HUMAN B CELL DIFFERENTIATION IN A MODEL SYSTEM

模型系统中的人类 B 细胞分化

基本信息

批准号：
2886969
负责人：
LORI Ruth COVEY
金额：
$ 11.87万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-09-15 至 2001-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2886969
关键词：
B lymphocyte CD40 molecule cell differentiation cytokine gene mutation gene rearrangement genetic transcription human genetic material tag human tissue immunoglobulin genes leukocyte activation /transformation polymerase chain reaction tissue /cell culture

项目摘要

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.

总体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，我们将使用一个由人类 B 细胞淋巴瘤细胞系 RAMOS 组成的模型系统 266，具有响应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 RAMOS B 细胞的初始激活可以通过上调来测量表面 CD23 的表达依赖于 CD40 配体 (CD40-L) D1.1 T 细胞的表面。我们将利用 RAMOS 266 的优势进行文化分化的能力来评估不同恒定区基因的转录激活对细胞因子和 T 细胞的反应有帮助。在这个项目中，T细胞的帮助将被定义为通过CD40-L的信号传导。初步实验将是使用 RT-PCR 对暴露于 CD40-L 后分离的 RAMOS mRNA 进行和/或细胞因子。我们将评估不同同种型的反应这些信号的类和子类并评估响应确定类别切换是否是通过定向或随机过程。我们的分析将包括测量种系（I- CH)转录本表达以及成熟表达(VDJ-CH) 成绩单。为了更全面地研究同种型转换的调控，我们将分析 RAMOS 266 单元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非转录反应开关变体的生产性等位基因进一步建立机制用于类切换的调节。最后，我们将改变 I 使用同源的重链 C gamma 1 区域的 gamma I 外显子重组。我们将确定非的重排状态与细胞因子和 T 细胞接触模拟后的生产性等位基因。