Figure S1.
Characterization of CD4 TRM in lungs and NT after IAV infection. (A) Gating strategy to identify OT-II+ CD4 TRM in the NT. (B) Representative microscopic images of OT-II+ (red) CD4+ (green) T cells in NALT and nasal septum. Hoechst is indicated in blue. The sections are derived from day 30 following PR8-OVA infection of mice that received OT-II CD4 T cells as described in Fig. 1 A. Scale bars are 50 μm for NALT and 20 μm for septum. Please note some nonspecific red and green staining of epithelial cell around the NALTs. (C and D) Expression of CD103 and CD11a (median fluorescence intensity) on OT-II CD4 TRM in NT and lungs and on total CD4 TEM of blood of CD45.2+ recipients on day 30 following IAV infection. The experiment was repeated thrice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by one-way ANOVA, with Tukey’s multiple comparison test. (D) Bar plot with individual data points indicating the percentage of I-Ab NP306–322 tetramer+, I-Ab HA91–107 tetramer+, and OT-II CD4 TRM in lung and NT on different dpi with PR8 OVA IAV i.n. Each data point indicates an individual mouse. The experiment was performed twice, and the results (mean + SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by one-way ANOVA on arcsine square-root transformed frequencies with Dunnett’s multiple comparison test. (E) Flow cytometry plot showing the percentage of OT-II+ CD4 TRM in the NT and lungs from different days after PR8 OVA infection. (F) Flow cytometry plot showing the percentage of I-Ab HA91–107 PE tetramer+ I-Ab HA91–107 BV421tetramer+ CD4 TRM and I-Ab human CLIP87–101 PE tetramer+ I-Ab human CLIP87–101 BV421 tetramer+ CD4 TRM in the NT of mice on day 60 after PR8 infection. (G) Representative flow cytometry plots of Ki67+ cells among I-Ab HA91–107 tetramer+ and I-Ab NP306–322 tetramer+ CD4 TRM of lungs and NT isolated on day 10, 30, and 60 after PR8 infection. (H and I) Frequency of OT-II CD4 TRM in the lung on day 22 following infection with PR8 or PR8-OVA. (H) Bar plot with individual data points showing the frequency of OT-II CD4 TRM. The experiment was performed twice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (I) A representative flow cytometry plot indicating the frequency of OT-II CD4 TRM. Refer to the image caption for details. Panel A shows gating strategy identifying O T 2 C D 4 T R M, Panel B shows microscopic images of O T 2 C D 4 cells in N A L T and septum, Panel C shows bar plots of C D 103 and C D 11 a expression levels, Panel D shows bar plots of tetramer positive and O T 2 C D 4 T R M frequencies, Panel E shows flow cytometry plots of O T 2 C D 4 T R M across timepoints, Panel F shows flow cytometry plots of antigen specific C D 4 T R M tetramer populations, Panel G shows flow cytometry plots of K i 67 positive antigen specific T R M, Panel H shows bar plots of O T 2 C D 4 T R M frequency in lungs, Panel I shows flow cytometry plots of O T 2 C D 4 T R M frequency.

Characterization of CD4 TRM in lungs and NT after IAV infection. (A) Gating strategy to identify OT-II+ CD4 TRM in the NT. (B) Representative microscopic images of OT-II+ (red) CD4+ (green) T cells in NALT and nasal septum. Hoechst is indicated in blue. The sections are derived from day 30 following PR8-OVA infection of mice that received OT-II CD4 T cells as described in Fig. 1 A. Scale bars are 50 μm for NALT and 20 μm for septum. Please note some nonspecific red and green staining of epithelial cell around the NALTs. (C and D) Expression of CD103 and CD11a (median fluorescence intensity) on OT-II CD4 TRM in NT and lungs and on total CD4 TEM of blood of CD45.2+ recipients on day 30 following IAV infection. The experiment was repeated thrice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by one-way ANOVA, with Tukey’s multiple comparison test. (D) Bar plot with individual data points indicating the percentage of I-Ab NP306–322 tetramer+, I-Ab HA91–107 tetramer+, and OT-II CD4 TRM in lung and NT on different dpi with PR8 OVA IAV i.n. Each data point indicates an individual mouse. The experiment was performed twice, and the results (mean + SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by one-way ANOVA on arcsine square-root transformed frequencies with Dunnett’s multiple comparison test. (E) Flow cytometry plot showing the percentage of OT-II+ CD4 TRM in the NT and lungs from different days after PR8 OVA infection. (F) Flow cytometry plot showing the percentage of I-Ab HA91–107 PE tetramer+ I-Ab HA91–107 BV421tetramer+ CD4 TRM and I-Ab human CLIP87–101 PE tetramer+ I-Ab human CLIP87–101 BV421 tetramer+ CD4 TRM in the NT of mice on day 60 after PR8 infection. (G) Representative flow cytometry plots of Ki67+ cells among I-Ab HA91–107 tetramer+ and I-Ab NP306–322 tetramer+ CD4 TRM of lungs and NT isolated on day 10, 30, and 60 after PR8 infection. (H and I) Frequency of OT-II CD4 TRM in the lung on day 22 following infection with PR8 or PR8-OVA. (H) Bar plot with individual data points showing the frequency of OT-II CD4 TRM. The experiment was performed twice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (I) A representative flow cytometry plot indicating the frequency of OT-II CD4 TRM.

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