The overall goal of these studies was to comprehensively and empirically evaluate CD4 T-cell specificity in peripheral lymphoid tissues following primary infection with influenza virus. We had a particular interest in determining whether CD4 T cells reactive to a subset of influenza proteins dominate the response, or if the response was equally distributed among the viral proteins expressed. Mouse strains expressing only single I-A MHC class II molecules were used to determine the impact of MHC class II allele variability on the distribution of specificities, and to simplify the determination of CD4 T-cell MHC restriction assignment. To accomplish this goal, C57BL/10 (I-Ab
) or SJL (I-As
) mice were infected intranasally with A/New Caledonia/20/99 influenza virus in PBS. This strain of influenza was isolated from humans and has been included in clinical vaccines in recent years (1
). Although it has not been adapted to mice, it replicates in the respiratory tract after intranasal infection of mice and is cleared within 10 d of infection (data not shown). At 8–10 d after inoculation with live virus, the mice were sacrificed and single-cell suspensions were prepared from the spleens. CD4 T cells were purified and then used in an IL-2 EliSpot assay, with syngeneic splenocytes depleted of T cells used as APC, and synthetic peptide used as antigen. Representative flow cytometry data demonstrating cell purity are shown in Supplementary Fig. 1
(see online supplementary material at http://www.liebertonline.com
). The CD4 T cells used were completely depleted of CD8 T cells (<1%), but had variable (15–40%) class II-positive cells remaining, mostly accounted for by B cells that are typically more resistant to complement-mediated lysis.
To evaluate the specificity of CD4 T cells elicited in response to primary influenza virus infection, we first obtained panels of 17-mer peptides overlapping by 11 amino acids that comprised the entire translated sequences of the HA, NA, NP, NS1, and M1 proteins. The protein length and number of peptides in each of these sets are shown in Supplementary Table 1
(see online supplementary material at http://www.liebertonline.com
). These proteins were chosen because together they encompass approximately half of the influenza genome. Importantly, they represent the major subcellular localizations of influenza virus proteins in infected cells, allowing us to determine if protein localization has any impact on the overall distribution of the epitopes identified. HA and NA are transmembrane proteins expressed both in infected cells and in the influenza virion. NP and NS1 are both expressed within the cytosol and nucleus of infected cells. While NP is expressed both in the virion and within the host cells, NS1 is excluded from the virion particle. The M1 matrix protein is the most abundant virion protein, and is expressed in cytosol and nuclei of infected cells, playing a major role in viral budding (9
). To evaluate if each of these proteins gained access to class II molecules and elicited CD4 T cells, these overlapping 17-mer synthetic peptides were tested for their ability to recall primed CD4 T cells isolated from previously infected mice using an EliSpot assay. The EliSpot assay has several advantages for epitope discovery, most notably that it allows rapid screening of multiple (>100) potential epitopes, and allows direct enumeration of antigen-specific T cells immediately ex-vivo,
without the need for further T-cell proliferation or persistence in culture (5
). We have found that the patterns of epitope distribution identified by IL-2 are similar to those identified by production of IFN-γ, but chose IL-2 for all studies reported here because some strains of mice produce fewer gamma-producing cells than IL-2-producing cells [(46
) and unpublished data], allowing IL-2 to be considered a more CD4-inclusive cytokine. Because the strains chosen express a single MHC class II molecule, the MHC restriction of any peptides that elicit a positive response will be known, and thus any discovered epitopes could potentially be used in subsequent studies to derive tetramers to label antigen-specific CD4 T cells, and more fully characterize the functional properties of these responding cells.
In order to determine whether the reactivity of CD4 T cells predictably tracked with particular influenza proteins, initial experiments tested CD4 T-cell reactivity to pools of peptides encompassing the entire sequences of the NP, NS1, M1, NA, and HA viral proteins. Overlapping peptides representing each protein were pooled together in a single sample and added to the cytokine EliSpot assay. We expected the two different MHC class II molecules to select for different influenza-derived peptides, as MHC molecules of different alleles have quite distinct peptide-binding motifs. However, if immunogenicity was primarily determined by a property of the viral protein, such as the protein's abundance or localization within an infected cell, we reasoned that the overall distribution of epitopes among the proteins should be similar between the two mouse strains. On the other hand, if selectivity of the individual MHC class II molecules determined specificity, a different distribution of epitopes among these proteins would be expected.
