In vivo proliferation of naïve and memory influenza-specific CD8(+) T cells

Abstract

The virus-specific CD8(+) T cell response has been analyzed through the development, effector, and recovery phases of primary and secondary influenza pneumonia. Apparently, most, if not all, memory T cells expressing clonotypic receptors that bind a tetrameric complex of influenza nucleoprotein (NP)(366-374) peptide+H-2D(b) (NPP) are induced to divide during the course of this localized respiratory infection. The replicative phase of the recall response ends about the time that virus can no longer be recovered from the lung, whereas some primary CD8(+)NPP(+) T cells may proliferate for a few more days. The greatly expanded population of CD8(+)NPP(+) memory T cells in the lymphoid tissue of secondarily challenged mice declines progressively in mean prevalence over the ensuing 100 days, despite the fact that at least some of these lymphocytes continue to cycle. The recall of cell-mediated immunity thus is characterized by massive proliferation of the antigen-specific CD8(+) set, whereas the extent of lymphocyte turnover in the absence of cognate peptide is variable, at a low level, and can be influenced by intercurrent infection.

Figure 1

Comparison of virus clearance and the acute response in the BAL after primary or secondary challenge with the HKx31 influenza A virus. Naïve (Primary) or PR8 (H1N1)-immune (Secondary) mice were infected i.n. with the HKx31 (H3N2) virus. The BAL samples from each group (n = 5–6) were pooled, stained for surface CD8 and NPP tetramer, and analyzed by flow cytometry. The BAL cell counts per mouse (□) were used, together with the flow cytometry data, to calculate average numbers for the total CD8⁺ (○) and NPP⁺ T cells (▵). Lung virus titers were determined as log₁₀EID₅₀ for groups of three mice (●).

Figure 2

Long-term quantitation of CD8⁺NPP⁺ T cells in the MLN and spleen. Groups of naïve (primary) or PR8-immune (secondary) mice were analyzed through the acute and memory stages of the immune response to the HKx31 virus. The values were determined for three to five individual mice at each time point. The results show the mean ± SD of percentage of NPP⁺ within the CD8⁺ set. A further set of experiments in which CD8⁺ T cells from secondarily challenged mice were stimulated in vitro with the NP_366–374 peptide in the presence of brefeldin A and then fixed and stained for the presence of cytoplasmic IFN-γ (2, 3) gave values for the MLN and spleen, respectively, of: day 29, 13 ± 1, 15 ± 10; day 42, 10 ± 10, 17 ± 5; day 62, 17 ± 9, 10 ± 9; day 100, 2 ± 1; 7 ± 4.

Figure 3

Profiles of in vivo BrdUrd incorporation for the BAL CD8⁺ populations recovered from naïve or PR8-immune mice at 8 days after respiratory challenge with the HKx31 virus. Groups of 10 immunologically naive (A and B) or PR8-immune (C) mice were infected i.n. with 10^6.8 EID₅₀ of HKx31. One group of 10 naïve mice was placed on normal drinking water (A), and all other mice were given BrdUrd for 8 days from the time of infection (B and C). The BAL cells were pooled from groups of five mice, depleted of macrophages by adherence to plastic, surface-stained with the NPP tetramer and anti-CD8, and then fixed and stained for BrdUrd. The FACS profiles were gated on CD8⁺ T cells in the lymphoblast gate. R1 is the background staining for BrdUrd that was determined in the group not given BrdUrd water. R3 contains BrdUrd^hi CD8⁺NPP⁺ and R2 contains BrdUrd^int (intermediate) CD8⁺NPP⁺ T cells.

Figure 4

Profiles of BrdUrd incorporation and loss for CD8⁺NPP⁺ T cells recovered from the MLN, spleen, and BAL of naïve and PR8 memory mice challenged with the HKx31 virus. Groups of naive (primary, 1°) or PR8-immune (secondary, 2°) B6 animals were given BrdUrd drinking water at the time of infection with HKx31 until day 5 (A, D, and G) or day 8 (B, E, and H), and then half the mice were sampled. The remaining mice were placed on normal drinking water for 3 days (A, D, and G) or 5 days (B, E, and H) and then sampled. Other mice (C, F, and I) were given BrdUrd water from day 12 postinfection and either sampled on day 16 or maintained on normal water until day 21. The CD8⁺ T cells were enriched from the MLN and spleen of five individual mice per group per time point. The BAL cells were collected and pooled from five mice per group. Samples were gated on the CD8⁺NPP⁺ (1°, solid bar; 2°, open bar) or T cells and analyzed for BrdUrd staining. The SD values for individuals are not shown for clarity of the figure.

Figure 5

Long-term turnover of virus-specific CD8⁺ T cells that proliferated during the course of the acute infection. The PR8-immune mice were infected i.n. with the HKx31 virus and given BrdUrd in the drinking water for 8 days, beginning on the day of secondary challenge. Thereafter, they were maintained on normal water. Typical BrdUrd-staining profiles for this pulse–chase analysis are presented for the CD8⁺NPP⁺ set recovered from the spleens of individuals sampled at 8 or 73 days after exposure to the HKx31 virus (A). The results presented in B are mean ± SD values for groups of four mice assayed at the end of the day 8 pulse and at 2, 3, 5, and 10 weeks after infection.