T cell receptor (TCR) antigen recognition induces the formation and stabilization of a specialized """"""""immunological synapse"""""""" at the T cell: antigen presenting cell (APC) junction. This junction is generated by the recruitment and exclusion of particular proteins from the contact area and is required for T cell activation. Advances in cell biology have revealed that the plasma membrane is composed of laterally associated """"""""lipid rafts"""""""" which float in a sea of otherwise glycerophospholipid rich membrane. Lipid rafts are enriched in a subset of cellular proteins and have been implicated in signal transduction, cytoskeletal reorganization and protein sorting. We hypothesize that lipid raft/non-raft partitioning provides the molecular basis for protein sorting which organizes the TCR and co stimulators, signal transducers and the actin cytoskeleton at the T cell:APC interface. Our recent studies discriminate two distinctly regulated lipid-raft-dependent T cell activation steps: 1) the TCR engagement-induced phosphorylation and concentration of TCR( and signal transducers within lipid rafts and 2) the TCR/CD48 co stimulation and Lck-SH3-domain-dependent migration of lipid rafts to the TCR contact cap. Whereas T cell activation (TCR induced IL-2 production) requires both steps, T cell inactivation (TCR-induced apoptosis) is unaffected by disruption of the second step. Therefore, we hypothesize that the TCR/mediated-mediated recruitment of lipid rafts to the TCR contact site facilitates the construction of a raft-based platform, which modulates the functional outcome of TCR engagement. To test the role of raft/non-raft membrane compartmentalization in organizing TCR signal transduction and the actin cytoskeleton at the T cell:APC contact cap, we will manipulate the raft/non raft partitioning of proteins involved in these processes and determine the effects on T cell signals and activation (Aim 1). To elucidate the molecular basis of TCR/co stimulation and the relationship between raft reorganization and the functional outcome of TCR engagement we will: a) characterize CD28, CD48 and LFA-1 costimulatory contributions to TCR lipid raft reorganization and signal transduction events (Aim 2); b) explore how subtle differences in TCR antigen binding properties affect antigen-induced raft reorganization, signals and the functional outcome of TCR engagement (Aim 3) and; c) determine if T cells manipulate lipid raft composition and dynamics throughout development to set thresholds for T cell activation (Aim 4). These studies will lead to a better understanding of how TCR signals are regulated to mediate distinct functional outcomes. Elucidation of lipid raft constituents and the molecular mediators of raft reorganization will provide novel targets for therapeutics aimed at independently modulating, individual TCR induced responses such as : 1) inhibiting unwanted T cell activation responsible for autoimmunity, graft rejection, and/or T cell transformation; 2) promoting peripheral tolerance in allograft reactive and autoimmune T cells and 3) potentiating tumor (or other) vaccines directed against sub optimally presented antigens.
