 T cells from lungs
and spleens. We studied the frequencies of cytokine-producing
CD4 T cells of the spleen and lung responding to peptide
stimulation. Among spleen lymphocytes, 0.73% and 1.19% of
CD4 T cells produced IL-2, and 0.34% and 0.58% of CD4 T
cells produced IFN-?, in response to peptides M209-223and
M225-39, respectively. Among lung lymphocytes, 0.61% and
0.86% of CD4 T cells produced IL-2, and 0.67% and 0.72% of
CD4 T cells produced IFN-?, in response to peptides M209-223
and M225-39, respectively (Fig. 5A). These data indicate that
there were more IL-2-producing than IFN-?-producing CD4 T
cells in the spleen but that these two populations had almost
the same frequency in the lungs. We studied the CD45RB
expression of CD4 T cells with a CD62L?phenotype. In
spleens, 57.1% of CD4 T cells were CD45RB?and 42.0% were
CD45RB?, while in lungs, 19.9% were CD45RB?and 79.9%
were CD45RB?(Fig. 5B). These findings suggest that the
majority of lung CD62L?CD4 T cells had encountered anti-
gen and that CD62L?CD4 T cells in spleens were more likely
to be antigen naïve.
Regulatory role of peptide-triggered CD4 T cells in the CD8
T-cell response. We precultured spleen lymphocytes with pep-
tides M209-223and M225-39before adding CD8 T-cell-specific
peptides M187-195and M282-90, and we measured cytokine pro-
duction by CD8 T cells (Fig. 6A). In lymphocytes from mice 7
days after RSV challenge, 3.0% and 3.7% of CD8 T cells
produced IFN-?, while 2.1% and 3.2% of CD8 T cells pro-
duced IL-2, in response to CD8 T-cell-specific peptide M187-195
when the cells were precultured with CD4 T-cell-specific pep-
tides M209-223and M225-39, respectively. Without prior CD4
FIG. 3. MHC class II restriction on CD4 T cells responding to M209-223
and M225-39. (A) Spleen lymphocytes from CB6F1, C57BL/6, and BALB/c
mice were cultured with the M209-223or M225-39peptide. (B) Spleen lympho-
cytes from CB6/F1 mice were cultured with anti-MHC class II molecule
antibodies at the indicated concentration for 1 h (‚, anti-I-Ad; ?, anti-I-Ak;
f, anti-I-Ab; F, anti-I-A&E). The M209-223and M225-39peptides were added
to cultures for another 5-h incubation. Cells were harvested and stained with
fluorochrome-conjugated antibodies for phenotype and cytokine assessment.
Results are means ? standard deviations for four mice, which are represen-
tative of data from three independent experiments.
FIG. 4. Identification of RSV M- and M2-specific CD4 T cells. Spleen lymphocytes were stained first either with I-Ab–M209-223–APC and
I-Ab–M226-39–PE or with I-Ab–hCLIP–APC and I-Ab–hCLIP–PE at 37°C for 60 min and then with fluorochrome-conjugated antibodies specific
to CD3, CD4, CD8, and PI at room temperature for another 10 min. Cell populations were gated on CD3?PI?, CD8?, and CD4?subsets or on
CD3?PI?, CD4?, and CD8?lymphocytes. Means ? standard deviations are shown for four mice from each group.
4938LIU ET AL.J. VIROL.
T-cell-specific peptide exposure, 4.5% of CD8 T cells produced
IFN-? and 4.4% produced IL-2 in response to peptide M187-195.
CD8 T cells showed a similar pattern of response to peptide
M82-90when they were pretreated with peptide M209-223. These
data indicate that pretreatment with the CD4-T-cell specific
peptide M209-223reduced the IFN-? and IL-2 production of
CD8 T cells responding to peptide M187-195or M2-82-90com-
pared to pretreatment with an irrelevant peptide and peptide
Next, we evaluated the phenotype of epitope-specific CD4 T
cells in lungs responding to primary RSV infection. On day 7
postchallenge, intracellular staining of tetramer-gated lung
lymphocytes showed that 28.5% of M209-223-specific CD4 T
cells and 4.7% of M225-39-specific CD4 T cells expressed FoxP3
(Fig. 6B). These data suggest that M209-223-specific CD4 T cells
may have both the functional properties and the phenotype of
regulatory T cells.
