A significant fraction of the human T cell repertoire does not recognize peptide-MHC complexes, but instead is activated by lipids complexed to CD1 antigen presenting molecules. CD1a, which is one of four surface-expressed lipid antigen presenting molecules in humans, is constitutively expressed at high levels on epidermal Langerhans cells. The localization of CD1ahigh Langerhans cells at the interface between skin immune system and skin microbiome, suggests an important role for the CD1a lipid antigen presenting system in skin immunity. This notion is further supported by our recent findings that CD1a-restricted T cells are common in blood and skin of healthy individuals, and that certain hydrophobic lipids present in human skin can act as antigens for these T cells. Among the lipid antigens we identified are free fatty acids, waxesters, triglycerides and squalene, all components of human sebum. This indicates a novel role for skin lipids beyond providing barrier function, namely as T cell antigens. Moreover, our skin is home to a large microbial population, which harbor close analogs of the mammalian lipids we identified as CD1a antigens, differing only in alkyl chain stereochemistry (cis/trans unsaturations) or fine structure (e.g. methyl branching, cyclopropanation). Prior studies have shown that minor structural changes in lipid chains can affect the strength of activation of CD1-restricted T cells, and we now plan to systemically address the following: 1) if CD1a-restricted TCRs bind with higher affinity to CD1a molecules loaded with microbial lipid structures than those loaded with mammalian analogs, using biophysical methods (Biacore), 2) determine the lipid binding preference of CD1a, by analyzing by LC/MS the eluted lipids from CD1a after co-incubation with defined mixtures of structurally related lipids, 3) determine the fine-specificity of skin-resident T cells using mammalian lipids and microbial analogs. Last, because affected skin in atopic dermatitis patients has been shown to display a shift in microbial flora as well as changes in lipid composition, we will determine if the frequency of CD1a-restricted T cells is increased in lesional compared to non-lesional skin of atopic dermatitis patients using CD1a tetramers. This research is in direct line with the applicant's goal of becoming an independent investigator in the field of skin immunobiology. The results of this proposal will provide valuable insights in the regulation of T cell activation by lipid antigens in the skin, and how shifts in lipid composition may affect T cell activation. This is highly relevant to the field of skin immunology, and opens the way to new lipid-based modes of intervention in skin diseases.