B-Cell Tolerance Mechanisms in Human SLE

描述(申请人提供)：这项建议的中心目标是阐明B细胞去除疗法和其他靶向B细胞疗法在系统性红斑狼疮(SLE)中有效并恢复耐受性的机制(S)。假设SLE的耐受性丧失是由于B细胞生存信号(例如，肿瘤坏死因子家族的BAFF-B细胞激活剂)相对于从骨髓(BM)涌出的过渡性B细胞(高BAFF/低数目的BM B细胞)的平衡的改变，从而降低了阴性选择的严格性。进一步推测，一个关键的失调信号是干扰素(IFN)，它在骨髓中局部产生，抑制骨髓B淋巴细胞的生成。另一方面，我们发现接受BCDT治疗的SLE患者中的一部分经历了B细胞耐受的恢复，并具有独特的B细胞重建模式，其特征是活跃的B细胞淋巴系和循环中的过渡细胞显著扩张。因此，在这组受试者中，恢复B细胞移行间隔内的动态平衡机制可能是恢复耐受性的关键，此外，移行B细胞可能发挥着在SLE中调节失调的生理调节作用，并在BCDT后恢复。该模型将通过以下特定目标进行检验：1.确定调节SLE移行B细胞室内稳态的因素，重点是BM B细胞的淋巴生成；2.确定B细胞耗竭治疗和BAFF或干扰素的靶向生物拮抗对移行B细胞的稳态和耐受性的影响；3.阐明移行B细胞的免疫调节作用以及移行B细胞的增殖对BCDT后SLE疾病改善的贡献。具体地说，在正常对照组、未治疗的SLE和BCDT、抗BAFF或抗干扰素治疗后的SLE中，将通过多参数流式细胞术检测BM B细胞亚群和细胞因子环境(通过Luminex、基因签名和基于流的信号)来检测BM淋巴生成及其调节。根据流式细胞术、Annexin-V结合、增殖抗原表达以及与干扰素和BAFF结合的复制历史，将确定BM中出现的过渡性B细胞的表型、存活、增殖和选择。耐受性将通过使用单细胞聚合酶链式反应和ELISPOT技术在从成熟过渡到成熟阶段期间自身反应性B细胞频率的降低来评估。过渡性B细胞表达抗炎细胞因子(IL10)和诱导T调节细胞表型的能力将被定义。这些研究将阐明SLE的潜在机制，B细胞在自身免疫中的作用，以及靶向治疗可能改善疾病的方式。 公共卫生相关性狼疮是一种慢性自身免疫性疾病，其特征是对自身的异常免疫反应。B细胞是狼疮的关键免疫细胞，部分原因是它们在自身抗体的产生中发挥核心作用，自身抗体是疾病过程的标志。这里提出的研究将帮助我们理解狼疮在B细胞发育和自身反应性B细胞的审查中发生的失调，以及B细胞耗尽和其他有针对性的生物治疗如何导致改善。这些研究将有助于我们了解B细胞在自身免疫中的多种功能，并为狼疮和其他自身免疫性疾病的治疗提供更好的策略。

DESCRIPTION (provided by applicant): The central goal of this proposal is to elucidate the mechanism(s) by which B cell depletion therapy (BCDT) and other targeted B cell therapies are efficacious and restore tolerance in systemic lupus erythematosus (SLE). It is hypothesized that loss of tolerance in SLE is due to an alteration in the balance of B cell survival signals (e.g. BAFF-B cell activator of the TNF family) relative to the numbers of transitional B cells emerging from the bone marrow (BM) (high BAFF/low numbers of emerging BM B cells), decreasing the stringency of negative selection. It is further postulated that a key dysregulated signal is interferon (IFN), with local production in the BM inhibiting BM B cell lymphopoiesis. On the other hand, we have found that a subset of SLE patients treated with BCDT experience restoration of B cell tolerance and have a unique pattern of B cell reconstitution characterized by exuberant B cell lymphopoiesis and a prominent expansion of circulating transitional cells. Thus, restoration of homeostatic mechanisms within the B cell transitional compartment may be critical for tolerance restoration in this group of subjects and, moreover, transitional B cells may play a physiological regulatory role that is dysregulated in SLE and restored after BCDT. This model will be tested through the following specific aims: 1. Define the factors regulating homeostasis of the transitional B cell compartment in SLE with a focus on BM B cell lymphopoiesis; 2. Determine the consequences of B cell depletion therapy and targeted biologic antagonism of BAFF or IFN on transitional B cell homeostasis and tolerance; and 3. Elucidate the immunoregulatory roles of transitional B cells and the contribution of a transitional B cell expansion to disease improvement in SLE after BCDT. Specifically, BM lymphopoiesis and its regulation will be examined by multi-parameter flow cytometry of BM B cell subsets and delineation of the cytokine milieu (IFN BAFF via luminex, gene signature, and flow based signaling) in normal controls, untreated SLE, and SLE after BCDT, anti-BAFF, or anti-IFN therapy. The phenotype, survival, proliferation, and selection of transitional B cells emerging from the BM will be ascertained based on flow cytometry, annexin-V binding, proliferation antigen expression, and replication history relative to IFN and engagement by BAFF. Tolerance will be assessed as a decrease in the frequency of autoreactive B cells during maturation from the transitional to the mature stage using single cell PCR and ELISPOT technology. The ability of transitional B cells to express anti-inflammatory cytokines (IL10) and induce a T regulatory cell phenotype will be defined. These studies will illuminate the mechanisms that underlie SLE, the role of B cells in autoimmunity, and the ways in which targeted therapy may improve disease. PUBLIC HEALTH RELEVANCE Lupus is a chronic autoimmune disease characterized by an abnormal immune response against self. B cells are a key immune cell in lupus in part because they play a central role in the production of auto-antibodies, a hallmark of the disease process. The research proposed here will help us understand the dysregulation that occurs in lupus in B cell development and censoring of autoreactive B cells and how B cell depletion and other targeted biologic therapies induce improvement. The knowledge gained from the present studies will help us understand the multiple functions of B cells in autoimmunity and develop better strategies for the treatment of lupus and other autoimmune diseases.