Consequences of immunodominant epitope deletion for minor influenza virus-specific CD8+-T-cell responses

Abstract

The extent to which CD8+ T cells specific for other antigens expand to compensate for the mutational loss of the prominent DbNP366 and DbPA224 epitopes has been investigated using H1N1 and H3N2 influenza A viruses modified by reverse genetics. Significantly increased numbers of CD8+ KbPB1(703)+, CD8+ KbNS2(114)+, and CD8+ DbPB1-F2(62)+ T cells were found in the spleen and in the inflammatory population recovered by bronchoalveolar lavage from mice that were first given the -NP-PA H1N1 virus intraperitoneally and then challenged intranasally with the homologous H3N2 virus. The effect was less consistent when this prime-boost protocol was reversed. Also, though the quality of the response measured by cytokine staining showed some evidence of modification when these minor CD8+-T-cell populations were forced to play a more prominent part, the effects were relatively small and no consistent pattern emerged. The magnitude of the enhanced clonal expansion following secondary challenge suggested that the prime-boost with the -NP-PA viruses gave a response overall that was little different in magnitude from that following comparable exposure to the unmanipulated viruses. This was indeed shown to be the case when the total response was measured by ELISPOT analysis with virus-infected cells as stimulators. More surprisingly, the same effect was seen following primary challenge, though individual analysis of the CD8+ KbPB1(703)+, CD8+ KbNS2(114)+, and CD8+ DbPB1-F2(62)+ sets gave no indication of compensatory expansion. A possible explanation is that novel, as yet undetected epitopes emerge following primary exposure to the -NP-PA deletion viruses. These findings have implications for both natural infections and vaccines.

FIG. 1.

Extent of compensation by CD8⁺ D^bPA₂₂₄⁺ and CD8⁺ K^bPB1₇₀₃⁺ T cells in mice primed and challenged with the mutant viruses. The B6 mice were infected i.p. (10⁸ EID₅₀) with PR (H1N1) and challenged i.n. (10⁷ EID₅₀) with HK (H3N2) RG (solid bars) and −NP (open bars), −PA (diagonally striped bars), and −NP−PA (diamond-patterned bars) mutant viruses. Spleen (A to C), BAL sample (D to F), and MLN (G to I) populations were sampled on day 0 (spleen only, at 8 weeks after i.p. priming), day 7, day 10, and day 30 (spleen only) after i.n. challenge. The numbers of virus-specific CD8⁺ T cells were calculated from total cell counts, and percent values were determined by staining with the D^bNP₃₆₆ (A, D, and G), D^bPA₂₂₄ (B, E, and H) and K^bPB1₇₀₃ (C, F, and I) tetramers. It is important to note that the scales for the y axis differ, reflecting the number of cells recovered from the site sampled. The results are expressed as means ± standard deviations, and statistical significance (relative to the HK-WT challenge) was determined by Student's t test (*, P ≤ 0.05, n = 5).

FIG. 2.

Localization of CD8⁺ K^bPB1₇₀₃⁺, CD8⁺ K^bNS2₁₁₄⁺, and CD8⁺ K^bM1₁₂₈⁺ IFN-γ⁺ T cells to the respiratory tract of virus secondarily challenged mice. This is the same experiment shown in Fig. 1. Individual BAL samples were stimulated with 1 μM concentrations of the PB1_703-711 (A), NS2_114-121 (B), and M1_128-135 (C) peptides for 5 h and stained for IFN-γ expression (PepC assay). The numbers of IFN-γ⁺ CD8⁺ T cells were calculated and expressed as means ± standard deviations. Differences from the WT group are indicated by an asterisk (P ≤ 0.05, by Student's t test).

FIG. 3.

Tetramer analysis of the K^bNS2₁₁₄-specific set in spleen and BAL fluid populations from secondarily challenged mice. The i.p. priming with PR viruses and i.n. challenge with HK viruses were done exactly as described in the legend to Fig. 1, and lymphocyte populations were sampled on day 10 after homologous challenge. The results (means ± standard deviations, n = 5) are expressed as the percentage of cells staining (A) and the numbers of epitope-specific cells (B). Significant differences from the WT group are indicated by an asterisk (P ≤ 0.05).

