Comparison of B Cell Differentiation in Mice and Humans

该建议集中在小鼠早期B谱系分化的未解决的问题上，特别是在小鼠中， 人类(Aim 1）已经鉴定了人B细胞发育的新模型，即EU 12细胞系， CD34+/|a重链”原B细胞自发地经历逐步分化， IgM+/IgD+ B细胞。该克隆将用于（i）检验克隆内V（D）J重组 多样化有助于有效产生原代B细胞库，（ii）定义 在B谱系分化期间的基因表达谱，（iii）测试修饰新鉴定的 和以前识别的前B细胞中的B谱系基因通过有义、反义和显性阴性 转基因，和（iv）检测连接细胞表面受体（preBCR，BCR，IL 7 R，CD 19，CD 32， 和CD 40）对这些B谱系细胞的生长和分化的影响。(Aim（2）一个新的、高度发展的、 将使用扩增免疫荧光法鉴定前B、前B和B细胞受体组分 在原代B细胞系细胞上，以确定人类和小鼠B细胞分化的相似性和差异 程序.在重新定义了重链、替代轻链、伊卡、轻链， 在前B细胞、前B细胞和B细胞这两个物种中，在B谱系分化过程中表达Igo/p 将分离亚群用于比较分析它们的基因表达谱和分化 潜力后面的实验将结合在分化分析中获得的新信息- EU 12细胞基因表达谱的相关变化。(Aim 3）补充的离体模型将是 用于阐述小鼠和人的早期B谱系分化事件。产生IL-7的气孔细胞 模型将用于描绘小鼠B谱系分化中的早期事件。小鼠基质细胞， 有和没有人基质细胞衍生因子1（SDF-1）、IL-7和Fc γ受体修饰 转基因，将用于评估人类淋巴祖细胞经历B谱系的能力 分化这种比较分析将确定B细胞分化的新特征，这些特征可能 具有重要的临床意义。

This proposal focuses on unresolved issues of early B lineage differentiation in mice and, especially, in humans. (Aim 1) A novel model of human B cell development has been identified, the EU12 cell line, in which CD34+/|a heavy chain" pro-B cells spontaneously undergo step-wise differentiation to become IgM+/IgD+ B cells. This clone will be used to (i) test the hypothesis that intraclonal V(D)Jrecombinatorial diversification contributes to the efficient generation of a primary B cell repertoire, (ii) define the changes in gene expression profile during B lineage differentiation, (iii) test the effects of modifying newly-identified and previously-recognized B lineage genes in pro-B cells by sense, antisense and dominant-negative transgenes, and (iv) examine the effects of ligating cell surface receptors (preBCR, BCR, IL7R, CD19, CD32, and CD40) on growth and differentiation of these B lineage cells. (Aim 2) A recently-developed, highly- amplified immunofluorescence method will be used to identify pro-B, pre-B and B cell receptor components on primary B lineage cells to define similarities and differences in the human and mouse B cell differentiation programs. After redefinition of when and where the \i heavy chains, surrogate light chains, icA,light chains, and Igo/p are expressed during B lineage differentiation in both species, pro-B, pre-B, and B cell subpopulations will be isolated for comparative analysis of their gene expression profiles and differentiation potential. The latter experiments will incorporate new information obtained in the analysis of differentiation- related changes in the gene expression profile of EU12 cells. (Aim 3) Complementary ex vivo models will be used to elaborate early B lineage differentiation events in mice and humans. An IL-7 producing stomal cell model will be employed to delineate early events in mouse B lineage differentiation. Mouse stromal cells, with and without modification by human stromal cell-derived factor 1 (SDF-1), IL-7 and Fey receptor transgenes, will be employed to evaluate the capacity of human lymphoid progenitors to undergo B lineage differentiation. This comparative analysis will define novel features of B cell differentiationthat are likely to have significant clinical implications.