![CXCR6–CXCL16 axis promotes NT CD4 TRM establishment. (A) Box plot showing the expression of Cxcr6 mRNA among CD4 TRM of the NT and lungs. Data are presented as median and interquartile range. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (B and C) Box plot showing the expression of Cxcr6 mRNA among different cell clusters of lungs and NT. Data are presented as median and interquartile range. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by Wilcoxon rank sum test with Benjamini–Hochberg correction. (C) Volcano plot for differential expressed genes in the NT in comparison with the lungs of the Th17 cluster. The dotted lines indicate fold change and adjusted P value cutoffs. (D) Bar plot with individual data points for the expression of CXCR6 (each median fluorescence intensity [MFI] normalized to mean MFI from CD4 TEM iv+ of NT) in CD4 TRM and iv+ CD4 TEM from the lungs and NT of naïve mice and PR8 IAV-infected mice (30 dpi) as indicated. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (E and F) Expression of CXCR6 on OT-II CD4 TRM of NT and lung on day 30 following PR8-OVA infection as indicated. (E) Representative histograms for CXCR6 expression from OT-II CD4 TRM of NT and lung are shown. (F) Bar plot with individual data points showing the expression of CXCR6 (each MFI normalized to mean MFI of NT OT-II CD4 TRM). The experiment was repeated thrice, 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. (G–K) Frequency of different cell populations within different organs derived from WT and Cxcr6−/− BM chimeric mice on day 30 after infection with PR8 IAV as indicated. (G) Schematic representation of the experimental setup. (H) Representative flow cytometry plots showing the percentage of WT and Cxcr6−/− CD4 TRM in NT and lungs. (I) Bar plot with individual data points for the percentage of WT and Cxcr6−/− CD4 TRM in NT and lungs as indicated. (J) Bar plot with individual data points for the percentage of I-Ab NP306–322 tetramer-specific WT and Cxcr6−/− CD4 TRM in NT and lungs as indicated. Samples that had <7 OT-II CD4 TRM were excluded from the analysis. The experiment was repeated thrice, 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. (K) Bar plot with individual data points showing the percentage of Cxcr6−/− and WT CD4 T cells in different organs of 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; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (L) A representative microscopic image of CXCL16 expression (magenta) and OT-II CD4 T cells (red and green) in the olfactory epithelium of the murine NT. NT is isolated on day 30 after PR8-OVA infection from mice that received OT-II CD4 T cells. Scale bar: 100 μm for main image and 50 μm for the inset. (M–O) Antigen-specific CD4 TRM in the lungs and NT of mice treated with isotype control or anti-CXCL16 antibody on day 10 after PR8 IAV infection. (M) Schematic representation of the experimental setup. (N) Representative flow cytometry plots indicating the percentage of I-Ab NP306–322 tetramer-specific CD4 TRM are shown. (O) Bar plot with individual data points indicating the absolute number of I-Ab NP306–322 tetramer-specific 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. Refer to the image caption for details.](https://cdn.rupress.org/rup/content_public/journal/jem/223/5/10.1084_jem.20251793/1/jem_20251793_fig6.png?Expires=1778597590&Signature=Hv6tTQpMFilyyGcgNBr03n2gOctIIlD00hmY7c6UX46BEs-lqNsHhIWmsi-HjA4waF5jzb~WxwexL8CVwjwIO2eqv5bniC9YjirXtOYG8cGmDtTa5jc9R7KCJUU4W6YonXYZeYu2tIVtafDZhsw1VY8es3lBna7buacqbnh58zXsQn6rCtIym1bUz032kU0Ve0L1TOPBg0TPDg7Hs9HxKZii2YoQIXCMRp7qx9a~Ov5AUSoey7iGgVxt4-7MDZJdxkcRSVHrDlXGKMVKYVGZy1IGopCPWmlW~DmYUQ~k3tiuHTaNl76WpXA1hHYXGo3eeV0AL6M18evmQiWf-LXKFw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A shows box plots of C x c r 6 expression in C D 4 T R M from nasal tissue and lungs. Panel B shows box plots of C x c r 6 expression across different C D 4 T cell clusters in tissues. Panel C shows volcano plots of differentially expressed genes in T h 1 7 cluster comparing nasal tissue and lungs. Panel D shows bar plots of C X C R 6 expression in C D 4 T R M and T E M across conditions. Panel E shows histograms of C X C R 6 expression on O T 2 (major histocompatibility