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. 2006 Jan 24;103(4):994-9.
doi: 10.1073/pnas.0510429103. Epub 2006 Jan 17.

A virus-specific CD8+ T cell immunodominance hierarchy determined by antigen dose and precursor frequencies

Nicole L La Gruta  1 , Katherine Kedzierska, Ken Pang, Richard Webby, Miles Davenport, Weisan Chen, Stephen J Turner, Peter C Doherty
Affiliations

A virus-specific CD8+ T cell immunodominance hierarchy determined by antigen dose and precursor frequencies

Nicole L La Gruta et al. Proc Natl Acad Sci U S A. .

Abstract

Immunodominance hierarchies are a substantial, but poorly understood, characteristic of CD8(+) T cell-mediated immunity. Factors influencing the differential responses to the influenza A virus nucleoprotein (NP(366-374)) and acid polymerase (PA(224-233)) peptides presented by H2D(b) have been analyzed by disabling (N5--> Q substitution) these peptides in their native configuration, then expressing them in the viral neuraminidase protein. This strategy of shifting epitopes within the same viral context resulted in an apparent equalization of D(b)NP(366) [epitope consisting of viral nucleoprotein (NP) amino acid residues 366-374 complexed with the H2D(b) MHC class I glycoprotein] and D(b)PA(224) (H2D(b)+PA(224-233)) epitope abundance after direct infection in vitro and induced reproducible changes in the magnitude of the D(b)NP(366)- and D(b)PA(224)-specific T cell subsets generated after infection of mice. Comparison of D(b)NP(366)- and D(b) PA(224)-specific CD8(+) T cell responses induced from the native configuration and from the viral neuraminidase stalk demonstrated that the size of both primary and secondary responses is influenced by relative epitope levels and that, at least after secondary challenge, the magnitude of responses is also determined by CD8(+) T cell precursor frequency. Thus, this immunodominance hierarchy is a direct function of antigen dose and T cell numbers. VSports手机版.

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Figures

Fig. 1.
Fig. 1.
Insertion of the NP366 or PA224 peptides into the viral NA has no effect on virus replication in the lung but alters DbNP366 and DbPA224 presentation levels on APCs. Four or five B6 mice were infected i.n. with 200 pfu of PR8 wt, NANP, or NAPA viruses, and lungs were sampled for virus titration 24 h later (A). EL4 cells were infected with PR8 wt, NANP, or NAPA viruses for 3 h, and RNA was extracted 5 h later. After reverse transcription, cDNA was amplified by real-time PCR by using SYBR chemistry and NP-, PA-, and NA-specific oligonucleotides. Data are shown as amount of signal relative to PA (B). BmDCs, DC2.4 cells, and EL4 cells were infected in vitro with the designated PR8 recombinant or wt viruses for 1 h at 37°C. After infection, APCs were added to short-term DbNP366- or DbPA224-specific CTL lines in the presence of brefeldin A at the times shown (C–H). The T cells were harvested 4 h later and analyzed for expression of CD8α and IFN-γ. Results are plotted as the percentage of the maximal response determined by stimulating CTL lines with EL4 cells at saturating peptide doses. Shown are the DbNP366-specific (C–E) and DbPA224-specific (F–H) CTL responses to epitopes presented by BmDCs (C and F), DC2.4 cells (D and G), and EL4 cells (E and H) after infection with the indicated viruses.
Fig. 2.
Fig. 2.
The magnitude of acute and memory DbNP366- and DbPA224-specific responses is altered after NANP and NAPA viral challenge. B6 mice were infected i.p. with NP+PA–, NANP, PA+NP–, or NAPA influenza A viruses, and splenocytes were sampled 10 days (A) or 55 days (B) later. Cells were stained with the DbNP366-PE or DbPA224-PE tetramers followed by anti-CD8α-FITC. *, P < 0.0005; **, P < 0.00001 comparing NP+PA– with NANP or PA+NP– with NAPA. #, P < 0.001; ##, P < 0.00001 comparing NP+PA– with PA+NP– or NANP with NAPA (Student's t test). The results in A are shown for individual mice from four separate experiments. Data in B are mean ± SD for groups of five mice.
Fig. 3.
Fig. 3.
Consequences of priming and boosting with recombinant viruses in B6 μMT mice. Mice were primed i.p. with 1.5 × 107 pfu of the NP+PA–, NANP, PA+NP–, or NAPA influenza A viruses and then challenged i.p. 5 weeks later with the indicated PR8 viruses (1.5 × 107 pfu). Splenocytes were sampled 8 days after secondary challenge, and T cells were stained with the DbNP366-PE (A) or DbPA224-PE (B) tetramers followed by anti-CD8α-FITC. Shown are the mean numbers of CD8+tetramer+ cells for, generally, groups of three to five mice. *, P < 0.05; **, P < 0.01 (Student's t test).
Fig. 4.
Fig. 4.
The magnitude of secondary recall is influenced by the level of epitope presentation. B6 mice were primed i.p. with 1.5 × 107 pfu of the HKx31 NP+PA–, PA+NP– variants (A) or with wt HKx31 virus (B), then challenged i.n. 6–8 weeks later with 200 pfu of the PR8 NP+PA–, NANP, PA+NP–, or NAPA viruses. Splenocytes were harvested 8 days later and stained with DbNP366-PE or DbPA224-PE tetramers followed by anti-CD8α-FITC. Shown are the mean splenic T cell numbers for groups of four to five mice. For comparison, data from the corresponding groups of B6 μMT mice (see Fig. 3) are shown in A as open bars. *, P < 0.05 comparing NP+PA– with NANP challenge or PA+NP– with NAPA challenge. #, P < 0.01 comparing NP+PA– with PA+NP– challenge (Student's t test).
Fig. 5.
Fig. 5.
A proposed model linking the contributions of precursor frequencies and epitope levels as determinants of CD8+ T cell response magnitude, and thus immunodominance hierarchies, after influenza virus infection.
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