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: 8555905
• 负责人: Susan Pierce
• 金额: $ 48.65万
• 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/8555905
• 关键词: Adult; Affinity; Antibodies; Antibody Formation; Antigen-Antibody Complex; Antigens; Area; Attention; Autoimmunity; B-Cell Activation; B-Cell Receptor Binding; B-Lymphocytes; Binding; Blood; CD19 gene; Cell Line; Cell Lineage; Child; Chronic; Collaborations; Complement; Complement 3d Receptors; Complex; Coupled; Cytoplasmic Tail; Down-Regulation; Environment; Event; Exclusion; Family; Fc Receptor; Fluorescence Resonance Energy Transfer; HIV; Human; Image; Imaging technology; Immune; Immune response; Immunoglobulin G; Immunoglobulin M; Immunoglobulin-Secreting Cells; Immunologic Memory; Individual; Infection; Integral Membrane Protein; Life; Ligation; Lipids; Lymphoid Tissue; Malaria; Mediating; Membrane; Membrane Microdomains; Memory; Memory B-Lymphocyte; Microscopic; Microscopy; Molecular; National Institute of Allergy and Infectious Disease; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Process; Proliferating; Receptor Activation; Receptor Signaling; Receptors, Antigen, B-Cell; Recruitment Activity; Regulation; Resolution; Rest; Role; Signal Transduction; Synapses; TLR9 gene; antigen binding; cellular imaging; member; molecular size; pathogen; receptor; response; single molecule; spatial relationship

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 made progress in defining the role of the inhibitory receptor, FcgammaRIIB, on regulating B cell receptor (BCR) signaling in human IgM-expressing naive B cells and in IgG-expressing memory B cells (MBCs). In addition we initiated studies to determine the function of the Fc like receptor 4 (FcRL4) present on a distinct subset of 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 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. In collaboration with Dr. Susan Moir in NIAID we provided evidence that down regulation of FcRL4 in B cells from HIV-infected high viremic individuals, restored their ability to respond through their BCRs to both proliferate and differentiate into antibody secreting cells. Taken together these findings are important in defining potential targets for reversing the deleterious effects of FcRL4 on immune responses during chronic pathogen infections. We also characterized the effect of 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. Over the last year we initiated studies to use super-high resolution imaging to describe, at the 10-50 nm level, the spatial relationship between the BCR, CD19 and FcgammaRIIB in resting B cells and in B cells in which the BCR is ligated alone by Ag or Fcgamma is ligated by immune complexes. Our high resolution images showed that BCR ligation leads to the accumulation of BCRs and CD19 into the immune synapse and the initiation of signaling. Ligation of the FcgammaRIIB results in its accumulation in the immune synapse and the exclusion of the BCR and CD19. Thus, even at this level of resolution the spatial organization of the B cell receptors appear to be regulated by mechanisms that do not require direct coligation.

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。 在过去的一年中，我们在确定抑制性受体Fc γ RIIB在人IgM表达幼稚B细胞和IgG表达记忆B细胞（MBC）中调节B细胞受体（BCR）信号传导的作用方面取得了进展。此外，我们启动了研究，以确定Fc样受体4（FcRL 4）的功能存在于一个独特的MBCs的子集，观察到大大扩展在HIV感染的病毒血症的个人和儿童和成人在疟疾流行地区。 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。 FcRL 4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，FcRL 4 + 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FcRL 4与BCR的共连接，而是依赖于FcRL 4胞质结构域中的两个免疫受体抑制基序（ITIM）。 值得注意的是，FcRL 4表达同时增强了通过先天免疫toll样受体9（TLR 9）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。 我们与NIAID的Susan Moir博士合作，提供了证据表明，HIV感染高病毒血症个体的B细胞中FcRL 4的下调，恢复了它们通过BCR增殖和分化为抗体分泌细胞的反应能力。 总之，这些发现对于定义用于逆转慢性病原体感染期间FcRL 4对免疫应答的有害作用的潜在靶标是重要的。 我们还表征了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MBCs比幼稚B细胞在BCR信号传导起始的每个步骤，包括询问含抗原的膜，形成亚显微镜下的BCR寡聚体和募集和激活信号传导相关的激酶，更强大。 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。 在过去的一年中，我们开始研究使用超高分辨率成像在10-50 nm水平上描述静息B细胞和B细胞中BCR、CD 19和Fc γ RIIB之间的空间关系，其中BCR通过Ag单独连接或Fc γ通过免疫复合物连接。我们的高分辨率图像显示，BCR连接导致BCR和CD 19积聚到免疫突触中并启动信号传导。Fc γ RIIB的连接导致其在免疫突触中的积累以及BCR和CD 19的排除。因此，即使在这种分辨率水平下，B细胞受体的空间组织似乎也受到不需要直接共连接的机制的调节。