Single-Cell Profiling of Splenic Immune Ageing and Chronic Stress Adaptations in Mice With Natural Microbiota

Immune ageing impairs adaptive and innate responses, yet the spleen remains underexplored by cross-cohort single-cell studies. We profiled splenocytes from young, old and chronically stressed old mice with natural microbiota using single-cell RNA sequencing. Ageing was characterised by reduced lymphocyte competence and elevated stress responses. Within Gzmk⁺ CD8⁺ T cells, young mice were enriched for Gzmk-high cells, whereas in old mice, both Gzmk-high and Gzmk-low cells showed greater heterogeneity and functional alterations: exhaustion in Gzmk-high and inflammation in Gzmk-low cells. Natural killer (NK) cells and macrophages exhibited reduced cytotoxic potential and sustained pro-inflammatory polarisation, respectively. Chronic stress caused modest compositional shifts that partially counteracted age-related changes. Furthermore, we integrated our dataset with six published datasets to build a comprehensive atlas with > 272,000 splenocytes. Atlas-level annotation showed reproducible compositional shifts across datasets. Conserved ageing signatures included loss of NK effector genes (Zeb2, Prf1), decline of naïve T quiescence (Lef1, Il7r) with stress induction (Rbm3, Socs3), gain of survival/pro-inflammatory/exhaustion genes (Bcl2, S100a6, Ccl5, Lag3) in effector T cells, and altered differentiation and regulation (Zbtb32, Zbtb20, Zfp318, Ighd, Cr2) in B cells. Our results define conserved features of splenic immunosenescence and provide an atlas for dissecting splenic immune alterations.