Figure 7.
Functional IAV-specific CD4 TRM exist in the nasopharynx of healthy human subjects. (A) Bar plot with individual data points showing the frequency of CD4 Tc in the NT and PBMC. The experiment was performed seven times, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; *P < 0.05 by unpaired two-tailed t test. (B) Bar plot with individual data points showing the frequency of naïve CD4 Tc (CD4+CCR7+CD45RA+), CD4 TCM (CD4+CCR7+CD45RA−), CD4 TEM (CD4+CCR7−CD45RA−), CD4 TEMRA (CD4+CCR7−CD45RA+), and CD4 TRM (CD4+CCR7−CD45RA−CD69+) in NT and blood. NS, not significant; ****P < 0.0001; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (C and D) Expression of CD103 and CXCR6 on NT CD4 TRM and CD4 TEM of PBMC derived from healthy human subjects. (C) Representative flow cytometry plots indicating the percentage of CD103 and CXCR6 expression on CD4 TRM and CD4 TEM from the same subject. (D) Bar plot with individual data points showing the percentage of CD103 and CXCR6 expression on CD4 TRM and CD4 TEM. Each data point indicates one subject. The experiment was performed five times, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; *P < 0.05 by unpaired two-tailed t test. (E) Percentage of NT CD4 TRM and CD4 TEM from PBMC-expressing IL-17a, IFN-γ, and TNFα. The cytokine expression is indicated from unstimulated cells and IAV NP peptide pool and M1 peptide pool-stimulated cells connected by a line. Each line on the graph indicates cytokine expression from the cells of each subject. NS, not significant; ****P < 0.0001; *P < 0.05 by two-sided Wilcoxon matched-pairs signed-rank test. (F) Bar plot with individual data points showing the percentage of NT CD4 TRM and CD4 TEM from PBMC-expressing IL-17a, IFN-γ, and TNF after stimulation with IAV NP peptide pool and M1 peptide pool (after subtraction of signals from the unstimulated control; negative values considered zero). The experiment was performed seven times, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (G) Representative flow cytometry plots showing the expression of IL-17a in NT CD4 TRM and CD4 TEM of PBMC, which are stimulated with IAV NP peptide pool and M1 peptide pool or left unstimulated. TEMRA, terminally differentiated effector memory T cells re-expressing CD45RA. Refer to the image caption for details. Panel A shows bar plots of C D 4 T cell frequencies comparing nasal tissue and peripheral blood mononuclear cells in healthy subjects. Panel B shows bar plots of C D 4 T cell subsets including naive T C M, T E M, T R M, and T E M R A across tissues. Panel C shows flow cytometry plots of C D 103 and C X C R 6 expression on C D 4 T R M and peripheral memory T cells. Panel D shows bar plots of C D 103 and C X C R 6 expression comparing nasal tissue and peripheral blood memory T cells. Panel E shows line graphs of cytokine expression including I L 17 a I F N gamma and T N F before and after stimulation. Panel F shows bar plots of cytokine producing C D 4 T cells comparing nasal tissue and peripheral blood after stimulation. Panel G shows flow cytometry plots of I L 17 a expression in stimulated and unstimulated C D 4 T cells across tissues.

Functional IAV-specific CD4 TRM exist in the nasopharynx of healthy human subjects. (A) Bar plot with individual data points showing the frequency of CD4 Tc in the NT and PBMC. The experiment was performed seven times, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; *P < 0.05 by unpaired two-tailed t test. (B) Bar plot with individual data points showing the frequency of naïve CD4 Tc (CD4+CCR7+CD45RA+), CD4 TCM (CD4+CCR7+CD45RA−), CD4 TEM (CD4+CCR7−CD45RA−), CD4 TEMRA (CD4+CCR7−CD45RA+), and CD4 TRM (CD4+CCR7−CD45RA−CD69+) in NT and blood. NS, not significant; ****P < 0.0001; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (C and D) Expression of CD103 and CXCR6 on NT CD4 TRM and CD4 TEM of PBMC derived from healthy human subjects. (C) Representative flow cytometry plots indicating the percentage of CD103 and CXCR6 expression on CD4 TRM and CD4 TEM from the same subject. (D) Bar plot with individual data points showing the percentage of CD103 and CXCR6 expression on CD4 TRM and CD4 TEM. Each data point indicates one subject. The experiment was performed five times, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; *P < 0.05 by unpaired two-tailed t test. (E) Percentage of NT CD4 TRM and CD4 TEM from PBMC-expressing IL-17a, IFN-γ, and TNFα. The cytokine expression is indicated from unstimulated cells and IAV NP peptide pool and M1 peptide pool-stimulated cells connected by a line. Each line on the graph indicates cytokine expression from the cells of each subject. NS, not significant; ****P < 0.0001; *P < 0.05 by two-sided Wilcoxon matched-pairs signed-rank test. (F) Bar plot with individual data points showing the percentage of NT CD4 TRM and CD4 TEM from PBMC-expressing IL-17a, IFN-γ, and TNF after stimulation with IAV NP peptide pool and M1 peptide pool (after subtraction of signals from the unstimulated control; negative values considered zero). The experiment was performed seven times, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (G) Representative flow cytometry plots showing the expression of IL-17a in NT CD4 TRM and CD4 TEM of PBMC, which are stimulated with IAV NP peptide pool and M1 peptide pool or left unstimulated. TEMRA, terminally differentiated effector memory T cells re-expressing CD45RA.

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