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: 8745551
• 负责人: Susan Pierce
• 金额: $ 25.17万
• 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/8745551
• 关键词: Actins; Affect; Affinity; Allergic Disease; Antibodies; Antigen-Presenting Cells; Antigens; Area; Autoimmune Diseases; Autoimmunity; B-Cell Neoplasm; B-Lymphocytes; BLNK gene; Burkitt Lymphoma; CD19 gene; CD4 Positive T Lymphocytes; Cells; Chronic; Coin; Collaborations; Cytoplasmic Tail; Cytoskeleton; Data; Environment; Equilibrium; Event; Exhibits; Failure; Goals; Growth; Helper-Inducer T-Lymphocyte; Image; Imaging technology; Immobilization; Immune; Immunoglobulin-Secreting Cells; Immunologic Deficiency Syndromes; Individual; Investigation; Laboratories; Life; Lipase; Lipid Bilayers; Lipids; Liquid substance; MAPK14 gene; MAPK8 gene; Malignant Neoplasms; Membrane; Membrane Microdomains; Microtubules; Modeling; Molecular Conformation; Mutation; NF-kappa B; National Cancer Institute; PLCgamma2; Pathway interactions; Patients; Phosphotransferases; Play; Process; Proliferating; Property; Receptor Signaling; Receptors, Antigen, B-Cell; Resolution; Role; Series; Signal Transduction; Structure of germinal center of lymph node; Surface; Surface Antigens; Syndrome; Torque; Tyrosine; antigen binding; cellular imaging; gain of function mutation; in vivo; large cell Diffuse non-Hodgkin' s lymphoma; mutant; receptor internalization; single molecule; systemic autoimmune disease; trafficking; 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. With the Staudt group we are also imaging ABC, Burkitts Lymphomas (BLs) and germinal center B cell (GBCs) tumors that are dependent on the BCR for survival and in addition are either dependent or not on the B cell coreceptors, CD19, for their survival. We will determine if the BCRs are clustered, whether the BCR and CD19 colocalize, if CD19 is active and phosphorylated and if these cells exhibit enhanced BCR and CD19 signaling by determining the colocalization of phosphorylated PI3K and Syk with the BCR and CD19. These studies have the potential to identify new targets for B tumor therapy. Our efforts in understanding B cell signaling mechanisms that contribute to autoimmunity have focused on a collaboration with Dr. Joshua Milner in Laboratory of Allergic Disease, to analyze B cells from patients with immunodeficiency and autoimmunity syndrome, cold urticarial. Dr. Milner discovered that affected individuals had gain of function mutations in PLCgamma2, a key lipase in the BCR signaling cascade. He coined this syndrome PLAID for PLCgamma2-associated antibody deficiency and immune dysregulation. We have determined that although BCR internalization from the surface following antigen binding occurs at a normal rate, the BCR fails to traffic to the MHC class III antigen-presenting compartment suggesting that these cells may be deficient in interactions with CD4+ helper T cells. We also observed altered BCR signaling in PLAID B cells with decreased levels of phosphorylated Syk, Btk, Igalpha, BLNK and PI3K involved in the early steps in the BCR signaling cascade and decreased levels of activated intermediate kinase including ERK, p38, JNK but not of NFKB that was in fact increased. By live cell imaging, the recruitment of the BCR to the contact area is more transient in PLAID B cells as compared to normal B cells and as compared to WT-PLCgamma2, mutant PLCgamma2 poorly co-localizes with the BCR. Taken together these data suggest that the failure to stably maintain BCRs and phosphorylated Btk in the antigen contact area in PLAID B cells contributes to the observed deficiency in downstream BCR signaling.

B细胞通过抗原与B细胞受体（BCR）结合而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信号启动的最早事件。我们的研究结果为抗原结合后一系列有序的离散事件提供了证据，这些事件最初是BCR固有的，然后依赖于BCR信号传导装置。当B细胞第一次遇到合并到流体脂质双层中的抗原时，我们对它们进行了成像，模拟了抗原提呈细胞的表面，这是B细胞在体内识别抗原的明显生理相关模式。我们的结果符合一个模型，即在缺乏抗原的情况下，bcr的外畴，特别是膜近端结构域，Cmu4或Cgamma3，不接受寡聚化。相反脂质双分子层上的结合抗原对BCR施加一个力或扭矩，使得膜近端结构域具有寡聚化能力。单分子跟踪分析表明，抗原结合bcr的随机碰撞导致其寡聚化和固定化。然后微团簇在大小和面积上都增长，从而增加了通往BCR簇的途径中的第一批激酶的招募。BCR微团簇的寡聚化和初始生长是BCR的内在事件，对BCR micg的亲和力和同型都很敏感。后来的簇生长取决于BCR信号级联中的激酶和肌动蛋白细胞骨架和微管网络。BCR微团簇扰乱了局部脂质环境，导致微团簇周围的脂筏瞬间聚结。BCR簇与筏脂相关的一个重要影响是BCR信号级联中第一个激酶的募集，即脂质-筏栓激酶（Lyn）。同时，BCR细胞质结构域经历从封闭到开放构象的转变，并在酪氨酸上被Lyn磷酸化。我们假设，BCR的突变或B细胞的变化影响这一过程中的任何一步，都可能导致激活和过度激活的阈值降低，例如在系统性自身免疫性疾病或导致B细胞肿瘤的慢性激活中。