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

B 细胞激活的共受体调节机制

• 批准号: 7732627
• 负责人: susan pierce
• 金额: $ 22.71万
• 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/7732627
• 关键词: ABL1 gene; Affect; Affinity; Alanine; Antibodies; Antigen-Antibody Complex; Antigens; Apoptosis; Attention; Autoimmunity; B-Cell Activation; B-Lymphocytes; Binding; Biochemical; Biochemical Genetics; Bone Marrow; CD81 gene; Cell Line; Cells; Cholesterol; Complement; Complex; Confocal Microscopy; Coupled; Cyclophosphamide/Fluorouracil/Prednisone; Cysteine; Dependence; Detergents; Environment; Event; Family; Fc Receptor; Fluorescence Resonance Energy Transfer; Heterogeneity; Human; ITIM; Image; Imaging technology; Immune response; Immunologic Memory; Length; Life; Lipids; Measurement; Mediating; Membrane; Membrane Microdomains; Memory B-Lymphocyte; Microscopy; Molecular; Morphologic artifacts; Mus; Pathway interactions; Pattern; Pharmaceutical Preparations; Phosphotransferases; Plasma Cells; Play; Proteins; Receptor Signaling; Receptors, Antigen, B-Cell; Regulation; Relative (related person); Resolution; Role; Signal Pathway; Signal Transduction; Sphingolipids; Surface; Time; Total Internal Reflection Fluorescent; antigen binding; c-abl Proto-Oncogenes; cellular imaging; crosslink; inhibiting antibody; interest; mutant; novel; palmitoylation; receptor; response

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 year 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. Using mutant CD81 in which the six cysteines that are modified by palmitoylation were changed to alanine, we demonstrated that palmitoylation was not necessary for interactions of CD81 with themselves but altered the pattern of expression on the surface and the downstream signaling from the BCR and CD19/CD21. 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) coupled with total internal reflection microscopy (TIRFM) or 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. 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 imaging 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 was prolonged by coengagement of the BCR and the CD19/CD21 coreceptor complex. Using live cell FRET TIRF imaging we recently provided direct evidence that the association of the BCR with raft lipids following antigen binding is blocked by 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. We have also initiated studies to describe the interactions of the BCR and FcgammaRIIB during B cell activation by TIRF microscopy. Surprisingly, we observed that the binding of antigen to the BCR resulted in the recruitment of the FcgammaRIIB to the clustered BCRs. We are interested in determining both the function of the recruitment and the underlying mechanism. 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.

