Primary CTL response magnitude in mice is determined by the extent of naive T cell recruitment and subsequent clonal expansion

Abstract

CD8+ T cell responses to viral infection are characterized by the emergence of dominant and subdominant CTL populations. The immunodominance hierarchies of these populations are highly reproducible for any given spectrum of virus-induced peptide-MHCI complexes and are likely determined by multiple factors. Recent studies demonstrate a direct correlation between naive epitope-specific CD8+ T cell precursor (CTLp) frequency and the magnitude of the response after antigen challenge. Thus, the number of available precursors in the naive pool has emerged as a key predictor of immunodominance. In contrast to this, we report here no consistent relationship between CTLp frequency and the subsequent magnitude of the immune response for 4 influenza virus-derived epitopes following intranasal infection of mice with influenza A virus. Rather, the characteristic, antigen-driven T cell immunodominance hierarchy was determined by the extent of recruitment from the available pool of epitope-specific precursors and the duration of their continued expansion over the course of the infection. These findings suggest possibilities for enhancing protective immune memory by maximizing both the size and diversity of typically subdominant T cell responses through rational vaccine design.

Figure 1. Influenza epitope–specific immune magnitudes do not correlate with naive precursor frequencies.

(A) Naive B6 mice were infected i.n. with influenza virus and spleens harvested 7, 8, or 10 days later for analysis of CD8⁺ D^bNP₃₆₆-, D^bPA₂₂₄-, D^bPB1-F2₆₂–, and K^bNS2₁₁₄-specific T cell responses. Shown are the mean total splenic numbers of CD8⁺tetramer⁺ cells for 4–5 mice ± SD. Results are representative of 2 independent experiments. (B) Representative dot plots of all D^bNP₃₆₆-, D^bPA₂₂₄-, D^bPB1-F2₆₂–, and K^bNS2₁₁₄-specific CD8⁺ T cells detected from spleen and all major LNs of naive mice using a magnetic enrichment and staining procedure (as described in Methods). Values indicate the number of tetramer⁺CD62L^hi cells within the gate shown. (C) Total numbers of epitope-specific CD8⁺ cells identified from spleen and all major LNs in naive B6 mice or OTI TCR Tg mice. Symbols represent data from individual mice obtained in 4 independent experiments. Horizontal bars indicate mean values.

Figure 2. Preferred TRBV usage in naive and immune epitope–specific CD8⁺ T cell populations.

(A) Representative dot plots of tetramer versus specific TRBV for CD8⁺ cells isolated from spleen and all major LNs of naive B6 mice after enrichment with the indicated tetramers. (B) Proportion of tetramer⁺CD8⁺ cells expressing the dominant TRBV in naive or immune B6 mice, as identified by flow cytometry (circles) or by single-cell RT-PCR for CD8 and TRBV (diamonds). Symbols represent data from individual mice obtained in at least 2 independent experiments.

Figure 3. Poor recruitment of CTLp specific for 1 subdominant epitope.

B6 mice were infected i.n. with influenza A virus and given BrdU in the drinking water at days 3 and 4 or days 5 and 6 after infection. Cells from spleen and major LNs were harvested the next day (day 5, panels A–D; day 7, panels E–H), enriched with specific tetramers, and analyzed for BrdU incorporation and CD44 expression. Shown are representative dot plots from a group of 5 individual mice with the proportion of CD8⁺CD3⁺CD4^–B220^–IA^b-F4/80^–tetramer⁺ cells shown for each quadrant. Data are representative of 2 independent experiments.

Figure 4. Reduced proliferation of subdominant epitope–specific CTLs late in infection.

(A) B6 mice were infected i.n. with influenza A virus and total cell tetramer cell numbers determined at days 5, 7, and 9 after infection. The fold expansion was determined by dividing the total number of CD8⁺tetramer⁺ CTL at each time point by the average number of naive CTLps identified from Figure 1. Data represent 5 mice per tetramer, per time point. Shown is the fold difference ± SD on a log₁₀ scale. (B) B6 mice were infected i.n. with influenza A virus and fed BrdU in their drinking water on days 3–5 after infection. Cells from spleen and major LNs were harvested at the end of the BrdU treatment on day 6, day 7, or day 8, enriched with specific tetramers, and analyzed for BrdU incorporation and CD44 expression. Shown is the mean proportion ± SD (n = 5 mice per group) of CD44⁺tetramer⁺BrdU⁺ CTL present for each epitope specificity. Data are representative of 2 independent experiments.

Figure 5. Expression of CD44 on epitope-specific CTLps from naive mice.

Epitope-specific CTLps were identified from naive mice as described in legend to Figure 1. The proportion of CD44^hi or CD44^lo epitope–specific CTLps are shown. Symbols represent data from individual mice. *P < 0.05 using Student’s t test with Bonferroni’s correction.