Fig. 4: The CD4⁺ T-cell response in the lung, not in the spleen, plays a critical role in cross-protection against heterosubtypic influenza virus.

a Experimental design. BALB/c mice (n = 10 per group) were immunized twice i.m. with H3N2 or H3N2 + NVT, or i.n. with H3N2 + NVT at two-week intervals. Two weeks after the last immunization, immune response analysis (n = 5 per group) (b, c, e) and H1N1 virus (A/Korea/2785/2009) challenge (n = 5 per group) (d) were performed. Schematic image was created in BioRender. Han, S. (2024) BioRender.com/e21s192. b After stimulating lung and spleen cells with H1N1 or H3N2, the IFN-γ⁺CD4⁺ T-cell response was determined by flow cytometry. c HAI antibody titers against H3N2 or H1N1 in serum were measured by HAI assay. An HAI antibody titer of 1:40 (red dotted line) was considered protective against influenza infection. d Mice were i.n. infected with 4 LD₅₀ of H1N1 virus, and body weight and survival were monitored daily. Weight-loss data are shown as mean value ± SD. Survival data are represented as Kaplan‒Meier survival curves, and the significant differences in survival rates were calculated by the log-rank test. e The proportion and number of CD4⁺ T_RM cells (CD4⁺CD44⁺CD62L^-CD69⁺) and CD8⁺ T_RM cells (CD8⁺CD44⁺CD62L^-CD69⁺) in the lungs were determined by flow cytometry. The gating strategy, frequency, and absolute number are shown. b, e Box and whisker plots show the median (center), 25th and 75th percentiles (box), and lowest and highest values (whiskers). b, c, e Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test. This study was performed independently at least three times, and one representative set is shown. BW, body weight; N.D., not detected; ns, not significant; NT, non-treated. Source data are provided as a Source Data file.