对抗原的B细胞应答受多种共受体调节，所述共受体向B细胞传递关于抗原的质量和正在进行的免疫应答的状态的信息。 在过去的一年里，我们将注意力集中在B细胞反应的两种有效调节剂上，即CD 19/CD 21复合物和FcgammaRIIB。 通过B细胞受体（BCR）的信号传导通过BCR和CD 19/CD 21复合物的共连接（通过补体固定抗原的结合）而被放大和延长。 低亲和力Fc受体Fc γ RIIB是一种有效的B细胞抑制性受体，因此在控制抗体介导的自身免疫中起核心作用。 确定这些辅助受体如何影响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。在过去的一年中，我们已经取得了进展，在确定的机制，积极的辅助受体，CD 19/CD 21复合物和抑制性受体，Fc γ RIIB，功能调节B细胞信号。 我们早期的研究提供了生物化学证据表明，当B细胞辅助受体CD 19/CD 21复合物通过补体标记抗原的结合与BCR结合时，通过延长BCR与富含鞘脂和胆固醇的膜微区（称为脂筏）的结合，增强BCR信号传导。 我们还提供了生物化学和遗传学证据，证明CD19/CD21复合物的CD81组分对于CD19/CD21复合物的筏稳定功能是必需的。 我们发现，当BCR和CD 19/CD 21复合物共连接时，CD 81与筏脂质相关，而在CD 81缺陷小鼠的B细胞中，共连接的BCR和CD 19/CD 21复合物不能与筏脂质相关或增强BCR信号传导。 我们进一步证明，在coligation CD81棕榈酰化，棕榈酰化是必不可少的筏稳定功能。 因此，我们定义了一种新的机制，通过这种机制，辅助受体影响受体的局部脂质环境，即通过诱导性脂化。 使用突变的CD81，其中6个半胱氨酸被棕榈酰化修饰为丙氨酸，我们证明棕榈酰化不是CD81与自身相互作用所必需的，但改变了表面上的表达模式以及来自BCR和CD19/CD21的下游信号传导。 我们还提供了生物化学证据，表明Fc γ RIIB与BCR共连接时使BCR与脂筏的结合不稳定。 在这些研究中，脂筏操作定义其相对洗涤剂不溶性，由于紧密包装的饱和链的筏脂质和他们的依赖胆固醇。 然而，使用清洁剂和胆固醇消耗药物充满了潜在的人为因素，包括创造我们着手研究的脂质异质性。 高分辨率荧光共振能量转移（FRET）结合全内反射显微镜（TIRFM）或共聚焦显微镜提供了机会，以量化的相互作用的BCR与筏脂质在活细胞中的时间和长度尺度，以捕捉抗原启动的B细胞活化的最早期的事件。 为了研究筏脂质与BCR的相互作用，我们产生了表达含有FRET供体荧光蛋白CFP和FRET受体蛋白YFP的BCR的细胞系，所述FRET供体荧光蛋白CFP和FRET受体蛋白YFP通过筏脂质或非筏脂质被拴系到膜上。 活B细胞的FRET共聚焦成像显示，在抗原结合的几秒钟内，BCR选择性地和瞬时地与脂筏结构相关联，并且这种关联通过BCR和CD 19/CD 21辅助受体复合物的共同参与而延长。 使用活细胞FRET TIRF成像，我们最近提供了直接的证据，即抗原结合后BCR与筏脂质的结合被Fc γ RIIB阻断。 因此，这些FRET测量提供了抗原诱导的BCR与活细胞中脂筏的结合以及CD 19/CD 21复合物和Fc γ RIIB对该非常早期事件的调节的第一个直接证据。 我们还启动了研究，通过TIRF显微镜来描述B细胞活化过程中BCR和Fc γ RIIB的相互作用。 令人惊讶的是，我们观察到抗原与BCR的结合导致Fc γ RIIB募集到成簇的BCR。 我们感兴趣的是确定招聘的功能和基本机制。 除了通过交联BCR和Fc γ RIIB启动的充分研究的抑制途径之外，我们提供了证据表明，当独立于BCR成簇时，Fc γ RIIB启动ITIM-、林恩-和SHIP-非依赖性途径，其通过涉及c-Abl家族激酶的机制触发细胞凋亡。 因此，Fc γ RIIB具有阻断BCR依赖性、抗原驱动的B细胞活化以及抗原非依赖性、BCR非依赖性B细胞活化的能力。 最近在小鼠中显示，长寿命的骨髓浆细胞表达Fc γ RIIB，并且通过免疫复合物单独接合Fc γ RIIB诱导这些浆细胞经历抗原非依赖性凋亡。 在过去的一年中，我们在人体中提供了Fc γ RIIB独立于BCR发挥作用以抑制浆细胞和幼稚B细胞但不抑制记忆B细胞的证据。 这些结果表明，BCR非依赖性Fc γ RIIB信号通路可能在人类中通过抑制抗体分泌PC和幼稚B细胞活化而不影响长寿命记忆B细胞库，在急性控制抗体水平方面发挥重要作用。

项目成果

The B cell inhibitory Fc receptor triggers apoptosis by a novel c-Abl family kinase-dependent pathway.

B 细胞抑制性 Fc 受体通过新型 c-Abl 家族激酶依赖性途径触发细胞凋亡。

• DOI: 10.1074/jbc.m505308200
• 发表时间: 2005
• 期刊: The Journal of biological chemistry
• 作者: Tzeng,Shiang-Jong;Bolland,Silvia;Inabe,Kazunori;Kurosaki,Tomohiro;Pierce,SusanK
• 通讯作者: Pierce,SusanK