IAV-specific NT CD4 TRM are induced by vaccination and provide protection. (A) I-A^b HA_91–107 tetramer-specific CD4 TRM in the NT and lungs of mice that were infected with X31 i.n. or left uninfected on day 30 following PR8 infection. The mice were treated with FTY720, and the organs were analyzed 6 days after X31 infection. Bar plot with individual data points indicating the percentage of I-A^b HA_91–107 tetramer⁺ cells among CD4 TRM in lungs and NT. The experiment was done twice, 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. (B) I-A^b NP_306–322 tetramer-specific CD4 TRM in the NT and lungs of mice 6 days after X31 infection. The mice were treated with FTY720 from day −2 to day 5 after infection. Left: Bar plot with individual data points indicating the percentage of I-A^b NP_306–322 tetramer⁺ cells among CD4 TRM of lungs and NT. The experiment was done twice, and the results (mean ± SEM) were pooled. Right: Representative flow cytometry plots indicating the percentages of I-A^b NP_306–322 tetramer-specific CD4 TRM are shown. (C) Percentage of CD4⁺ and CD8⁺ T cells in the blood of mice on day 3 following secondary X31 infection from different groups as indicated in the Fig. 3 D. The experiment was done twice and the results (mean ± SEM) were 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. (D) Bar plot with individual data points showing the frequency of I-A^b NP_306–322 tetramer⁺ cells among CD3⁺ T cells in the NT of mice treated with isotype control antibody or anti-CD4 and anti-CD8 antibody. The experiment was performed with four to five mice per group. The result is shown as mean ± SEM. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (E) Percentage of relative infection of MDCK cells by X31 IAV after incubation with serum derived on day 30 after infection of mice with PR8 or X31. The serum from mice immunized with irrelevant protein (COVID spike or streptavidin) was used as controls. The experiment was performed twice, and the results (mean ± SEM) are pooled. (F) Bar plot with individual data points indicating the absolute number of I-A^b NP_306–322⁺ CD4 TRM in lungs and NT of mice on day 14 and day 30 after infection with PR8 intratracheally. The experiment was performed with five mice per group. The result is shown as mean ± SEM. 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. (G–I) I-A^d M2e_2–17 tetramer⁺ CD4 TRM in the NT of BALB/c on day 10 after third immunization with CTA1-DD or CTA1-3M2e-DD i.n. (G) Schematic representation of the experimental setup. (H) Representative flow cytometry plots indicating the percentage of I-A^d M2e_2–17 tetramer⁺ CD4 TRM from different treatment groups. Staining with I-A^d Human CLIP_87–101 tetramer (CLIP control) is used as the negative control. (I) Bar plot with individual data points indicating the percentage of I-A^d M2e_2–17 tetramer⁺ cells among all CD4 TRM. The experiment was repeated twice, 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. (J–M) I-A^d M2e_2–17 tetramer⁺ CD4 TRM in the NT and lungs of BALB/c on day 30 after last immunization with CTA1-3M2e-DD i.n. (J) Schematic representation of the experimental setup. (K) Bar plot with individual data points indicating the percentage of I-A^d M2e_2–17 tetramer⁺ cells among all CD4 TRM of lungs and NT. The experiment was repeated twice, 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) Representative flow cytometry plots indicating the percentage of I-A^d M2e_2–17 tetramer⁺ CD4 TRM and I-A^d human CLIP_87–101 tetramer⁺ CD4 TRM from lungs and NT. (M) Bar plot with individual data points indicating the percentage of I-A^d M2e_2–17 tetramer⁺ cells among all CD4 TEM of MLN and cLN in mice immunized as shown in Fig. S3 J. The experiment was repeated twice, 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. (N–P) Survival rate and weight loss of CTA1-3M2e-DD or CTA1-DD-immunized BALB/c mice that were infected with PR8 in URT-restricted manner and were treated with or without FTY720 and anti-CD4 antibody or anti-CD8 antibody or isotype control antibody. (N) Schematic representation of the experimental setup. (O) Kaplan–Meier survival curves of mice from different groups after PR8 infection. The experiment was repeated thrice, and the results were pooled. *P < 0.05 by log-rank Mantel–Cox test. Significant differences were found between CTA1DD versus CTA1M2eDD+Iso (*) and CTA1DD versus CTA1M2eDD+Iso+FTY720 (*). Comparisons between CTA1M2eDD+Iso versus CTA1M2eDD+a-CD4+FTY720/CTA1M2eDD+a-CD8+FTY720/CTA1M2eDD+a-CD4+a-CD8+FTY720 were run but nonsignificant. (P) Weight loss curve for mice from different treatment groups as indicated. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. Statistical comparisons between treatment groups (NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05) were performed using a linear mixed-effects model with comparisons of weight across treatment groups (averaged over time) using estimated marginal means and pairwise comparisons with Tukey’s honest significant difference adjustment. Statistical comparisons across groups for each day are reported in Table S2. (Q) Scatter plot indicating viral titers (TCID₅₀/g) in NT and lungs of immunized mice as shown in Fig. S3 N on day 3 following infection with PR8 IAV. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. No significant difference was found between the groups by one-way ANOVA, with Dunnett’s multiple comparison test.