Human breastmilk memory T cells throughout lactation manifest activated tissue-oriented profile with prominent regulation
Elise S. Saager, Arthur H. van Stigt, Butstabong Lerkvaleekul, Lisanne Lutter, Anneke H. Hellinga, M. Marlot van der Wal, Louis J. Bont, Jeanette H.W. Leusen, Belinda van’t Land, Femke van Wijk, the Protection against Respiratory tract infections through human Milk Analysis (PRIMA) group
Elise S. Saager, Arthur H. van Stigt, Butstabong Lerkvaleekul, Lisanne Lutter, Anneke H. Hellinga, M. Marlot van der Wal, Louis J. Bont, Jeanette H.W. Leusen, Belinda van’t Land, Femke van Wijk, the Protection against Respiratory tract infections through human Milk Analysis (PRIMA) group
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Research Article Immunology

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

Abstract

Breastfeeding provides important immunological benefits to the neonate, but how the different immunoactive components in breastmilk contribute to immunity remains poorly understood. Here, we characterized human breastmilk T cells using single-cell RNA-Seq and flow cytometry. Breastmilk contained predominantly memory T cells, with expression of immune signaling genes, high proliferation, and an effector Th1/cytotoxic profile with high cytokine production capacities. Elevated activation was balanced by an enriched Treg population and immune regulatory markers in conventional memory T cells. Gene and surface expression of tissue-residency markers indicate that breastmilk T cells represented 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 Tregs, might imply an additional role in neonatal immune support. 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.

Authors

Elise S. Saager, Arthur H. van Stigt, Butstabong Lerkvaleekul, Lisanne Lutter, Anneke H. Hellinga, M. Marlot van der Wal, Louis J. Bont, Jeanette H.W. Leusen, Belinda van’t Land, Femke van Wijk, the Protection against Respiratory tract infections through human Milk Analysis (PRIMA) group

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Figure 6

Tissue-residency and tissue-homing profiles in breastmilk T cells.

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Tissue-residency and tissue-homing profiles in breastmilk T cells. (A an...
(A and B) Distribution of tissue-resident memory T cell subsets based on protein expression of the TRM markers CD69 and CD103 (FACS) expressed as the percentage of CD4+CD45RA– (PBMC n = 8, BM 1 week n = 2, BM 1 month n = 11, BM 3 months n = 17, BM 6 months n = 12) (A) and CD8+CD45RA– T cells (PBMC n = 8, BM 1 week n = 2, BM 1 month n = 11, BM 3 months n = 16, BM 6 months n = 6) (B) with pairwise comparisons between BM time points and PBMC. (C) UMAP showing clustering and gene expression (log-normalized) projections for the TRM-related genes CD69, RUNX3, and CXCR6 and the homing receptors integrin α4 (ITGA4), integrin β1 (ITGB1), and integrin β7 (ITGB7) in 1 month postpartum breastmilk (single-cell RNA-Seq, n = 7). (D) Frequency of CXCR6+ cells (FACS) as the percentage of CD8+CD45RA– T cells compared between the 4 different CD8+ TRM subsets within breastmilk (colored dots) and with the total population in control PBMC (gray dots) (PBMC n = 8, BM n = 18). (E–G) Surface expression of different homing receptors (FACS) with pairwise comparisons among PBMC and BM across different time points. Frequencies of α4β1+ cells as the percentage of CD4+FOXP3–CD45RA– T cells (PBMC n = 7, BM 1 week n = 5, BM 1 month n = 19, BM 3 months n = 19, BM 6 months n = 4) (E), α4β7+ cells as the percentage of CD8+CD45RA– T cells (F), and cells double-positive for α4β7 and CLA as the percentage of CD8+CD45RA– T cells (G). FACS data include cells from breastmilk of 1 week and 1, 3, and 6 months postpartum compared with PBMC of age-matched female control donors (PBMC n = 7, BM 1 week n = 6, BM 1 month n = 17, BM 3 months n = 15, BM 6 months n = 3). (A, B, and E–G) Pairwise comparisons among PBMC and BM time points were tested using the Kruskal-Wallis test followed by Dunn’s test for multiple comparisons. (D) For comparisons between breastmilk T cell subsets, the Friedman test was used followed by Bonferroni-corrected pairwise Wilcoxon ranked-sum post hoc testing. *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean ± SD. Gray lines connect data points of different time points from the same breastmilk donor. TRM, tissue-resident memory T cell; BM, breastmilk.