B-Cell Tolerance Mechanisms in Human SLE

描述（由申请方提供）：本提案的中心目标是阐明B细胞耗竭疗法（BCDT）和其他靶向B细胞疗法有效并恢复系统性红斑狼疮（SLE）耐受性的机制。据推测，SLE中耐受性丧失是由于相对于从骨髓（BM）中出现的过渡性B细胞数量（高BAFF/低数量的出现的BM B细胞），B细胞存活信号（例如TNF家族的BAFF-B细胞激活剂）的平衡改变，从而降低了阴性选择的严格性。进一步假设关键的失调信号是干扰素（IFN），其在BM中的局部产生抑制BM B细胞淋巴细胞生成。另一方面，我们发现用BCDT治疗的一部分SLE患者经历了B细胞耐受性的恢复，并具有独特的B细胞重建模式，其特征在于旺盛的B细胞淋巴细胞生成和循环移行细胞的显著扩增。因此，B细胞过渡区室内的稳态机制的恢复对于该组受试者的耐受性恢复可能是关键的，而且，过渡B细胞可能发挥在SLE中失调并在BCDT后恢复的生理调节作用。该模型将通过以下具体目标进行测试：1.明确SLE中过渡期B细胞区室的稳态调节因素，重点关注BM B细胞淋巴细胞生成; 2.确定B细胞耗竭疗法和BAFF或IFN的靶向生物拮抗作用对过渡性B细胞稳态和耐受性的后果;和3.阐明过渡性B细胞的免疫调节作用以及过渡性B细胞扩增对BCDT后SLE疾病改善的贡献。具体而言，将通过BM B细胞亚群的多参数流式细胞术和正常对照、未治疗的SLE和BCDT、抗BAFF或抗IFN治疗后的SLE中的细胞因子环境（通过luminex、基因签名和基于流动的信号传导的IFN BAFF）的描绘来检查BM淋巴细胞生成及其调节。将基于流式细胞术、膜联蛋白-V结合、增殖抗原表达和相对于IFN的复制历史以及BAFF的接合来确定从BM出现的过渡性B细胞的表型、存活、增殖和选择。使用单细胞PCR和ELISPOT技术，将耐受性评估为从过渡期到成熟期成熟期间自身反应性B细胞频率的降低。将确定过渡性B细胞表达抗炎细胞因子（IL 10）和诱导T调节细胞表型的能力。这些研究将阐明SLE的发病机制、B细胞在自身免疫中的作用以及靶向治疗改善疾病的途径。 狼疮是一种慢性自身免疫性疾病，其特征是针对自身的异常免疫反应。B细胞是狼疮中的关键免疫细胞，部分原因是它们在自身抗体的产生中起着核心作用，这是疾病过程的标志。这里提出的研究将帮助我们了解狼疮中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细胞耗竭和其他靶向生物疗法如何诱导改善。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.