The Mechanism of Co-Receptor Regulation of B-cell Activation

B细胞对抗原的反应受多种辅助受体的调节，这些辅助受体向B细胞传递有关抗原质量和正在进行的免疫反应状态的信息。在过去的回顾期间，我们将注意力集中在两个有效的B细胞反应调节因子上，即CD19/CD21复合体和FcGammaRIIB。B细胞受体(BCR)和CD19/CD21复合体通过结合补体结合，信号被放大和延长。低亲和力Fc受体FcGammaRIIB是一种有效的B细胞抑制受体，因此在控制抗体介导的自身免疫中起着核心作用。确定这些辅助受体如何影响BCR诱导的信号转导，将从根本上帮助我们理解B细胞被激活的机制。在过去的一年里，我们在确定积极的辅助受体CD19/CD21复合体和抑制受体FcGammaRIIB调节B细胞信号的机制方面取得了进展。 我们早期的研究提供了生化证据，即B细胞辅助受体CD19/CD21复合体，当通过与补体标记的抗原结合而与BCR结合时，延长并增强BCR信号，部分是通过延长BCR与富含鞘脂和胆固醇的膜微域的结合，称为脂筏。我们还提供了生化和遗传学证据，证明CD19/CD21复合体的CD81组分对于CD19/CD21复合体的RAFT稳定功能是必不可少的。我们发现CD81在BCR和CD19/CD21复合体结合时与RAFT脂结合，而在CD81缺陷小鼠的B细胞中，CD81结合BCR和CD19/CD21复合体不能与RAFT脂结合或增强BCR信号转导。我们进一步证明CD81在连接时是棕榈酰化的，并且棕榈酰化对于它的RAFT稳定功能是必不可少的。因此，我们定义了一种新的机制，即通过诱导脂肪作用，辅助受体影响受体的局部脂环境。 我们还提供了生化证据，表明当FcGamma RIIB与BCR连接时，BCR与脂筏的结合不稳定。在这些研究中，通过脂筏脂类饱和链条的紧密堆积和对胆固醇的依赖，在操作上定义了脂筏脂筏的相对洗涤剂不溶性。然而，洗涤剂和降胆固醇药物的使用充满了潜在的假象，包括造成我们开始研究的脂质异质性。高分辨率荧光共振能量转移(FRET)共聚焦显微镜提供了在活细胞中定量BCR与RAFT脂类相互作用的机会，在捕捉抗原启动的B细胞激活的最早事件所需的时间和长度范围内。使用活细胞FRET共聚焦成像，我们最近提供了BCR与RAFT脂类在抗原结合后选择性关联的直接证据。 为了研究RAFT脂类与BCR的相互作用，我们建立了表达BCR的细胞系，该细胞系含有FRET供体荧光蛋白CFP和FRET受体蛋白YFP，通过RAFT脂类或非RAFT脂类拴在膜上。活的B细胞的FRET共聚焦显微镜显示，在抗原与BCR结合的几秒钟内，BCR选择性地和脂筏结构相关联，并且这种关联比钙离子流动的触发早了几秒钟。通过BCR与用于增强BCR信号的CD19/CD21共受体复合体的共同作用，抗原结合的BCR与脂筏探针的结合时间延长。相反，BCR与脂筏探针的联系被BCR与强大的抑制性受体FcGammaRIIB的共同作用所阻断。因此，这些FRET测量为抗原诱导的BCR与活细胞中的脂筏的关联以及CD19/CD21复合体和FcGammaRIIB对这一非常早期的事件的调节提供了第一个直接证据。 除了通过交联BCR和FcGammaRIIB启动的已被广泛研究的抑制途径外，我们提供的证据表明，当FcGammaRIIB独立于BCR聚集时，FcGammaRIIB启动一条ITIM、LYN和SHIP无关的途径，通过涉及c-Abl家族激酶的机制触发细胞凋亡。因此，FcGammaRIIB具有阻断BCR依赖的、抗原驱动的B细胞激活以及抗原非依赖的、BCR非依赖的B细胞激活的能力。最近在小鼠身上发现，长寿的骨髓浆细胞表达FcGammaRIIB，免疫复合体单独与FcGammaRIIB结合可诱导这些浆细胞经历非抗原依赖性的凋亡。在过去的一年里，我们在人类中提供了证据，表明FcGammaRIIB的功能独立于BCR，可以抑制浆细胞和幼稚的B细胞，但不能抑制记忆B细胞。这些结果表明，BCR非依赖的FcGammaRIIB信号通路可能在人类通过抑制抗体分泌PC和激活幼稚B细胞而不影响长寿记忆B细胞库而迅速控制抗体水平方面发挥重要作用。

The B cell response to antigen is regulated by a variety of co-receptors that convey information to the B cell about the quality of the antigen and the status of the ongoing immune response. Over the last review period we focused our attention of two potent regulators of B cell responses, namely the CD19/CD21 complex and the FcgammaRIIB. Signaling through the B cell receptor (BCR) is both amplified and prolonged by coligation of the BCR and the CD19/CD21 complex through the binding of complement fixed antigens. The low affinity Fc receptor, FcgammaRIIB, is a potent B cell inhibitory receptor and as such plays a central role in controlling antibody-mediated autoimmunity. Determining how these co-receptors 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 and the inhibitory receptor, FcgammaRIIB, function to regulate B cell signaling. Our earlier studies provided biochemical evidence that the B cell coreceptor, the CD19/CD21 complex, 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 also provided biochemical and genetic evidence that the CD81 component of the CD19/CD21 complex was essential for the raft stabilizing function of the CD19/CD21 complex. We showed that CD81 associates with raft lipids upon coligation of the BCR and the CD19/CD21 complex and that in B cells from CD81-deficient mice coligated BCR and CD19/CD21 complexes failed to associate with raft lipids or enhance BCR signaling. We further demonstrated that upon coligation CD81 was palmitoylated and that the palmitoylation was essential for its raft-stabilizing function. Thus, we