When CD4 T-cell specificity was examined using this method, dramatic differences were noted in the distribution of responses among the different influenza proteins in the two mouse strains examined (). In the C57BL/10 strain (), the predominant CD4 T-cell specificity was for NP and NA. Strikingly, there were very few CD4 T cells specific for HA and M1 in this strain, and only a modest number of CD4 T cells specific for the small NS1 protein. In contrast, in the SJL strain () the epitopes were distributed primarily among the HA and NP proteins, with readily detectable CD4 T-cell responses specific for NA and M1, but with very few cells recruited by peptides representing NS1. Together, these findings suggested that the reactivity of CD4 T cells was not determined to a significant extent by the localization of the protein within the infected cell, as both the NP protein (cytosolic and nuclear) and the HA and NA (membrane-associated) proteins recruited significant numbers of CD4 T cells, depending on the strain being examined. In addition, these findings suggested that the epitope distribution among different viral proteins could be strongly influenced by MHC class II polymorphism.
FIG. 1. Analyzing anti-influenza CD4 T-cell reactivity toward different viral proteins using pools of synthetic peptides representing the entire protein sequences of HA, NA, NP, M1, and NS1. Individual 17-mer peptides representing the entire sequences of the (more ...)
To further explore what accounted for the strain-dependent differences seen in the distribution of the CD4 T-cell specificities among the influenza proteins, the epitopes within the HA, NS1, NP, NA, and M1 proteins were directly identified and quantified in both strains. When screening for epitopes in smaller proteins (NS1 and M1), peptides were tested individually, with any peptide eliciting on average >30 CD4 T cells per 1
cells above background considered positive. In order to quickly screen larger proteins (HA, NA, and NP), pools of non-overlapping peptides were created and organized into a matrix design as described by Tobery and Caulfield (62
). Here, non-overlapping peptides were pooled such that an epitope would trigger intersecting rows and columns as positive, allowing us to more rapidly localize epitopes within these larger proteins. Shown in Supplementary Fig. 2B and D
are examples of the peptide-pooling matrix analyses performed with peptides representing the NP protein, with layouts of the peptide-pooling matrix shown (the numbers indicate an individual peptide's position in the matrix) (see online supplementary material at http://www.liebertonline.com
). Shown in Supplementary Fig. 2A and C
are examples of NP-specific EliSpots obtained using these matrices, with the results presented as the number of antigen-dependent IL-2 spots above background detected per 1
CD4 T cells (see online supplementary material at http://www.liebertonline.com
). Any pool that elicited on average >40 CD4 T cells per 1
cells over background was considered for further analysis, while negative pools were presumed to have only very weak or no CD4 T-cell epitopes. Peptides contained in pools within negative rows or columns were eliminated from further consideration, and the other peptides were tested individually in subsequent studies.
Shown in are the results of CD4 cytokine EliSpots using individual peptides from each of the influenza virus proteins tested. Epitopes that elicited on average between 30 and 150 IL-2 spots per 1
CD4 T cells were considered subdominant, while peptides that elicited greater than 150 but less than 300 IL-2 spots per 1
T cells were considered dominant, and peptides that elicited greater than 300 IL-2 spots per 1
T cells were considered to be “super-dominant.” All peptides were tested in at least 2 individual experiments, and positive peptides were tested a greater number of times ().
FIG. 2. Screening of candidate individual epitopes in the NP, NA, NS1, M1, and HA proteins using EliSpot assays. Panels A–E show the peptides in the C57BL/10 (red) and SJL (blue) mouse strains screened as individual peptides in the NP, NA, NS1, M1, and (more ...)