FIG. 4.

Characteristics of the primary virus-specific CD8⁺-T-cell response. The B6 mice were infected i.n. with 10⁷ EID₅₀ of the HK variants. The spleen (A to D) and BAL fluid (E) populations were sampled on day 10 at the peak of the acute response, and the early phase of memory was measured on day 30 (A to D) for the spleen. The numbers of tetramer⁺ CD8⁺ T cells were calculated from total cell counts and the percentages of cells staining. The results are expressed as means ± standard deviations, and significant differences from the HK-WT values are indicated by an asterisk (P ≤ 0.05).

FIG. 5.

Prevalence of IFN-γ⁺ TNF-α⁺ CD8⁺ K^bPB1₇₀₃⁺ and CD8⁺ K^bNS2₁₁₄⁺ T cells in a secondary response. The mice were primed and challenged as described in the legend to Fig. 1. The levels of TNF-α⁺ and IFN-γ⁺ production were determined by intracellular cytokine staining subsequent to peptide stimulation (see legend to Fig. 2), and the IFN-γ⁺ TNF-α⁺/IFN-γ⁺ ratios were determined for the BAL fluid and spleen populations. The results are expressed as means ± standard deviations, and significant differences from the findings for the WT group are indicated by an asterisk (P ≤ 0.05).

FIG. 6.

Intensity of IFN-γ and TNF-α staining for peptide-stimulated CD8⁺ K^bPB1₇₀₃⁺ and CD8⁺ K^bNS2₁₁₄⁺ T cells. The MFI values are shown for IFN-γ⁺ (A to D) and TNF-α⁺ (E to H) in T cells from spleen (A, B, E, and F) and BAL samples (C, D, G, and H). The statistical analysis compares values significantly different from those for the WT, and the asterisk denotes P ≤ 0.05.

FIG. 7.

Comparison of the prevalence of epitope-specific and nonspecific CD8⁺ T cells. The total CD8⁺-cell counts (A and B) were subdivided to compare the size of the virus-specific (C and D) IFN-γ⁺ CD8⁺-T-cell sets from Table 1. (A and C) Spleen populations; (B and D) BAL sample populations. The asterisk indicates P ≤ 0.05, where the −NP, −PA, and −NP−PA groups were compared to WT by a two-sided exact P value for the two-sample Wilcoxon rank-sum test.

FIG. 8.

ELISPOT analysis of the influenza virus-specific CD8⁺-T-cell response. The total virus-specific response assay was performed following primary (A) or secondary (B) infection with the WT, −NP, −PA, and −NP−PA influenza viruses. Purified CD8⁺ splenocytes from infected mice were incubated for 48 h with MHC class II^+/+ (B6) and MHC class II^−/− (IA^b−/−) APCs that were infected in vitro with WT HKx31 influenza virus. The numbers of IFN-γ-producing cells are expressed as spots per million CD8⁺ T cells. The results are shown as means ± standard errors (n = 4), and the asterisk indicates values that differed significantly from the WT result (P < 0.05). The analysis was repeated (data not shown) with comparable results.

FIG. 9.

Prevalence of CD8⁺ PB1-F2₆₂ population in the primary and secondary response. The IFN-γ⁺ CD8⁺ PB1-F2₆₂⁺-T-cell numbers (per mouse) in spleen (A and C) and BAL sample (B and D) populations were determined by the PepC assay following primary (day 10, panels A and B) and secondary (days 7 and 10, panels C and D) i.n. challenge. (A and C) Results from spleen and BAL samples for primary infection; (C and D) data from spleen and BAL samples for secondary infection. The HKx31 WT and mutant viruses were given i.n. while the homologous PR viruses were given i.p. 6 weeks previously to those mice that were secondarily challenged. The values for spleen that were significantly different from those for the WT are identified by an asterisk (P < 0.05). Spleen samples were tested individually, and the BAL samples were pooled from the four mice. The experiment was repeated with comparable results (data not shown).