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: 8156981
• 负责人: Susan Pierce
• 金额: $ 40.84万
• 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/8156981
• 关键词: B-Cell Activation; Regulation; receptor

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 have made progress in defining the mechanisms by which the inhibitory receptor, FcgammaRIIB, function to regulate the initiation of BCR signaling. In addition we initiated studies to determine the function of the Fc like receptor 4 (FcRL4) present on a distinct subset of memory B cells (MBCs) observed to be greatly expanded in HIV infected viremic individuals and in children and adults in malaria-endemic areas. Our progress using high resolution live cell imaging to delineate the very early antigen driven events in B cell activation has provided a new context in which the impact of coreceptors can be evaluated. Using high resolution fluorescence resonance energy transfer (FRET) coupled with total internal reflection microscopy (TIRFM) and single molecule tracking we provided evidence for an ordered process that occurs within seconds to minutes of the BCR binding antigen. Antigen bound BCRs form immobile clusters that then grow in size by molecular trapping. The clusters perturb the local lipid environment causing lipid rafts to coalesce around the BCR clusters. As a consequence of the membrane perturbation the first kinase in the pathway, Lyn, that is tethered to the membrane by raft lipids is brought into close molecular proximity to the BCR clusters. Simultaneously, Lyn phosphorylates the Ig alpha beta cytoplasmic domain of the BCR and the Ig alpha beta chains undergo a conformational change from a closed to an open form. Syk is recruited to the phosphorylated BCR and the signaling cascades are triggered. Over the last year we investigated the effect of coligating the FcgammaRIIB and BCR by the binding of immune complexes (ICs) on these early events. We discovered that the binding of ICs induced the colocalization of the BCR and FcgammaRIIB in microscopic clusters but that within these clusters the BCR and Fcgamma RIIBs were not in close molecular proximity as measured by FRET. Nonetheless, binding of ICs resulted in a block in the earliest events in BCR signaling beginning with the oligomerization of the BCR into immobile signaling active clusters. Following IC binding the BCRs failed to associate with a lipid raft probe in contrast to the FcgammaRIIB that stably associated with the raft lipid probe. The ability of the FcgammaRIIB to block the initiation of BCR signaling appeared to be dependent on its ability to associate with raft lipids as loss of function mutant FcgammaRIIBs that are unable to associate with lipid rafts did not block BCR signaling. Taken together these studies indicate that the FcgammaRIIB affects BCR signaling at a much earlier point than previously appreciated. Over the last year we initiated studies to define the function of FcRL4, a member of an ancient family of transmembrane proteins, the FcRLs, that share ancestors with the classical FcRs, like FcgammaRIIB, and are preferentially expressed in the B cell lineage. FcRL4 is expressed on a distinctive subset of MBCs located in mucosal lymphoid tissues in healthy individuals. In addition, FcRL4+ MBCs are greatly expanded in numbers in the blood of HIV-infected viremic individuals and in individuals chronically re-infected with malaria. We provided evidence that the expression of FcRL4 in human B cell lines inhibits antigen-induced BCR signaling at the level of Syk phosphorylation. Inhibition did not require coligation of the FcRL4 with the BCR but depended on the two immunoreceptor inhibitory motifs (ITIMs) in FcRL4s cytoplasmic domain. Remarkably, FcRL4 expression simultaneously enhanced signaling through the innate immune toll-like receptor 9 (TLR9). These findings suggest that FcRL4 may act as molecular switch in B cells to dampen adaptive immune signaling and enhance signaling in response to chronic antigenic stimulation.

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，我们在确定抑制性受体Fc γ RIIB调节BCR信号传导起始的机制方面取得了进展。此外，我们启动了研究以确定存在于记忆B细胞（MBC）的不同亚群上的Fc样受体4（FcRL 4）的功能，所述记忆B细胞（MBC）在HIV感染的病毒血症个体以及疟疾流行地区的儿童和成人中被观察到大大扩增。 我们使用高分辨率活细胞成像描绘B细胞活化中非常早期的抗原驱动事件的进展提供了一个新的背景，其中可以评估辅助受体的影响。 使用高分辨率荧光共振能量转移（FRET）结合全内反射显微镜（TIRFM）和单分子跟踪，我们提供了证据，在BCR结合抗原的数秒至数分钟内发生的有序过程。 抗原结合的BCR形成不动的簇，然后通过分子捕获使其尺寸增大。 这些簇扰乱了局部脂质环境，导致脂筏在BCR簇周围聚结。 作为膜扰动的结果，途径中的第一个激酶林恩通过筏脂质与膜连接，使其分子与BCR簇紧密接近。 同时，林恩使BCR的IG α β胞质结构域磷酸化，并且IG α β链经历从封闭形式到开放形式的构象变化。 Syk被募集到磷酸化的BCR，并触发信号级联反应。 在过去的一年中，我们研究了通过免疫复合物（IC）的结合将Fc γ RIIB和BCR共连接对这些早期事件的影响。 我们发现，IC的结合诱导BCR和Fc γ RIIB在微观簇中的共定位，但是在这些簇中，如通过FRET测量的，BCR和Fc γ RIIB并不处于紧密的分子接近。 尽管如此，IC的结合导致BCR信号传导中的最早事件的阻断，所述最早事件开始于BCR寡聚化成固定的信号传导活性簇。 IC结合后，BCR未能与脂筏探针结合，而Fc γ RIIB与脂筏脂质探针稳定结合。 Fc γ RIIB阻断BCR信号传导起始的能力似乎取决于其与筏脂质缔合的能力，因为不能与脂筏缔合的功能丧失突变体Fc γ RIIB不阻断BCR信号传导。 总之，这些研究表明Fc γ RIIB在比先前认识到的早得多的点影响BCR信号传导。 在过去的一年里，我们开始研究FcRL 4的功能，FcRL 4是一个古老的跨膜蛋白家族FcRL的成员，与经典的FcR（如Fc γ RIIB）共享祖先，并优先在B细胞谱系中表达。 FcRL4在位于健康个体的粘膜淋巴组织中的MBC的独特子集上表达。 此外，FcRL4+ MBC在HIV感染的病毒血症个体和慢性再感染疟疾的个体的血液中的数量大大增加。 我们提供的证据表明，FcRL 4在人B细胞系中的表达在Syk磷酸化水平上抑制抗原诱导的BCR信号传导。 抑制作用不需要FcRL4与BCR的共连接，而是依赖于FcRL4胞质结构域中的两个免疫受体抑制基序（ITIM）。 值得注意的是，FcRL4表达同时增强了通过先天免疫toll样受体9（TLR9）的信号传导。 这些发现表明，FcRL 4可能作为B细胞中的分子开关，以抑制适应性免疫信号传导并增强对慢性抗原刺激的信号传导。