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

项目总结 CRAC(钙释放激活的钙通道)是激活钙-NFAT(核因子)的关键通道 激活的T细胞)T细胞中的信号通路。Ca~(2+)-NFAT通路缺陷导致免疫缺陷 人类。此外，包括环孢菌素A和FK506在内的该途径的阻断剂被广泛用于抑制 T细胞在临床上的功能，进一步强调了这一途径的重要性。的主要问题是 Ca~(2+)-NFAT通路的治疗利用是抑制这一通路导致系统免疫 抑制由于其在T细胞功能中的广泛作用。我们的目标是阐明CRAC的调控机制 局部效应T细胞中的通道，这些通道已经迁移到组织并形成治疗性 方法选择性抑制这些细胞中细胞因子的产生。在自身免疫的开始阶段，幼稚的T 细胞被准备好，并在淋巴结处分化为效应T细胞。启动的效应性T细胞然后迁移到 炎症的部位，在那里他们遇到自我抗原浓缩的环境，并产生最大水平的 炎性细胞因子包括干扰素-γ、白介素17和粒-巨噬细胞集落刺激因子。我们的初步数据现在揭示了一部小说 CRAC通道在局部效应T细胞反应中的选择性调节机制，而不是T细胞 引爆。我们发现，CRAC通道的孔亚基ORAI1的表面表达是有限的，并且有很大的 部分蛋白质(60%)被困在细胞内的小泡中。在效应性T细胞中，T细胞受体(TCR) 刺激触发ORAI1+囊泡向质膜的运输，这对持续的 钙离子进入，从而产生强大的效应器T细胞反应。从目标屏幕上，我们确定了两个 这些ORAI1+囊泡的成分，NKD2(裸皮同源物2)，一个信号适配器，以及Rab44，a 新的Rab GTP酶。机制上，NKD2在TCR刺激下被激活，并协调ORAI1+ 通过Rab44进行囊泡运输。NKD2和Rab44在效应器T细胞中大量表达和缺失 在不影响T细胞发育的情况下，Nkd2特异性地降低了局部效应T细胞的反应 体内引爆。综上所述，当前提案的主要假设是，来自 胞内到质膜的池对于局部所需的高和持续的钙信号至关重要 效应器T细胞反应。这项提案的具体目标是1)揭示TCR信号在 触发ORAI1+囊泡运输，2)确定参与运输的分子机制 ORAI1/NKD2+囊泡，包括Rab44，以及3)阐明ORAI1+囊泡的生理作用 使用神经元性炎症的动物模型运输局部效应T细胞反应。

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.