Imaging and Function of the Immunological Synapse

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DESCRIPTION (provided by the applicant): T lymphocytes see antigen only as small peptides bound to MHC molecules on the surface of antigen presenting cells (APC). In addition to the T cell antigen receptor (TCR) and peptide-loaded MHC, dozens of other cell surface molecules play necessary or modulating roles in determining the outcome of the T ceIl/APC interaction. Antigen recognition is accompanied by large-scale, cytoskeleton-dependent rearrangements of these molecules to form an organized contact interface between the T cell and the APC termed the "immunological synapse." This application is based on the hypothesis that synapse formation functions as a checkpoint for full T cell activation, integrating information on the number and quality of TCR ligands as well as the nature and activation state of the APC (van der Merwe et al. 2000 Semin. Immunol. 12:5). To test this hypothesis, state-of-the-art video microscopy will be used to follow calcium signals and synapse formation between murine TCR transgenic T cells and fibroblasts bearing fluorescent MHC molecules loaded with covalently attached antigenic peptides to determine whether accumulation of MHC molecules in the synapse is peptide-specific, and if so, to determine when sorting occurs during synapse formation. The same techniques will be used to determine whether MHC molecules loaded with antagonist peptides also accumulate in the synapse and how they may affect synapse formation or function. Mice transgenic for fluorescent MHC molecules loaded with covalently attached antigenic peptide will be made and used to study synapse formation, function, and accessory molecule requirements between T cells and physiological antigen presenting cells (dendritic cells and B cells) in different states of activation and maturation. The proposed experiments will contribute to a basic understanding of T cell/APC interactions, which in turn will have health-related applications in transplantation, autoimmunity, allergy, cancer immunotherapy, and control of infectious diseases.