Panel A shows flow cytometry plots of C D 4 T R M cytokine expression including I F N gamma I L 2 and T N F with stimulation comparison. Panel B shows bar plots of C D 4 T R M cytokine responses across N P H A N A and N S 1 peptides indicating immunodominance patterns. Panel C shows bar plots of P D 1 and I C O S normalized expression comparing activation states of C D 4 T R M in nasal tissue and lungs.
IAV-specific CD4 TRM of the NT are functional and exhibit immunodominance hierarchy similar to the lungs. (A) Flow cytometry plots indicating the percentage of CD4 TRM expressing IFN-γ, IL-2, and TNF with and without stimulation with IAV NP peptide pool. The organs are isolated on day 30 following PR8 IAV infection of mice. (B) Bar plot with individual data points indicating the percentage of CD4 TRM-expressing IFN-γ, IL-2, and TNF after stimulation with IAV NP peptide pool, IAV HA peptide pool, IAV NA peptide pool, and IAV NS1 peptide pool. The organs are isolated on day 30 following PR8 IAV infection of mice. The experiment was performed thrice, and the results (mean ± SEM) were pooled. NS, not significant; ***P < 0.001; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (C) Bar plot with individual data points showing the expression of PD-1 and ICOS (MFI normalized to average MFI of respective markers on NT CD4 TRM) on CD4 TRM of lungs and NT on day 30 following infection with PR8 IAV. The experiment was repeated twice, and the results (mean ± SEM) are pooled. NS, not significant; ***P < 0.001; *P < 0.05 by unpaired two-tailed t test. MFI, median fluorescence intensity.