Transitional B cell selection during peripheral B lymphopenia and reconstitution

描述（由申请人提供）：BLyS在整合B细胞稳态与基于特异性的耐受机制中起关键作用。这通过操纵BLyS或其受体的作用以及BLyS/BLyS受体家族与体液自身免疫之间的联系来证明。最近的数据和我们的初步研究结果表明，过渡性选择中存活的新形成的B细胞的比例可以根据稳态需求而变化。因此，当对新形成的淋巴细胞的需求很高时，阴性选择的严格性被放松，允许自身反应性克隆型成熟并进入滤泡和边缘区池。这些观察结果表明，一旦停止消融外周血池的治疗，改变的稳态需求将损害选择和稳态的正常平衡。因此，我们假设在骨髓输出减少或外周B淋巴细胞减少后的淋巴重建过程中，选择性阈值将放松。我们进一步假设，这将是由可用的BLyS和BLyS受体水平，并出现自身反应特异性，因此可以通过控制可用的BLyS调制。本文的研究将探讨这些想法。在目标1中，我们将确定骨髓输出减少和外周B淋巴细胞减少如何影响过渡性B细胞通量。这些实验将采用BrdU标记结合淋巴自动重建、药物诱导的外周B淋巴细胞减少症和混合骨髓嵌合体研究，以确定这些条件下细胞的通量和命运。在目标2中，我们将确定在外周淋巴细胞减少症或BM输出减少期间，过渡库选择的严格性是否放松。我们将通过监测过渡和成熟亚群的库组成、多样性和特异性的变化来评估选择严格性。此外，我们将直接测试是否自身反应性特异性通常消除在过渡检查点提供进入成熟区室。这些实验将在正常和转基因小鼠中使用流式细胞术、有限稀释和单细胞特异性分析。在目标3中，我们将确定是否可以通过调节BLyS水平或外周池补充期间的反应性来恢复正常选择。我们将评估在正常和自身免疫模型中，通过使用可溶性泰爱IG或中和抗BLyS抗体进行体内治疗，降低的BLyS水平是否会恢复正常的选择性严格性。

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.