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。在过去的一年里，我们在确定抑制性受体FcGammaRIIB调节BCR信号启动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)的一个不同亚群上的Fc样受体4(FcRL4)的功能，观察到在HIV感染的病毒型个体以及疟疾流行地区的儿童和成人中，MBC显著扩大。 我们使用高分辨率活细胞成像来描绘B细胞激活中非常早期的抗原驱动事件的进展，为评估辅助受体的影响提供了新的背景。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，我们提供了发生在BCR结合抗原几秒到几分钟内的有序过程的证据。抗原结合的BCR形成固定的簇，然后通过分子捕获增大大小。这些簇扰乱了局部的脂质环境，导致脂筏聚集在BCR簇周围。作为膜扰动的结果，通路中的第一个激酶Lyn被RAFT脂类拴在膜上，使其接近BCR簇的分子位置。同时，Lyn使BCR的Igαβ胞浆结构域磷酸化，Igαβ链经历了从封闭到开放的构象变化。SYK被招募到磷酸化的BCR上，并触发信号级联。 在过去的一年里，我们研究了通过免疫复合物(IC)的结合来连接FcGammaRIIB和BCR对这些早期事件的影响。我们发现ICs的结合诱导了Bcr和FcGammaRIIB在微观簇中的共定位，但FRET测量表明，在这些簇中，BcR和FcGamma RIIB并不是紧密的分子距离。尽管如此，ICs的结合导致了BCR信号的最早事件的阻断，开始于BCR的寡聚化成固定的信号活性簇。在IC结合后，BCR不能与脂筏探针结合，而FcGammaRIIB稳定地与脂筏探针结合。FcGammaRIIB阻断BCR信号的启动的能力似乎依赖于它与RAFT脂结合的能力，因为不能与脂筏结合的功能缺失突变体FcGammaRIIB不能阻断BCR信号。综上所述，这些研究表明，FcGammaRIIB影响BCR信号的时间比之前估计的要早得多。 在过去的一年里，我们开始了定义FcRL4功能的研究，FcRL4是一个古老的跨膜蛋白家族FcRLS的成员，它与经典的FCRs，如FcGammaRIIB有着共同的祖先，并优先在B细胞系中表达。FcRL4在健康个体粘膜淋巴组织中的一种特殊的MBCs亚群上表达。此外，在感染艾滋病毒的病毒症患者和长期再次感染疟疾的患者中，FcRL4+单核细胞在血液中的数量大大增加。我们提供的证据表明，FcRL4在人B细胞系中的表达在Syk磷酸化水平上抑制了抗原诱导的BCR信号转导。抑制作用不需要FcRL4与BCR结合，而是依赖于FcRL4胞浆区的两个免疫受体抑制基序(ITIMs)。值得注意的是，FcRL4的表达同时通过天然免疫Toll样受体9(TLR9)增强了信号转导。这些发现表明，FcRL4可能作为B细胞的分子开关，抑制获得性免疫信号，并增强对慢性抗原刺激的信号。

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.