The goal of this proposal is to dissect the functions of Rap1 and its molecular partners in the regulation of T cell responses. Rap1, originally identified by its ability to reverse Ras-mediated transformation, is now known to regulate cytoskeletal reorganization, adherens junction positioning, integrin activation and cell adhesion. In T lymphocytes, transient activation and localization of Rap1 at the immunological synapse is one of the physiologic consequences of TCR ligation. In contrast, sustained, forced expression of active Rap1 inhibits T cell activation and IL-2 transcription. Our studies proposed here, are based on observations made during the previous funding period of this application: First, we have identified and characterized RIAM, a new molecular partner of Rap1 that regulates integrin activation in lymphocytes and platelets, and we determined that RIAM interacts with components of the T cell signaling machinery. Second, we developed experimental evidence that Rap1 regulates plasma membrane compartmentalization of Ras signaling. In T cells, TCR and CD28- mediated Ras activation occurs at the Golgi apparatus and requires phospholipase Cγ, whereas LFA-1- mediated Ras activation occurs at the plasma membrane and requires phospholipase D but not phospholipase Cγ. Rap1 may compartmentalize Ras signaling at the plasma membrane because it induces LFA-1 activation. Third, using Rap1-deficient and Rap1-GTP transgenic mice we have determined that Rap1 may be involved in the generation of thymic and peripheral Treg. These exciting results will become the basis of the studies proposed in this application.
Our Specific Aims are: 1. To investigate the role of Rap1 and its novel partner, RIAM, in regulating T cell migratory and interaction dynamics. We will examine the role of Rap1 and RIAM in regulating T cell: APC migration dynamics and activation kinetics in and the differentiation of T effector cells after T cell: APC interaction in vivo. 2. To investigate the role of Rap1 and its novel partners in regulating molecular and functional T cell responses in vitro. We will investigate the role of Rap1 in regulating formation of the TCR signalosome, compartmentalized Ras activation and functional outcome of T cell responses. 3. To investigate the role of Rap1 in regulating expression of Foxp3. We will investigate whether Rap1 promotes generation of Treg by enhancing LFA-1 mediated adhesion or by modulating PI3K/Akt, NFAT/Smad3 and Notch pathways. Our in vitro and in vivo studies will complement each other in understanding the effects of Rap1 at the molecular/signaling level and in the intact host.
The work proposed in this application will study the mechanisms that regulate T cell responses mediated via the small GTPase, Rap1. This knowledge will suggest how therapeutic manipulation of Rap1-mediated events could modulate immune responses either for suppression, as in autoimmunity, inflammatory diseases, allergy, graft rejection and GVHD, or for augmentation against pathogens and tumor antigens.
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