I-Ab and I-As Restricted Epitopes
The results of epitope mapping using CD4 T cells obtained from C57BL/10 mice revealed that the diversity of the repertoire was very limited. Most of the proteins that were studied contained only a limited number of epitopes; however, while some proteins contained only subdominant epitopes, others had several highly dominant epitopes that accounted for the majority of the anti-influenza reactivity in the CD4 T-cell repertoire. When candidate positive NP peptides were screened individually (), nine epitopes were identified, of which four were “superdominant.” Two of these superdominant epitopes were in overlapping peptides, and thus likely belong to a single unique CD4 T-cell epitope. Individual peptide screening of the NA protein revealed that the response observed in the large peptide pools could be accounted for by eight epitopes out of a total of 78 peptides tested, with the responses ranging from around 30 to 200 recruited CD4 T cells (). In NS1 (), four subdominant epitopes were identified, each of which recruited approximately 80 CD4 T cells per million cells, while in M1 (), three minor epitopes were discovered that recruited around 50 CD4 T cells per million cells. A similar pattern of only four epitopes was observed for HA, all of which elicited on average less than 150 CD4 T cells per million cells (). Based on these data, we concluded that there was a narrow distribution of epitopes restricted to the I-Ab MHC class II molecule in C57BL/10 mice that accounted for the overall reactivity detected in the pools, with less than 15 epitopes eliciting CD4 T cells at a frequency of greater than 100 per million cells tested, or 0.01%. The CD4 T-cell response specific for NP and NA dominated the immune response to influenza, in large part due to the strongly immunodominant epitopes present in these proteins.
When we examined the CD4 T-cell specificity from I-As-restricted cells obtained from SJL mice, we again found that the diversity of the repertoire was quite limited. Similarly to C57BL/10 mice, candidate NP epitopes were individually screened and 10 peptide epitopes were identified, many of which were dominant or superdominant (). Several of these (16/17, 45/46, and 74/75) were present in overlapping peptides and thus likely represent a single CD4 T-cell epitope (). However, in contrast to what was found in C57BL/10 mice, 13 peptide epitopes were identified in HA, four of which elicited greater than 300 CD4 T cells per million cells (). In screening for reactivity towards NA in SJL mice, only four epitopes were discovered, and these elicited a cytokine response from only a modest number of CD4 cells (). CD4 T cells specific for NS1 and M1 were identified by screening individual peptides, with only one subdominant epitope identified in NS1 that elicited on average around 150 spots per million CD4 T cells (). In M1, there was one subdominant and two dominant epitopes present (), all of which elicited less than 300 CD4 T cells per million cells. Based on these data, we were able to conclude that similarly to the case in C57BL/10 mice, there was also a relatively narrow distribution of epitopes restricted to the I-As molecule in SJL mice. However, in this strain the CD4 T-cell response specific for HA dominated the immune response to influenza, again in large part due to the strongly immunodominant epitopes present.
In order to ensure that the differences in CD4 T-cell protein specificity noted between the two mouse strains examined were not the result of differences in the genetic background of the mice leading to, for example, different rates of influenza clearance in vivo, we obtained and infected B10.S mice with A/New Caledonia influenza. B10.S mice are MHC congenic with C57BL/10 mice, and like SJL mice, they express the H-2s haplotype genes and thus the I-As protein. Screening EliSpot assays were done in these mice using the same matrices previously used to discover epitopes in the SJL mouse strain. No striking differences were found in the patterns of positive and negative pools restricted to the I-As molecule in the HA, NA, or NP matrices (, respectively). Also, select I-As-restricted epitopes were screened in parallel in SJL and B10.S mice, with no major dissimilarities in the immunodominance pattern of positive epitopes noted (). Overall, these results indicate that the differences in CD4 T-cell protein specificity found between the C57BL/10 and SJL mouse strains are the result of the MHC molecule expressed, rather than the result of differences in non-MHC-related genetic background genes that might affect factors such as viral gene expression or influenza growth in vivo.
FIG. 3. Comparison of the HA, NA, and NP peptide-pooling matrices and individual peptides in the SJL and B10.S mouse strains. HA (A), NA (B), and NP (C) peptide-pooling matrices were screened in EliSpot assays using CD4-enriched T cells obtained from previously (more ...)
Based on these results, we concluded that there was a relatively low degree of diversity in the primary influenza-specific CD4 T-cell response restricted to both the I-Ab and I-As MHC class II molecules. summarizes the epitopes identified in both strains, and when known, the core epitopes as determined through the use of truncated peptides (data not shown). All of the proteins that were screened had only a limited number of epitopes, of which several were strongly immunodominant. Given the limited diversity of the overall response, it was these strongly immunodominant epitopes that determined the overall protein specificity of the responding CD4 T cells. Interestingly, this distribution was quite distinct in the two mouse strains examined in this study, depending on the MHC class II molecule presenting the peptides in the host.