Project Summary Food allergy (FA) is a growing public health concern. The effects of maternal immune responses on the induction of regulatory T (Treg) cell-mediated tolerance in offspring are poorly understood. We have recently found that maternal sensitization with allergen [ovalbumin (OVA) or peanut] prevented FA responses in murine offspring in response to epicutaneous sensitization and oral challenge with the same allergen, as indicated by a decrease in the levels of food anaphylaxis, allergen-specific immunoglobulin (Ig) E, serum mouse mast cell proteinase 1, and intestinal mast cell expansion. This protection was associated with an increase in the levels of IgG and allergen immune complexes (IgG-IC) in breast milk. Neonatal Fc receptor (FcRn)-dependent transfer of maternal IgG-IC via breast milk and IgG-IC presentation by offspring CD11c+ dendritic cells induced allergen-specific Treg cells in offspring. Breastfeeding by OVA-sensitized mothers, or maternal IgG-IC supplementation induced neonatal tolerance. Consistently, human breast milk collected from non-atopic mothers contained IgG-IC and induced tolerance in humanized FcRn mice. These results indicate that maternal immune factors, especially IgG- IC, in breast milk are critical for the induction of Treg cells and control food-specific tolerance in neonates. Wild- type (WT) offspring adopted and nursed by OVA-exposed Rag2-/- mothers showed failure of tolerance towards FA as compared to WT controls, associated with a decrease in the frequencies of allergen-specific Treg cells in offspring. IgG-IC supplementation of OVA-exposed Rag2-/- mothers failed to rescue neonatal food tolerance in adopted WT offspring, suggesting that IgG-IC require additional maternal milk factors to successfully induce neonatal food tolerance. Transfer of maternal cells to infants through milk has been recognized, however, the presence of maternal Treg cells in milk are poorly described. We found that maternal Treg cells were transferred to offspring via milk and accumulated in the intestine and spleen of offspring. Depletion of maternal Treg cells during breastfeeding period resulted in failure of neonatal tolerance in the presence of IgG-IC. Further, we found that Treg cells are present in human milk. These results suggest that maternal Treg transfer via milk has a significant role in shaping the neonatal immune system and the susceptibility to FA. The goals of this proposal are to establish the phenotype of maternal Treg cells in milk and to decipher the role of these cells in regulating food-specific tolerance in neonates. We hypothesize that maternal Treg cells transferred through milk act in synergy with maternally transferred allergens (IgG-IC) during a specific time window in the perinatal period to successfully induce neonatal food tolerance. We also hypothesize that maternal milk Treg cells exhibit unique phenotype and gene expression profiles as compared to systemic Treg cells. Lastly, we hypothesize tha...