Histological characterization and spatial profiling of age-induced tertiary lymphoid structures in mouse lung parenchyma

: Tertiary lymphoid structures are ectopic lymphoid aggregates traditionally associated with inflammation or injury. Their presence in uninjured, aged murine lungs remains unexplored. This study investigates age-induced TLS formation, morphology and cellular composition, comparing these structures to those induced by bleomycin treatment. Lungs from healthy mice aged two, 18, and 24 months were analyzed using histological staining, histomorphometry and high-plex immunofluorescence. TLSs were identified and spatially classified (perivascular, peribronchial, parenchymal). We performed a single-cell phenotype analysis that revealed distinctive alterations in the immune repertoire identifying lymphoid neogenesis in healthy elderly lungs. BLM-induced TLSs in young (2-month-old) mice were also examined. Age-related TLS formation was evident, with a significant increase in both density and size at 18 and 24 months compared to two months. Peribronchial TLSs were larger and more circular with age, while perivascular TLSs showed higher T cell density. Immunofluorescence revealed diverse immune cell populations, including B cells, T cells and macrophages, organized in location-specific patterns. BLM-induced TLSs were larger and less compact than those in aged lungs, correlating with fibrotic severity (R² = 0,92). This study reveals that TLSs develop in murine lungs with age, exhibiting distinct spatial organization and immune cell compositions. Compared to damage-induced TLSs, age-related TLSs are more compact and structured. These findings highlight the role of TLSs in age-related immune surveillance and suggest their potential involvement in inflammaging and chronic lung conditions. It will be crucial to further investigate their role and determine whether their formation is associated with respiratory disease and age-related immune dynamics.