Human breastmilk memory T cells throughout lactation manifest an activated and tissue-oriented profile with prominent regulation

Breastfeeding provides important immunological benefits to the neonate, but how different immunoactive components in breastmilk contribute to immunity remains poorly understood. Here, we characterized human breastmilk T cells using single cell RNA sequencing and flow cytometry. Breastmilk contained predominantly memory T cells with immune response signaling, proliferation and an effector Th1/cytotoxic profile with high cytokine production capacities. High activation markers were balanced by an enriched Treg population and immune regulatory markers in conventional memory T cells. Gene and surface expression of tissue-residency markers indicated that breastmilk T cells represent tissue-adapted rather than circulatory T cells. In addition, breastmilk T cells had a broad homing profile and higher activation markers in these migratory subsets. The partly overlapping transcriptome profile between breastmilk and breast tissue T cells, particularly cytotoxic T cells, might support a role in local immune defense in the mammary gland. However, unique features of breastmilk, such as regulatory T cells, might imply an additional role in neonatal immune defense. We found some correlations between the breastmilk T-cell profile and clinical parameters, most notably with maternal and household factors. . Together, our data suggest that breastmilk contains an adapted T cell population that exerts their function in specific tissue-sites. scRNAseq data of plate-sorted T cells (sort-seq) from 1 month postpartum breastmilk (n=7) were clustered together to reveal T-cell populations and characteristic DEG present in human breastmilk.