Figure S5.
Additional characterization of experimental models, related to Figs. 6, 7, and 8. (A) Percentage of CD45+ donor cells (both Cxcr6−/− and WT) and recipient cells in the blood of BM chimera on day 67 following BM transplantation. Left panel: Bar plot with individual data points for the percentage of donor cells and recipient cells. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by one-way ANOVA, with Tukey’s multiple comparison test. Right panel: A representative flow cytometry plot showing the percentage of donor cells and recipient cells. (B) Bar plot with individual data points showing the percentage of donor and recipient cells among CD4 T cells in different organs in the BM chimera on day 30 after infection with PR8. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by two-way ANOVA, with Tukey’s multiple comparison test. (C) Representative microscopic image of CXCL16 expression (magenta) in the olfactory epithelium of the murine NT is shown. Two negative controls for CXCL16 staining showing NT stained (1) with α-rabbit secondary IgG Texas red only and (2) stained with isotype control and α-rabbit secondary IgG Texas red. Hoechst is indicated in grey. NT is isolated on day 30 after PR8-OVA infection from mice that received OT-II CD4 T cells. Top images are stitched to show the whole NT. Scale bar: 500 μm for top panels and 100 μm for bottom. (D) Gating strategy to identify CD4 TEM (CD4+CCR7−CD45RA−), CD4 TCM (CD4+CCR7+CD45RA−), naïve CD4 Tc (CD4+CCR7+CD45RA+), CD4 TEMRA(CD4+CCR7−CD45RA+), and CD4 TRM (CD4+CCR7−CD45RA−CD69+) in NT and blood. (E) RORγt+CD4+ T cells in the PPs of WT C57BL/6 mice, CD4creRorcfl/wt, and CD4creRorcfl/fl mice. Left panel: Bar plot with individual data points showing the frequency of RORγt+CD4+ T cells among all CD3+ T cells. The experiment was done twice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. Right panel: A representative flow cytometry plot for the percentage of RORγt+CD4+ T cells in different groups. (F) Microscopy images (magnified) for TUNEL+ cells in the nasal septum (respiratory region) from CD4creRorcfl/wtand CD4creRorcfl/fl mice (same as Fig. 8 G). Scale bar: 50 μm. (G and H) Microscopy for TUNEL+ cells and viral titer (TCID50/g) from organs of mice infected with PR8 and reinfected with X31 IAV on day 30 following PR8 IAV infection. The mice were treated with isotype or IL-17a/f antibody. (G) Bar plot with individual data points showing number of TUNEL+ cells in the nasal septum (respiratory region) on day 4 after X31 IAV infection. The experiment was repeated twice. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. (H) Viral titer (TCID50/g) from NT and lungs on day 3 after X31 IAV infection. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. TEMRA, terminally differentiated effector memory T cells re-expressing CD45RA. Refer to the image caption for details. Panel A shows bar plots and flow cytometry plots of donor and recipient chimerism in blood after bone marrow transplantation. Panel B shows bar plots of donor and recipient C D 4 T cell distribution across organs in chimeric mice. Panel C shows microscopic images of C X C L 16 expression in nasal tissue with isotype and secondary antibody controls. Panel D shows gating strategy plots identifying C D 4 T cell subsets including T E M T C M T R M and naive populations. Panel E shows bar plots and flow cytometry plots of R O R gamma t positive C D 4 T cells across different genotypes. Panel F shows microscopic images of TUNEL positive cells in nasal septum comparing different genetic backgrounds. Panel G shows bar plots of TUNEL positive cell counts in nasal tissue following infection and antibody treatment. Panel H shows bar plots of viral titers in nasal tissue and lungs after reinfection and cytokine blockade.

Additional characterization of experimental models, related to Figs. 6, 7, and 8. (A) Percentage of CD45+ donor cells (both Cxcr6−/− and WT) and recipient cells in the blood of BM chimera on day 67 following BM transplantation. Left panel: Bar plot with individual data points for the percentage of donor cells and recipient cells. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by one-way ANOVA, with Tukey’s multiple comparison test. Right panel: A representative flow cytometry plot showing the percentage of donor cells and recipient cells. (B) Bar plot with individual data points showing the percentage of donor and recipient cells among CD4 T cells in different organs in the BM chimera on day 30 after infection with PR8. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by two-way ANOVA, with Tukey’s multiple comparison test. (C) Representative microscopic image of CXCL16 expression (magenta) in the olfactory epithelium of the murine NT is shown. Two negative controls for CXCL16 staining showing NT stained (1) with α-rabbit secondary IgG Texas red only and (2) stained with isotype control and α-rabbit secondary IgG Texas red. Hoechst is indicated in grey. NT is isolated on day 30 after PR8-OVA infection from mice that received OT-II CD4 T cells. Top images are stitched to show the whole NT. Scale bar: 500 μm for top panels and 100 μm for bottom. (D) Gating strategy to identify CD4 TEM (CD4+CCR7−CD45RA−), CD4 TCM (CD4+CCR7+CD45RA−), naïve CD4 Tc (CD4+CCR7+CD45RA+), CD4 TEMRA(CD4+CCR7−CD45RA+), and CD4 TRM (CD4+CCR7−CD45RA−CD69+) in NT and blood. (E) RORγt+CD4+ T cells in the PPs of WT C57BL/6 mice, CD4creRorcfl/wt, and CD4creRorcfl/fl mice. Left panel: Bar plot with individual data points showing the frequency of RORγt+CD4+ T cells among all CD3+ T cells. The experiment was done twice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. Right panel: A representative flow cytometry plot for the percentage of RORγt+CD4+ T cells in different groups. (F) Microscopy images (magnified) for TUNEL+ cells in the nasal septum (respiratory region) from CD4creRorcfl/wtand CD4creRorcfl/fl mice (same as Fig. 8 G). Scale bar: 50 μm. (G and H) Microscopy for TUNEL+ cells and viral titer (TCID50/g) from organs of mice infected with PR8 and reinfected with X31 IAV on day 30 following PR8 IAV infection. The mice were treated with isotype or IL-17a/f antibody. (G) Bar plot with individual data points showing number of TUNEL+ cells in the nasal septum (respiratory region) on day 4 after X31 IAV infection. The experiment was repeated twice. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. (H) Viral titer (TCID50/g) from NT and lungs on day 3 after X31 IAV infection. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. TEMRA, terminally differentiated effector memory T cells re-expressing CD45RA.

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