B cell Receptor Dysregulation in Cancer and Autoimmune Disease

B细胞通过与B细胞受体(BCR)的抗原结合而被激活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启动BCR信号的最早事件。我们的结果为抗原结合后的一系列离散事件提供了证据，这些事件最初是BCR固有的，然后变得依赖于BCR信号装置。当B细胞第一次遇到结合到液体脂质双层中的抗原时，我们对B细胞进行了成像，模拟了抗原呈递细胞的表面，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，在该模型中，在没有抗原的情况下，BCR的胞外结构域，特别是膜近端结构域Cmu4或CGamma3，不能接受寡聚。相对的脂质双层上的结合抗原对BCR施加力或扭矩，从而使膜近端区域成为寡聚活性区域。单分子跟踪分析表明，抗原结合的BCR的随机碰撞导致了它们的寡聚和固定化。然后，微团簇在大小和面积上都会增长，导致在通往BCR簇的途径中第一批激酶的募集增加。齐聚和BCR微团簇的初始生长是BCR的内在事件，对BCRs MiG的亲和力和同型都很敏感。后来簇的生长依赖于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微团簇扰乱了局部的脂质环境，导致脂筏在微团簇周围瞬间聚集。BCR簇与RAFT脂类关联的一个重要影响是BCR信号级联中的第一个激酶LYN的招募，即脂筏系留激酶。同时，BCR胞质结构域经历了从封闭构象到开放构象的转变，并被Lyn在酪氨酸上磷酸化。我们假设，影响这一过程中任何一步的BCR突变或B细胞变化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。 在NCI与Louis Staudt博士的合作中，我们提供了证据，证明在依赖BCR生存的ABC亚型DLBCL B细胞肿瘤中，BCR本质上处于静止状态，信号活跃。由于其他不依赖BCR生存的B细胞肿瘤不会自发形成信号活性的BCR微簇，这些数据强烈表明自发的BCR聚集在B细胞肿瘤的发生中发挥了作用。我们正在继续这项合作，以确定这些肿瘤中导致自发bcr聚集和信号传递的变化。这些结果很重要，因为它们可能为这些肿瘤的治疗提供新的策略。 Staudt和他的同事观察到，依赖BCR的B细胞肿瘤也依赖于TLR信号适配器MYD88。值得注意的是，近30%的bcr依赖型B细胞肿瘤在进化不变残基上有MYD88 TLR/TIR结构域的功能突变。突变体MYD88似乎通过自发激活核因子-B来促进肿瘤存活。我们将使用活细胞成像技术研究这些肿瘤中TLRs和BCRs的细胞内相互作用。 我们还确定了B细胞骨髓瘤的一个亚群表达BCR，并依赖这些细胞生存。使用活细胞全内反射显微镜，我们确定这些BCR是结构性信号。这一新的观察结果可能为骨髓瘤治疗的发展提供新的策略。

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.