The Regulation of B-cell Activation

B 细胞激活的调节

• 批准号: 7315093
• 负责人: susan pierce
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7315093
• 关键词: Regulation; cell; Activation

B cell receptor (BCR) triggered B cell activation is regulated by a variety of factors that control the outcome of the B cell's encounter with antigen including the developmental state of the B cell, the engagement of coreceptors and Toll-like receptors (TLR) and viral infection. Determining how these factors influence BCR-induced signaling should add fundamentally to our understanding of the mechanism by which B cells are activated. Over the last year we have made progress in defining the mechanisms by which the positive coreceptor, the CD19/CD21 complex, the inhibitory receptor, FcgammaRIIB1, and the TLR, TLR9 function to regulate signaling. The B cell coreceptors CD19/CD21 when coligated to the BCR through the binding of complement tagged antigens prolongs and enhances BCR signaling in part by prolonging the association of the BCR with sphingolipid- and cholesterol-rich membrane microdomains, termed lipid rafts. We determined that the ability of the CD19/CD21 complex to function in rafts was dependent on a tetraspanin CD81 that is a component of the CD19/CD21 complex. Thus, in B cells from CD81-deficient mice and B cells expressing chimeric CD19 receptors that fail to associate with CD81, the CD19/CD21 complex when coligated to the BCR failed to stabilize the BCR in rafts. Many proteins that associate with lipid rafts do so by virtue of their acylation in particular by their palmitoylation, a reversible acylation event. We determined that CD81 becomes palmitoylated in the lipid rafts following crosslinking of the BCR and the CD19/CD21 complex and that palmitoylation is essential for the function of CD81. CD81 contains six cysteines within its cytoplasmic face that are potential sites of palmitoylation. To assess the contribution of these cysteines to CD81 function CD81 molecules containing mutated cysteine residues were expressed in B cell lines in which the endogenous wild type CD81 expression was knocked down by RNAi technology. These mutants showed defects in signaling following coligation of the BCR and the CD19/CD21 complex verifying the importance of these cysteine residues in the function of CD81. Constructs of CD81 that contained FRET donor fluorescent proteins were used in conjunction with FRET acceptor fluorescent proteins targeted to raft microdomains to demonstrate by FRET confocal microscopy in living cells that the cysteine residues of CD81 were essential for the association of CD81 with raft lipids following the coligation of the BCR and the CD19/CD21 complex. Progress was also made in determining how the FcgammaRIIB, a potent negative regulator of BCR signaling when coligated to the BCR, signals for apoptosis when crosslinked to itself. We learned that the FcgammaRIIB when crosslinked to itself becomes associated with lipid rafts and signals for apoptosis by a mechanism dependent on c-Abl but independent of both the phosphatase SHIP and the FcgammaRIIB?s ITIM motifs that are required for FcgammaRIIB?s inhibition of BCR signaling. Results of recent studies have provided evidence that crosslinking the FcgammaRIIB1 blocks the differentiation of memory B cells to antibody secreting plasma cells. Thus, the addition of mAb specific for FcgammaRIIB1 to human CD27+ memory B cells blocks the TLR-9 agonist-induced differentiation of memory B cells to plasma cells. These findings are significant in defining a new target for FcgammaRIIB1 mediated regulation. Studies are in progress to determine at which point in the differentiation of memory cells to plasma cells the FcgammaRIIB1 functions. Lastly, BCR signaling is greatly amplified by TLR-9 agonists. Recent studies have suggested that antigens that contain the TLR-9 agonist, CpG oligonucleotides, reduce the threshold for B cell activation and may, as a consequence, play a role in autoimmune disease by allowing the activation of low affinity self-reactive B cells. However, TLR-9 is only present in B cells in intracellular endosomal compartments and the cellular mechanisms by which CpG-containing antigens augment BCR signaling through TLR-9 are not well understood. Our recent studies using a combination of confocal and electron microscopy provided evidence that BCR signaling results in the recruitment of TLR9-containing endosomes to the intracellular multivesicular bodies to which the BCR transports CpG-containing antigens. These vesicles appear to be the site of augmented BCR/TLR-9 signaling. This novel mechanism for TLR-9 enhancement of BCR signaling may be important in providing new targets for therapy for autoimmune antibody responses.

