Mechanism underlying regulation of Ca2+ signaling in local effector T cells

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PROJECT SUMMARY CRAC (Ca2+ release-activated Ca2+) channels are pivotal for activation of the Ca2+-NFAT (nuclear factor of activated T cells) signaling pathway in T cells. Defects in the Ca2+-NFAT pathway lead to immune deficiency in humans. In addition, blockers for this pathway including cyclosporin A and FK506 are widely used to suppress T cell functions in the clinic, further emphasizing the importance of this pathway. The major problem with therapeutic exploitation of the Ca2+-NFAT pathway is that inhibition of this pathway leads to systemic immune suppression due to its broad role in T cell functions. Our goal is to elucidate mechansims regulating CRAC channels specifically in local effector T cells that are already migrated to the tissue and develop a therapeutic method to selectively suppress cytokine production in those cells. During the onset of autoimmunity, naïve T cells are primed and differentiate to effector T cells at the lymph nodes. Primed effector T cells then migrate to the site of inflammation, where they encounter self-antigen-enriched milieu and produce maximal levels of inflammatory cytokines including IFN-gamma, IL-17 and GM-CSF. Our preliminary data now reveal a novel mechanism underlying regulation of CRAC channels selectively in local effector T cell responses, not in T cell priming. We find that surface expression of ORAI1, the pore subunit of CRAC channels is limited and a large fraction of the protein (60%) is intracellularly trapped within vesicles. In effector T cells, T cell receptor (TCR) stimulation triggers trafficking of ORAI1+ vesicles to the plasma membrane, which is important for sustained Ca2+ entry and thereby robust effector T cell response. From a targeted screen, we have identified two components of these ORAI1+ vesicles, NKD2 (naked cuticle homolog 2), a signaling adaptor, and Rab44, a novel Rab GTPase. Mechanistically, NKD2 gets activated upon TCR stimulation and orchestrates ORAI1+ vesicle trafficking via Rab44. Both NKD2 and Rab44 are abundantly expressed in effector T cells and deletion of Nkd2 specifically decreased local effector T cell responses without influencing T cell development and priming in vivo. Taken together, the main hypothesis of the current proposal is that ORAI1 trafficking from the intracellular pool to the plasma membrane is crucial for high and sustained Ca2+ signaling required for local effector T cell responses. The specific objectives of this proposal are 1) uncover the role of TCR signals in triggering of ORAI1+ vesicle trafficking, 2) identify the molecular machinery involved in trafficking of the ORAI1/NKD2+ vesicles, that includes Rab44, and 3) elucidate the physiological role of ORAI1+ vesicle trafficking in local effector T cell responses using animal models of neuronal inflammation.