B cell Receptor Dysregulation in Cancer and Autoimmune Disease

癌症和自身免疫性疾病中的 B 细胞受体失调

• 批准号: 8556031
• 负责人: Susan Pierce
• 金额: $ 19.43万
• 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/8556031
• 关键词: Actins; Affect; Affinity; Antigen-Presenting Cells; Antigens; Area; Autoimmune Diseases; B-Cell Neoplasm; B-Lymphocyte Subsets; B-Lymphocytes; Cells; Chronic; Collaborations; Cytoplasmic Tail; Cytoskeleton; Data; Environment; Equilibrium; Event; Goals; Growth; Image; Imaging technology; Immobilization; Immunoglobulin-Secreting Cells; Individual; Investigation; Life; Lipid Bilayers; Lipids; Liquid substance; Malignant Neoplasms; Membrane; Membrane Microdomains; Microscopy; Microtubules; Modeling; Molecular Conformation; Multiple Myeloma; Mutation; National Cancer Institute; Pathway interactions; Phosphotransferases; Play; Process; Proliferating; Property; Receptor Signaling; Receptors, Antigen, B-Cell; Resolution; Role; Series; Signal Transduction; Surface Antigens; Torque; Tyrosine; antigen binding; cellular imaging; gain of function mutation; in vivo; large cell Diffuse non-Hodgkin' s lymphoma; mutant; novel; single molecule; systemic autoimmune disease; therapy development; treatment strategy; tumor; tumorigenesis

B cells are activated to proliferate and differentiate into antibody secreting cells by antigen binding to the B cell receptor (BCR). We have applied high resolution live cell imaging technologies to the investigation of the earliest events in the antigen-driven initiation of BCR signaling. Our results provided evidence for an ordered series of discrete events following antigen binding that are initially BCR intrinsic and then become dependent on the BCR signaling apparatus. We imaged B cells as they first encountered antigen incorporated into fluid lipid bilayers, mimicking the surface of an antigen presenting cell, the apparent physiologically relevant mode of antigen recognition for B cells in vivo. Our results fit a model in which in the absence of antigen the BCRs ectodomain, particularly the membrane proximal domain, Cmu4 or Cgamma3, is not receptive to oligomerization. Binding antigen on the opposing lipid bilayer exerts a force or torque on the BCR such that the membrane proximal domain becomes oligomerization competent. The random bumping of antigen-bound BCRs results in their oligomerization and immobilization as shown by single molecule tracking analyses. The microclusters then grow in both size and area resulting in an increased recruitment of the first kinases in the pathway to the BCR clusters. Oligomerization and the initial growth of the BCR microclusters are BCR intrinsic events that are sensitive to both the affinity and the isotype of the BCRs mIg. Later cluster growth depends on kinases in the BCR signaling cascades and the actin cytoskeleton and microtubule network. The BCR microclusters perturb the local lipid environment causing the transient coalescing of lipid rafts around the microclusters. An important repercussion of the association of the BCR clusters with raft lipids is the recruitment of the first kinase in the BCR signaling cascade, the lipid-raft tethered kinase, Lyn. Simultaneously, the BCR cytoplasmic domains undergo a transition from a closed to an open conformation and are phosphorylated on tyrosines by Lyn. We hypothesize that mutations in the BCR or changes in the B cell that affect any step in this process could result in a lowered threshold for activation and hyperactivation such as in systemic autoimmune disease or in chronic activation leading to B cell tumors. In collaboration with Dr. Louis Staudt in the NCI we provided evidence that in the ABC subtype of DLBCL B cell tumors that are dependent on the BCR for survival the BCRs are constitutively in immobile, signaling active clusters. Because other B cell tumors that did not depend on their BCRs for survival did not spontaneously form signaling active BCR microclusters, these data strongly suggest that spontaneous BCR clustering plays a role in B cell tumorigenesis. We are continuing this collaboration to identify the changes in these tumors that result in spontaneous BCR clustering and signaling. These results are important as they may provide new strategies for treatment of these tumors. Staudt and colleagues observed that the BCR-dependent B cell tumors were also dependent on MYD88 the TLR signaling adaptor. Remarkably nearly 30% of the BCR-dependent B cell tumors had a gain of function mutation in the MYD88 TLR/TIR domain at an evolutionary invariant residue. The mutant MYD88 appeared to promote tumor survival by spontaneously activating NF-B. We will investigate the intracellular interaction of TLRs and BCRs in these tumors using live cell imaging. We have also determined that a subset of B cell myeloma tumors express BCRs and are dependent on these for their survival. Using live cell total internal reflection microscopy we determined that these BCR were constitutively signaling. This novel observation may provide for new strategies of the development of therapies for myelomas.

