Analyses of the specificity of CD4 T cells during the primary immune response to influenza virus reveals dramatic MHC-linked asymmetries in reactivity to individual viral proteins

Abstract

Influenza is a contagious, acute respiratory disease that is a major cause of morbidity and mortality throughout the world. CD4 T cells play an important role in the immune response to this pathogen through the secretion of antiviral cytokines, and by providing help to CD8 T cells and B cells to promote the development of immunological memory and neutralizing antibody responses. Despite these well-defined roles in the anti-influenza response, our understanding of CD4 T-cell diversity and specificity remains limited. In the study reported here, overlapping peptides representing 5 different influenza viral proteins were used in EliSpot assays to enumerate and identify the specificity of anti-influenza CD4 T cells directly ex vivo following infection of mice with influenza virus, using two strains that express unrelated MHC class II molecules. These experiments evaluated whether the reactivity of CD4 T cells generally tracked with particular influenza proteins, or whether MHC preferences were the predominant factor dictating anti-CD4 T-cell specificity in the primary immune response. We made the unexpected discovery that the distribution of CD4 T-cell specificities for different influenza proteins varied significantly depending on the single class II molecule expressed in vivo. In SJL mice, the majority of epitopes were specific for the HA protein, while the NP protein dominated the response in C57BL/10 mice. Given the diversity of human MHC class II molecules, these findings have important implications for the ability to rationally design a vaccine that will generate a specific CD4 T-cell immune response that is effective across diverse human populations.

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 above proteins were pooled such that each peptide in the pools was at a final concentration of 2 μM. CD4 T-cell-enriched populations from previously infected mice were plated with the indicated peptide pool and the number of reactive cells was quantified by IL-2 EliSpots, as described in the materials and methods section. Data are shown as the average number of spots per 10⁶ CD4-enriched T cells, with error bars representing the standard error over all experiments. Reactivity of CD4 T cells from C57BL/10 (“B10”) mice are shown in panels A and B, while the reactivity of CD4 T cells from SJL mice are shown in panels C and D. (Color image is available online at www.liebertonline.com/vim.)

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 HA proteins, as indicated in each panel. Peptides were used at a final concentration of 10 μM, and the number of reactive CD4 T cells was quantified by the number of IL-2 EliSpots per 10⁶ CD4 T cells, as described in the Materials and Methods section. All peptides were tested in at least two individual experiments, with the data presented as the average number of spots per 10⁶ CD4-enriched T cells, and error bars representing the standard error over all experiments. Note that the standard error for peptide NP 46 in SJL mice in panel A is not fully depicted on the graph shown, as indicated by an asterisk, but is equal to 130 spots per 10⁶ CD4 T cells. Peptides were determined to be negative using either peptide-pooling matrices or by screening individual peptides.

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 infected SJL (white bars) and B10.S (black bars) mice, with each peptide in the pooling matrix at a final concentration of 10 μM. The pattern of positive and negative peptide pools was compared between the two mouse strains. Data are presented as the percentage of total spots, with error bars representing the standard error over at least two individual assays. Panel D shows a comparison of select I-A^s-restricted epitopes in an EliSpot assay using CD4-enriched T cells isolated from previously infected SJL (white bars) and B10.S (black bars) mouse strains in parallel. Data are presented as the average number of spots per 10⁶ CD4-enriched T cells, with error bars representing the standard error over all assays performed.