Integrated transcriptomic and immunological mapping reveals peri-pubertal immune reprogramming in rat mammary gland...

Postnatal mammary gland development is a hormone-dependent process involving extensive epithelial expansion and stromal remodeling. Although endocrine regulation of mammary morphogenesis is well established, the role of the immune system during normal mammary gland development remains incompletely understood. Here, we characterized immune remodeling across pre-puberty, peri-puberty, and adulthood in the rat mammary gland by integrating bulk transcriptomics, immune-stromal deconvolution, pathway enrichment analysis, cytokine and chemokine profiling, and immunolocalization. Our analyses identified peri-puberty as a distinct phase of immune-stromal remodeling. Pre-puberty was enriched in innate immune populations and immune establishment programs associated with tissue organization. In contrast, peri-puberty displayed increased adaptive immune signatures and activation of chemokine pathways involved in leukocyte recruitment and spatial organization. Notably, this transition occurred alongside lower tissue concentrations of several inflammatory cytokines, suggesting that immune recruitment during peri-puberty reflects a developmentally programmed signaling state rather than classical inflammation. By adulthood, immune composition and signaling profiles stabilized, consistent with immune homeostasis and surveillance. Transcript-protein integration revealed limited concordance between developmental changes in cytokine and chemokine transcripts and their corresponding proteins. No paired RNA-protein associated remained significant after multiple-testing correction, suggesting that regulation beyond transcription may contribute to soluble mediator profiles. Together, these findings demonstrate that postnatal mammary gland development is accompanied by stage-specific immune programming and establish a systems-level framework for understanding developmental immunology in the mammary gland.