Atopic constitution starts shortly after birth with atopic dermatitis (AD) and sensitization to foods, which is an important marker for the potential development of clinical food allergy (FA). A farming lifestyle has been reproducibly associated with a reduced risk of asthma and atopic disease at school-age. Sustained microbial exposure experienced when living on farms is proposed to confer protection through priming of innate immune populations/receptors, whereas increased monocyte activation and hyper-responsive cytokine response to lipopolysaccharide (LPS) stimulation was associated with increased risk of allergic asthma. Epigenetic modifications in myeloid cells can lead to a change in functional potential to a second stimulus, either an enhanced or suppressed response. This long-lived modification in immune potential, a concept known as trained immunity, can lead to an array of functional outcomes, similar to the T cell differentiation outcomes that they influence. Farmlife protection is also proposed to be mediated through expansion of regulatory T (Treg) cells. Through epigenetic mechanisms or direct interaction with immune cells, microbial metabolites promote generation of Tregs. This can reinforce tolerance through maintenance of epithelial barrier integrity, which is known to be compromised in skin in AD, facilitating epicutaneous allergen sensitization and in gastrointestinal sites in FA. Despite several large birth cohorts, the early mechanisms and biomarkers of AD and FA are poorly characterized, although the emerging strategies for prevention call for a need to identify those at risk. Our preliminary studies in the “Zooming in to Old Order Mennonites” (ZOOM)1 birth cohort identified novel memory effector Th2 subpopulations in infancy as a marker of development of allergic sensitization in infants born to Rochester urban families (ROC). In comparison, our studies found a higher frequency of gut-homing (β7+) Tregs and Tregs that express TIGIT, an inhibitory co-stimulatory molecule, in infants from Old Order Mennonites (OOM), a traditional agrarian community protected against atopic diseases. In addition, ROC infants demonstrated a hyper-inflammatory monocyte response in cord blood. Utilizing samples and clinical data collected under the already enrolled ZOOM1 cohort (n=160) and to be enrolled expansion cohort ZOOM 2 (n=120), we will be testing our central hypothesis that infants who will develop atopic diseases generate Th2- skewed T cell populations in early infancy, whereas the protected infants develop gut-homing suppressive Tregs, and monocytes hyporesponsive to LPS and viral targets. We have three specific aims: Aim 1 will assess infant adaptive and innate immune markers in farming and urban lifestyles associated with protection and atopic disease outcomes. Aim 2 will evaluate the role of T and innate cell transcriptome and epigenome on atopic disease outcomes. Aim 3 will measure the association between gut barrier function, far...