T Cell Signal Transduction in Membrane Domains and its Alterations by Modification of Membrane Lipids

Lipids modulate the immune response by affecting T cell activation. Stimulation of T cells via the transmembrane T cell receptor(TCR)/CD3-complex or costimulatory glycosyl phosphatidylinositol(GPI)-anchored proteins seems to involve sphingolipid and cholesterol-enriched membrane domains (SCMDs) which are functional rafts within the plasma membrane. Previous work from our laboratory revealed that modification of membrane lipids, namely enrichment with polyunsaturated fatty acids (PUFAs) and lowering of cellular cholesterol, inhibits T cell signal transduction and is accompanied by structural alterations of SCMDs in quiescent PUFA-enriched T cells. However, the molecular changes which occur in SCMDs following T cell activation, and the interference of lipid modifications with the proceeding T cell signaling cascade have yet to be elucidated. This project, therefore, aims at further elucidating the mechanisms underlying the immunomodulatory effects of lipids by analyzing T cell signal transduction as it occurs in membrane domains and possible alterations of these locally confined signaling events by modification of membrane cholesterol and fatty acid composition. To this end, membrane lipids of Jurkat T cells are to be modified by enrichment with PUFAs or by metabolic depletion of cellular cholesterol, and lipid-modified and control cells will be stimulated via the transmembrane TCR/CD3 complex or GPI-anchored CD59. Since protein tyrosine phosphorylation is a key event in T cell signal transduction, the occurrence of tyrosine phosphorylated proteins in biochemically distinct parts of the plasma membrane, namely SCMDs, the intermediate fraction previously described by us, and the remaining mass of the plasma membrane, will be analyzed by density gradient centrifugation. Particular tyrosine phosphorylated proteins will be identified and possible alterations in the distribution of signaling proteins and the efficiency of their phosphorylation as induced by lipid modifications will be observed. In addition, protein-protein complexes known to occur during T cell signaling will be localized in density gradients and the influence of lipid modifications will be studied. In order to circumvent the use of detergents, SCMDs will be isolated from lipid-modified and control cells also by a detergent-free method. Such isolated SCMDs will be stimulated in vitro in order to directly define a role of membrane domains in T cell signal transduction as well as the influence of lipids on SCMD signaling function. Immunofluorescence studies will complete the information on the distribution of tyrosine phosphorylated proteins in stimulated lipid-modified T cells. In conclusion, the results of this project may promote understanding of signal transduction mechanisms in T cells and may provide more general information about the influence of lipids on the immune response.

Lipids modulate the immune response by affecting activation of immune cells such as T lymphocytes. Particularly polyunsaturated fatty acids (PUFA) of the "omega-3" series that are abundantly found in marine fish oils inhibit T lymphocyte activation. Hence, omega-3 PUFA are also clinically applied in addition to classical immunosuppressive drugs, e.g., in the treatment of inflammatory joint and bowel disorders. Activation of T lymphocytes involves specialized areas of the cell membrane. These so-called lipid rafts are built up by a particular lipid composition rich in cholesterol and saturated fatty acids and harbor a variety of molecules with critical functions in cell activation. In this project we analyzed the mechanisms how PUFA affect T lymphocyte activation in vitro. We found a novel molecular mechanism by which PUFA inhibit T cell activation. Treatment with PUFA provokes displacement of a variety of molecules that are involved in T cell activation from lipid rafts to other parts of the cell membrane. Here we demonstrated that PUFA displace molecules from rafts by altering raft lipid composition rather than affecting anchoring mechanisms of proteins. The inhibitory action of PUFA could be pinpointed to a single T cell activation molecule called linker for activation of T cells (LAT) whose displacement from lipid rafts by PUFA provokes diminished activation of T cells. To proof this concept, we demonstrated that relocalization of LAT alone in lipid rafts by molecular techniques is sufficient to restore crucial activation events in PUFA-treated T lymphocytes. Further studies revealed that the inhibitory action of PUFA on T cell activation is surprisingly selective also for later activation events that are necessary to successfully initiate an immune response. T cell activation occurs in vivo by so-called antigen-presenting cells that digest foreign molecules ("antigens") and present them in a proper way to T cells by forming the "immunological synapse". Our experiments revealed that formation of the immunological synapse was disturbed by PUFA treatment of T cells in a rather specific manner. Disruption of synapse formation not only potentiates the defects in T cell activation mediated by PUFA but also widens their impact on activation pathways that are not intrinsically altered in T cells. In conclusion this project has greatly enhanced our understanding of the immunomodulatory action of PUFA by disclosing a novel mechanisms and by characterizing T cell activation defects in detail.