通过抗原与B细胞受体（BCR）的结合，B细胞被激活以增殖并分化为抗体分泌细胞。 我们已经应用高分辨率活细胞成像技术来研究抗原驱动的BCR信号传导起始的最早期事件。 我们的研究结果提供了证据，一系列有序的离散事件后，抗原结合，最初是BCR固有的，然后成为依赖于BCR信号装置。 我们成像B细胞，因为它们第一次遇到抗原纳入流体脂质双层，模仿抗原呈递细胞的表面，体内B细胞的抗原识别的明显生理相关模式。 我们的研究结果符合一个模型，其中在抗原的BCR胞外域，特别是膜近端结构域，Cmu 4或Cgamma 3，是不接受寡聚化。 相对的脂质双层上的结合抗原对BCR施加力或扭矩，使得膜近端结构域变得具有寡聚化能力。 抗原结合BCR的随机碰撞导致其寡聚化和固定化，如单分子追踪分析所示。 然后微簇在大小和面积上都生长，导致在通向BCR簇的途径中第一激酶的募集增加。 寡聚化和BCR微簇的初始生长是BCR固有事件，其对BCR mIg的亲和力和同种型都敏感。 随后的簇生长依赖于BCR信号级联中的激酶以及肌动蛋白细胞骨架和微管网络。 BCR微团扰动局部脂质环境，引起微团周围脂筏的瞬时聚结。 BCR簇与筏脂质结合的一个重要反应是BCR信号级联中第一个激酶的募集，即脂筏束缚激酶林恩。 同时，BCR胞质结构域经历从封闭构象到开放构象的转变，并通过林恩在酪氨酸上磷酸化。 我们假设BCR突变或B细胞变化影响该过程中的任何步骤，可能导致激活和过度激活阈值降低，如系统性自身免疫疾病或慢性激活导致B细胞肿瘤。 与NCI的Louis施陶德德博士合作，我们提供的证据表明，在依赖BCR生存的ABC亚型DLBCL B细胞肿瘤中，BCR组成性地处于不动的信号活性簇中。 由于其他不依赖BCR生存的B细胞肿瘤不会自发形成信号传导活性BCR微簇，因此这些数据强烈表明自发BCR聚集在B细胞肿瘤发生中起作用。 我们正在继续这项合作，以确定这些肿瘤中导致自发BCR聚集和信号传导的变化。 这些结果很重要，因为它们可能为治疗这些肿瘤提供新的策略。 施陶德德及其同事观察到，BCR依赖性B细胞肿瘤也依赖于TLR信号衔接子MYD 88。 值得注意的是，近30%的BCR依赖性B细胞肿瘤在MYD 88 TLR/TIR结构域的进化不变残基处具有功能获得突变。 突变体MYD 88似乎通过自发激活NF-B来促进肿瘤存活。 我们将使用活细胞成像研究这些肿瘤中TLR和BCR的细胞内相互作用。 我们还确定了一部分B细胞骨髓瘤表达BCR，并依赖于它们的生存。 使用活细胞全内反射显微镜，我们确定这些BCR组成型信号。 这一新的观察可能为骨髓瘤治疗的发展提供新的策略。