SUMMARY Sharks are members of the oldest class of living Vertebrates with adaptive immunity based on immunoglobulin, T cell receptors, and the major histocompatibility complex. Eight class I lineages have recently been uncovered by the Flajnik lab, the classical class I (UAA) and seven nonclassical lineages (UBA to UHA). Unexpectedly, two of these MHC-linked shark nonclassical class I (Class Ib) molecules show striking similarities to the human nonclassical class I molecules CD1 and HLA-E. Preliminary data on genetics, expression, and sequence similarity strongly in the Flajnik lab suggest that UFA is the orthologue of CD1. Structural modeling and in silico biophysical analyses in the Adams lab are consistent with UFA’s clear similarity to CD1, revealing a strongly hydrophobic binding site. The second shark class Ib molecule, UDA, is predicted to bind peptides like classical class I, yet it is monomorphic, single-copy, and has properties similar to mammalian HLA-E/Qa-1. We will biochemically define UFA and UDA ligands and elucidate their structures. We will express and purify recombinant versions of UDA and UFA for biochemical analysis including ligand elution and preparation of tetramers to identify the shark cells recognizing these nonclassical class I molecules. Highly purified forms of recombinant proteins will be used for structural analysis through X-ray crystallography. Using in-house technologies, we will generate nanobody binding reagents to these proteins for structural analysis and also complement the antisera and monoclonal antibodies already generated for tissue-expression analysis. By in situ hybridization and antibody/nanobody staining we will define UDA/UFA cellular expression and examine their upregulation in cells under stress. UDA and UFA tetramers will reveal which lymphocytes interact with UDA/UFA and, with single-cell analysis, the adaptive and/or innate receptors that interact with them. We will perform biochemical analysis of shark primary cells to characterize UDA/UFA biosynthetic pathways. In this proposal, therefore, we will combine the expertise and experience of the two investigators, Martin Flajnik, who has studied the genetics, biochemistry, and function of the shark immune system for 35 years and generally the evolution of adaptive immunity, and Erin Adams, a structural biologist with deep knowledge of the human class I system and evolutionary biology. In summary, we hypothesize that CD1 and HLA-E analogues arose at the Big Bang of adaptive immunity 500 million years ago, suggesting that peptide-binding by the classical class I, lipid-binding by “CD1,” and a peptide-binding by a jack-of-all-trades “HLA-E-like” nonclassical class I were significant features in the earliest adaptive immune system.