Transitional B cell selection during peripheral B lymphopenia and reconstitution

描述（由申请人提供）：BLyS在整合B细胞稳态和特异性耐受机制方面起着关键作用。操纵BLyS或其受体的影响，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细胞在过渡选择中存活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，负选择的严格性就会放松，使自身反应性克隆型成熟并进入滤泡区和边缘区池。这些观察结果表明，在停止消融外周池的治疗后，改变的内稳态需求将损害正常的选择和内稳态平衡。因此，我们假设在骨髓输出减少或外周B淋巴减少后的淋巴重建过程中，选择性阈值将被放宽。我们进一步假设，这将受到可用的BLyS和BLyS受体水平的控制，并且因此可以通过控制可用的BLyS来调节自身反应特异性的出现。本文的研究将解决这些问题。在目的1中，我们将确定移行性B细胞通量如何受到骨髓输出减少和外周B淋巴减少的影响。这些实验将采用BrdU标记结合淋巴自身重建、药物诱导的外周B淋巴减少和混合骨髓嵌合体研究来确定这些条件下细胞的通量和命运。在目的2中，我们将确定在周围淋巴细胞减少或脑脊髓炎输出减少时，过渡性保留液选择的严格性是否放松。我们将通过监测过渡和成熟亚群中曲目组成、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点消除的自身反应性特异性是否被允许进入成熟的隔室。这些实验将使用流式细胞术，限制稀释和单细胞特异性分析在正常和转基因小鼠。在目标3中，我们将确定是否可以通过调整BLyS水平或外围池补充时的响应性来恢复正常的选择。我们将评估在正常和自身免疫模型中，通过可溶性TACI Ig或中和抗BLyS抗体在体内治疗，减少的可用BLyS水平是否会恢复正常的选择严格性。

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.