Figure 3.
NT CD4 TRM provide protection against heterosubtypic challenge. (A–C) I-Ab NP306–322 tetramer and I-Ab HA91–107 tetramer-specific CD4 TRM in the NT and lungs of mice that were infected with X31 i.n. or left uninfected on day 30 following PR8 infection. The mice were treated with FTY720, and the organs were analyzed 6 days after X31 infection. (A) Schematic representation of the experimental setup. (B) Bar plot with individual data points indicating the percentage of I-Ab NP306–322 tetramer + cells among CD4 TRM of lungs and NT. The experiment was done twice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (C) Representative flow cytometry plots indicating the percentages of I-Ab NP306–322 tetramer and I-Ab HA91–107 tetramer-specific CD4 TRM are shown. (D) Schematic representation of primary and secondary infection of mice, their treatment with anti-CD4 antibody, anti-CD8 antibody, or/and FTY720 on the indicated days, and collection of NT. (E) Left panel: Bar plot with individual data points showing the frequency of CD4 Tc and CD8 Tc among all live cells in the NT of mice treated with isotype control antibody or anti-CD4 and anti-CD8 antibody. The experiment was performed with four to five mice per group. The result is shown as (mean ± SEM). NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. Right panel: A representative flow cytometry plot for the frequency of CD4 and CD8 Tc among total live cells. (F) Scatter plot indicating viral titers (TCID50/g) in NT of mice on day 3 following secondary infection with X31 IAV. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by one-way ANOVA with Dunnett’s multiple comparison test. The outliers were identified and removed using Grubbs’ method (alpha = 0.05). One outlier each removed from all groups except for groups: PR8 + anti-CD8 + FTY720 and PR8 + anti-CD4. (G–J) Viral titer and frequency of I-Ab NP306–322+ CD4 TRM in the NT and lungs of mice who have undergone i.t. or i.n. PR8 infection followed by i.n. infection with X31 on day 30 after primary infection. The mice were treated with FTY720+/− anti-CD4 antibody or left untreated. (G) Schematic representation of the experimental set up. (H) Bar plot with individual data points indicating the frequency of I-Ab NP306–322 tetramer+CD4iv−CD44+CD62L− cells in the NT and lungs of mice on day 3 after secondary X31 infection +FTY720 treatment versus mice without secondary X31 infection and not treated with FTY720 as shown in Fig. 3 G. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (I) Representative flow cytometry plots indicating percentages of I-Ab NP306–322 tetramer+CD4iv−CD44+CD62L− cells in NT and lung of mice with and without secondary X31 infection (related to H). (J) Scatter plot showing viral titers (TCID50/g) in NT and lungs of mice on day 3 following secondary infection with X31 IAV who were infected with PR8 i.n. or intratracheally 30 days prior to X31 infection. The mice were treated with FTY720 i.p. as indicated in Fig 3 G. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by one-way ANOVA, with Dunnett’s multiple comparison test. Refer to the image caption for details. Panel A shows schematic diagram of infection timeline with P R 8 priming X 31 challenge F T Y 7 20 treatment and tissue harvest, Panel B shows bar plots of tetramer positive C D 4 T R M frequencies in nasal tissue and lungs following heterosubtypic infection, Panel C shows flow cytometry plots of I A b N P and H A tetramer specific C D 4 T R M populations, Panel D shows schematic diagram of primary and secondary infections with antibody depletion and F T Y 720 treatment timeline, Panel E shows bar plots and flow cytometry plots of C D 4 and C D 8 T cells after antibody mediated depletion, Panel F shows scatter plots of viral titers in nasal tissue following secondary infection under different depletion and treatment conditions, Panel G shows schematic diagram of infection routes intranasal and intratracheal with secondary challenge and F T Y 720 treatment, Panel H shows bar plots of tetramer positive C D 4 T R M frequencies in nasal tissue and lungs after secondary infection, Panel I shows flow cytometry plots of tetramer positive C D 4 T R M in nasal tissue and lungs with or without challenge, Panel J shows scatter plots of viral titers comparing infection routes and treatment conditions in nasal tissue and lungs.

NT CD4 TRM provide protection against heterosubtypic challenge. (A–C) I-Ab NP306–322 tetramer and I-Ab HA91–107 tetramer-specific CD4 TRM in the NT and lungs of mice that were infected with X31 i.n. or left uninfected on day 30 following PR8 infection. The mice were treated with FTY720, and the organs were analyzed 6 days after X31 infection. (A) Schematic representation of the experimental setup. (B) Bar plot with individual data points indicating the percentage of I-Ab NP306–322 tetramer + cells among CD4 TRM of lungs and NT. The experiment was done twice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (C) Representative flow cytometry plots indicating the percentages of I-Ab NP306–322 tetramer and I-Ab HA91–107 tetramer-specific CD4 TRM are shown. (D) Schematic representation of primary and secondary infection of mice, their treatment with anti-CD4 antibody, anti-CD8 antibody, or/and FTY720 on the indicated days, and collection of NT. (E) Left panel: Bar plot with individual data points showing the frequency of CD4 Tc and CD8 Tc among all live cells in the NT of mice treated with isotype control antibody or anti-CD4 and anti-CD8 antibody. The experiment was performed with four to five mice per group. The result is shown as (mean ± SEM). NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. Right panel: A representative flow cytometry plot for the frequency of CD4 and CD8 Tc among total live cells. (F) Scatter plot indicating viral titers (TCID50/g) in NT of mice on day 3 following secondary infection with X31 IAV. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by one-way ANOVA with Dunnett’s multiple comparison test. The outliers were identified and removed using Grubbs’ method (alpha = 0.05). One outlier each removed from all groups except for groups: PR8 + anti-CD8 + FTY720 and PR8 + anti-CD4. (G–J) Viral titer and frequency of I-Ab NP306–322+ CD4 TRM in the NT and lungs of mice who have undergone i.t. or i.n. PR8 infection followed by i.n. infection with X31 on day 30 after primary infection. The mice were treated with FTY720+/− anti-CD4 antibody or left untreated. (G) Schematic representation of the experimental set up. (H) Bar plot with individual data points indicating the frequency of I-Ab NP306–322 tetramer+CD4iv−CD44+CD62L− cells in the NT and lungs of mice on day 3 after secondary X31 infection +FTY720 treatment versus mice without secondary X31 infection and not treated with FTY720 as shown in Fig. 3 G. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (I) Representative flow cytometry plots indicating percentages of I-Ab NP306–322 tetramer+CD4iv−CD44+CD62L− cells in NT and lung of mice with and without secondary X31 infection (related to H). (J) Scatter plot showing viral titers (TCID50/g) in NT and lungs of mice on day 3 following secondary infection with X31 IAV who were infected with PR8 i.n. or intratracheally 30 days prior to X31 infection. The mice were treated with FTY720 i.p. as indicated in Fig 3 G. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by one-way ANOVA, with Dunnett’s multiple comparison test.

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