MONOCLONAL ANTI-IGM REGULATION OF HUMAN B CELL FUNCTION

描述（改编自申请人的摘要）：拟议的工作 试图定义亲和性的物理化学和生物化学基础- 通过mIgM对人B细胞的依赖性阳性和阴性选择 信号通路 这将通过使用一套良好的- 具有不同结合位点的表征的鼠抗IgM mAb 亲和力和实验操作价，作为模型抗原 可以与所有IgM加上人类B细胞结合。 (1)这些研究将 评估交联形成速率，或者替代地， 交联解离，是亲和力依赖的限制参数， 限制性mIgM条件下mIgM介导的信号转导 受体密度，如发生在B细胞激活的晚期， mIgM介导的信号是完整细胞周期进程所必需的。 这将涉及初始解离动力学的比较。 配体的单价接合（k1）、交联形成的速率，以及 交联解离速率，早期酪氨酸 激酶活化和蛋白酪氨酸磷酸化。 (2)研究 将评估不同的信号通路是否可能是差异性的， 受mIgM配体亲和力和配体价态的影响，通过 评估早期酪氨酸磷酸化的程度 不同的蛋白质受到这些参数的不同影响。 （三） 这些研究将评估是否存在物理化学结合 诱导B细胞进入S期的要求与那些 需要上调bcl-2，并增加合成的一些 或所有显示调节G1到S相变的蛋白质 在其他真核细胞中，即，细胞周期蛋白和CDK激酶。 (4)工作 将评估是否成熟的B细胞，接受不足的mIgM- G1到S相变的介导信号，被引导到 活化相关的凋亡或无反应性。 (5)mIgM：配体结合 诱导未成熟B淋巴细胞无反应性和凋亡的条件 将使用B细胞表达 人μ链的膜形式。 (6)作为最后一个主要目标，研究 将评估B细胞mIgM和其他B细胞的共连接是否 粘附分子（即，CD 21、CD 22和VLA-4）通过相同的分子 底物的亲和力和/或化合价要求， 诱导B细胞增殖。 如果是这样，进一步的研究将确定 共连接是否导致增强的配体结合动力学;增强的 或修饰的受体近端信号转导; 细胞凋亡; bcl-2水平升高;和/或一些或 所有负责G1到S相变的蛋白质。 采取 总之，拟议的研究应该提供相当多的新见解 mIgM和其他抗体的定量和定性占有率 B细胞表面的辅助受体将信号 用于B细胞无反应性、B细胞缺失或B细胞克隆增殖。 因为在（a）中，依赖亲和力的B细胞选择很重要 对入侵病原体的免疫反应， 疫苗，（B）对自身抗原的B细胞自身免疫，（c）克隆的 某些B细胞恶性肿瘤的演变，这些见解应该导致 加强对上述现象的监管干预。

DESCRIPTION (Adapted from the applicant's abstract): The proposed work seeks to define the physicochemical and biochemical basis for affinity- dependent positive and negative selection of human B cells via the mIgM signaling pathway. This will be accomplished by using a set of well- characterized murine anti-IgM mAbs with differing binding site affinities, and experimentally manipulated valencies, as model antigens that can engage with all IgM plus human B cells. (1) The studies will evaluate whether rate of crosslink formation, or alternatively, rate of crosslink dissociation, is the limiting parameter in affinity-dependent, mIgM-mediated signal transduction under conditions of limiting mIgM receptor density, such as occurs late in B cell activation when new mIgM-mediated signals are required for full cell cycle progression. This will involve comparisons of the dissociation kinetics for initial monovalent engagement of ligand (k1), rates for crosslink formation, and rates for crosslink dissociation, with the kinetics of early tyrosine kinase activation and protein tyrosine phosphorylation. (2) The studies will evaluate whether distinct signaling pathways may be differentially affected by ligand affinity for mIgM and ligand valency, through assessing the degree to which the early tyrosine phosphorylation of distinct proteins is differentially affected by these parameters. (3) The studies will evaluate whether the physicochemical binding requirements for inducing B cell S phase entry are identical to those required for upregulation of bcl-2, and for increased synthesis of some or all of the proteins shown to regulate the G1 to S phase transition in other eukaryotic cells, i.e., cyclins and cdk kinases. (4) The work will evaluate whether mature B cells, which receive insufficient mIgM- mediated signals for the G1 to S phase transition, are channeled into activation-related apoptosis or anergy. (5) The mIgM:ligand binding requirements for inducing anergy and apoptosis in immature B lymphocytes will be further assessed using transgenic mice whose B cells express the membrane form of human mu chain. (6) As a final major aim, the studies will evaluate whether co-ligation of B cell mIgM and other B cell adhesion molecules (i.e., CD21, CD22, and VLA-4) by the same molecular substrate, can reduce the affinity and/or valency requirements for inducing B cell proliferation. If so, additional studies will determine whether co-ligation results in enhanced ligand binding kinetics; enhanced or modified receptor-proximal signal transduction; diminished levels of apoptosis; enhanced levels of bcl-2; and/or enhanced levels of some or all the proteins responsible for the G1 to S phase transition. Taken together, the proposed studies should provide considerable new insights into how the quantitative and qualitative occupancy of mIgM and other ancillary receptors on the surface of B cells translates into signals for B cell anergy, B cell deletion, or B cell clonal proliferation. Because affinity-dependent selection of B cells is important in (a) immune responses to intruding pathogens and deliberately administered vaccines, (b) B cell autoimmunity to self antigens, (c) the clonal evolution of certain B cell malignancies, these insights should lead to enhanced regulatory intervention of the above phenomena.