Developmental B Cell Tolerance and Negative Selection

描述(申请人提供)：未成熟的B细胞代表 B淋巴细胞发育的重要窗口，因为正是在这个阶段 表达自身反应特异性的细胞被识别和消除。在……里面 分离，未成熟的B细胞在BCR参与下发生凋亡 而成熟的B细胞启动激活程序并抵抗 细胞凋亡。这些数据表明，人类不同的命运反应 BCR参与后的未成熟和成熟期B细胞是由于固有的 信号事件。我们之前的研究表明，虽然BCR参与 成熟B细胞导致胞浆内钙离子水平升高 伴随蛋白激酶C激活的(Ca2+)i (PKC)，未成熟的B细胞通过升高细胞内(Ca~(2+))i来应答BCR的参与。 PKC活性相对缺失。我们假设，在没有PKC的情况下 激活，钙离子依赖的信号转导事件在 BCR诱导未成熟B细胞凋亡。我们的初步结果表明 BCR诱导的未成熟B细胞的凋亡反应相对 与(Ca~(2+))i的增加无关，而是暗示 内质网(ER)钙库在此过程中可能起决定性作用 回应。内质网钙离子储存耗尽导致蛋白质转运受阻 这伴随着一种被称为未折叠蛋白的细胞应激反应 回应(普遍定期审议)。UPR由一条独特的“内质网-核”信号通路组成 旨在检测内质网中错误折叠的蛋白质水平，并重塑 减少它的分泌途径。重要的是，普遍定期审议具有内置的“故障安全”功能 机制；如果错误折叠的蛋白质的积累不减少，一个 随之而来的是细胞凋亡反应。这项研究的中心假设是 这个应用是内质网应激的水平和/或持续时间和 相关的UPR决定了B细胞对BCR信号的命运。至 具体检验这一假设，我们建议：1)评估钙离子的变化 未成熟和成熟B细胞BCR交联后的动态平衡，2) 评估普遍定期审议在规范BCR引起的命运决定方面的潜在作用， 3)评估错误折叠蛋白的积累是否影响B细胞的发育 通过UPR介导的事件，以及4)确定PKC激活对 BCR诱导的钙稳态改变和UPR诱导。理解 这些发育调控的bcr诱导命运的分子基础 决策将提供对这些过程可能如何被颠覆的洞察 自身免疫性疾病状态。

DESCRIPTION (provided by applicant): The immature B cell represents an important window in B lymphocyte development, for it is at this stage that cells expressing self-reactive specificities are identified and eliminated. In isolation, immature B cells undergo apoptosis in response to BCR engagement while mature B cells initiate activation programs and are resistant to apoptosis. These data suggest that the differential fate responses of the immature and mature stage B cells following BCR engagement are due to intrinsic signaling events. Our previous studies demonstrated that while BCR engagement of mature B cells results in an elevation in intracytoplasmic Ca2+ levels (Ca2+) i that is accompanied by a concomitant activation of protein kinase C (PKC), immature B cells respond to BCR engagement by elevating (Ca2+) i in the relative absence of PKC activation. We hypothesize that, in the absence of PKC activation, Ca2+ -dependent signaling events play a causative role in the BCR-induced apoptosis of immature B cells. Our preliminary results indicate that the BCR-induced apoptotic response of immature B cells is relatively independent of an increase in (Ca2+)i and instead suggest that a depletion of endoplasmic reticulum (ER) Ca + stores may play a determinant role in this response. Depletion of ER Ca2+ stores results in a block in protein transport that is accompanied by a cellular stress response known as the unfolded protein response (UPR). The UPR comprises a unique "ER to nucleus" signaling pathway designed to sense the level of misfolded protein in the ER and remodel the secretory pathway to reduce it. Importantly, the UPR has a built in "fail-safe" mechanism; if the accumulation of misfolded protein is not reduced, an apoptotic response ensues. The central hypothesis of the studies proposed in this application is that the level and/or duration of ER stress and the associated UPR determines the fate of the B cell to BCR signaling. To specifically test this hypothesis, we propose to: 1) assess alterations in Ca2+ homeostasis following BCR crosslinking in immature and mature stage B cells, 2) assess the potential role of the UPR in regulating BCR-induced fate decisions , 3) assess whether accumulation of misfolded protein affects B cell development through UPR-mediated events, and 4) determine the effects of PKC activation on BCR-induced alterations in Ca2+ homeostasis and UPR induction. Understanding the molecular basis for these developmentally regulated BCR-induced fate decisions will provide insight into how these processes may be subverted in autoimmune disease states.