MONOCLONAL ANTI-IGM REGULATION OF HUMAN B CELL FUNCTION

描述(改编自申请人的摘要)：建议的作品 寻求定义亲和力的物理化学和生化基础- 人B细胞通过mIgM依赖的阳性和阴性选择 信号通路。这将通过使用一套油井- 不同结合部位的鼠源性抗IgM单抗的鉴定 亲和力和实验操纵的价态，作为模型抗原 它可以与所有的IgM和人类B细胞结合。(1)研究将会 评估是否有交联剂形成的速率，或者可选的， 交联链解离，是亲和力依赖的极限参数， 限制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介导的信号是整个细胞周期进程所必需的。 这将涉及对初始的离解动力学的比较。 配体(K1)的单价结合，交联键的形成速率，以及 交联剂解离速率与早期酪氨酸动力学 蛋白酪氨酸磷酸化和蛋白酪氨酸活化。(2)研究 将评估不同的信号通路是否可能存在差异 受mIgM的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。(3) 这些研究将评估物理化学结合是否 诱导B细胞进入S时相的要求与 Bcl-2的上调和一些新的 或所有显示为调节G1到S相变的蛋白质 在其他真核细胞中，即细胞周期蛋白和细胞周期蛋白激酶。(4)作品 将评估成熟的B细胞，其接受不足的mIgM- 从G1到S相变的中介信号，被引导到 激活相关的细胞凋亡或无能。(5)mIgM：配体结合 诱导未成熟B淋巴细胞无能和凋亡的条件 将使用其B细胞表达该基因的转基因小鼠进行进一步评估 人膜形式的Mu链。(6)作为最终的主要目标，研究 将评估B细胞mIgM和其他B细胞是否共连接 黏附分子(即CD21、CD22和VLA-4)通过相同的分子 底物，可以降低亲和力和/或价态要求 诱导B细胞增殖。如果是这样的话，其他研究将确定 共连接是否导致配基结合动力学增强；增强 或修饰的受体近端信号转导； 细胞凋亡；bcl-2水平升高；和/或某些或某些 所有负责G1向S相变的蛋白质。已被占用 总之，拟议的研究应该会提供相当多的新见解。 如何定量和定性地占据mIgM和其他 B细胞表面的辅助受体转化为信号 B细胞无能、B细胞缺失或B细胞克隆性增殖。 因为亲和力依赖的B细胞选择在(A)中很重要 对入侵病原体的免疫反应和故意给药 疫苗，(B)B细胞对自身抗原的自身免疫，(C)克隆 某些B细胞恶性肿瘤的进化，这些见解应该会导致 加大对上述现象的监管干预力度。

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.