Calcium Regulation of NF-kB Activation in Lymphocytes

项目总结 抗原受体诱导的钙信号调节淋巴细胞的发育和效应器功能，但 我们对这些信号的动态及其转录结果的理解仍然存在重大差距 以及细胞命运是如何由不同的受体诱导的钙波形决定的。此表中的支持数据 应用和即将发表的一篇论文揭示了钙离子通过 特异性地调节TCR，而不是肿瘤坏死因子，诱导核因子-κB p65和c-Rel激活。我们的发现P65和c-Rel 磷酸化可作为钙离子依赖的检查点提供一个框架，用于理解 体内的钙信号模式控制着驱动淋巴细胞发育和 功能。TCR诱导的钙内流的中枢是内质网跨膜钙感应信号STIM1和STIM2 蛋白质，以及STIM激活的钙通道CRac/Orai。T细胞前体细胞缺乏STIM1/2的小鼠表现出 NTreg诱导的选择性缺陷和这些小鼠的免疫表型几乎无法区分 与c-Rel缺乏的小鼠不同。此外，由于nTreg的开发是由CD28协同刺激推动的，因此 结合高亲和力/亲和力的TCR连接，我们之前已经表明CD28对此有偏见 在一系列抗原受体连接狂热中，朝向高幅度钙离子的确定性信号尖峰 假设TCR诱导的钙动力学的CD28调节产生一个定量独特的波形，该波形 驱动依赖于c版本的nTreg生成。根据我们即将发布的出版物和新的支持数据，我们 假设TCR诱导单个p65和c-rel Ser/Thr残基的钙依赖磷酸化是 由不同的钙离子阈值和波形调节，从而提供了一种机制，通过这种机制 不同的Ca~(2+)信号差异调节控制淋巴细胞的NF-κB依赖基因表达 发展和功能。为了解决这一假设，我们将：1)定义钙离子的性质和功能- 调节p65和c-Rel的磷酸化，2)决定Ca~(2+)如何控制NF-κB驱动的转录 3)明确了胸腺nTreg发育的钙依赖机制。我们将使用一个定制 建立了“钙钳”灌流系统，以施加钙的幅度，脉冲宽度，频率和 总输入以定义Ca~(2+)波形如何控制NF-B激活和长期的顺序检查点 利用一种新的遗传编码的钙指示剂(GCaMP6f)进行成像测量以确定如何 抗原诱导的钙信号参与调节nTreg的发育。两家公司的综合专业知识 团队、系统和方法以及为这些研究开发的量化工具将提供新的 以及钙依赖的磷酸化如何调节转录的详细机制 核因子-B在T细胞中的特异性，并将为增强 不充分，抑制自身反应，或重定向不适当的免疫反应。

PROJECT SUMMARY Antigen receptor-induced calcium (Ca2+) signals regulate lymphocyte development and effector functions, but major gaps persist in our understanding of the dynamics of these signals, their transcriptional consequences and how cell fates are governed by distinct receptor-induced Ca2+ waveforms. Supporting data in this application and a pending publication reveal entirely novel and indispensable mechanisms by which Ca2+ specifically tunes TCR, but not TNF, induced NF-κB p65 and c-Rel activation. Our findings that p65 and c-Rel phosphorylation may serve as Ca2+ dependent checkpoints provide a framework for understanding how distinct patterns of Ca2+ signaling in vivo control the transcriptional programs that drive lymphocyte development and function. Central to TCR-induced Ca2+ entry are the ER transmembrane Ca2+ sensing STIM1 and STIM2 proteins, and the STIM-activated Ca2+ channel CRAC/Orai. Mice whose T cell progenitors lack STIM1/2 exhibit a selective defect in nTreg induction and the immune phenotype of these mice is virtually indistinguishable from that of mice with a c-Rel deficiency. Moreover, as nTreg development is driven by CD28 costimulation in conjunction with high affinity/avidity TCR ligation, and we have previously shown that CD28 biases this deterministic signal toward high amplitude Ca2+ spikes over a range of antigen receptor ligation avidities, we hypothesize that CD28 tuning of TCR induced Ca2+ dynamics generates a quantitatively unique waveform that drives c-Rel-dependent nTreg generation. Based on our pending publication and new supporting data, we hypothesize that TCR induced Ca2+-dependent phosphorylation of individual p65 and c-Rel Ser/Thr residues is regulated by distinct Ca2+ thresholds and waveforms, thereby providing a mechanism by which quantitatively distinct Ca2+ signals differentially regulate NF-κB-dependent gene expression controlling lymphocyte development and functions. To address this hypothesis, we will: 1) Define the nature and function of Ca2+- regulated phosphorylation of p65 and c-Rel, 2) determine how Ca2+ controls NF-κB-driven transcriptional specificity, and 3) define the Ca2+ dependent mechanisms of thymic nTreg development. We will use a custom built “Ca2+ clamp” perfusion system to impose variations in the Ca2+ amplitude, pulse width, frequency, and total input to define how Ca2+ waveforms control sequential checkpoints in NF-B activation and long-term imaging measurements utilizing a novel genetically encoded Ca2+ indicator (GCaMP6f) to determine how antigen-induced Ca2+ signals contribute to the regulation of nTreg development. The combined expertise of the team, the systems and approaches, and the quantitative tools developed for these studies will provide a new and detailed mechanistic understanding of how calcium dependent phosphorylation regulates transcriptional specificity of NF-B in T cells and will provide insights into strategies and therapeutic targets for enhancing insufficient, suppressing auto-reactive, or redirecting inappropriate immune responses.