Multi-layer transcriptomic characterization of age-related immune dynamics

Despite the pivotal role of mRNA isoform diversity in governing immune cell function, current investigations into peripheral immune aging predominantly focused on gene-level expression, obscuring deeper regulatory layers of transcriptome complexity. Here, we leveraged a 5′ scRNA-seq atlas comprising approximately 2.5 million PBMCs from 378 healthy donors. We demonstrate that immune aging is characterized by profound, non-linear transcriptional reprogramming that extends beyond gene-level shifts to include fine-tuned regulation of alternative transcription initiation and splice site selection. By quantifying the transcriptional activity of cis-regulatory elements, we resolved their contributions to age-related expression dynamics. Notably, we identified a subset of endogenous retroviruses that are reactivated in older individuals, some of which served as alternative promoters driving the production of chimeric transcripts. Furthermore, our analysis revealed EDA as a top-ranked gene consistently upregulated with age across multiple independent cohorts. Increasing EDA expression in in vitro-stimulated naïve CD4 ⁺ T cells from young individuals recapitulated aged phenotypes. This comprehensive resource elucidates the multi-layered transcriptomic landscape of the aging immune system and facilitates the identification of novel drivers of immune aging.