Gene Expression Profiles of MYC+ and MYC- mouse Germinal Center B cells. Mus musculus

Germinal centers (GC) arise within B cell follicles upon antigenic challenge. In the dark zones (DZ) of GCs, B cells proliferate and hypermutate their immunoglobulin genes, and mutants with increased affinity are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells, or re-enter the DZ for further refinement. However, the molecular circuits governing GC positive selection are not known. Here, we show that the GC reaction requires the biphasic regulation of c-MYC expression, involving its transient induction during early GC commitment, its repression by BCL6 in DZ B cells, and its re-induction in a subpopulation of positively selected LZ B cells destined to DZ re-entry. Accordingly, acute disruption of MYC function in vivo leads to GC collapse, indicating an essential role in GC physiology. These results have implications for our understanding of GC selection and the role of MYC deregulation in B cell lymphomas. We used microarrays to determine the global gene expression programs that distinguish MYC+ GC B cells from their MYC- negative counterparts. Overall design: GFPMYC+ and GFPMYC- GC B cell subpopulations were collected by Fluorescence Activated Cell Sorting (FACS) from B cell enriched fractions of splenic mononuclear cell pools of GFPMYC knock-in mice (12 days after SRBC immunization). 5-20ng of total RNA (RIN>9) for each sample was used as a template for linear cDNA amplification (Ovation RNA amplification Kit, NuGen). cDNA was labeled using the Encore Biotin Labeling Kit (NuGen) and hybridized to Affymetrix Mouse 430.2 gene expression arrays.

生发中心（Germinal Centers, GC）起源于抗原激发后的B细胞滤泡。在GC的暗区（Dark Zone, DZ）中，B细胞发生增殖并对其免疫球蛋白基因进行体细胞超突变；而亲和力提升的突变体则在亮区（Light Zone, LZ）中被正向选择，进而分化为浆细胞与记忆细胞，或重新进入暗区完成进一步的亲和力成熟。然而，调控GC正向选择的分子环路尚未明确。本研究证实，GC反应需要c-MYC表达的双相调控：包括其在GC早期定向分化阶段的瞬时诱导、在暗区B细胞中被BCL6抑制，以及在注定重新进入暗区的正向选择亮区B细胞亚群中实现重新诱导。据此，体内MYC功能的急性缺失会导致GC崩溃，表明其在GC生理过程中发挥关键作用。本研究结果有助于理解GC选择过程，以及MYC失调在B细胞淋巴瘤中的作用。我们通过基因表达微阵列鉴定了区分MYC阳性与MYC阴性GC B细胞的全局基因表达程序。

总体设计：从经绵羊红细胞（SRBC）免疫12天后的GFPMYC敲入小鼠脾脏单核细胞混合悬液的B细胞富集组分中，通过荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）分离得到GFPMYC阳性与GFPMYC阴性的GC B细胞亚群。每份样本使用5-20ng总RNA（RNA完整性指数RIN>9）作为模板，进行线性cDNA扩增（使用Ovation RNA扩增试剂盒，NuGen公司）。随后使用Encore生物素标记试剂盒（NuGen公司）对cDNA进行标记，并杂交至Affymetrix小鼠430.2基因表达芯片。