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

ABSTRACT 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.