HUMAN B CELL DIFFERENTIATION IN A MODEL SYSTEM

其总体目标是了解免疫球蛋白的调节。 人类B细胞中的类开关重组。具体来说，我们将使用 由人B细胞淋巴瘤细胞系Ramos组成的模型系统 266，具有进行同型分化反应的能力 细胞因子和T细胞介导的信号。此外，还发现 Ramos B细胞的初始激活可以通过上调来测量 并依赖于细胞表面表达的CD40配体(CD40-L D1.1T细胞表面。我们将利用拉莫斯266的S 在培养中经历分化的能力以评估 不同恒定区基因在转录水平的激活 对细胞因子和T细胞的反应有帮助。在这个项目中，T细胞帮助将 被定义为通过CD40-L传递信号。最初的实验将是 用RT-PCR法检测CD_(40)-L作用后分离的Ramos基因 和/或细胞因子。我们将评估不同同工型的反应。 类和子类，并根据以下条件评估响应 来确定类切换是通过定向的还是通过 随机过程。我们的分析将包括测量生殖系(I- CH)转录本表达及成熟蛋白(VDJ-CH)的表达 成绩单。为了更全面地研究同型转换的规律， 我们将分析Ramos 266细胞的开关变体，并确定它们的 用于进一步切换事件的容量。此外，我们计划评估 重排事件与非核糖体的转录反应 生产等位基因的开关变异，以进一步建立机制 用于班级切换的调节。最后，我们将变异i 重链C-γ1区的γI外显子的同源研究 重组。我们将确定非重排的状态 模拟后产生等位基因与细胞因子和T细胞接触。

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.