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

• 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
• 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; 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

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严重取决于短期抗体分泌细胞（ASC）和生发中心的形成。尽管同时产生ASC和GC的重要性，但对于在初级B细胞分裂期间能够与这些不同命运的初始差异的分子变化知之甚少。生发中心B细胞的初始形成需要提高转录阻遏物Bcl6的产生。另一方面，AC的形成涉及PAX5的减少，然后增加其他几种转录因子，包括Blimp-1。已知BCl-6和Blimp-1相互拮抗，从而沿着互斥的分化途径限制和推动细胞。虽然是 现在已经确定，改变转录因子的表达模式介导向一个谱系的转移，并远离以前在幼稚B细胞中稳定的转录程序的转移，这仍然是一个谜，它仍然是一个谜，在某些细胞中，PAX5主导的B细胞程序如何中断，而BCL6在其他细胞中增加了。尽管对这些途径和GC建立的差异很重要，但对这些更先进的差异阶段之前的多个分区中发生的过程几乎没有什么了解。在这里，我们建议研究在GC或ASC转录程序的已知指标出现之前的分化进程。使用经过验证的策略，该策略按细胞分裂的程度和受BCl6和Pax5调控的基因的表达进行分类，我们将在其最初出现期间进一步表征细胞子集的特征，并定义谱系所承诺的B细胞的直接前体。 AIM 1：在体内谱系承诺之前定义B细胞的进行性分化。免疫后不久将评估抗原特异性B细胞的转录因子的出现和已知与初始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仪和/或QRT-PCR来量化，并将CD38与CD38相关，CD23表型将被确定。通过细胞分裂数和表型排序的B细胞子集的RNA测序将确定转录变化，包括microRNA转录，该变化紧接在谱系发育中最早已知的分子移位之前。 AIM 2：通过子细胞分析确定谱系承诺子集的遗产。为了定义生发中心B细胞的前体，将在体外简要培养排序的子集，可以在体外完成随后的分裂的后期，并评估其BCl6的表达。类似地分类和固定的B细胞，在细胞周期的G2/M期富含细胞，将通过共聚焦显微镜直接离体成像，以在表面标记的分布和结合女儿中的转录因子的分布中进行对称程度。 公共卫生相关性：对病原体和疫苗的有效免疫反应严重取决于形成生发中心以形成高亲和力记忆B细胞和浆细胞。该应用提出了实验，该实验将回答有关生发中心B细胞开发的启动的几个基本问​​题。