IAV-specific CD4 TRM are formed at the URT and are dependent on presence of cognate antigen. (A) Schematic diagram showing the procedure of transfer of OVA-specific (OT-II) CD4 T cells, infection of mice with PR8-OVA, and subsequent harvesting of organs. (B and C) CD45.1⁺ or tdTomato⁺ OT-II CD4 TRM (gated on live CD3⁺CD4iv⁻CD4 tissue⁺CD44⁺CD62L⁻CD69⁺ as shown in Fig. S1 A) in different organs of CD45.2⁺ recipients on day 30 following PR8-OVA infection. (B) Representative flow cytometry plots indicating the percentages of CD45.1⁺ OT-II CD4 TRM. The data are representative of one experiment out of three independent experiments. (C) Bar graph indicating the absolute number of CD45.1⁺ or tdTomato⁺ OT-II CD4 TRM. 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 two-way ANOVA, with Tukey’s multiple comparison test. (D) Representative microscopic images of OT-II⁺ (red) CD4⁺ (green) T cells in different sections of the NT and NALT out of two independent experiments. The sections are derived from day 30 following PR8-OVA infection of mice that received OT-II CD4 T cells as described in Fig. 1 A. A stitched microscopic image showing different regions of the NT and NALT is shown at the top right panel. Scale bar: 1 mm for the main image, 100 μm for insets I and II, and 20 μm for inset III. OE, olfactory epithelium; S, nasal septum. (E and F) Frequency of CD103⁺ and CD11a⁺ cells among OT-II CD4 TRM in NT and lungs (gated on live CD3⁺CD4iv⁻CD4 tissue⁺CD44⁺CD62L⁻CD69⁺), and among total CD4 effector memory T cells (CD3⁺CD4 iv⁺CD44⁺CD62L⁻) in blood of CD45.2⁺ recipients on day 30 following IAV infection. (E) Representative flow cytometry plots indicating the percentages of CD103⁺ and CD11a⁺ cells. The data are representative of one experiment out of two independent experiments. (F) Bar plot with individual data points indicating the percentages of CD103⁺ and CD11a⁺ cells. Each data point indicates an individual mouse. 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 one-way ANOVA, with Dunnett’s multiple comparison test. (G–I) Kinetics of I-A^b NP_306–322 tetramer, I-A^b HA_91–107 tetramer-specific CD4 TRM, and OT-II⁺ CD4 TRM in the NT and lungs on indicated days following PR8 or PR8-OVA IAV infection. (G) Schematic representation of the experimental setup. (H) Representative flow cytometry plots indicating the percentages of I-A^b NP_306–322 tetramer and I-A^b HA_91–107 tetramer-specific CD4 TRM are shown. (I) Bar plot with individual data points indicating the absolute numbers of I-A^b NP_306–322 tetramer, I-A^b HA_91–107 tetramer-specific CD4 TRM, and OT-II⁺ CD4 TRM. Each data point indicates an individual mouse. The experiment was performed twice, and the results (mean + SEM) were plotted. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by one-way ANOVA on log(x + 1)-transformed counts, with Dunnett’s multiple comparison test. (J) Bar plot with individual data points showing the percentage of Ki-67⁺ cells among I-A^b NP_306–322 tetramer⁺ and I-A^b HA_91–107 tetramer⁺ CD4 TRM in lung and NT isolated on day 10, 30, and 60 after PR8 infection. The experiment was performed 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. (K–M) Frequency of OT-II CD4 TRM in the NT on day 22 following infection with PR8 or PR8-OVA. (K) Schematic representation of the experimental setup. (L) A representative flow cytometry plot indicating the frequency of OT-II CD4 TRM. (M) Bar plot with individual data points showing the frequency of OT-II 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.