TRANSGENIC ANALYSIS OF BCL-X IN B CELL IMMUNITY

淋巴细胞的程序性细胞死亡或凋亡是调节免疫系统内环境平衡的关键机制。例如，当B细胞在发育过程中移动时，无法有效重排重链Ig等位基因的细胞以及具有强烈自我反应的Ig受体的细胞会被凋亡删除。在生发中心(GC)反应中，未能积累体细胞突变以提高Ig受体对抗原亲和力的B细胞会因细胞凋亡而丢失。在这笔赠款的支持下，我们实验室的工作重点是了解抗凋亡基因bclx在B细胞发育和激活过程中调节生存的作用。我们的研究表明，Bclxl在三个不同的检查点上具有独特的作用：重链等位基因首次重排的前B细胞阶段，轻链重排和受体编辑期间的前B/未成熟B阶段，以及GC B细胞免疫应答期间。在下一个资金阶段，我们建议测试关于Bclxl在Pre-B/未成熟B阶段和GC反应期间的功能的两个主要假说：1)在Pre-B晚期/早期未成熟B细胞阶段，Bclxl提供了一个编辑窗口，允许自我反应细胞有机会将自反应轻链受体交换为一个自反应性较低的轻链受体；2)在GC反应中，在抗原阳性选择的细胞中，Bclxl表达上调，这是亲和力成熟所必需的。为了验证这些假说，我们将首先探讨在体内和体外的功能获得转基因模型中，在骨髓轻链受体编辑过程中，Bcl-xl在调节自身反应性B细胞存活方面的作用。我们还将建立抗HEL轻链敲入小鼠，在更多的生理中枢受体编辑的背景下分析Bclxl的功能。最后，我们建议使用携带BCL-x基因的小鼠和表达B细胞特异性Cre重组酶的小鼠来研究BCL-x功能丧失对B细胞的影响。这些动物将使我们能够测试bclx缺陷的B细胞是否有能力重新排列骨髓中的轻链和受体编辑，并在GC反应中经历亲和力成熟。包括系统性红斑狼疮在内的许多自身免疫性疾病的特征是对自身抗原的耐受性丧失。系统性红斑狼疮的自身免疫B细胞反应似乎是由生发中心的体细胞突变驱动的，这种疾病的患者表现出轻链基因上过度的受体编辑。更好地理解在前B期/未成熟B期和GC中调节生存的机制可能会为启动和维持自身免疫提供重要的新见解。

Programmed cell death, or apoptosis, of lymphocytes is a key mechanism in the homeostatic regulation of immunity. For example, as B cells move through development, apoptosis deletes from the repertoire cells unable to productively rearrange a heavy chain Ig allele, as well as those with strongly self- reactive Ig receptors. During germinal center (GC) responses, B cells that fail to accumulate somatic mutations that improve the affinity of the Ig receptor for antigen are lost by apoptosis. The work in our laboratory supported by this grant has focused on understanding the role of the anti-apoptotic gene bcl-x in regulating survival during B cell development and activation. Our studies suggest that Bcl-XL has a unique role at 3 distinct checkpoints: the pro-B cell stage when heavy chain alleles first begin to rearrange, the pre-B/immature B stage during the period of light chain rearrangement and receptor editing, and during GC B cell immune responses. In the next funding period we propose to test two major hypotheses regarding the function of Bcl-XL at the pre-B/immature B stage and during GC responses: 1) At the late pre-B/early immature B cell stage, Bcl-XL provides an editing "window" which allows self-reactive cells the opportunity to exchange a self-reactive light chain receptor for one with less auto-reactivity; 2) During GC responses, Bcl-XL is upregulated in cells positively selected by antigen, and is required for affinity maturation. To test these hypotheses we will first explore the role of Bcl-XL in regulating survival of self-reactive B cells during bone marrow light chain receptor editing in gain-of-function transgenic models both in vivo and in vitro. We will also generate an anti-HEL light chain knock-in mouse in which to analyze the function of Bcl-XL in the setting of more physiologic central receptor editing. Finally, we propose to examine the influence of a loss-of-function of bcl-x in B cells using mice carrying floxed bcl-x genes and mice expressing B cell-specific Cre recombinase. These animals will allow us to test whether B cells deficient in bcl-x have the ability to rearrange light chains and receptor edit in the bone marrow, and undergo affinity maturation in GC reactions. Many autoimmune diseases, including systemic lupus erythematosus, are characterized by the loss of tolerance to self antigens. The autoimmune B cell response in SLE appears to be driven by somatic mutation in germinal centers, and patients with this disease show evidence of excessive receptor editing at light chain loci. A better understanding of the mechanisms that regulate survival at the pre-B/immature B stage and in GCs may provide important new insights into the initiation and maintenance of autoimmunity.