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

项目摘要 CRAC（Ca 2+释放激活的Ca 2+）通道对于Ca 2 +-NFAT（细胞核因子）的激活是关键的。 活化的T细胞）在T细胞中的信号通路。Ca 2 +-NFAT途径的缺陷导致免疫缺陷， 人类此外，包括环孢菌素A和FK 506在内的该途径的阻断剂广泛用于抑制细胞凋亡。 T细胞在临床中的功能，进一步强调了这一途径的重要性。的主要问题 Ca 2 +-NFAT途径的治疗性开发是该途径的抑制导致全身性免疫应答。 由于其在T细胞功能中的广泛作用，抑制。我们的目标是阐明调节CRAC的机制 通道，特别是在局部效应T细胞，已经迁移到组织和发展的治疗 选择性抑制这些细胞中细胞因子产生的方法。在自身免疫发病期间， 细胞被引发并在淋巴结处分化为效应T细胞。然后，致敏的效应T细胞迁移至 炎症部位，在那里它们遇到自身抗原富集的环境并产生最大水平的 炎性细胞因子包括IFN-γ、IL-17和GM-CSF。我们的初步数据显示 在局部效应T细胞应答中，而不是在T细胞应答中，选择性调节CRAC通道的潜在机制 启动我们发现，CRAC通道的孔亚基ORAI 1的表面表达是有限的，并且大量表达于细胞表面。 部分蛋白质（60%）被胞内捕获在囊泡内。在效应T细胞中，T细胞受体（TCR） 刺激触发ORAI 1+囊泡向质膜的运输，这对于维持细胞的增殖是重要的。 Ca 2+进入并由此产生稳健的效应T细胞应答。从目标屏幕上，我们已经确定了两个 这些ORAI 1+囊泡的组分，NKD 2（裸角质层同源物2），一种信号衔接子，和Rab 44，一种 小说《拉布·戈蒂默》从机制上讲，NKD 2在TCR刺激后被激活，并协调ORAI 1 + 囊泡通过Rab 44运输。NKD 2和Rab 44都在效应T细胞中大量表达， Nkd 2特异性降低局部效应T细胞应答而不影响T细胞发育， 体内预充。综上所述，目前建议的主要假设是，从 细胞内池的质膜是至关重要的高和持续的Ca 2+信号所需的局部 效应T细胞应答。该提案的具体目标是：1）揭示TCR信号在 触发ORAI 1+囊泡运输，2）鉴定参与ORAI 1+囊泡运输的分子机制， 包括Rab 44的ORAI 1/NKD 2+囊泡，和3）阐明ORAI 1+囊泡的生理作用 使用神经元炎症的动物模型运输局部效应T细胞应答。

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.