Membrane Microdomains And B- Cell Signaling

B细胞抗体反应是由抗原与克隆分布的B细胞抗原受体(BCR)结合而触发的。BCR的作用是启动信号级联，并将抗原运送到细胞内以供呈递，这两个过程都是B细胞激活的基本过程。在过去的几年里，关于BCR抗原结合触发的复杂信号级联的生物化学以及抗原被运输进行处理的细胞内途径，人们已经了解了很多。信号和抗原靶向似乎都是通过膜相关的Src家族成员Lyn对BCR进行磷酸化来启动的。然而，使抗原结合的BCR与LYN接触的B细胞激活的起始事件尚不清楚。现在看来，富含胆固醇和鞘磷脂的膜微域，称为脂筏，既是BCR信号传递的平台，也是抗原运输的平台。在静息细胞中，BCR被排除在浓缩LYN的RAFT之外，但在多价抗原结合时，BCR寡聚并与RAFT结合，在那里它被LYN磷酸化，信号被启动。BCR的移位到RAFT中不需要BCR信号中最早的两个事件，即LYN对BCR的磷酸化或BCR与肌动蛋白细胞骨架的结合。因此，BCR与RAFT的结合以及由此产生的信号的启动似乎仅依赖于BCR的寡聚。在RAFT结合后，结合到BCR上的抗原被内化以供处理。在RAFT中信号的起始之后是RAFT聚集，并最终形成一个被称为免疫突触的高度组织化的结构，BCR信号可能从这个突触延长。从我们对B细胞受体与RAFT关系的研究中出现了一个令人兴奋的主题，即BCR RAFT关联受到多种因素的调节，这些因素控制着B细胞的结局-S与抗原的接触，包括B细胞的发育状态，辅助受体的结合和病毒感染。我们已经了解到，BCR稳定地与脂筏结合的能力在发育过程中发生了变化，并且这种变化与B细胞与抗原结合的结果有关。值得注意的是，BCR与未成熟B细胞中的RAFT无关，BCR抗原结合会导致B细胞的死亡而不是激活。我们还了解到，增强BCR反应的辅助受体可以延长BCR在筏子中的驻留时间。相反，减弱bcr信号的B细胞受体会破坏筏子中bcr的稳定性。最后，在人类B细胞中建立潜伏感染的爱泼斯坦-巴尔病毒阻断了bcr？S进入木筏的通道，从而阻断了抗原激活感染细胞的能力。在过去的一年里，我们在确定辅助受体调节bcr与脂筏的关联并因此调节信号的机制方面取得了进展。我们重点研究了两个重要的B细胞辅助受体，即CD19/CD21和FcGammaRIIB，CD19/CD21是BCR信号的重要正向调节因子，FcGammaRIIB是BCR信号的有效负调节因子。当CD19/CD21通过补体标记抗原与BCR结合时，可以延长BCR在RAFT中的停留时间和发出的信号。我们确定CD19/CD21复合体在RAFT中发挥作用的能力依赖于CD19/CD21复合体的一种成分--Tetraspanin CD81。因此，在CD81缺陷小鼠的B细胞和表达嵌合CD19受体但未能与CD81结合的B细胞中，CD19/CD21复合体与BCR结合时无法稳定筏子中的BCR。我们正在研究在微域中稳定信号复合体的能力是否是Tetraspanins的共同特征。在成熟的B细胞中，FcGammaRIIB通过免疫复合物与BCR结合，是BCR信号的有效负调节因子，在未成熟的B细胞中，FcGammaRIIB可以诱导细胞凋亡。我们过去一年的结果提供了证据，表明FcGamma RIIB在脂筏内调节这些功能。研究正在进行中，以确定FcGammaRIIB的哪些结构特征控制其在成熟和未成熟B细胞中划分为微域的能力。

B cell antibody responses are triggered by the binding of antigen to the clonally distributed B cell antigen receptors (BCRs). The BCR serves to initiate signal cascades and to transport antigens into the cell for presentation, both essential processes in B cell activation. Over the last several years a great deal has been learned about the biochemistry of the complex signal cascades triggered by BCR antigen engagement and the intracellular pathway by which antigen is transported for processing. Both signaling and antigen targeting appear to be initiated by phosphorylation of the BCR by a membrane associated member of the Src family kinase, Lyn. However, the initiating event in B cell activation that brings the antigen bound BCR into contact with Lyn is not known. It now appears that cholesterol- and sphingolipid-rich membrane microdomains, termed lipid rafts, serve as platforms for both BCR signaling and antigen trafficking. In resting cells the BCR is excluded from rafts that concentrate Lyn but upon multivalent antigen binding, the BCR oligomerizes and associates with rafts where it is phosphorylated by Lyn and signaling is initiated. The translocation of the BCR into rafts does not require two of the earliest events in BCR signaling, namely the phosphorylation of the BCR by Lyn or association of the BCR with the actin cytoskeleton. Thus, the association of the BCR with rafts and the resulting initiation of signaling appear to be dependent only on the oligomerization of the BCR. Following raft association antigen bound to the BCR is internalized for processing. The initiation of signaling in the rafts is followed by raft clustering and ultimately by the formation of a highly organized structure termed an immunological synapse from which BCR signaling may be prolonged. An exciting theme that emerged from our studies of the relationship of the BCR with rafts is one in which BCR raft association 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 viral infection. We have learned that the ability of the BCR to stably associate with lipid rafts changes during development and that the changes correlate with the outcome of antigen engagement by the B cell. Significantly, the BCR does not associate with rafts in immature B cells and BCR antigen binding results in the death of the B cell rather than activation. We also learned that coreceptors that function to enhance BCR response prolong the residency of the BCR in rafts. Conversely, B cell receptors that attenuate BCR signaling destabilize the BCR in rafts. Lastly, Epstein Barr Virus that established a latent infection in human B cells blocks the BCR?s access to rafts and thus blocks the ability of antigen to activate infected cells. Over the last year we have made progress in defining the mechanisms by which coreceptors function to regulate the association of the BCR with lipid rafts and as a consequence regulate signaling. We have focused on two important B cell coreceptors namely, CD19/CD21, an essential positive regulator of BCR signaling, and FcgammaRIIB, a potent negative regulator of BCR signaling. CD19/CD21 when coligated to the BCR through the binding of complement tagged antigens prolongs BCR residency in and signaling from 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 fails to stabilize the BCR in rafts. We are investigating whether the ability to stabilize signaling complexes in microdomains is a common feature of tetraspanins. The FcgammaRIIB is a potent negative regulator of BCR signaling in mature B cells when coligated to the BCR through the binding of immune complexes and in immature B cells induces apoptosis. Our results over the last year provide evidence that the Fcgamma RIIB mediate these functions from within lipid rafts. Studies are in progress to determine which structural features of the FcgammaRIIB controls its ability to partition into microdomains in mature and immature B cells.