Identification of a CD4 T-cell epitope in the hemagglutinin stalk domain of pandemic H1N1 influenza virus and its antigen-driven TCR usage signature in BALB/c mice

Abstract

The stalk region of the influenza virus hemagglutinin is relatively well conserved compared with the globular head domain, which makes it a potential target for use as a universal vaccine against influenza. However, the role of CD4 T cells in the hemagglutinin stalk-specific immune response is not clear. Here we identified a mouse CD4 T-cell epitope that encompasses residues HA2_113-131 from the hemagglutinin stalk domain after a sub-lethal infection of influenza. In response to stimulation with the identified epitope, splenocytes derived from the infected mice showed significant polyfunctionality as shown by IL-2, TNF-α and IFN-γ production as well as degranulation. Moreover, mice immunized with the peptide corresponding to this CD4 T-cell epitope exhibited interindividual sharing of the CD4 T-cell receptor β sequences, and they had a higher survival rate following a challenge with a lethal dose of pandemic H1N1 influenza virus. Thus, our data demonstrated a crucial role of hemagglutinin stalk-specific CD4 T cells in the host immune response against influenza virus infection.

Figure 1

Identification of an HA stalk-specific CD4 T-cell epitope and memory HA stalk-specific CD4 T-cell immune responses. (a) IFN-γ ELISPOT analysis of splenocytes obtained 5 and 10 weeks after pH1N1 sublethal infection (n=8 and n=12, respectively). Cells were stimulated with a set of overlapping 15-mer peptides spanning the HA stalk protein. Stimulations were performed with individual peptides. (b) IFN-γ ELISPOT analysis of splenocytes obtained 10 weeks post pH1N1 sublethal infection (n=4) with the depletion of CD8^± T cells and CD49b^± NK cells (CD8⁻49b⁻ SP) or without depletion (control SP). ns, non-significant. (c) Splenocytes were harvested 10 weeks after pH1N1 sublethal infection and stimulated with a mixture of peptides 105, 106 and 107 or were stimulated with negative control peptides. Responses were visualized using an ICS assay. Representative flow cytometry profiles are shown. (d) The magnitude and polyfunctionality of CD4 T-cell responses. All of the possible combinations of the responses are shown on the X-axis, whereas the percentages of the functionally distinct cell populations within the total CD4 T-cell population are shown on the Y-axis. Rows: C, CD107a; I, IFN- γ 2, IL-2; T, TNF-α. Responses are grouped and color coded on the basis of the number of functions. ^*P<0.05. Error bars represent the s.d.

Figure 2

Detection of CD154-expressing CD4 T cells following HA stalk peptide immunization. (a) Scheme of peptide immunization. Mice were primed and boosted with peptides containing an HA stalk epitope (Pep_Immun group) or PBS (Mock group) in Alum. Two weeks after the final boost, splenocytes from mice were harvested for antigen-specific detection. (b) Splenocytes were re-stimulated for 12 h with or without the presence of peptides (Pep_restim. or No_restim.) and then stained for CD4 and CD154. One representative flow cytometry profile of four experiments is shown. (c) The frequency of CD154 expression in CD4 T-cell populations was measured. ^*P<0.05. Error bars represent the s.d.

Figure 3

Comparison of the TCRβ repertoires of CD4 T cells. (a) Vβ usage analysis. Each bar represents the mean frequency of a Vβ gene segment in the spleens of mice from the Pep_Immun group or the Mock group. (b) CDR3 spectratyping analysis. Each bar represents the relative frequency of a particular CDR3 length (in amino acids) measured across each individual spleen sample. Upper panel: Pep_Immun group (P1-P4); lower panel: Mock group (M1-M4). (c) Sharing of clones between each sample computed by averaging across the two ratios of shared reads over total reads for each sample.

Figure 4

Protective immunity. Mice immunized with HA stalk peptides (Pep_Immun), mice immunized with HA stalk peptides and depleted of CD4 T cells (Pep_Immun_CD4^dp), and mice immunized with PBS in alum (Mock) were challenged by 5 × MLD50 of mouse-adapted pH1N1 virus (A/Luxembourg/46/2009) two weeks after the final boost (n=12 mice/group, representative of two distinct experiments). (a) Body weight was monitored daily. (b) The percent survival rate after the virus challenge is shown by a Kaplan–Meier survival plot and was analyzed with the log-rank test and pairwise comparisons.