B 细胞受体 (BCR) 触发的 B 细胞激活受到多种因素的调节，这些因素控制 B 细胞与抗原相遇的结果，包括 B 细胞的发育状态、辅助受体和 Toll 样受体 (TLR) 的结合以及病毒感染。确定这些因素如何影响 BCR 诱导的信号传导应该从根本上增加我们对 B 细胞激活机制的理解。去年，我们在确定正向辅助受体 CD19/CD21 复合物、抑制性受体 FcgammaRIIB1 以及 TLR、TLR9 调节信号传导的机制方面取得了进展。 B 细胞辅助受体 CD19/CD21 当通过补体标记抗原的结合与 BCR 结合时，会部分通过延长 BCR 与富含鞘脂和胆固醇的膜微域（称​​为脂筏）的结合来延长和增强 BCR 信号传导。我们确定 CD19/CD21 复合物在筏中发挥作用的能力依赖于作为 CD19/CD21 复合物的组成部分的四跨膜蛋白 CD81。因此，在来自 CD81 缺陷小鼠的 B 细胞和表达无法与 CD81 结合的嵌合 CD19 受体的 B 细胞中，CD19/CD21 复合物与 BCR 结合时未能稳定筏中的 BCR。许多与脂筏结合的蛋白质是通过它们的酰化作用，特别是通过它们的棕榈酰化作用（一种可逆的酰化事件）来实现这一点。我们确定，BCR 和 CD19/CD21 复合物交联后，CD81 在脂筏中发生棕榈酰化，并且棕榈酰化对于 CD81 的功能至关重要。 CD81 在其细胞质表面含有六个半胱氨酸，它们是棕榈酰化的潜在位点。为了评估这些半胱氨酸对 CD81 功能的贡献，在 B 细胞系中表达含有突变半胱氨酸残基的 CD81 分子，其中内源野生型 CD81 表达被 RNAi 技术敲低。这些突变体在 BCR 和 CD19/CD21 复合物连接后显示出信号传导缺陷，验证了这些半胱氨酸残基在 CD81 功能中的重要性。将含有 FRET 供体荧光蛋白的 CD81 构建体与靶向筏微结构域的 FRET 受体荧光蛋白结合使用，通过活细胞中的 FRET 共聚焦显微镜证明，在 BCR 和 CD19/CD21 复合物连接后，CD81 的半胱氨酸残基对于 CD81 与筏脂质的关联至关重要。 在确定 FcgammaRIIB（与 BCR 结合时 BCR 信号传导的有效负调节因子）如何在与其自身交联时发出凋亡信号方面也取得了进展。我们了解到，FcgammaRIIB 当与其自身交联时，通过依赖于 c-Abl 的机制与脂筏和细胞凋亡信号相关，但不依赖于 FcgammaRIIB 抑制 BCR 信号传导所需的磷酸酶 SHIP 和 FcgammaRIIB 的 ITIM 基序。最近的研究结果提供了证据，表明交联 FcgammaRIIB1 会阻止记忆 B 细胞向分泌抗体的浆细胞分化。因此，向人 CD27+ 记忆 B 细胞添加 FcgammaRIIB1 特异性 mAb 可阻断 TLR-9 激动剂诱导的记忆 B 细胞向浆细胞的分化。这些发现对于确定 FcgammaRIIB1 介导的调节的新靶点具有重要意义。正在进行研究以确定 FcgammaRIIB1 在记忆细胞分化为浆细胞的哪个点发挥作用。 最后，TLR-9 激动剂极大地放大了 BCR 信号传导。最近的研究表明，含有 TLR-9 激动剂、CpG 寡核苷酸的抗原可降低 B 细胞激活的阈值，因此可能通过激活低亲和力自身反应性 B 细胞而在自身免疫性疾病中发挥作用。然而，TLR-9 仅存在于细胞内内体区室的 B 细胞中，并且含 CpG 的抗原通过 TLR-9 增强 BCR 信号传导的细胞机制尚不清楚。我们最近使用共聚焦和电子显微镜相结合的研究提供了证据，证明 BCR 信号传导导致含有 TLR9 的内体募集到细胞内多泡体，BCR 将含有 CpG 的抗原转运到细胞内多泡体。这些囊泡似乎是 BCR/TLR-9 信号传导增强的部位。这种 TLR-9 增强 BCR 信号传导的新机制可能对于为自身免疫抗体反应治疗提供新靶标很重要。