Study of RSV-specific CD4 T cells and their effects on other
immune competent cells is essential to understanding RSV
pathogenesis and protective immunity. CB6F1 hybrid mice
that express both H-2band H-2dMHC class I molecules have
allowed detailed investigation of CD8 T-cell responses in RSV
infection. Those CD8 T cells can respond to RSV M and M2
epitopes, including Db-restricted M187-195, Kd-restricted M282-90,
and M2127-135(28, 30, 39), and they provide the opportunity to
define the rules for epitope hierarchy and to understand the
determinants of CD8 T-cell effector function. Identifying RSV-
specific CD4 T-cell epitopes in the CB6F1 mouse provides a
new tool for the study of antigen-specific CD4 T cells and their
interactions with epitope-specific CD8 T cells during infection
or immunization and will improve our understanding of viral
pathogenesis and immunity.
Using overlapping peptides (1, 6, 25, 26, 33), we identified
the minimal epitope sequences M213-223(FKYIKPQSQFI) and
M227-37(YFEWPPHALLV). The M213-223epitope is near the
C terminus of the RSV M protein, 17 amino acids downstream
from the dominant CD8 T-cell epitope M187-195. The M227-37
epitope is at the N terminus of the RSV M2 protein, 44 amino
FIG. 5. Phenotype and cytokine profiles of CD4 T cells from the
lung and spleen. (A) Spleen and lung lymphocytes from CB6F1 mice
were cultured with the M209-223and M225-39peptides for 5 h. Cells were
harvested and stained with fluorochrome-conjugated antibodies for
cytokine assessment. (B) Spleen and lung lymphocytes from CB6F1
mice were stained with fluorochrome-conjugated antibodies specific to
CD3, CD4, CD8, CD45RB, and CD62L, and with PI to exclude dead
cells. The frequencies of CD62L?cells with and without CD45RB
expression in the CD4?population were compared. The mean fre-
quencies (for cells from five mice) of CD4 T cells producing cytokines
and expressing CD45RB were compared by a Student t test. The data
shown are from one experiment representative of two.
FIG. 6. Regulatory function of RSV epitope-specific CD4 T cells.
(A) First, the effect of peptide-stimulated CD4 T cells on CD8 T-cell
responses was assessed. Spleen lymphocytes from CB6F1 mice were
cultured with the M209-223, M225-39, or OVA323-339peptide overnight.
The M187-195and M282-90peptides, representing CD8 T-cell epitopes,
were then added and cultured for an additional 5 h. Cells were har-
vested and stained with fluorochrome-conjugated antibodies for cyto-
kine assessment. The data show the proportions of cytokine-producing
CD8 T cells from five mice compared by a Student t test in one
experiment representative of two with similar results. (B) Next, FoxP3
expression was evaluated in RSV epitope-specific CD4 T cells. Lung
lymphocytes from RSV-infected mice were stained first with I-Ab–
M209-223–APC and I-Ab–M226-39–PE as described in the Fig. 4 legend
and then with fluorochrome-conjugated antibodies specific to CD3,
CD4, and CD8 at room temperature for another 10 min. Cells were
fixed, permeabilized, and stained for FoxP3 according to the manufac-
turer’s instructions. Cells were first gated on CD3?, CD8?, and CD4?
subsets. Frequencies shown are the percentages of FoxP3-positive
M209-223and M226-39tetramer-specific CD4 T cells [upper right quad-
rant/(upper right ? lower right quadrants)]. Means ? standard devi-
ations are shown for five mice (P ? 0.002).
VOL. 83, 2009 RSV M- AND M2-SPECIFIC CD4 T CELLS4939
acids upstream from the dominant CD8 T-cell epitope (M282-90)
and 89 amino acids upstream from the subdominant CD8 T-cell
The two peptides M209-223and M225-39, which contain the
identified core sequences, are CD4 specific. They induced CD4
T-cell responses in vitro in a dose-dependent manner, peaking
at a concentration of 2 ?g/ml. CD8 T cells in the same culture
did not respond to the peptides at the concentrations tested.
