Novel mechanisms of Ca2+ signaling in B lymphocytes

描述(由申请人提供)：B淋巴细胞在宿主防御中起关键作用；然而，其激活和分化的关键机制尚不清楚。例如，细胞内的钙离子被认为是中枢的，但人们对它是如何调控的知之甚少。我们对淋巴细胞中的钙离子的许多了解都是从T细胞的研究中学到的。例如，钙释放激活钙通道(CRAC)被认为是T细胞中主要的，也可能是唯一的抗原受体操纵的钙通道，已被详细研究，但尚未在原代B细胞中被描述。事实上，有限的先前研究和我们的初步数据表明，B细胞中的钙信号转导机制比T细胞中所描述的更复杂，CRAC通道是否在B细胞免疫功能中发挥类似的不可或缺的作用现在似乎不太可能。我们发现B细胞不仅利用CRAC通道，还利用额外的钙离子非选择性阳离子通道(NSCC)。我们进一步证明，这些NSCCs被与先天性免疫反应相关的刺激激活，包括称为病原体相关分子模式(PAMPs)的Toll样受体(TLR)的病毒和细菌激动剂，以及机械力。这些反应在T细胞中没有观察到。我们推测，B细胞和T细胞之间的这些差异反映了这两个淋巴细胞群体识别抗原的方式的根本差异。B细胞通过与其表面的免疫球蛋白分子接触直接识别抗原。相反，T细胞对抗原的识别是间接的，依赖于与抗原提呈细胞(APC)的允许相互作用。作为对自身免疫的一种保护措施，仅识别抗原不足以触发激活；需要第二个信号来指示危险的存在或不存在--TLR配体，如病原体特有的双链RNA、脂多糖、LTA、未甲基化的CpG DNA。T细胞的大部分决策是由APC做出的，而B细胞在整合共刺激信息方面是独立的，这将需要我们已经发现的更复杂的信号转导。因此，这一应用的核心基本思想之一是，B细胞的适当反应将反映由BCR和天然刺激激活的重叠通路对不同的钙通道(NSC和CRAC)的协调激活。我们将使用单细胞膜片钳记录和钙成像方法来生物物理地定义这些由BCR和先天刺激激活的通道，并对这种方法进行新的修改，以揭示协调其激活和功能的复杂的细胞内信号通路。我们的结果将对理解免疫能力、免疫缺陷和自身免疫的基础具有重要意义。

DESCRIPTION (provided by applicant): B lymphocytes play a critical role in host defenses; however critical mechanisms of their activation and differentiation are poorly understood. For example, intracellular Ca2+ is recognized as being central, yet relatively little is known about how it is regulated. Much of what we know about Ca2+ in lymphocytes has been learned from studies of T cells. For example, Ca2+-Release Activated Calcium (CRAC) channels, which are believed to be their major, and possibly only, antigen receptor-operated Ca2+ channel in T cells, has been studied in great detail, but it has not yet been described in primary B cells. In fact, limited previous studies and our preliminary data demonstrate that the mechanisms of Ca2+ signaling in B cells are more complex than described for T cells, and whether CRAC channels play a similar indispensable role in B cell immunological functions now seems unlikely. We show that B cells utilize CRAC channels, but also additional Ca2+-permeant Non-Selective Cation Channels (NSCC). We further demonstrate that these NSCCs are activated by stimuli associated with innate immune responses including viral and bacterial agonists of Toll like receptors (TLR) called Pathogen-Associated Molecular Patterns (PAMPs), and by mechanical forces. These responses are not observed in T cells. We speculate that these dissimilarities between B and T cells reflect the fundamental difference in the way that these two lymphocyte populations recognize antigens. B cells recognize antigens directly through contact with immunoglobulin molecules at their surface. In contrast, antigen recognition by T cells is indirect and depends upon permissive interactions with antigen presenting cells (APC's). As a safeguard against autoimmunity, recognition of antigen alone is not sufficient to trigger activation; a second signal indicating the presence or absence of danger - TLR ligands such as double-stranded RNA, LPS, LTA, unmethylated CpG DNA that are unique to pathogens - is required. Much of the decision making for a T cell is made by the APC, while the B cell is on its own in terms of integrating costimulatory information and this will require the more complex signaling that we have uncovered. One of the central underlying ideas of this application, therefore, is that appropriate responses of B cells will reflect the coordinated activation of distinct Ca2+ permeant (NSC and CRAC) channels by the overlapping pathways activated by the BCR and innate stimuli. We will use single cell patch clamp recording and calcium imaging methods to biophysically define these channels activated by BCR and innate stimuli, but also a novel modification of this approach to unravel the complex intracellular signaling pathways that coordinate their activation and functions. Our results will have fundamental importance for understanding the basis of immune competence, immune deficiency, and autoimmunity.