PROJECT SUMMARY Children raised in traditional farm environments rich in microbes are protected from asthma. Our 2016 New Engl J Med papers characterized the “farm effect” in two U.S. farming populations, the Amish and the Hutterites. Despite similar genetic ancestries and lifestyles, Amish and Hutterites follow distinct farming practices (traditional among the Amish, industrialized among the Hutterites) and show striking disparities in asthma prevalence (4- times lower in the Amish), immune responses and environmental microbial exposures (6.7-fold higher among the Amish). Notably, inhalation of Amish (but not Hutterite) farm dust extracts (AFDE) dramatically reduced allergen-induced airway hyperresponsiveness (AHR), broncho-alveolar lavage (BAL) eosinophilia and serum IgE, and these effects depended on innate immune signaling. These studies showed that the farm environment protects from asthma, but did not address the mechanisms of protection. Recently we found that the lungs of AFDE-treated mice (both Balb/c and C57BL/6) harbor a large population of Vγ4+γδ17 T cells (≈25% of lung T cells). These cells (which in other models have allergy-suppressive properties) were induced by both autoclaved and non-autoclaved AFDE in Balb/c and C57BL/6 mice of both genders, and were invariably present whenever AFDE inhibited experimental asthma. Lung γδ/Vγ4 transcriptional signatures were robustly associated with inhibition of AHR and BAL eosinophilia, and γδ17 T cells were absent in Myd88/Trif-/- and Hutterite dust extract- treated mice, which were not protected from asthma. These preliminary data lead us to hypothesize that lung γδ17 T cells elicited by AFDE inhalation play a role in asthma protection. This hypothesis will be tested through complementary in vivo approaches. Aim 1 will determine whether γδT cells (total or γδ17) are required for the asthma-protective effects of AFDE inhalation. Mice that lack γδT or γδ17 T cells because of genetic deficiency (Tcrδ -/- and Blk-/-) or antibody-mediated depletion will be assessed for their capacity to be protected from experimental allergic asthma upon AFDE inhalation. Aim 2 will determine whether transfers of γδ17 T cells isolated from the lungs of AFDE-exposed mice are sufficient to protect unexposed animals from allergic asthma. Total γδT cells or Vγ4+ γδ17 T cells isolated from the lungs of AFDE-treated mice will be adoptively transferred into allergen-treated WT, Tcrδ -/- and Blk-/- (γδ17-deficient) mice. Allergic inflammation phenotypes in recipient mice will be assessed as in Aim 1. The asthma-protective role of γδT cell-derived IL17 will also be tested by transferring γδT cells from Il17a-deficient mice. Aim 3 will characterize circulating γδ17 T cells from Amish and Hutterite children for their proportions, phenotypes and relation with immune profiles. Mass cytometry/CyTOF and our previous data on the children's immune profiles will be leveraged to assess whether circulating γδ17 T cell proportions are ...