The long-term goal of this proposal is to dissect the functions of Rap1 and its molecular targets 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 and 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 increase of active Rap1 is detected in anergic cells. Forced expression of active Rap1 inhibits T cell activation and IL-2 transcription. Our studies proposed here are based on preliminary results generated during the previous funding period of this application: 1) Using the yeast two-hybrid system we detected that Rap1-GTP interacts with AF-6 in T cells. AF-6 regulates actin remodeling proximal to junctional complexes and promotes clustering of its associated proteins at cellular junctions. Thus, AF-6 is an attractive candidate to mediate the effects of Rap1 on integrin activation and immunological synapse formation. 2) By suppression subtractive hybridization, we determined that Rap1-GTP induces a distinct gene expression pattern in T cells. Among Rap1-GTP-induced genes were MKP-7 dual specificity phosphatase, and CD103. MKP-7 has selective activity for MAP kinases p38 and JNK that are required for induction and activation of NFAT and AP-1 transcription factors. CD103 defines a unique subset of Treg, which are the most potent immunosuppressors in vivo. 3) We have generated Rap1-GTP (E63)-Transgenic mice. These mice have increased CD103+CD4+CD25+ Treg and display impaired recall T cell responses to antigenic stimulation. These exciting results will become the basis of the studies proposed in this application.
Our Specific Aims are: 1. To investigate the role of the Rap-1 associated molecule AF-6 and the Rap1-induced molecules MKP-7 and CD103 in T cell responses in vitro. We will examine the involvement of AF-6 in formation of the immunological synapse, localization of Rap1 at the synapse and in Rap1-mediated integrin activation. We will examine the effects of MKP-7 on activation of p38 and JNK, expression and transactivation of NFAT, phosphorylation of c-Jun, transactivation of AP-1, and transcription of IL-2. Finally, we will determine the role of CD103 on T cell activation and cytokine production in a cell autonomous and non-autonomous manner. 2. To investigate the role of Rap1 in T cell responses in vitro and in vivo by using mice transgenic for constitutively active Rap1. Our preliminary data with Rap1-GTP-Tg mice point to a negative role of Rap1-GTP in vivo in regulating effector T cell responses. The studies proposed here will extend our preliminary observations and will examine the effects of Rap1 on the activation and differentiation of na?ve CD4+ cells, function of effector CD4+ T cells, function of CD4+ helper T cells, induction of T cell tolerance in vivo to a peptide antigen and alloantigen, and function of CD8+ cytolytic cells. Thus 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. ? ?
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