defined a novel mechanism by which a co-receptor influences the local lipid environment of a receptor namely by inducible lipidation. We also provided biochemical evidence that the Fcgamma RIIB when coligated to the BCR destabilized the association of the BCR with lipid rafts. In these studies lipid rafts were operationally defined by their relative detergent insolubility, due to the tight packing of the saturated chains of the raft lipids and by their dependence on cholesterol. However, the use of detergents and cholesterol-depleting drugs are fraught with potential artifacts including creating the lipid heterogeneities we set out to study. High resolution fluorescence resonance energy transfer (FRET) confocal microscopy offered the opportunity to quantify the interactions of the BCR with raft lipids in live cells over the time and length scale necessary to capture the earliest events in antigen-initiated B cell activation. Using live cell FRET confocal imaging we recently provided direct evidence for the selective association of the BCR with raft lipids following antigen binding. To study the interactions of raft lipids with BCRs we generated cell lines that expressed a BCR containing the FRET donor fluorescent protein CFP and the FRET acceptor protein, YFP, tethered to the membrane by either raft lipids or by non-raft lipids. FRET confocal microscopy of living B cells revealed that within seconds of antigen binding the BCR selectively and transiently associated with the lipid raft constructs and that this association preceded the triggering of Ca++ fluxes by several seconds. The association of the antigen bound BCR with the lipid-raft probe was prolonged by coengagement of the BCR and the CD19/CD21 coreceptor complex that serves to enhance BCR signaling. Conversely, the association of the BCR with the lipid-raft probe was blocked by the coengagement of the BCR with the potent inhibitory receptor, the FcgammaRIIB. Thus, these FRET measurements provided the first direct evidence for the antigen-induced association of the BCR with lipid rafts in living cells and the regulation of this very early event by the CD19/CD21 complex and the FcgammaRIIB. In addition to the well studied inhibitory pathway initiated by crosslinking the BCR and FcgammaRIIB, we provided evidence that when clustered independently of the BCR, the FcgammaRIIB initiates an ITIM-, Lyn- and SHIP-independent pathway that triggers apoptosis through a mechanism that involves c-Abl family kinases. Thus, the FcgammaRIIB has the ability to block the BCR-dependent, antigen-driven activation of B cells as well as antigen-independent, BCR-independent B cell activation. It was recently shown in mice that long lived bone marrow plasma cells express the FcgammaRIIB and that engaging the FcgammaRIIB alone by immune complexes induced these plasma cells to undergo antigen-independent apoptosis. Over the last year we provided evidence in humans that the FcgammaRIIB functions independently of the BCR to inhibit plasma cells and naive B cells but not memory B cells. These results suggest that the BCR-independent FcgammaRIIB signaling pathway may play an important role in humans in acutely controlling antibody levels by inhibiting antibody secreting PCs and the activation of naive B cells without affecting the long-lived memory B cell pool.