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免疫稳态调节的关键机制。例如，当B细胞在发育过程中移动时，细胞凋亡从库中删除了不能有效重排重链Ig等位基因的细胞，以及那些具有强自反应性Ig受体的细胞。在生发中心（GC）应答过程中，未能积累体细胞突变以改善Ig受体对抗原的亲和力的B细胞因凋亡而丢失。在这项资助下，我们实验室的工作重点是了解抗凋亡基因bcl-x在B细胞发育和激活过程中调节存活的作用。我们的研究表明，Bcl-XL在3个不同的检查点具有独特的作用：重链等位基因首次开始重排的前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。在下一个资助期，我们将测试关于Bcl-XL在前B/未成熟B阶段和GC反应期间功能的两个主要假设：1)在前B/早期未成熟B细胞阶段晚期，Bcl-XL提供了一个编辑“窗口”，使自反应细胞有机会将自反应轻链受体交换为具有较低自反应性的受体；2)在GC应答过程中，Bcl-XL在抗原阳性选择的细胞中表达上调，是亲和力成熟所必需的。为了验证这些假设，我们将首先在体内和体外的功能获得转基因模型中探索Bcl-XL在骨髓轻链受体编辑过程中调节自身反应性B细胞存活的作用。我们还将构建抗hel轻链敲入小鼠，以分析Bcl-XL在更多生理性中枢受体编辑设置中的功能。最后，我们建议使用携带固定bcl-x基因的小鼠和表达B细胞特异性Cre重组酶的小鼠来检测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缺乏bcl-x的B细胞是否具有在骨髓中重排轻链和受体编辑的能力，并在GC反应中进行亲和成熟。许多自身免疫性疾病，包括系统性红斑狼疮，以丧失对自身抗原的耐受性为特征。SLE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，并且该疾病患者在轻链位点显示出过度的受体编辑的证据。更好地了解B前/未成熟B阶段和GCs的生存调节机制可能为自身免疫的启动和维持提供重要的新见解。

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.