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

描述(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。尽管同时产生ASC和GC很重要，但人们对在初级B细胞分裂过程中导致这些不同命运的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增加转录抑制物Bcl6的产生。另一方面，急性冠脉综合征的形成涉及Pax5的减少和包括blimp-1在内的其他几个转录因子的增加。已知bcl6和blimp-1可以相互拮抗彼此的表达，从而抑制和推动细胞沿着相互排斥的分化途径前进。尽管它 目前已经证实，转录因子表达模式的改变介导了向一个谱系的转变，而不是以前在幼稚B细胞中稳定的转录程序，但如何在一些细胞中Pax5主导的B细胞程序被中断，而在另一些细胞中Bcl6增加仍然是一个谜。在这些更高级的分化阶段之前的多个分裂过程中，人们知之甚少，尽管它们对这些途径的分化和GC的建立很重要。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。使用一种有效的策略，根据细胞分裂的程度和受Bcl6和Pax5调控的基因的表达对细胞进行分类，我们将进一步描述细胞亚群在最初出现时的特征，并定义致力于B细胞谱系的直接前体。目的1：在体内确定B细胞的渐进性分化，然后进行谱系承诺。抗原特异性B细胞将在免疫后不久被评估转录因子的出现和已知与最初的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进行定量，并与细胞分裂数相关，并确定CD38、CD23的表型。按细胞分裂数和表型排序的B细胞亚群的RNA测序将识别转录变化，包括microRNA转录，这些变化直接先于谱系发育中已知的最早的分子变化。目的2：通过子代细胞分析确定血统亚群的遗传。为了确定生发中心B细胞的前体细胞，分离的亚群将在体外短暂培养，允许完成下一轮的体外分裂(S)，并评估它们对bcl6的表达。同样分选和固定的B细胞，富含处于细胞周期G2/M期的细胞，将直接在体外通过共聚焦显微镜成像，以了解连接子体中表面标记和转录因子分布的对称性程度。

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.