Protection and compensation in the influenza virus-specific CD8+ T cell response

Abstract

Influenza virus-specific CD8+ T cells generally recognize peptides derived from conserved, internal proteins that are not subject to antibody-mediated selection pressure. Prior exposure to any one influenza A virus (H1N1) can prime for a secondary CD8+ T cell response to a serologically different influenza A virus (H3N2). The protection afforded by this recall of established CD8+ T cell memory, although limited, is not negligible. Key characteristics of primary and secondary influenza-specific host responses are probed here with recombinant viruses expressing modified nucleoprotein (NP) and acid polymerase (PA) genes. Point mutations were introduced into the epitopes derived from the NP and PA such that they no longer bound the presenting H2Db MHC class I glycoprotein, and reassortant H1N1 and H3N2 viruses were made by reverse genetics. Conventional (C57BL/6J, H2b, and Ig+/+) and Ig-/- (muMT) mice were more susceptible to challenge with the single NP [HKx31 influenza A virus (HK)-NP] and PA (HK-PA) mutants, but unlike the Ig-/- mice, Ig+/+ mice were surprisingly resistant to the HK-NP/-PA double mutant. This virus was found to promote an enhanced IgG response resulting, perhaps, from the delayed elimination of antigen-presenting cells. Antigen persistence also could explain the increase in size of the minor KbPB1703 CD8+ T cell population in mice infected with the mutant viruses. The extent of such compensation was always partial, giving the impression that any virus-specific CD8+ T cell response operates within constrained limits. It seems that the relationship between protective humoral and cellular immunity is neither simple nor readily predicted.

Fig. 1.

The in vitro growth characteristics (A) and profiles of weight loss (B) and survival (C–F) are shown for the HK-RG (triangles), HK-NP (squares), HK-PA (diamonds), and HK-NP/-PA (circles) viruses. (A) Madin–Darby canine cells were infected with a multiplicity of infection of 0.01, and viral titers from culture supernatants were measured by plaque assay at various time points after infection. (B) Groups of five B6 mice were infected i.n. with 10^6.3 eID₅₀ of the wt and mutant viruses and weighed at daily intervals together with uninfected controls (inverted triangles). The termination of the data set on day 7 reflects that evidence of severe morbidity was apparent in the groups given the mutant viruses. Survival profiles are shown for B6 (C and D), BALB/c (E), and μMT (F) mice infected i.n. with 10^6.0 (C and E) or 10^5.0 (D and F) eID₅₀ of the HK-variants.

Fig. 2.

Naive mice were infected i.n. with 10⁵ eID₅₀ of the HK-RG (open bars), HK-NP (filled bars), HK-PA (striped bars), or HK-NP/-PA (diamond bars) viruses. Lungs were sampled on days 3, 5, 7, 8, and 10, and then after infection they were frozen (-70°C) and later homogenized for virus titration by allantoic inoculation in embryonated hen's eggs. Virus titers are expressed as log₁₀ eID₅₀ per lung.

Fig. 3.

Mice primed i.p. 8 weeks previously with 10⁸ eID₅₀ of the PR-RG and mutant viruses were challenged i.n. with 10^6.3 eID₅₀ of the homologous HK variants. At various time points after infection, the splenic CD8⁺ T cell response was measured by staining with the D^bNP₃₆₆ (A and D), D^bPA₂₂₄ (B and E), and K^bPB1₇₀₃ (C and F) tetramers. The results are expressed as the percentage of cells staining (A–C) and the numbers of epitope-specific cells (D–F) that were calculated from the percentage values and the total cell counts (data not shown). The results are expressed as mean ± SD, and significant differences from the HK-RG are shown as * (P < 0.05) and † (P < 0.01). X denotes 100% death.

Fig. 4.

Mice previously primed with PR-RG and mutant viruses were challenged i.n. with 10^5.0 eID₅₀ of the homologous viruses. The virus-specific CD8⁺ T cell counts (see Fig. 3 legend) were measured for the spleen (A–C) and BAL (D–F) populations at various time points after challenge. The results are expressed as mean ± SD, and significant differences from the HK-RG are shown as * (P < 0.05) and † (P < 0.01).

Fig. 5.

Groups of seven naive B6 mice were infected i.n. with 10^6.0 eID₅₀ of the HK-RG (open bars), HK-NP (filled bars), HK-PA (striped bars), or HK-NP/-PA (diamond bars), and virus-specific IgG titers were determined by ELISA for serum samples taken on days 6 (A), 7 (B), and 9 (C) after infection. Titers are presented as the highest dilution that yielded an OD three times greater than that for a 1:100 dilution of preimmune sera. The results are expressed as mean ± SD, and significant differences from the HK-RG are shown as * (P < 0.01). The histograms are labeled as described in the Fig. 2 legend.