Fig. 6: Chemokines induced by NVT contribute to lung CD4⁺ T_RM cell formation, but direct codelivery of antigens is essential.

a, b WT (n = 5) and IFNAR1^−/− mice (n = 5) were immunized i.n. with NVT + H3N2. One day later, lung lysates and serum were collected. a Pooled lung lysates and serum samples were analyzed using a mouse cytokine kit. The signals were normalized to the pixel density of the indicated reference spots. b Concentrations of CCL2, CCL5, CXCL9, and CXCL10 in lung lysates and sera were measured via ELISA. c BALB/c Mice (n = 5 per group) were immunized i.n. with H3N2 or NVT. One day later, the levels of CCL2, CCL5, CXCL9, and CXCL10 in lung lysates and sera were assessed via ELISA. b, c Data are expressed as dot plots, with horizontal lines representing the medians. The two-sided Mann–Whitney U test was used for statistical analysis of differences between two groups. d, e BALB/c mice were immunized i.m. with H3N2 (n = 20), H3N2 + NVT (n = 10), or i.n. with H3N2 + NVT (n = 10) twice at two-week intervals. Two weeks later, mice injected i.m. with H3N2 (n = 10) or H3N2 + NVT (n = 10) were boosted i.n. with NVT. d On day 42, the proportion of lung CD4⁺ T_RM cells from vaccinated mice (n = 5 per group) was determined via flow cytometry. e The other vaccinated mice (n = 5 per group) were infected i.n. with 4 LD₅₀ of H1N1 virus, and survival was monitored daily. Survival data are represented as Kaplan‒Meier survival curves, and the significant differences in survival rates were calculated by the log-rank test. f BALB/c mice were immunized i.n. with H3N2 + NVT (n = 15) or i.m. with H3N2 (n = 20). Two weeks later, the mice were immunized i.m. with H3N2 or i.n. with H3N2 + NVT, H3N2, or NVT, and the proportion of lung CD4⁺ T_RM cells was determined via flow cytometry on day 42. d, f Box and whisker plots showing the median (center), 25th and 75th percentiles (box), and lowest and highest values (whiskers). Statistical analyses were performed using one-way ANOVA with the Tukey’s multiple comparisons test. This study was performed once. ns, not significant. Source data are provided as a Source Data file.