Fluorochrome-conjugated peptide–I-Abmolecule tetramers
identified CD4 T cells specific to RSV M and M2 proteins in
the lymphocytes of infected mice. The binding of the class II
tetramer was highly CD4 T-cell specific. Neither CD8 T cells
from immunized mice nor a significant number of CD4 T cells
from naïve mice were labeled by the MHC class II tetramers.
The two peptides stimulated specific CD4 T cells from both the
spleen and the lung. The number of CD4 T cells responding to
the peptides increased in the spleen and lung from days 4
through 10 post-RSV infection, suggesting that specific CD4 T
cells may have expanded in both lymphoid and peripheral
tissues after virus infection. MHC class II molecule I-Abis
associated with the presentation of both peptides to CD4 T
cells, because only CD4 T cells from the CB6F1(I-Ab/d) and
C57BL/6 (I-Ab) parental strains responded to these two pep-
tides, and a monoclonal antibody to I-Abblocked the CD4
T-cell response in a dose-dependent manner. Other antibodies
to MHC class II molecules, such as anti-I-Ad, anti-I-Ak, and
anti-I-E, had no effect on CD4 T-cell cytokine production.
CD4 T cells from the spleen and the lung showed phenotypic
differences after RSV infection, suggesting differential traffick-
ing of functionally distinct populations. In the spleen, there
were more CD45RB?than CD45RB?cells in the CD62L?
CD4 T-cell population, suggesting that the majority of acti-
vated CD4 T cells had not yet encountered antigen. In con-
trast, the majority of CD62L?CD4 T cells in the lung were
CD45RB?, and less than 20% were CD45RB?. This pattern of
low CD45RB expression was also maintained in the total pop-
ulation of CD4 T cells in lungs (data not shown). These data
are consistent with the location of RSV infection, which is
restricted to the respiratory tract. Although CD4 T cells from
the lung and spleen could both produce IFN-? and IL-2 upon
peptide stimulation, their cytokine profiles were different.
There are more IL-2-producing than IFN-?-producing CD4 T
cells in the spleen, but these two cytokine-producing popula-
tions had similar frequencies in the lung. This correlates with
the differentiation of CD45RB expression in the spleen and
lung and is consistent with previous reports that CD45RB?
CD4 T cells produce more IFN-? than CD45RB?CD4 T cells
(7). IL-2 has little direct effector function but is an indicator of
cells with higher capacity for survival and proliferation that can
later evolve effector responses. CD4 T cells producing IL-2 are
also more likely to differentiate into memory cells, and CD4 T
cells producing only IFN-? tend to be short-lived (22, 41, 50,
51). But those CD45RB?CD4 T cells may play a critical
effector role in the lung, since IFN-? is a major factor in the
defense against viral infection. Other studies of RSV infection
have also shown that effector CD4 T cells accumulate in lungs
after infection. Antigen-activated CD4 T cells that migrate
from lymphoid tissue to the lung are short-lived, while memory
CD4 T cells reside in the spleen or other lymphoid tissue (49).
We found that M209-223-specific CD4 T cells have a higher
frequency of FoxP3 expression than M225-39-specific CD4 T
cells. This is consistent with our observation that M209-223-
triggered CD4 T cells reduce RSV-specific CD8 T-cell re-
sponses to M187-195and M282-90, based on less IFN-? and IL-2
production, while M225-39-triggered CD4 T cells do not have a
significant effect on CD8 T-cell function. These observations
suggest that many M209-223-specific CD4 T cells have the phe-
notype and functional properties associated with T regulatory
cells. Epitope-specific T regulatory cells have been identified
previously both in mice (47) and in humans (15), but demon-
strating a correlation of FoxP3 expression with regulatory func-
tion using two different pathogen-specific class II tetramers ex
vivo is novel.
The specific roles of RSV M- and M2-specific CD4 T cells in
regulating the immune response to RSV infection and immu-
nization are under further investigation. Our work in defining
the specificity of T-cell responses to RSV and producing novel
reagents will allow more detailed investigation of pathogenesis,
immunity, and regulation of T-cell responses, which will ad-
vance vaccine development.
We thank the NIAID-sponsored tetramer core facility at Emory
University (Atlanta, GA) for assisting in the construction of the class
II tetramers. We also thank Jan Lukszo at the NIAID peptide core
facility (Bethesda, MD) for help in obtaining peptides.
This work was supported entirely by intramural NIAID funding.
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