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

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。 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，导致脂筏聚集在BCR簇周围。 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，我们表征了抑制性受体Fc γ RIIB接合对人幼稚和MBC活化的影响。 使用高分辨率成像，我们表明人MBC在BCR信号传导启动的每个步骤都比初始B细胞更稳健，包括询问含抗原的膜、亚显微镜下BCR寡聚体的形成以及信号传导相关激酶的募集和激活。 尽管它们对抗原有强烈的应答，但MBC对Fc γ RIIB介导的抑制仍然高度敏感。 这些观察对于理解记忆抗体反应的调节是重要的。 我们还在人B细胞中证明，BCR在含抗原双层上的B细胞界面处的快速蓄积触发了磷酸化CD 19的独立蓄积，伴随着磷酸化PI 3 K的蓄积和共定位，PI 3 K是BCR-CD 19信号传导途径中的关键组分。值得注意的是，BCR和Fc γ RIIB的共连接阻断了BCR的积累，以及磷酸化的CD 19和PI 3 K。这些结果表明Fc γ RIIB抑制BCR信号传导的另一个方面。我们将继续与Ezio Bonvini博士和他在Macrogenics的同事合作进行这些研究。Bonvinis博士的团队开发了双功能抗体DARTS，允许BCR和CD 19或BCR和Fc γ RIIB的二聚化。BCR-Fc γ RIIB DARTS在阻断自身免疫性疾病中的B细胞活化和杀死BCR依赖性B细胞肿瘤方面显示出功效。我们正在使用高分辨率活细胞成像来探索这些DARTS对BCR信号传导的影响。 在过去的一年中，我们在建立超高分辨率成像以在10-50 nm水平上描述静息的人B细胞和B细胞中BCR、CD 19和Fc γ RIIB之间的空间关系方面取得了进展，其中BCR通过Ag单独连接或Fc γ通过免疫复合物连接。我们还表征了这些受体在表达幼稚人IgM的B细胞和表达IgG的记忆B细胞上的空间分布。

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. 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 characterized the effect of the inhibitory receptor FcgammaRIIB engagement on the activation of human naive and MBCs. Using high-resolution imaging we showed that human MBCs are more robust than naive B cells at each step in the initiation of BCR signaling, including interrogation of antigen containing membranes, formation of sub-microscopic BCR oligomers and recruitment and activation of signaling-associated kinases. Despite their robust response to antigen, MBCs remain highly sensitive to FcgammaRIIB-mediated inhibition. These observations are important for understanding the regulation of memory antibody responses. We also demonstrated in human B cells that the rapid accumulation of BCRs at the interface of the B cell on antigen-containing bilayer triggers the independent accumulation of phosphorylated CD19 accompanied by the accumulation and colocalization of phosphorylated PI3K, a key component in the BCR-CD19 signaling pathway. Remarkably, coligation of the BCR and FcgammaRIIB blocked the accumulation of the BCR, and phosphorylated CD19 and PI3K. These results suggest an additional facet of the inhibition of BCR signaling by the FcgammaRIIB. We will continue these studies in collaboration with Dr. Ezio Bonvini and his colleagues at Macrogenics. Dr. Bonvinis group has developed bi-functional antibodies coined DARTS that allow the dimerization of the BCR and CD19 or the BCR and FcgammaRIIB. The BCR-FcgammaRIIB DARTS are showing efficacy in blocking B cell activation in autoimmune disease and killing BCR-dependent B cell tumors. We are exploring the effect of these DARTS on BCR signaling using high resolution live cell imaging. Over the last year we have made progress in establishing super-high resolution imaging to describe, at the 10-50 nm level, the spatial relationship between the BCR, CD19 and FcgammaRIIB in resting human B cells and in B cells in which the BCR is ligated alone by Ag or Fcgamma is ligated by immune complexes. We are also characterizing the spatial distribution of these receptors on naive human IgM expressing B cells and in IgG-expressing memory B cells.