Human B Cell Differentiation in a Model System

描述(申请人提供)：我们已经确认了一种B细胞免疫缺陷，其特征是对CD4O和IL-4的反应有缺陷 发信号。1例年轻女性患者(PT#L)的B细胞表达正常 CD4O，但显然在CD4O介导的功能子集中存在缺陷，包括 开关重组所需的早期信号。但是，在特定的 在体外条件下，PT#1 B细胞可以恢复功能反应，并经历转换以表达下游抗体类别或同种抗体。我们的预赛 数据支持一种模型，在该模型中，信号分子或转录因子 导致核因子-kB活化的CD4O信号转导通路受到影响。 这一假设得到了我们发现的PT#1 B细胞的支持 EB病毒(EBV)转化细胞表面不表达CD23 严重依赖于病毒潜伏感染膜的分子 蛋白LMP)1以及随后由该蛋白激活的核因子-kB。 此外，LMP1篡夺了CD4O信号通路，以维持细胞 转型。令人惊讶的是，PT#1 EBV转化的B细胞松动了他们的 在稀薄培养条件下生长时的转化潜力 提示LMP1活性受损。因此，三行相关的数据 强烈提示pt#1缺陷位于CD4O信号通路中。 这导致了核因子-kB的激活和特定细胞基因的转录 参与了B细胞的激活。我们建议使用原始的和转换的 来自pt#1的B细胞，以表征导致B细胞的潜在缺陷 功能障碍。最初的努力将集中在TRAF的特征上 最近端和最远端的核因子-kB的表达和功能 CD4O信号级联中的信号事件分别。这个 EB病毒转化的pT#L B细胞将用于分析LMP1通过 CD4O途径。此外，这些细胞将用于转基因研究，这些研究 旨在对PT#L缺陷进行补充，并对核因子-kappaB进行分析 响应性的推动者。细胞外信号转导途径的特征 不同的成长条件将为这一关系提供信息 这些信号通路与细胞转化之间的关系。最后，pt#L T细胞 在提供辅助功能以控制B细胞方面有细微的缺陷， 表现为对重链基因的不适当转录 单独的CD4O信号。为了理解这一点，我们建议进行实验。 功能缺陷。

DESCRIPTION (provided by applicant): We have identified a B-cell immunodeficiency that is distinguished by defective responses to CD4O and IL-4 signaling. B-cells from a young female patient (pt#l) have normal expression of CD4O but are clearly deficient in a subset of CD4O-mediated functions including early signals required for switch recombination. However, under specific in vitro conditions pt#1 B-cells can regain functional responsiveness and undergo switching to express downstream antibody classes or isotypes. Our preliminary data support a model whereby a signaling molecule or transcription factor in the CD4O signal transduction pathway leading to NF-kB activation is affected. This hypothesis is supported by our finding that pt#1 B-cells that are transformed by Epstein-Barr virus (EBV) do not express CD23 a cell surface molecule that is critically dependent on the viral latent infection membrane protein LMP)1 and the subsequent activation of NF-kB by this protein. Furthermore, LMP1 usurps the CD4O signaling pathway in order to maintain cell transformation. Surprisingly, the pt#1 EBV-transformed B-cells loose their transforming potential when grown in dilute culture conditions which also suggests that LMP1 activity is compromised. Thus, three related lines of data strongly suggest that the pt#1 defect is located in the CD4O signaling pathway that leads to NF-kB activation and the transcription of specific cellular genes involved in B-cell activation. We propose to use primary and transformed B-cells from pt#1 to characterize the underlying defect Leading to B-cell dysfunction. Efforts will be initially focused on the characterization of TRAF and NF-kB expression and function which are the most proximal and distal signaling events in the CD4O signaling cascade, respectively. The EBV-transformed pt#l B-cells will be used to analyze signaling of LMP1 via the CD4O pathway. Also, these cells will be used in transfection studies that are aimed at complimenting the pt#l defect and in the analysis of of NF-kappaB responsive promoters. Characterization of signaling pathways under the different growth conditions will provide information into the relationship between these signaling pathways and cell transformation. Finally, pt#l T-cells have subtle defects in helper function provided to control B-cells that manifest in inappropriate transcription of the heavy chain locus in response to CD4O signaling alone. Experiments are proposed to understand the basis of this functional defect.