Figure 8.
NT harbors Th17 CD4 TRM that reduce local pathology. (A and B) Frequency of IL-17a+ cells among CD4 TRM of NT and lungs upon restimulation with IAV immunodominant NP306–322 peptide in comparison with unstimulated cells. The organs are isolated on day 30 following PR8 IAV infection of mice and from naïve mice. (A) A representative flow cytometry plot showing the percentage of IL-17a in lungs and NT. (B) Bar plot with individual data points indicating the frequency of IL-17a+ cells among all CD4 TRM. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (C) Percentage of IL-17a+ cells among CD4 TRM of the lungs and NT that are stimulated with NP306–322 peptide in the presence or absence of brefeldin A. Left panel: Graph showing expression of IL-17a from the cells that are stimulated in the presence or absence of brefeldin A connected by a line. Each line on the graph indicates the cells from individual mouse. The experiment was repeated twice, and the results are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by two-sided Wilcoxon matched-pairs signed-rank test. Right panel: Representative flow cytometry plots showing the expression of IL17-a among CD4 TRM of lungs and NT. (D and E) Viral titers in the NT of CD4creRorctfl/fl and CD4creRorcfl/wt mice on day 3 following secondary infection with X31 IAV. The mice were infected with PR8 and reinfected with X31 IAV on day 30 after primary infection and treated with fingolimod i.p. as indicated. (D) A schematic representation of the experimental setup. (E) Bar plot with individual data points showing viral titers (TCID50/g) in the NT of the two groups. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (F–H) Microscopy for TUNEL+ cells in the nasal septum (respiratory region) derived from CD4creRorctfl/fl and CD4creRorcfl/wt mice. The mice were infected with PR8 and reinfected with X31 IAV on day 30 following PR8 IAV infection. The tissues were isolated on day 4 and day 8 following X31 IAV infection. (F) A schematic representation of the experimental setup. (G) Representative microscopic images for TUNEL+ cells (brown) in the nasal septum from mice on day 4 after X31 IAV infection are shown. Scale bar: 100 µm for the main image, 50 μm for insets. A magnified version of this image is shown in Fig. S5 F. (H) Bar plot with individual data points showing the number of TUNEL+ cells per mm2 in the respiratory region of the nasal septum. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (I) Microscopy for PR8 mCherry (yellow) in the nasal septum (respiratory region) of mice infected with PR8 mCherry virus. Scale bar: 50 μm for the main image, 10 μm for insets. The NT was isolated on day 3 after infection. Refer to the image caption for details. Panel A shows flow cytometry plots of I L 17 a producing C D 4 T R M in nasal tissue and lungs with stimulation conditions. Panel B shows bar plots of I L 17 a positive C D 4 T R M frequencies comparing naive and infected conditions. Panel C shows line graphs and flow cytometry plots of I L 17 a expression with or without brefeldin treatment. Panel D shows schematic diagram of infection and reinfection experimental timeline with treatment conditions. Panel E shows bar plots of viral titers in nasal tissue comparing different genetic backgrounds. Panel F shows schematic diagram of infection and tissue collection timeline for pathology analysis. Panel G shows microscopic images of TUNEL positive cells in nasal tissue indicating tissue damage. Panel H shows bar plots of TUNEL positive cell counts per area comparing experimental groups. Panel I shows microscopic images of P R 8 m Cherry signal in nasal tissue indicating viral localization.

NT harbors Th17 CD4 TRM that reduce local pathology. (A and B) Frequency of IL-17a+ cells among CD4 TRM of NT and lungs upon restimulation with IAV immunodominant NP306–322 peptide in comparison with unstimulated cells. The organs are isolated on day 30 following PR8 IAV infection of mice and from naïve mice. (A) A representative flow cytometry plot showing the percentage of IL-17a in lungs and NT. (B) Bar plot with individual data points indicating the frequency of IL-17a+ cells among all CD4 TRM. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (C) Percentage of IL-17a+ cells among CD4 TRM of the lungs and NT that are stimulated with NP306–322 peptide in the presence or absence of brefeldin A. Left panel: Graph showing expression of IL-17a from the cells that are stimulated in the presence or absence of brefeldin A connected by a line. Each line on the graph indicates the cells from individual mouse. The experiment was repeated twice, and the results are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by two-sided Wilcoxon matched-pairs signed-rank test. Right panel: Representative flow cytometry plots showing the expression of IL17-a among CD4 TRM of lungs and NT. (D and E) Viral titers in the NT of CD4creRorctfl/fl and CD4creRorcfl/wt mice on day 3 following secondary infection with X31 IAV. The mice were infected with PR8 and reinfected with X31 IAV on day 30 after primary infection and treated with fingolimod i.p. as indicated. (D) A schematic representation of the experimental setup. (E) Bar plot with individual data points showing viral titers (TCID50/g) in the NT of the two groups. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (F–H) Microscopy for TUNEL+ cells in the nasal septum (respiratory region) derived from CD4creRorctfl/fl and CD4creRorcfl/wt mice. The mice were infected with PR8 and reinfected with X31 IAV on day 30 following PR8 IAV infection. The tissues were isolated on day 4 and day 8 following X31 IAV infection. (F) A schematic representation of the experimental setup. (G) Representative microscopic images for TUNEL+ cells (brown) in the nasal septum from mice on day 4 after X31 IAV infection are shown. Scale bar: 100 µm for the main image, 50 μm for insets. A magnified version of this image is shown in Fig. S5 F. (H) Bar plot with individual data points showing the number of TUNEL+ cells per mm2 in the respiratory region of the nasal septum. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (I) Microscopy for PR8 mCherry (yellow) in the nasal septum (respiratory region) of mice infected with PR8 mCherry virus. Scale bar: 50 μm for the main image, 10 μm for insets. The NT was isolated on day 3 after infection.

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