MECHANISM OF HUMAN B CELL SELECTION AND V GENE DIVERSIFICATION

V基因超突变的抗原选择及相关过程 对抗体谱系有重大影响。这些过程起作用 在B细胞发育的阶段，对最常见的形式也是至关重要的 人类淋巴瘤的病症。该项目的特点是对应的 使用两个独特的实验系统的细胞和分子过程 在前一个授权期内开发的。 一种病毒抗原，hiv gp120，被发现选择性地结合和激活。 通过VH3表达的保守序列获得一个大的B细胞亚群 免疫球蛋白基因家族，通过与新的非CDR框架的明显相互作用 网站。在临床感染HIV期间，VH3B细胞群是 最初扩展和激活，然后选择性克隆删除 与CD4T细胞的丧失平行。这些发现可以是 被概念化为B细胞系超抗原的先例。 这一概念的推论是gp120的生物紊乱- 结合B细胞克隆可能是体液免疫的重要因素 在这些患者中，功能障碍和B细胞淋巴瘤的风险增加 个人。在提议的项目中，我们将首先定义 Gp120-VH3 Ig相互作用的结构基础。抗体基序 将定义结合所需的gp120表位，并具有选择性 这种相互作用的多肽或蛋白质拮抗剂将会产生。 第二，我们将确定艾滋病毒的细胞生物学过程- 诱导VH3B细胞耗竭。我们将定义他们对 Gp120，以及候选VH3耗尽机制的有效性。 第三，我们将评估这种超抗原的潜在作用 通过检测HIV相关淋巴肿大的患病率来研究二者的相互作用 在HIV相关性淋巴瘤和散发性淋巴瘤中，这种VH3Ig亚群。 建立了一种V基因超突变的实时检测方法。 检测突变活性的基因特异性聚合酶链式反应DNA修复试验 在原代B细胞或细胞系的内源性Ig VDJ片段中。我们的 研究反映了基因转化过程的优势，即 以顺序和阶段特定的方式为VDJ选择性地激活 高突变阳性(生发中心)B细胞的片段。在 建议的工作，我们的第一个目标是确定结构特征 这些事件是通过基因组分离和基因特征来实现的 转换轨迹和候选供体基因。第二，原生细胞 化验将使用酶特异性抑制剂和一组 寻找主要生化和细胞的免疫刺激 可能激活或抑制V基因转换的生理过程。 第三，将使用超突变分析来筛选和表征 生发中心B细胞系具有结构性或可诱导性高突变 活动。

Antigenic selection and the associated process of V gene hypermutation have a major influence on the antibody repertoire. These processes act at a stage in B-cell development also critical for the most common forms of human lymphomas. This project characterizes the corresponding cellular and molecular processes using two unique experimental systems developed in the previous grant period. A viral antigen, HIV gp120, was found to selectively bind and activate a large B-cell subset through conserved sequences expressed by the VH3 Ig gene family, through apparent interaction with novel non-CDR framework sites. During clinical HIV infection, the VH3 B-cell population is initially expanded and activated, followed by a selective clonal deletion paralleling the loss of CD4 T cells. These findings can be conceptualized as a precedent for superantigens of the B-cell lineage. The corollary of this concept is that the biologic disturbance of gp120- binding B-cell clones may be an important factor in the humoral immune dysfunction and elevated risk of B-cell lymphomagenesis in these individuals. In the proposed project, we will first define the structural basis of the gp120-VH3 Ig interaction. The antibody motifs and gp120 epitopes required for binding will be defined, and selective peptide or protein antagonists of this interaction will be generated. Second, we will determine the cell biologic processes accounting for HIV- induced VH3 B-cell depletion. We will define their direct response to gp120, and the effectiveness of candidate VH3 depletion mechanisms. Third, we will evaluate the potential role of this superantigen interaction in HIV-associated lymphomagenesis by testing the prevalence of this VH3 Ig subset among HIV-associated versus sporadic lymphomas. A real-time assay for V gene hypermutation has been developed, based on a gene-specific PCR DNA repair assay which detects mutational activity in endogenous Ig VDJ segments of primary B-cells or cell lines. Our studies reflect the predominance of a gene conversion process which is selectively active in a sequence and stage-specific manner for VDJ segments in hypermutation-positive (germinal center) B cells. In the proposed work, our first goal is to ascertain the structural features of these events by genomic isolation and characterization of the gene conversion tracks and candidate donor genes. Second, the primary cell assay will be manipulated with enzyme specific inhibitors and a panel of immunologic stimuli to search for predominant biochemical and cell physiologic processes which may activate or silence V gene conversion. Third, the hypermutation assay will be used to screen and characterize germinal center B-cell lines with constitutive or inducible hypermutation activity.