The CD8 coreceptor is a critical molecule for T cell activation and development forming a trimolecular complex with the T cell receptor, and peptide-MHC class I (pMHC1). As the CD8ab heterodimer is 100 fold more efficient than CD8aa as a coreceptor, we are studying the role of CD8b in coreceptor function. We identified critical residues on the CDR-like loops of the immunoglobulin-like domain CD8b protein required for binding to pMHC1 as well as mutants that led to enhanced binding. Based on our studies we proposed that CD8ab has two binding modes for interaction with MHC class I. Understanding the molecular basis for CD8ab-pMHCI interaction is hampered due to a lack of structural information. In aim 1, we will determine structurally how CD8ab and pMHCI interact by NMR spectroscopy and test our hypothesis that there are two binding modes. In aim 2, we will further characterize the murine CD8b enhancing mutants by making affinity measurements with surface plasmon resonance studies and will compare the enhancement of coreceptor activity with agonist, weak agonist, and antagonist peptides using FRET analysis of CD8b-TCR interaction. In addition, we will identify human CD8b enhancing mutants using an approach that involves random mutagenesis at individual amino acid residues in CDR- loops, transfection of plasmids into COS7 cells for transient expression, and FACS sorting for cells expressing CD8b mutants with enhanced binding to an HLA class I tetramer. The human CD8b protein has multiple isoforms with different cytoplasmic tails arising from alternative splicing, unlike murine CD8b. We found differential expression at the level of mRNA of the four human CD8b splice variants (M1-4) during development, after activation, and in memory subsets. and surface expression of one of the isoforms (M-2) was regulated by ubiquitination. We hypothesize that these isoforms contribute to differential regulation of CD8 T cell responses in humans. In aim 3, we will study functional differences between the isoforms and determine the molecular basis for those differences. Different murine and human cell lines will be used including human CD8 T cells from an anti-melanoma clone and an anti-viral cytomegalovirus specific line. Motifs within the cytoplasmic tails suggesting molecular mechanisms, e.g. phosphorylation, dileucine, Grb2 binding, will be studied by creating mutations in the motifs and performing functional assays, association with adaptor proteins, coreceptor internalization, and signal transduction. The outcome of the proposed studies will provide insights into a critical protein-protein interaction, CD8-pMHC1, for the CD8 cytotoxic T cells that kill tumor cells and cells infected with intracellular pathogens. The information we obtain will serve as a basis for designing an "optimized"CD8b with enhanced binding to pMHC1 and/or optimal signaling that potentially could be used for adoptive cellular immunotherapy against tumors. PUBLIC HEALTH RELEVANCE: The long-term objective is to isolate an enhanced human CD8b mutant, and to determine the functional significance of human CD8b splice variants with different cytoplasmic tails in order to design an optimized CD8b protein that potentially can be used for adoptive cellular immunotherapy.