Neonatal immune tolerance promoted by breastfeeding is mediated by regulatory T cells and is associated with a reduction in release of inflammatory cytokines
Having observed that breastfeeding promotes the expansion of Tregs and suppresses proliferative responses against maternal antigen, we next investigated if this immune tolerance was dependent on the presence of Tregs. To this end, we repeated the MLRs on a set of 6 mother-and-baby dyads where babies were exclusively breastfed following the depletion of CD25+ cells (Figure 5a). Pregnancy is associated with peripheral accumulation of Tregs and the proliferation of maternal CD3+ cells in response to neonatal antigens increased both at birth (20.4 vs 59.7% median proliferating cells) and at 3 weeks of age (34.1 vs 57.1% median proliferating cells) following depletion of CD25+ cells (Figure 5b). The same pattern was observed for CD4+ but not in CD8+ cells. Of note, the proliferation of neonatal CD3+ cells in response to maternal antigens increased after depletion of CD25+ cells in samples taken at 3 weeks of age (71.4 vs 85.1% median proliferating cells) but this was not seen with the use of cord blood cells (Figure 5c). The same pattern was observed for CD4+ but not in CD8+ cells.
We also measured the concentration of cytokines in MLR supernatants (n = 11) produced by neonatal T cells of exclusively breastfed neonates in response to maternal antigens. The concentration of IFN-γ and TNF-α was found to be lower at 3 weeks of age compared to birth, whereas no difference was observed in case of the other cytokines tested (IL-4, IL-6, IL-8, IL-10, IL-17). The maximal IFN-γ and TNF-α producing capacity of neonatal T cells, tested by culturing with CD3/CD28 activator beads, was higher both at birth and at 3 weeks of age compared to the level seen in response to maternal antigens at 3 weeks (Figure 5d). As such, breastfeeding is seen to suppress the inflammatory Th1 cytokine response of neonatal T cells in response to maternal antigen stimulation.