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Keywords:

  • Human milk immunology;
  • T lymphocyte subsets;
  • flow cytometry;
  • markers of activation;
  • leukocyte common antigen isoforms

Abstract

Since activated macrophages and cytokines are found in human milk (HM), a flow cytometry study was conducted to determine whether T cells in HM display phenotypic markers of recent or previous activation. HM was collected during the first 3 d of lactation. The Paint-a-Gate program was used to optimize gating on the lymphocyte population. A mean ± 1 SD of 4 ± 3% of total HM leukocytes were lymphocytes and 96 ± 3% were macrophages and granulocytes (N=33 subjects). HM lymphocyte populations were further analyzed in five subjects. T calls (CD3+) represented 83 ± 11% and B cells (CD19+) were 6 ± 4% of HM lymphocytes. The mean CD4/CD8 ratio of T cells in HM was 0.88 (range 0.40–1.25). This ratio was significantly decreased compared to the peripheral blood (PB) of control adults (P < 0.02) and postpartum women (P < 0.02), due mostly to a significant increase in CD8+ CD3+ cells in HM compared to the PB of control adults (P < 0.002) and postpartum women (P < 0.05). T cells bearing markers of recent activation were significantly increased in HM compared to the PB of control adults: 85 ± 7% of CD3+ cells in HM were HLA-DR+ (controls, 10 ± 4%; P < 0.001), and 15 ± 6% of CD3+ cells in HM were IL-2R+ (controls, 6 ± 2%; P < 0.001). Subpopulations of CD4+ and CD8+ cells in HM defined by the T200 isoforms CD45RA and CD45RO were markedly altered compared to PB, indicating a striking shift from virginal to antigen-primed (memory) T cells in HM and suggesting certain functional capacities for these cells. Virtually all CD4+ cells (99.8 ± 0.4%) and 92 ± 5% of CD8+ cells in HM were CD45R0+ (vs. 71 ± 12% and 50 ± 10%, respectively, in the PB of postpartum women; P < 0.001). CD4+ and CDS8+ cells expressing CD45RA were correspondingly markedly reduced (P < 0.001). This phenotypic pattern of T cells in HM may result from T cell activating substances in HM and/or selective homing of T cells to the breast. Conversely, activated T cells in HM may be responsible for cytokines in HM. These activated and memory T cell populations may be transferred to the infant via breastfeeding.