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。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，我们表征了抑制性受体FcGammaRIIB参与对人类NAIVE和MBCs激活的影响。利用高分辨率成像，我们发现在启动BCR信号的每个步骤中，人的MBCs比原始的B细胞更健壮，包括询问含有抗原的膜，形成亚微观的BCR寡聚体，以及募集和激活信号相关激酶。尽管单核细胞对抗原有很强的反应，但它们对FcGammaRIIB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很重要。 我们还在人类B细胞中证明了BCR在含有抗原的双层上的B细胞界面上的快速积聚触发了磷酸化CD19的独立积聚，同时伴随着BCR-CD19信号通路的关键成分-磷酸化PI3K的积聚和共存。值得注意的是，BCR和FcGammaRIIB的结合阻止了BCR的积累，并使CD19和PI3K磷酸化。这些结果提示了FcGammaRIIB抑制BCR信号的另一个方面。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博士团队已经开发出双功能抗体，这种抗体创造了DART，可以使BCR和CD19或BCR和FcGammaRIIB二聚化。BCR-FcGammaRIIB飞镖在阻断自身免疫性疾病中的B细胞激活和杀死BCR依赖的B细胞肿瘤方面显示出有效性。我们正在使用高分辨率活细胞成像来探索这些DART对BCR信号的影响。 在过去的一年里，我们在建立超高分辨率成像方面取得了进展，以在10-50 nm的水平上描述静止的人B细胞中BCR、CD19和FcGammaRIIB之间的空间关系，以及在B细胞中BCR被抗原单独连接或FcGamma被免疫复合物连接的空间关系。我们还研究了这些受体在表达免疫球蛋白M的幼稚B细胞和表达免疫球蛋白的记忆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.