B-Cell Tolerance Mechanisms in Human SLE

描述（由申请人提供）：本提案的中心目标是阐明B细胞耗损疗法（BCDT）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。假设SLE的耐受性丧失是由于B细胞存活信号（例如TNF家族的BAFF-B细胞激活剂）相对于骨髓（BM）中出现的移行性B细胞数量的平衡改变（BAFF高/ BM中出现的B细胞数量低），从而降低了负选择的严格性。进一步假设，一个关键的失调信号是干扰素（IFN），在骨髓中局部产生抑制骨髓B细胞淋巴生成。另一方面，我们发现一部分接受BCDT治疗的SLE患者恢复了B细胞耐受性，并且具有独特的B细胞重建模式，其特征是B细胞淋巴生成旺盛，循环移行细胞显著扩增。因此，B细胞过渡室内稳态机制的恢复可能对这组受试者的耐受性恢复至关重要，此外，过渡B细胞可能发挥生理调节作用，这种作用在SLE中失调，在BCDT后恢复。该模型将通过以下具体目标进行测试：1。明确SLE患者移行性B细胞室内平衡的调节因素，重点关注骨髓性B细胞淋巴生成；2. 确定B细胞耗竭治疗和BAFF或IFN靶向生物拮抗对移行性B细胞稳态和耐受性的影响；和3。阐明移行性B细胞的免疫调节作用以及移行性B细胞扩增对BCDT后SLE疾病改善的贡献。具体来说，骨髓淋巴生成及其调控将通过骨髓B细胞亚群的多参数流式细胞术和细胞因子环境（IFN BAFF通过luminex、基因标记和基于流量的信号传导）的描述在正常对照、未治疗的SLE和BCDT、抗BAFF或抗IFN治疗后的SLE中进行检查。通过流式细胞术、膜联蛋白v结合、增殖抗原表达以及与IFN和BAFF结合相关的复制史，可以确定BM中出现的移行性B细胞的表型、存活、增殖和选择。利用单细胞PCR和ELISPOT技术，通过观察从过渡期到成熟期成熟过程中自身反应性B细胞频率的降低来评估耐受性。移行性B细胞表达抗炎细胞因子（IL10）和诱导T调节性细胞表型的能力将被确定。这些研究将阐明SLE的机制，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。

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.