The Regulation of B-cell Activation

B细胞受体（BCR）触发的B细胞活化受控制B细胞与抗原相遇的结果的多种因素调节，包括B细胞的发育状态、辅助受体和Toll样受体（TLR）的接合以及病毒感染。确定这些因素如何影响BCR诱导的信号传导，将从根本上增加我们对B细胞激活机制的理解。在过去的一年里，我们在确定积极的辅助受体，CD 19/CD 21复合物，抑制性受体，Fc γ RIIB 1，TLR，TLR 9调节信号传导的机制方面取得了进展。 当B细胞共受体CD 19/CD 21通过补体标记抗原的结合与BCR共连接时，通过延长BCR与富含鞘脂和胆固醇的膜微结构域（称为脂筏）的结合，部分地增强BCR信号传导。我们确定了CD 19/CD 21复合物在筏中发挥作用的能力依赖于四跨膜蛋白CD 81，四跨膜蛋白CD 81是CD 19/CD 21复合物的一个组分。因此，在来自CD 81缺陷小鼠的B细胞和表达不能与CD 81结合的嵌合CD 19受体的B细胞中，当与BCR共连接时，CD 19/CD 21复合物不能稳定筏中的BCR。许多与脂筏结合的蛋白质通过其酰化，特别是通过其棕榈酰化（可逆的酰化事件）而这样做。我们确定，在BCR和CD 19/CD 21复合物交联后，CD 81在脂筏中变为棕榈酰化，并且棕榈酰化对于CD 81的功能是必需的。CD 81在其细胞质面内含有六个半胱氨酸，这些半胱氨酸是棕榈酰化的潜在位点。为了评估这些半胱氨酸对CD 81功能的贡献，在B细胞系中表达含有突变的半胱氨酸残基的CD 81分子，其中通过RNAi技术敲低内源性野生型CD 81表达。这些突变体在BCR和CD 19/CD 21复合物的共连接后显示出信号传导缺陷，证实了这些半胱氨酸残基在CD 81功能中的重要性。将含有FRET供体荧光蛋白的CD 81构建体与靶向筏微结构域的FRET受体荧光蛋白结合使用，以通过活细胞中的FRET共聚焦显微镜来证明CD 81的半胱氨酸残基对于BCR和CD 19/CD 21复合物的coligation之后CD 81与筏脂质的缔合是必需的。 在确定Fc γ RIIB（当与BCR共连接时，BCR信号传导的有效负调节剂）当与自身交联时如何发出细胞凋亡信号方面也取得了进展。我们了解到，FcgammaRIIB交联时，成为与脂筏和信号细胞凋亡的机制依赖于c-Abl，但独立的磷酸酶SHIP和FcgammaRIIB？s FcgammaRIIB所需的ITIM基序？抑制BCR信号传导。最近的研究结果提供了证据，表明交联Fc γ RIIB 1阻断记忆B细胞向抗体分泌浆细胞的分化。因此，向人CD 27+记忆B细胞中加入对Fc γ RIIB 1特异的mAb阻断了TLR-9激动剂诱导的记忆B细胞向浆细胞的分化。这些发现在定义Fc γ RIIB 1介导的调控的新靶点方面具有重要意义。正在进行研究以确定Fc γ RIIB 1在记忆细胞向浆细胞分化的哪个点起作用。 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寡核苷酸的抗原降低了B细胞活化的阈值，因此可能通过允许低亲和力自身反应性B细胞的活化而在自身免疫性疾病中发挥作用。然而，TLR-9仅存在于B细胞的细胞内内体区室中，并且含CpG的抗原通过TLR-9增强BCR信号传导的细胞机制还不清楚。我们最近使用共聚焦显微镜和电子显微镜的组合的研究提供了证据，BCR信号转导导致含TLR 9的内体募集到细胞内多泡体，BCR将含CpG的抗原转运到该多泡体。这些囊泡似乎是增强的BCR/TLR-9信号传导的位点。TLR-9增强BCR信号传导的这种新机制可能在为自身免疫抗体应答的治疗提供新靶点方面很重要。

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.