Analysis of B cell transcriptome shifts prior to lineage divergence in vivo

体内谱系分歧之前 B 细胞转录组变化的分析

基本信息

批准号：
8356988
负责人：
ANN M HABERMAN
金额：
$ 16.6万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8356988
关键词：
Adoptive Transfer Affinity Antibodies Antigens Appearance B cell differentiation B-Cell Development B-Lymphocyte Subsets B-Lymphocytes CD22 gene Cell Cycle Cell Separation Cell division Cells Commit Confocal Microscopy Cytokinesis Daughter Development Ensure Evolution Flow Cytometry Gene Expression Gene Expression Profile Genetic Transcription Image Immune response Immunization Immunoglobulin-Secreting Cells Immunoglobulins In Vitro Invaded Label Location Mediating Memory B-Lymphocyte MicroRNAs Mitosis Molecular Pathway interactions Pattern Phase Phenotype Plasma Cells Population Process Production Proteins RNA Sequences Sorting - Cell Movement Staging Structure of germinal center of lymph node Surface T-Lymphocyte Time Transcription Repressor/Corepressor Vaccines daughter cell in vivo pathogen programs research study transcription factor

项目摘要

DESCRIPTION (provided by applicant): Effective immune responses to pathogens and vaccines critically depend on the formation of both short-term antibody secreting cells (ASC) and germinal centers. Despite the importance of generating both ASC and GCs, very little is known about the molecular changes that enable the initial divergence to these disparate fates during primary B cell divisions. Initial formation of germinal center B cells requires an elevated production of the transcriptional repressor Bcl6. Formation of ACS, on the other hand, involves a decrease in Pax5 followed by an increase of several other transcription factors including BLIMP-1. Bcl-6 and BLIMP-1 are known to antagonize each others expression, thus constraining and propelling cells along mutually exclusive pathways of differentiation. Although it is now well established that altered expression patterns of transcription factors mediates the shifts toward one lineage and away from what was previously a stable transcriptional program in naive B cells, it remains a mystery how the Pax5 dominated B cell program is interrupted in some cells and Bcl6 increased in others. Very little is know about the processes that occur during the multiple divisions preceding these more advanced stages of differentiation, despite their importance to the divergence of these pathways and GC establishment. Here we propose to investigate the progression of differentiation that precedes the appearance of known indicators of the GC or ASC transcriptional program. Using a validated strategy that sorts cells by the extent of cell division and expression of genes regulated by Bcl6 and Pax5, we will further characterize the cell subsets during their initial emergence and define the immediate precursors to lineage committed B cells. Aim 1: Define the progressive differentiation of B cells prior to lineage commitment in vivo. Antigen specific B cells will be assessed shortly after immunization for the emergence of transcription factor and phenotypic changes known to be associated with initial AFC and GC lineage formation. Expression levels of relevant molecules will be quantified by flow cytometry and/or qRT-PCR and correlates to cell division number and CD38, CD23 phenotype will be determined. RNA sequencing of B cell subsets sorted by cell division number and phenotype will identify transcriptional changes, including microRNA transcription, that immediately precede the earliest known molecular shifts in lineage development. Aim 2: Determine the heritage of lineage committed subsets through daughter cell analysis. To define the precursors of germinal center B cells, sorted subsets will be briefly cultured in vitro, allowed to complete the subsequent round(s) of division in vitro and assessed of their expression of Bcl6. Similarly sorted and fixed B cells, enriched for cells in the G2/M phase of the cell cycle, will be imaged via confocal microscopy directly ex vivo for extent of symmetry in the distribution of surface markers and transcription factors in conjoined daughters. PUBLIC HEALTH RELEVANCE: Effective immune responses to pathogens and vaccines critically depends on the formation of germinal centers to form high affinity memory B cells and plasma cells. This application proposes experiments that will answer several fundamental questions about the initiation of germinal center B cell development.

描述（由申请人提供）：对病原体和疫苗的有效免疫反应关键取决于短期抗体分泌细胞（ASC）和生发中心的形成。尽管产生 ASC 和 GC 都很重要，但对于在原代 B 细胞分裂过程中导致这些不同命运最初分化的分子变化知之甚少。生发中心 B 细胞的初始形成需要转录抑制因子 Bcl6 的产生增加。另一方面，ACS 的形成涉及 Pax5 的减少，随后包括 BLIMP-1 在内的其他几种转录因子的增加。已知 Bcl-6 和 BLIMP-1 可以相互拮抗对方的表达，从而限制和推动细胞沿着相互排斥的分化途径前进。虽然它目前已经确定，转录因子表达模式的改变会介导向一种谱系的转变，并远离初始 B 细胞中先前稳定的转录程序，但 Pax5 主导的 B 细胞程序如何在某些细胞和 Bcl6 中被中断仍然是一个谜。其他人增加。人们对这些更高级的分化阶段之前的多重分裂期间发生的过程知之甚少，尽管它们对于这些途径的分歧和 GC 的建立很重要。在这里，我们建议研究 GC 或 ASC 转录程序的已知指标出现之前的分化进展。使用经过验证的策略，根据细胞分裂的程度以及 Bcl6 和 Pax5 调控的基因表达对细胞进行分类，我们将进一步表征细胞亚群在其最初出现期间的特征，并定义谱系定型 B 细胞的直接前体。目标 1：定义 B 细胞在体内谱系定型之前的渐进分化。免疫后不久将评估抗原特异性 B 细胞中转录因子的出现以及已知与初始 AFC 和 GC 谱系形成相关的表型变化。相关分子的表达水平将通过流式细胞术和/或qRT-PCR进行定量，并与细胞分裂数相关，并且将确定CD38、CD23表型。按细胞分裂次数和表型分类的 B 细胞亚群的 RNA 测序将识别转录变化，包括 microRNA 转录，这些变化紧接在谱系发育中最早已知的分子转变之前。目标 2：通过子细胞分析确定谱系定型子集的遗传。为了定义生发中心 B 细胞的前体，将分选的亚群在体外进行短暂培养，使其在体外完成后续轮次的分裂，并评估其 Bcl6 的表达。类似地分类和固定的 B 细胞，富集细胞周期 G2/M 期的细胞，将通过共聚焦显微镜直接离体成像，以获得连体子代中表面标记和转录因子分布的对称程度。公共卫生相关性：对病原体和疫苗的有效免疫反应关键取决于生发中心的形成，以形成高亲和力记忆 B 细胞和浆细胞。本申请提出的实验将回答有关生发中心 B 细胞发育启动的几个基本问题。