Nuclear Receptors in B Cell Tolerance and Humoral Immune Responses

项目摘要/摘要 这项提议的长期目标是利用孤儿核激素Nr4a家族的生物学优势 选择性地操控抗原特异性B细胞反应的受体。如果成功，这可能会改变我们的 免疫介导性疾病的疫苗接种和治疗方法。 Nr4a家族成员(Nur77、Nurr1和Nor1)由三个基因(分别为Nr4a1-3)编码，分别是 在淋巴细胞中被抗原(Ag)刺激而快速诱导。尽管它们被认为具有配基的功能- 独立的、具有结构性活性的转录因子、小分子激动剂和拮抗剂配体已经被 开发出来的，使它们可以下药。我们已经证明了Nr4a1表达的荧光报告(Nur77- EGFP、BAC、Tg)与Ag刺激的强度成比例，也与自然条件下的自我反应性相关 在健康小鼠和三种不同的bcr-tg模型中出现B细胞。我们最近已经证明了Nur77 抑制B细胞对慢性和急性Ag刺激的生存和扩张，施加一种新的 B细胞耐受层。重要的是，Nr4a家族的DNA在结构上有相当大的同源性- 因此，它们在T细胞中表现出深刻的功能冗余，在那里它们也扮演着 发挥重要的监管作用。这种冗余可能与它们在B细胞中的功能高度相关( 主要表达Nr4a1和Nr4a3)，但从未在实验中得到解决。这表示一个 我们知识上的重大差距限制了对这些因素的充分治疗利用。 在这里，我们报告了新产生的具有B细胞特异性缺失的Nr4a1和Nr4a3的小鼠显示出主要的断裂 在B细胞耐受中，包括自发生发中心和自身抗体的产生。这些 缺陷与对Ag刺激的反应中新的靶基因的深刻超诱导有关-包括 招募T细胞所需的几个基因帮助-即使一个功能正常的Nr4a也在很大程度上被掩盖了 等位基因仍然存在。因此，我们假设Nr4a家族在Ag下游介导负反馈 通过转录抑制关键靶基因的刺激。我们建议这样做有两个作用：(A) 在没有信号2(共刺激)的情况下抑制接收信号1(抗原)的自我反应克隆，以及(B) 抑制免疫优势克隆独占正常体液免疫反应，以保存 克隆多样性。在这项拨款中，我们建议： (1)明确Nr4a家族在调节中枢和外周B细胞耐受中的作用，并确定 在BCR、TG和自然产生的自身反应性B细胞中调节这些功能的转录机制。 (2)明确Nr4a家族在调节B细胞克隆间竞争和克隆多样性中的作用。 主要依赖T的体液免疫反应及其对限制免疫优势的贡献。 (3)明确新发现的Nr4a靶基因BATF在这些过程中的作用。

Project Summary/Abstract This proposal's long-term goal is to take advantage of the biology of the Nr4a family of orphan nuclear hormone receptors to selectively manipulate antigen-specific B cell responses. If successful, this could transform our approach to vaccination and treatment of immune-mediated diseases. Nr4a family members (Nur77, Nurr1, and Nor1) are encoded by three genes (Nr4a1-3, respectively) that are rapidly induced by antigen (Ag) stimulation in lymphocytes. Although they are thought to function as ligand- independent, constitutively active transcription factors, small molecule agonist and antagonist ligands have been developed, rendering them druggable. We have shown that a fluorescent reporter of Nr4a1 expression (Nur77- eGFP BAC Tg) scales with the intensity of Ag stimulation, and also correlates with self-reactivity in naturally occurring B cells in healthy mice and in three different BCR Tg models. We have recently shown that Nur77 curbs the survival and expansion of B cells in response to chronic and acute Ag stimulation, imposing a novel layer of B cell tolerance. Importantly, the Nr4a family harbor considerable structural homology in their DNA- binding domain and, as a result, exhibit profound functional redundancy in T cells, where they also play an important regulatory role. Such redundancy is likely highly relevant to their function in B cells (which predominantly express Nr4a1 and Nr4a3) yet has never been experimentally addressed. This represents a major gap in our knowledge that limits full therapeutic exploitation of these factors. Here we report that newly generated mice with B cell-specific deletion of Nr4a1 and Nr4a3 show a major break in B cell tolerance, including the development of spontaneous germinal centers and autoantibodies. These defects correlate with profound super-induction of novel target genes in response to Ag stimulation – including several genes required for recruitment of T cell help – that is largely masked when even one functional Nr4a allele remains. We therefore hypothesize that the Nr4a family mediates negative feedback downstream of Ag stimulation via transcriptional repression of key target genes. We propose that this serves two functions: (a) to restrain self-reactive clones that receive signal 1 (antigen) in the absence of signal 2 (co-stimulation), and (b) restrain immunodominant clones from monopolizing normal humoral immune responses, in order to preserve clonal diversity. In this grant, we propose: (1) To define the role of the Nr4a family in regulating central and peripheral B cell tolerance, and identify transcriptional mechanisms that mediate these functions in BCR Tg and naturally occurring self-reactive B cells. (2) To define the role of the Nr4a family in regulating B cell inter-clonal competition and clonal diversity during the primary T-dependent humoral immune response and its contribution to limiting immunodominance. (3) To define the role of the newly identified Nr4a target gene Batf in mediating these processes.