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Journal of Virology, May 2009, p. 4934-4941, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02140-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 M_213-223 (FKYIKPQSQFI) and M2_27-37 (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-A^b-restricted pattern. Construction of fluorochrome-conjugated peptide-I-A^b class 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, M_209-223 peptide-activated CD4 T cells reduced IFN- and IL-2 production in M- and M2-specific CD8 T-cell responses to D^b-M_187-195 and K^d-M2_82-90 peptides more than M2_25-39 peptide-stimulated CD4 T cells. This correlated with the fact that I-A^b-M_209-223 tetramer-positive cells responding to primary RSV infection had a much higher frequency of FoxP3 expression than I-A^b-M2_26-39 tetramer-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-A^b tetramers allows detailed analysis of their roles in RSV pathogenesis and immunity.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 mandatory 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 interleukin 2 (IL-2) from CD4 T cells can restore CD8 T-cell function in lungs (10) and that IL-2 supplementation can increase 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 immunopathogenesis 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) immunization-mediated enhanced illness upon subsequent natural 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-immunized 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). Increased disease severity has also been associated with polymorphisms 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 association of RSV and allergic inflammation, which is largely mediated by CD4 T cells. Understanding the influence of CD4 T cells on CD8 T-cell responses and other immunological effector 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-A^b. Fluorochrome-conjugated peptide-I-A^b molecule tetrameric 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-challenged 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 stimulation. These observations suggest a regulatory role for CD4 T cells in the host response to RSV infection. The development of novel MHC class II tetramer reagents allows the characterization 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 infections.

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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 previously described (5). Expression was verified by Western blotting. HEK 293 cells were transfected with p8400 containing the M/M2 gene according to the Invitrogen (Carlsbad, CA) Lipofectamine 2000 protocol. Cells were lysed with Invitrogen (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 antibody and with a peroxidase-conjugated AffiniPure rabbit anti-goat immunoglobulin 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 µg 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 anesthetized intramuscularly with ketamine (40 µg/g of body weight) and xylazine (6 µg/g of body weight) and were inoculated intranasally with 10⁷ PFU of live RSV in 100 µl of Eagle's minimal essential medium with 10% fetal bovine serum.

Synthetic peptides. Fifteen-mer peptides, overlapping by 11 amino acids, spanning 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 OVA_323-339 (ISQAVHAAHAEINEAGR) was from AnaSpec (San Jose, CA). All peptides were dissolved into dimethyl sulfoxide and adjusted to the concentrations 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 µg/ml streptomycin. 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 Biosciences (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 fluorochrome-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-A^b (clone KH74), anti-I-A^d (clone 39-10-8), anti-I-A^k (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-A^b-M_209-223-APC and I-A^b-M2_26-39-PE tetramers were prepared by the MHC tetramer core facility of NIAID (Atlanta, GA). I-A^b-hCLIP conjugated with APC or PE was used as a control.

In vitro stimulation and study of cytokine expression. The isolation of lymphocytes from mouse spleens and lungs has been described previously (40). Isolated lymphocytes were cultured with an appropriate peptide at 2 µg/ml for 1 h, together with 1 µg/ml of costimulatory antibodies to CD28 and CD49d (BD Bioscience). Brefeldin A (BD Bioscience) at 1 µg/ml was added to the culture for another 4 h of culturing. Cultured cells were collected and stained with fluorochrome-conjugated antibodies to cell phenotype markers and with EMA for 10 min at room temperature, followed by 10 min of exposure to light for photocovalent 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% paraformaldehyde. 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 antibodies 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 antibodies without brefeldin A for 24 h. Supernatants were harvested and stored at –70^oC 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.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 configured 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 IL-2 (Fig. 1C). CD8 T cells from the same culture did not respond to these peptides (data not shown).

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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 µg/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 OVA323-339 peptide).

Peptides 53 (M_209-223 [NKGAFKYIKPQSQFI]) and 54 (M_213-227 [FKYIKPQSQFIVDLG]) are from the RSV M protein 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 M_209-223 and M_231-227, we identified an 11-mer peptide, M_213-223 (FKYIKPQSQFI), that retained more than 60% potency for stimulating CD4 T cells. Depleting a single amino acid from either end dramatically affected the potency of stimulation. Peptide 69 (M2_25-39 [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, M2_27-37 (YFEWPPHALLV), that retained more than 60% potency to stimulate CD4 T cells (Table 2).

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TABLE 1. Defining the minimal CD4 T-cell epitope of RSV M protein

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TABLE 2. Defining the minimal CD4 T-cell epitope of RSV M2 protein

Characteristics of RSV M- and M2-specific CD4 epitopes. We titrated the M_209-223 and M2_25-39 peptides and found that spleen CD4 T cells could respond to the peptides at concentrations as low as 0.03 µg/ml. At a peptide concentration of 2 µg/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 µg/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.

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FIG. 2. Kinetics of production of cytokines by CD4 T cells in response to peptides M209-223 and M225-39. (A) Spleen lymphocytes isolated from mice at 7 days postchallenge were cultured with peptide M209-223 (), M225-39 (), or OVA323-339 (•) 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 OVA323-339 at 2 µg/ml. Cells were harvested and stained with fluorochrome-conjugated antibodies for cytokine assessment. CD4 T-cell responses are shown as means ± standard deviations for five mice from one experiment representative of three.