complex class 2) C D 4 T R M in tissues. Panel F shows bar plots of normalized C X C R 6 expression on O T 2 C D 4 T R M in nasal tissue and lungs. Panel G shows schematic diagram of bone marrow chimera experimental design with infection and analysis timeline. Panel H shows flow cytometry plots of W T and C x c r 6 deficient C D 4 T R M in tissues. Panel I shows bar plots of W T and C x c r 6 deficient C D 4 T R M frequencies in nasal tissue and lungs. Panel J shows bar plots of tetramer specific W T and C x c r 6 deficient C D 4 T R M frequencies. Panel K shows bar plots of W T and C x c r 6 deficient C D 4 T cells across multiple organs. Panel L shows microscopic images of C X C L 16 expression and O T 2 C D 4 T cells in olfactory epithelium. Panel M shows schematic diagram of antibody treatment experimental timeline for C X C L 16 blockade. Panel N shows flow cytometry plots of tetramer specific C D 4 T R M after antibody treatment. Panel O shows bar plots of tetramer specific C D 4 T R M numbers after C X C L 16 blockade.
CXCR6–CXCL16 axis promotes NT CD4 TRM establishment. (A) Box plot showing the expression of Cxcr6 mRNA among CD4 TRM of the NT and lungs. Data are presented as median and interquartile range. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (B and C) Box plot showing the expression of Cxcr6 mRNA among different cell clusters of lungs and NT. Data are presented as median and interquartile range. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by Wilcoxon rank sum test with Benjamini–Hochberg correction. (C) Volcano plot for differential expressed genes in the NT in comparison with the lungs of the Th17 cluster. The dotted lines indicate fold change and adjusted P value cutoffs. (D) Bar plot with individual data points for the expression of CXCR6 (each median fluorescence intensity [MFI] normalized to mean MFI from CD4 TEM iv+ of NT) in CD4 TRM and iv+ CD4 TEM from the lungs and NT of naïve mice and PR8 IAV-infected mice (30 dpi) as indicated. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (E and F) Expression of CXCR6 on OT-II CD4 TRM of NT and lung on day 30 following PR8-OVA infection as indicated. (E) Representative histograms for CXCR6 expression from OT-II CD4 TRM of NT and lung are shown. (F) Bar plot with individual data points showing the expression of CXCR6 (each MFI normalized to mean MFI of NT OT-II CD4 TRM). The experiment was repeated thrice, 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. (G–K) Frequency of different cell populations within different organs derived from WT and Cxcr6−/− BM chimeric mice on day 30 after infection with PR8 IAV as indicated. (G) Schematic representation of the experimental setup. (H) Representative flow cytometry plots showing the percentage of WT and Cxcr6−/− CD4 TRM in NT and lungs. (I) Bar plot with individual data points for the percentage of WT and Cxcr6−/− CD4 TRM in NT and lungs as indicated. (J) Bar plot with individual data points for the percentage of I-Ab NP306–322 tetramer-specific WT and Cxcr6−/− CD4 TRM in NT and lungs as indicated. Samples that had <7 OT-II CD4 TRM were excluded from the analysis. The experiment was repeated thrice, 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. (K) Bar plot with individual data points showing the percentage of Cxcr6−/− and WT CD4 T cells in different organs of 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; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (L) A representative microscopic image of CXCL16 expression (magenta) and OT-II CD4 T cells (red and green) in the olfactory epithelium of the murine NT. NT is isolated on day 30 after PR8-OVA infection from mice that received OT-II CD4 T cells. Scale bar: 100 μm for main image and 50 μm for the inset. (M–O) Antigen-specific CD4 TRM in the lungs and NT of mice treated with isotype control or anti-CXCL16 antibody on day 10 after PR8 IAV infection. (M) Schematic representation of the experimental setup. (N) Representative flow cytometry plots indicating the percentage of I-Ab NP306–322 tetramer-specific CD4 TRM are shown. (O) Bar plot with individual data points indicating the absolute number of I-Ab NP306–322 tetramer-specific 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.