Long-Term Maintenance of Virus-Specific Effector Memory CD8⁺ T Cells in the Lung Airways Depends on Proliferation

Sendai virus NP_324–332/K^b-specific memory CD8⁺ T cells in the lung airways and spleen are maintained by homeostatic proliferation. C57BL/6 mice were intranasally infected with 500 EID₅₀ Sendai virus and then given BrdU by continuous feeding in the drinking water starting at either 1 mo (A) or 12 mo (B) after infection. On various days after BrdU initiation, cells were isolated from the lung airways (○) or spleen (▪) and stained with Sen-NP_324–332/K^b tetramer-PE, anti-CD8-Tricolor, and anti-BrdU-FITC Abs. The data are shown as BrdU incorporation among CD8⁺/tetramer⁺ gated T cells and the error bars indicate the SD of three individual mice. The full time course was done once, but the day 6 and day 21 time points were repeated several times with similar results.

Sendai virus-specific memory CD8⁺ T cells persist in the lung airways for >2 wk. C57BL/6 mice were infected intranasally with Sendai virus and on day 16 after infection CFSE (80 μl) was introduced directly into the lung airways via the trachea. On days 1 (A and B), 4 (C), and 15 (D) after CFSE labeling, cells were isolated from the lung airways (BAL) and stained with Sen-NP_324–332/K^b tetramer-PE and anti-CD8-Tricolor. The data in A are a histogram of CFSE fluorescence with the frequency of CFSE⁺ cells among the lymphocyte population. The data in B–D are gated on CFSE⁺ lymphocytes and show Sen-NP_324–332/K^b tetramer vs CD8 staining. The percentages in these panels indicate the frequencies of tetramer⁺ cells among CD8⁺ T cells. The frequencies of CFSE⁺ lymphocytes in the spleen and MLN at 1 day after CFSE labeling were <0.7%. The data are representative of four individual experiments.

Sendai virus NP_324–332/K^b-specific memory CD8⁺ T cells in the lung airways persist predominantly in the G₀-G₁ stage of the cell cycle. BAL and spleen cells were isolated from C57BL/6 mice at various times after Sendai virus infection and stained with Sen-NP_324–332/K^b and anti-CD8-Tricolor. The cells were then stained with Hoechst 33342 and analyzed as described in Materials and Methods. The data show the percentages of Sen-NP_324–332/K^b tetramer⁺/CD8⁺ (A) cells or Sen-NP_324–332/K^b tetramer-negative/CD8⁺ (B) cells that were in G₂-S phase of the cell cycle. The line represents the average of two experiments.

Sendai virus-specific CD8⁺ T cells isolated from the lung airways do not proliferate after intratracheal transfer into naive mice. BAL cells were collected from C57BL/6 (Thy1.2⁺) mice at day 31 after Sendai virus infection, labeled with CFSE, and transferred into naive C57BL/6 (Thy1.1⁺) mice via the trachea. On day 8 posttransfer, cells isolated from the spleen, MLN, BAL, or lung tissue were analyzed for donor cell Thy1.2 expression (left panels). In addition, at days 1, 2, 4, and 8 after transfer, BAL cells were isolated and stained with Sen-NP_324–332/K^b tetramer-PE, anti-CD8-CyChrome, and anti-Thy1.2-allophycocyanin (right panels). The data are gated on Thy1.2⁺/CD8⁺ lymphocytes and are representative of five independent experiments. The absolute numbers of donor cells recovered from the BAL was quite variable between experiments. However, in every experiment, donor cells were only recovered from the BAL, and not other tissues.