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

描述（由申请方提供）：对病原体和疫苗的有效免疫应答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两者的重要性，但对于在原代B细胞分裂期间使这些不同命运的初始分歧成为可能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阻遏物Bcl 6的升高的产生。另一方面，ACS的形成涉及Pax 5的减少，随后包括BLIMP-1在内的几种其他转录因子的增加。已知Bcl-6和BLIMP-1相互拮抗表达，从而沿着沿着相互排斥的分化途径约束和推动细胞。虽然 现在已经很好地确定了转录因子的表达模式的改变介导了向一个谱系的转变，而远离了幼稚B细胞中以前稳定的转录程序，但Pax 5主导的B细胞程序如何在一些细胞中被中断而Bcl 6在其他细胞中增加仍然是一个谜。很少有人知道的过程中发生的多个分裂之前，这些更先进的分化阶段，尽管他们的重要性，这些途径的分歧和GC的建立。在这里，我们建议调查分化的进展之前，GC或ASC转录程序的已知指标的外观。使用一个有效的策略，分类细胞的细胞分裂的程度和表达的基因调节Bcl 6和Pax 5，我们将进一步表征细胞亚群在其最初出现，并定义直接的前体细胞谱系定型B细胞。目的1：明确B细胞在体内谱系定型前的进行性分化。免疫后不久，将评估抗原特异性B细胞是否出现已知与初始AFC和GC谱系形成相关的转录因子和表型变化。将通过流式细胞术和/或qRT-PCR定量相关分子的表达水平，并确定其与细胞分裂数和CD 38、CD 23表型的相关性。通过细胞分裂数和表型分类的B细胞亚群的RNA测序将鉴定转录变化，包括microRNA转录，其紧接在谱系发育中最早的已知分子转变之前。目的2：通过子细胞分析确定谱系定型亚群的遗传。为了确定生发中心B细胞的前体，将在体外短暂培养分选的亚群，使其在体外完成随后的一轮或多轮分裂，并评估它们的Bcl 6表达。类似地分选和固定的B细胞，富集细胞周期的G2/M期的细胞，将通过共聚焦显微镜直接离体成像，以获得连体子体中表面标志物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应答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细胞发育起始的几个基本问题。

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.