Gut symbiotic microbiota–derived CD1d ligands and their immunomodulatory mechanisms PROJECT SUMMARY The symbiotic microbiota has co-evolved with the mammalian host for millennia, and the host has developed a sophisticated system for distinguishing pathogens from commensal microbes. Unlike pathogens, many of whose molecules trigger robust inflammatory and immune reactions, symbiont-derived molecular factors have been believed to be “silent,” even though they reside within the host at a very high density. However, recent studies strongly suggest that molecular factors of symbiotic origin actively contribute to host immune regulation and protection from excessive inflammation. We have previously identified and characterized a unique class of lipids (alpha-galactosylceramides) from the human gut symbiont Bacteroides fragilis that can modulate host immune development early in life. Our preliminary results show that these molecules (BfaGCs) are presented by the nonclassical MHC class I–like molecule CD1d in a structurally conserved manner similar to that documented for prototypic CD1d ligands. Of considerable interest, unlike CD1d agonists such as KRN7000, BfaGCs function as a regulator of natural killer T (NKT) cells, a specific T cell subtype restricted by the CD1d–lipid antigen complex. Synthetic BfaGC molecules can induce distinct immunomodulatory signals from NKT cells and can function as a regulator of NKT cell proliferation in the colon. Furthermore, targeted lipidomic profiling of gut symbionts has identified lipid species structurally related to BfaGCs in multiple gut symbionts, implying that gut symbionts can collectively synthesize potential NKT cell regulators. We propose an investigation of molecular immunomodulatory mechanisms that underlie the activity of gut symbiont–derived lipid ligands. We aim to determine (1) the distinct NKT population recognized by the CD1d-BfaGC complex and their immunomodulatory responses to BfaGCs, (2) the specific molecular-level interactions between CD1d and BfaGCs, (3) the structure and immunomodulatory activity of previously uncharacterized lipid species of symbiont origin and (4) the modulation of inflammatory responses by symbiont-derived CD1d ligands in vivo. The proposed studies will provide valuable knowledge of the molecular mechanisms by which symbiotic microbiota-derived molecules modulate the host immune system and help the development of potential immunotherapeutics.