Functional shifts in immune composition follow lactation stage in human milk

Human milk contains diverse immune and non-immune cellular components that change dynamically from early to established lactation. We sought to determine if the milk immune microenvironment exhibits stage-associated changes.

We performed multi-modal profiling using single-cell RNA sequencing, high-dimensional flow cytometry, and soluble protein assays on a longitudinal cohort of paired maternal peripheral blood samples and milk. Samples were collected across three distinct phases: colostrum, transitional, and mature milk.

Integrated analysis revealed that human milk contains transcriptionally distinct immuno and epithelial cellular populations compared with maternal peripheral blood. Early lactation (colostrum) demonstrated greater neutrophil enrichment of antimicrobial-associated transcriptional programs, including degranulation- and NETosis-associated signatures. Later lactation stages (transition and mature) demonstrated relatively greater effector-memory-associated T-cell transcriptional signatures. Stage-associated differences in soluble immune mediator profiles were also observed, with early recruitment-associated cytokines (CXCL8, CXCL13) declining across lactation while IL-7 increased in mature milk. Cell-cell communication analysis identified inferred lactocyte-associated signalling pathways involving MHC and growth factor-related interactions that differed across lactation stages.

Human milk demonstrates substantial stage-associated remodelling of both cellular and soluble immune components across lactation. Early milk was enriched for neutrophil-associated antimicrobial and inflammatory transcriptional programs, whereas later lactation stages demonstrated stronger effector-memory-associated T-cell signatures together with distinct soluble immune mediator profiles. These findings support the concept that human milk represents a dynamic immune environment that changes across lactation and may expose infants to distinct immune signals during early postnatal life. Further studies will be required to determine the functional significance of these stage-associated immune features and their implications for neonatal health.