Figure 2.
NT CD4 TRM express cytokines upon antigen stimulation. (A and B) Expression of intracellular cytokines in CD4 TRM of NT and lungs upon restimulation with IAV NP peptide pool. The organs are isolated on day 30 following PR8 IAV infection of mice or from naïve mice. (A) Flow cytometry plots indicating the percentage of CD4 TRM-expressing IFN-γ, IL-2, and TNF. (B) Bar plot with individual data points showing the percentage of CD4 TRM-expressing IFN-γ, IL-2, and TNF. 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. (C and D) Frequency of IFNγ+ cells among all lung and NT CD4 TRM isolated on day 30 after PR8 infection and that are stimulated with peptide pool (IAV NP versus SARS-CoV-2 N) pulsed splenocytes. (C) Representative flow cytometry plots showing the expression of IFNγ+ cells among all lung and NT CD4 TRM. (D) Graph indicating the IFNγ+ cells among CD4 TRM stimulated by IAV NP versus SARS-CoV-2 N pulsed splenocytes connected by a line. Each line indicates an individual mouse. The experiment was performed three times, and the results were pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by two-sided Wilcoxon matched-pairs signed-rank test. (E and F) Expression of ICOS and PD-1 on CD4 TRM on day 30 after PR8 IAV infection. (E) Representative flow cytometry plots of CD4 TRM-expressing ICOS and PD-1 in lungs and NT. (F) Bar plot with individual data points showing the percentage of CD4 TRM-expressing ICOS and PD-1. 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 unpaired two-tailed t test. Refer to the image caption for details. Panel A shows flow cytometry plots of cytokine producing C D 4 T R M in nasal tissue and lungs, Panel B shows bar plots of I F N gamma I L 2 and T N F expression in C D 4 T R M, Panel C shows flow cytometry plots of I F N gamma positive C D 4 T R M after stimulation, Panel D shows line graphs of I F N gamma responses comparing different antigen stimulations, Panel E shows flow cytometry plots of I C O S and P D 1 expression on C D 4 T R M, Panel F shows bar plots of I C O S and P D 1 expressing C D 4 T R M.

NT CD4 TRM express cytokines upon antigen stimulation. (A and B) Expression of intracellular cytokines in CD4 TRM of NT and lungs upon restimulation with IAV NP peptide pool. The organs are isolated on day 30 following PR8 IAV infection of mice or from naïve mice. (A) Flow cytometry plots indicating the percentage of CD4 TRM-expressing IFN-γ, IL-2, and TNF. (B) Bar plot with individual data points showing the percentage of CD4 TRM-expressing IFN-γ, IL-2, and TNF. 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. (C and D) Frequency of IFNγ+ cells among all lung and NT CD4 TRM isolated on day 30 after PR8 infection and that are stimulated with peptide pool (IAV NP versus SARS-CoV-2 N) pulsed splenocytes. (C) Representative flow cytometry plots showing the expression of IFNγ+ cells among all lung and NT CD4 TRM. (D) Graph indicating the IFNγ+ cells among CD4 TRM stimulated by IAV NP versus SARS-CoV-2 N pulsed splenocytes connected by a line. Each line indicates an individual mouse. The experiment was performed three times, and the results were pooled. NS, not significant; ****P < 0.0001; **P < 0.01; *P < 0.05 by two-sided Wilcoxon matched-pairs signed-rank test. (E and F) Expression of ICOS and PD-1 on CD4 TRM on day 30 after PR8 IAV infection. (E) Representative flow cytometry plots of CD4 TRM-expressing ICOS and PD-1 in lungs and NT. (F) Bar plot with individual data points showing the percentage of CD4 TRM-expressing ICOS and PD-1. 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 unpaired two-tailed t test.

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