HUMAN B CELL DIFFERENTIATION IN A MODEL SYSTEM

总的目标是了解免疫球蛋白的调节 人类B细胞中的类别转换重组。 具体来说，我们将使用 由人B细胞淋巴瘤细胞系拉莫斯组成的模型系统 266，其具有在应答中经历同种型分化的能力 细胞因子和T细胞介导的信号。此外，还发现， 拉莫斯B细胞的初始激活可以通过上调 CD 23依赖于CD 40配体（CD 40-L）， D1.1 T细胞的表面。我们将利用拉莫斯266的优势 在文化中进行分化的能力， 不同恒定区基因的转录激活， 细胞因子和T细胞的帮助。在这个项目中，T细胞帮助将 被定义为通过CD 40-L的信号传导。初步实验将在 使用RT-PCR对暴露于CD 40-L后分离的拉莫斯mRNA进行 和/或细胞因子。我们将评估不同同种型的反应 类和子类，并评估这些信号的响应， 确定类切换是否通过定向或 随机过程我们的分析将包括测量生殖系（I- CH）转录本表达以及成熟（VDJ-CH）的表达 成绩单为了更全面地研究同种型转换的调节， 我们将分析拉莫斯266细胞的开关变体，并确定它们的 进一步切换事件的能力。此外，我们计划评估 重排事件和转录反应的非- 开关变体的生产性等位基因，以进一步建立一种机制 用于调节类切换。最后，我们会改变I 重链C γ 1区的γ I外显子，使用同源 重组我们将确定非- 在用细胞因子和T细胞接触刺激后产生等位基因。

The overall goal of this is to understand the regulation of immunoglobulin class switch recombination in human B cells. Specifically, we will use a model system that is comprised of a human B cell lymphoma cell line, RAMOS 266, that has the capacity to undergo isotype differentiation in response to cytokine and T cell-mediated signals. In addition, it was found that initial activation of the RAMOS B cells could be measured by up-regulation of surface CD23 and was dependent on the CD40 ligand (CD40-L) expressed on the surface of the D1.1 T cells. We will take advantage of RAMOS 266's capacity to undergo differentiation in culture to assess the transcriptional activation of the different constant region genes in response to cytokines and T cell help. In this project, T cell help will be defined as signaling through the CD40-L. Initial experiments will be carried out using RT-PCR on RAMOS mRNA isolated after exposure to CD40-L and/or cytokines. We will assess the response of the different isotype classes and subclasses to these signals and evaluate the response in terms of establishing whether class switching is proceeding by a directed or stochastic process. Our analyses will include measuring the germline (I- CH) transcript expression as well as the expression of mature (VDJ-CH) transcripts. To study more completely the regulation of isotype switching, we will analyze switch variants of RAMOS 266 cells and determine their capacity for further switch events. Additionally, we plan to evaluate the rearrangement events and the transcriptional response on the non- productive alleles of the switch variants to further establish a mechanism for the regulation of class switch. And finally, we will mutate the I gamma I exon of the heavy chain C gamma 1 region using homologous recombination. We will identify the rearrangement status of the non- productive allele after simulation with cytokines and T cell contact.