Transitional B cell selection during peripheral B lymphopenia and reconstitution

描述(由申请人提供)：BLyS在整合B细胞稳态和基于特异性的耐受机制方面发挥着关键作用。操纵BLyS或其受体的效果以及BLyS/BLyS受体家族与体液自身免疫之间的联系证明了这一点。最近的数据和我们的初步结果表明，新形成的B细胞在过渡期选择中存活的比例可以根据体内平衡需求的不同而变化。因此，当对新形成的淋巴细胞的需求很高时，负面选择的严格程度就会放松，允许自身反应性克隆类型成熟并进入滤泡和边缘区池。这些观察表明，一旦停止消融周围池的治疗，改变的动态平衡需求将损害选择和动态平衡的正常平衡。因此，我们假设，在骨髓输出量减少或外周B淋巴细胞减少后的淋巴重建过程中，选择性阈值将会放松。我们进一步假设，这将由可用的BLyS和BLyS受体水平控制，因此，自身反应特异性的出现可以通过控制可用的BLyS来调节。这里的研究将解决这些想法。在目标1中，我们将确定骨髓输出量减少和外周B淋巴细胞减少如何影响过渡性B细胞的吞吐能力。这些实验将使用BrdU标记与淋巴自我重建、药物诱导的外周B淋巴细胞减少和混合骨髓嵌合体研究相结合，以确定在这些条件下细胞的吞吐量和命运。在目标2中，我们将确定在外周血淋巴细胞减少或骨髓输出量减少期间，过渡谱系选择的严格程度是否放松。我们将通过监测过渡子集和成熟子集的曲目组成、多样性和特异性的变化来评估选择的严格性。此外，我们将直接测试通常在过渡检查站消除的自动反应特性是否允许进入成熟的隔间。这些实验将在正常和转基因小鼠中使用流式细胞术、限制稀释和单细胞特异性分析。在目标3中，我们将确定在外周血池补充期间，是否可以通过调整BLyS水平或反应性来恢复正常选择。我们将评估降低可用BLyS水平是否会通过在正常和自身免疫模型中用可溶性TACI Ig或中和抗BLyS抗体在体内治疗来恢复正常的选择性严谨性。

DESCRIPTION (provided by applicant): BLyS plays a critical role in integrating B cell homeostasis with specificity-based tolerance mechanisms. This is evidenced by the effects of manipulating BLyS or its receptors, as well as the links between the BLyS/BLyS receptor family and humoral autoimmunity. Recent data and our preliminary results show that the proportion of newly formed B cells surviving transitional selection can be varied based on homeostatic demands. Thus, the stringency of negative selection is relaxed when the demand for newly formed lymphocytes is high, allowing autoreactive clonotypes to mature and enter the follicular and marginal zone pools. These observations suggest that upon cessation of treatments that ablate peripheral pools, altered homeostatic demands will compromise the normal balance of selection and homeostasis. Accordingly, we hypothesize that during lymphoid reconstitution following curtailed marrow output or peripheral B lymphopenia, selective thresholds will be relaxed. We further hypothesize that this will be governed by available BLyS and BLyS receptor levels, and that the emergence of autoreactive specificities can thus be modulated by controlling available BLyS. The studies herein will address these ideas. In aim 1, we will determine how transitional B cell throughput is influenced by reduced marrow output and peripheral B lymphopenia. These experiments will employ BrdU labeling coupled with lymphoid autoreconstitution, drug-induced peripheral B lymphopenia, and mixed marrow chimera studies to determine the throughput and fate of cells under these conditions. In aim 2 we will determine whether the stringency of transitional repertoire selection is relaxed during peripheral lymphopenia or reduced BM output. We will assess selection stringency by monitoring changes in repertoire composition, diversity and specificity across transitional and mature subsets. In addition, we will directly test whether autoreactive specificities normally eliminated at the transitional checkpoint are afforded entry to mature compartments. These experiments will use flow cytometry, limiting dilution and single cell specificity analyses in normal and transgenic mice. In aim 3, we will establish whether normal selection can be restored by adjusting BLyS levels or responsiveness during replenishment of peripheral pools. We will assess whether reduced available BLyS levels will restore normal selective stringency by in vivo treatment with soluble TACI Ig or neutralizing anti-BLyS antibody in normal and autoimmune models.