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-A^d/b), BALB/c (I-A^d), and C57BL/6 (I-A^b) 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-A^b, but not with anti-I-A^d, anti-I-A^k, or anti-I-E, decreased cytokine production by CD4 T cells from CB6F1 mice in a dose-dependent manner (Fig. 3B; data for anti-I-E not shown).

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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 M209-223 or M225-39 peptide. (B) Spleen lymphocytes 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; , anti-I-Ab; •, anti-I-A&E). The M209-223 and M225-39 peptides 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 representative of data from three independent experiments.

Quantitative assessment of cytokines in the supernatants of spleen lymphocytes after stimulation with peptide M_209-223 or M2_25-39 revealed significant amounts of IL-1, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, and RANTES (data not shown), in addition 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 M_209-223 and M2_25-39 peptides.

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-A^b-M_209-223- and 1.55% I-A^b-M2_25-39-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-A^b-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).

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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.

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 M_209-223 and M2_25-39, 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 M_209-223 and M2_25-39, 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 antigen and that CD62L^– CD4 T cells in spleens were more likely to be antigen naïve.

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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 M209-223 and M225-39 peptides 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 frequencies (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.

Regulatory role of peptide-triggered CD4 T cells in the CD8 T-cell response. We precultured spleen lymphocytes with peptides M_209-223 and M2_25-39 before adding CD8 T-cell-specific peptides M_187-195 and M2_82-90, and we measured cytokine production 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 produced IL-2, in response to CD8 T-cell-specific peptide M_187-195 when the cells were precultured with CD4 T-cell-specific peptides M_209-223 and M2_25-39, respectively. Without prior CD4 T-cell-specific peptide exposure, 4.5% of CD8 T cells produced IFN- and 4.4% produced IL-2 in response to peptide M_187-195. CD8 T cells showed a similar pattern of response to peptide M_82-90 when they were pretreated with peptide M_209-223. These data indicate that pretreatment with the CD4-T-cell specific peptide M_209-223 reduced the IFN- and IL-2 production of CD8 T cells responding to peptide M_187-195 or M2_-82-90 compared to pretreatment with an irrelevant peptide and peptide M_25-39.

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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 OVA323-339 peptide overnight. The M187-195 and M282-90 peptides, representing CD8 T-cell epitopes, were then added and cultured for an additional 5 h. Cells were harvested and stained with fluorochrome-conjugated antibodies for cytokine 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 manufacturer's instructions. Cells were first gated on CD3+, CD8–, and CD4+ subsets. Frequencies shown are the percentages of FoxP3-positive M209-223 and M226-39 tetramer-specific CD4 T cells [upper right quadrant/(upper right + lower right quadrants)]. Means ± standard deviations are shown for five mice (P = 0.002).

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 M_209-223-specific CD4 T cells and 4.7% of M2_25-39-specific CD4 T cells expressed FoxP3 (Fig. 6B). These data suggest that M_209-223-specific CD4 T cells may have both the functional properties and the phenotype of regulatory T cells.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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-2^b and H-2^d MHC 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 D^b-restricted M_187-195, K^d-restricted M2_82-90, and M2_127-135 (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 M_213-223 (FKYIKPQSQFI) and M2_27-37 (YFEWPPHALLV). The M_213-223 epitope is near the C terminus of the RSV M protein, 17 amino acids downstream from the dominant CD8 T-cell epitope M_187-195. The M2_27-37 epitope is at the N terminus of the RSV M2 protein, 44 amino acids upstream from the dominant CD8 T-cell epitope (M2_82-90) and 89 amino acids upstream from the subdominant CD8 T-cell epitope (M2_127-135).

The two peptides M_209-223 and M2_25-39, 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 µg/ml. CD8 T cells in the same culture did not respond to the peptides at the concentrations tested. Fluorochrome-conjugated peptide-I-A^b molecule 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-A^b is associated with the presentation of both peptides to CD4 T cells, because only CD4 T cells from the CB6F1(I-A^b/d) and C57BL/6 (I-A^b) parental strains responded to these two peptides, and a monoclonal antibody to I-A^b blocked the CD4 T-cell response in a dose-dependent manner. Other antibodies to MHC class II molecules, such as anti-I-A^d, anti-I-A^k, 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 trafficking 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 activated CD4 T cells had not yet encountered antigen. In contrast, 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 population 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 populations 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 M_209-223-specific CD4 T cells have a higher frequency of FoxP3 expression than M2_25-39-specific CD4 T cells. This is consistent with our observation that M_209-223-triggered CD4 T cells reduce RSV-specific CD8 T-cell responses to M_187-195 and M2_82-90, based on less IFN- and IL-2 production, while M2_25-39-triggered CD4 T cells do not have a significant effect on CD8 T-cell function. These observations suggest that many M_209-223-specific CD4 T cells have the phenotype 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 demonstrating a correlation of FoxP3 expression with regulatory function 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 immunization 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 advance 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.

 
* 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: bgraham{at}nih.gov

Published ahead of print on 4 March 2009.

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Journal of Virology, May 2009, p. 4934-4941, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02140-08
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