Fig. 8: Infection-experienced lung structural cells display enhanced viral control and can reactivate T cells.

A C57BL/6 mice were infected with influenza A virus (IAV)-WSN on day 0 and treated with 400μg isotype control or 200μg each of anti-CD4 (GK1.5) and anti-CD8 (2.43) on days 28 and 30 when mice were (re)-infected with IAV-X31. Lung IAV titers were examined 2 days post-IAV infection, and data were combined from two experiments: primary (n=9) mice, recall IgG (n=9), and recall aCD4/aCD8 (n=11). B Experimental design of IAV infection, in vitro re-challenge and co-culture, schematic created in BioRender; Worrell, J. (2025) https://biorender.com/hwavd50. Lung CD45-negative cells were isolated from naive or IAV-WSN-infected mice 30 days earlier. After 24 hours, the cells were infected with IAV-X31 and the cells were examined by flow cytometry after a further 24 hours or co-cultured with T cells from the spleens of day 9 IAV-X31-infected mice infected. C Percentage of CD44hi CD4/CD8 T cells that were CD25+ or CD69+ , normalized to the mean of the no infection control within each of the two experiments, n = 4 mice per group in each experiment. D Representative FACS plots and data for IAV-Nucleoprotein (NP) for each cell type, graphed data are the average of two technical replicates per mouse. Cells were gated on live, single, CD45-negative EpCAM1+ (epithelial cells), CD140+ (fibroblasts), and CD31+ (blood endothelial cells). Numbers in plots indicate the percentages of cells that are IAV-NP + . E Supernatants from the infected cultures were tested for multiple immune mediators by Luminex 24 hours after in vitro infection. Data in D, E combined from the same two experiments: Primary: n = 8, Re-infection: and n = 7; in D one sample in the IAV-memory/IAV-31 group was lost due to a technical error during acquisition. In all graphs, each symbol represents a mouse, and the bars show means ± SEM for normally distributed data and median with interquartile range for non-normally distributed data. A Data are not normally distributed. CD4 data are not normally distributed, and CD8 data are normally distributed. D Epithelial cell and BEC data are normally distributed, and fibroblast data are not normally distributed. E All data are normally distributed. A–D non-normally distributed data tested with a one-way Kruskal-Wallis test followed by a Dunn’s multiple comparisons test. C, D normally distributed data tested by a one-way ANOVA followed by Šidák’s multiple comparison test. E Data tested by a one-way t-test. Source data for A, C–E data are provided as a Source Data file.