Transfer of Maternal Immune Cells by Breastfeeding: Maternal Cytotoxic T Lymphocytes Present in Breast Milk Localize in the Peyer's Patches of the Nursed Infant

Abstract

Despite our knowledge of the protective role of antibodies passed to infants through breast milk, our understanding of immunity transfer via maternal leukocytes is still limited. To emulate the immunological interface between the mother and her infant while breast-feeding, we used murine pups fostered after birth onto MHC-matched and MHC-mismatched dams. Overall, data revealed that: 1) Survival of breast milk leukocytes in suckling infants is possible, but not significant after the foster-nursing ceases; 2) Most breast milk lymphocytes establish themselves in specific areas of the intestine termed Peyer's patches (PPs); 3) While most leukocytes in the milk bolus were myeloid cells, the majority of breast milk leukocytes localized to PPs were T lymphocytes, and cytotoxic T cells (CTLs) in particular; 4) These CTLs exhibit high levels of the gut-homing molecules α4β7 and CCR9, but a reduced expression of the systemic homing marker CD62L; 5) Under the same activation conditions, transferred CD8 T cells through breast milk have a superior capacity to produce potent cytolytic and inflammatory mediators when compared to those generated by the breastfed infant. It is therefore possible that maternal CTLs found in breast milk are directed to the PPs to compensate for the immature adaptive immune system of the infant in order to protect it against constant oral infectious risks during the postnatal phase.

Fig 1. Transfer of breast milk leukocytes to suckling pups.

(A) Breeding of wild type (WT) C57BL/6 and C57BL/6-GFP^tg (GFP^tg) mice was coordinately mated. At day 0–2, WT neonates were transferred to be continually nursed by GFP^tg dams until their weaning (21 days). PPs and other organs from suckling infants were surgically excised, purified, and put into cell suspensions for flow cytometry analysis. FACS plots show the level of GFP expression by CD45.2+ splenocytes of GFP^tg (left, positive control) and WT (right, negative control) mice. (B) Leukocytes were identified using the following orientating gates: The first orientating gate was selected using a SSC vs. CD45 plot where the cutoff for CD45+ cells was set using FMO control. Subsequently, annexin V vs. propidium iodide (PI) plot of CD45+ cells separated events into a major gate of annexin V^- PI^- events which represents viable CD45+ cells. Subsequent specific gate for other populations that were preselected as CD45+ annexin V^- PI^- are shown in CD45 vs. GFP plots. Dot Plots show the presence of breast milk leukocytes (GFP+CD45.2+) in the peyer’s patches (PPs), spleen (SPL), thymus (THY), mesenteric lymphnodes (MLN), and intestinal mucosa (IM). Percent of transferred leukocytes was assessed 18 days post-fostering. Panels show WT pups breastfed by WT mothers (upper, negative control) and WT pups breastfed by MHC-matched GFP^tg dams (lower). (C) Bar graphs summarize the presence of GFP+ cells in organs as a % of total CD45.2+ cells at day 6, 14, and 18 post-fostering, and one week post-weaning (day 28). Data were obtained from three experiments using a total of 24 pups with n = 6–10 pups per experiment (DAY 6); three experiments using a total of 16 pups with n = 4–6 pups per experiment (DAY 14); five experiments using a total of 26 pups with n = 4–6 pups per experiment (DAY 18); and three experiments using a total of 9 mice with n = 3 animals per experiment (DAY 28). (D) BALB/c neonates were transferred to be foster-nursed by GFP^tg dams until their weaning. Bar graph summarizes the presence of GFP+ cells as a % of total CD45.2+ cells in organs at day 18 post-fostering and one week post-weaning (day 30). Data were generated from three experiments using a total of 12 pups (DAY 18) and 6 mice (Day 30) with n = 2–5 animals per experiment. (C and D) Data are shown as means of % GFP+ CD45.2+ cells ± s.e.m. Error bars represent s.e.m. A 2-tailed Student’s t test distribution with paired groups was evaluated for statistical significance. P > 0.05 was considered not significant (NS), *P < 0.05 was considered significant, and **P < 0.005.

Fig 2. Characterization of breast milk leukocytes.

