ROLE OF BLYS IN PERIPHERAL B CELL SELECTION AND SURVIVAL

描述(申请人提供)：肿瘤坏死因子家族成员，BLyS，在外周B细胞的动态平衡和选择中起关键作用。扩增、存活率和分化率在这些效应中扮演的相对角色，以及BLyS如何影响每个外周成熟亚群中的细胞，目前尚不清楚。通过对正常和突变小鼠的研究，我们已经证明BLyS至少通过两种方式控制外周B细胞数量：通过改变成功完成过渡发育的细胞的比例，以及通过在成熟B细胞中充当长寿的主要决定因素。拟议的研究将进一步探讨BLyS影响外周B细胞分化、选择和寿命的机制。在第一个目标中，我们将评估BLyS介导的效应是否仅反映了生存率的提高，或者它们是否也涉及对分化的直接影响。我们将通过对A/WySnJ缺陷纯合子的Bcl-xL转基因小鼠的细胞荧光和体内标记研究，以及对由A/WySnJ或正常干细胞重组的宿主进行分析来解决这个问题。我们最近发现，BLyS受体的表达随着成熟度的变化而变化，并可由BCR信号调节，从而将BLyS介导的生存与BCR驱动的选择联系起来。在第二个目标中，我们将在体外用抗Ig或抗原处理后，检测所有未成熟和成熟B细胞亚群的BLyS受体表达和BLyS反应性。此外，我们将通过动力学和剂量反应研究，以及评估其他Ig亚型或共刺激分子与Bcmd/BR3表达的潜在偶联的研究，进一步表征BCR介导的BLyS受体的表达。在第三个目标中，我们将研究BCR和Bcmd/BR3的偶联与成熟B细胞活性和成功的过渡细胞成熟所必需的BCR表达之间的关系。我们将确定异位表达的Bcmd/BR3是否在BCR表达被有条件地消融的小鼠中提供B细胞存活，以及BCR消融是否产生Bcmd/BR3的下调。我们还将确定结构性Bcmd/BR3表达是否阻碍过渡性选择；B细胞成熟所需的BCR刺激程度是否可以通过改变可用的BLyS水平来改变。在第四个目标中，我们将通过限制稀释和对A/J与A/WySnJ小鼠的HA特异性反应的精细特异性分析，研究Bcmd/BR3如何影响对流感血凝素(HA)反应的新出现的谱系中的克隆型多样性和组成。

DESCRIPTION (provided by applicant): The tumor necrosis factor family member, BLyS, plays a key role in the homeostasis and selection of peripheral B cells. The relative roles played by expansion, survival, and differentiation rates in these effects, as well as how BLyS influences cells within each peripheral maturation subset, remains unknown. Through studies of normal and mutant mice, we have shown that BLyS controls peripheral B cell numbers in at least two ways: by varying the proportion of cells that successfully complete transitional development, and by serving as the primary determinant of longevity among mature B cells. The proposed studies will further examine the mechanisms through which BLyS influences peripheral B cell differentiation, selection, and lifespan. In the first aim, we will assess whether BLyS mediated effects solely reflect enhanced survival, or if they also involve direct influences on differentiation. We will address this question through cytofluorimetric and in vivo labeling studies of Bcl-xl transgenic mice homozygous for the A/WySnJ defect, as well a analysis of hosts reconstituted with either A/WySnJ or normal stem cells transfected with viral constructs containing Bcl-xl. We have recently discovered that BLyS receptor expression shifts with maturation and can be regulated by BcR signaling, thus linking BLyS-mediated survival with BcR-driven selection. In the second aim, we will examine BLyS receptor expression and BLyS-responsiveness in all immature and mature B cell subsets following treatment in vitro with anti-Ig or antigen. In addition, we will further characterize BcR-mediated BLyS receptor expression with kinetic and dose response studies, as well as studies where the potential coupling of other Ig isotypes or costimulatory molecules with Bcmd/BR3 expression is assessed. In the third aim, We will examine how the coupling of BcR and Bcmd/BR3 is related to the requisite of BcR expression for mature B cell viability, and for successful transitional cell maturation. We will determine whether ectopically expressed Bcmd/BR3 affords B cell survival in mice where BcR expression is conditionally ablated, and whether BcR ablation yields down-regulation of Bcmd/Br3. We will also determine whether constitutive Bcmd/BR3 expression thwarts transitional selection; whether the degree of BcR stimulation required for B cell maturation can be altered by varying available BLyS levels. In the fourth aim, we will examine how Bcmd/BR3 influences clonotype diversity and composition in the emerging repertoire responsive to influenza hemagglutinin (HA), through limiting dilution and fine specificity analyses of HA specific responses in A/J vs. A/WySnJ mice.