(A) Different GFP+ cell subtypes were identified in milk bolus and PPs of C57BL/6 pups nursed by GFP^tg dams based on the co-expression of GFP and CD45.2 in combination with CD3, CD8, CD4, CD11b, CD19, and Gr1. Bar graphs show the average contribution of cell subset(s) to the total of GFP+CD45.2+ cells in milk bolus (left) and PPs (right). (B) Bar graphs show the % of each cell subset (gated on total CD45.2+ GFP+ cells) in milk bolus versus PPs: CD11b+ (CD3-CD19-CD11b+, upper left), GR1+ (CD3-CD19-Gr-1+, upper middle), CD19+ (CD3-CD19+, upper right), CD3+ (CD19-CD11b-CD3+, lower left), CD8+ (CD3+CD8+, lower middle), and CD4+ (CD3+CD4+, lower right) cells. Data are shown as means of % cell subset gated on GFP+CD45.2+ cells ± SD. Error bars represent SD. (A and B) Data were obtained from four experiments using a total of 16 (milk bolus) and 25 (PPs) animals with a combined material of 3 to 5 (milk) and 5 to 8 (PPs) mice per experiment. (C) Contour Plots show expression of α4β7, CCR9, and CD62L by breast milk CD8+GFP+ T cells that localize in PPs of suckling pups vs. blood CTLs of the birth mother. (D) Bar graphs show the % of CD8+CCR9+, CD8+α4β7+, and CD8+CD62L+ in PPs (gated on milk CD3+CD8+GFP+ cells) versus adult blood (gated on total CD3+CD8+ cells). Data are shown as means of % cell subset gated on CD8+GFP+ (PPs) or CD8+ (blood) T lymphocytes ± SD. Error bars represent SD. Bar graphs summarize the data from four experiments using a total of 25 (PPs) and 5 (blood) animals with a combined material of 5 to 8 (PPs) and 1 to 2 (blood) mice per experiment. A 2-tailed Student’s t test distribution with paired groups was evaluated for statistical significance. *P < 0.05 was considered significant, **P < 0.005, ***P < 0.0005, and P > 0.05 was considered not significant (NS).

Fig 3. Production of granzyme B and pro-inflammatory cytokines by breast milk and infant T cells.

PPs from CD45.1 pups breastfed by CD45.2 congenic dams for 18 days were collected, put into cell suspensions, and processed for T cell purification. (A) Enriched T lymphocytes were stimulated overnight with PMA and ionomycin. Bar graphs show % cytokine- and GRMB-producing maternal cells (transferred milk CD45.2+) vs. infant (host CD45.2-) T lymphocytes. Data were obtained from three experiments shown as means of % CD3+ T cells ± s.e.m. Error bars represent s.e.m. **P < 0.005, ***P < 0.0005 using student’s two-tailed t test. (B-D) Enriched T lymphocytes were activated with anti-CD3 /anti-CD28 Abs or control Igs for four days in the presence of 200U/mL of IL-2. T cell responses associated with IFN‐γ, TNF-α, Granzyme B (GZMB), and IL-18 production were examined by intracellular staining and analyzed by FACS. The first orientating gate was selected using a SSC vs. CD3 plot where the cutoff for CD3+ cells was set using FMO control. Subsequent gating of a CD3 vs. CD8 plot of total CD3+ cells separated events into a selected population of CD3+CD8+ cells. Subsequent specific gates identified infant (host CD45.2-) and maternal (transferred breast milk CD45.2+) cells that were producing cytokines and GZMB. (B) Contour Plots show outcomes from activation assays with anti-CD3/anti-CD28 (upper panel) and isotype (lower panel) Abs. Graphs show functional outcomes (C) with anti-CD3/anti-CD28 Abs (n = 5) and (D) isotype controls (n = 3). The expression of each product was shown as means of % CD45.2+ CD8+ (maternal) or CD45.2- CD8+ (infant) T cells ± s.e.m. (B-D) A 2-tailed Student’s t test distribution with paired groups using equal number of samples was evaluated for statistical significance. *P < 0.05 was considered significant, **P < 0.005, ***P < 0.0005, and P > 0.05 was considered not significant (NS). (E) Peripheral blood CD8 T cells collected from birth mothers (CD45.1+) were activated with anti-CD3/anti-CD28 Abs. Four days later, cytokine and GZMB production was measured as in b-d. FACS Plots show CD8+CD45.1+ cells that were producing IFN‐γ, TNF-α, GZMB, and IL-18. (F) Bar graph summarizes the data from three experiments using two dams per experiment. Data are shown as means of % CD8+ CD45.1+ cells ± SD. Error bars represent SD. ****P < .0005 using a 2-tailed Student’s t test.