Regulation of CD4 T cell tolerance by the NR4A family of nuclear receptors

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 在免疫介导性疾病中选择性地操纵抗原特异性T细胞反应的受体。 NR4A成员(NUR77、NURR1和Nor1)由三个基因(分别是Nr4a1-3)编码，它们是 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。我们和其他人已经表明，NR4A Tf的表达随 Ag刺激强度，在经历负选择的胸腺细胞中高度上调，在 T细胞(Tregs)，以及自身反应、无能或耗竭的T细胞。由于重叠的表达模式 和DNA结合区的相当大的结构同源性，NR4A转录因子显示出深刻的功能 冗余；多个但不是单个的NR4A家族成员的胸腺缺失(Nr4a1和Nr4a3；>；；Nr4a2， 这是最低限度的表达)导致严重的Treg缺乏和一种表现性的“皮屑样”疾病 Foxp3-/-小鼠。因此，以前不可能揭开额外的冗余功能 这个家族在胸腺选择和常规成熟的CD4T细胞(Tconv)。这是一个很大的差距 据我们所知，这限制了对这些因素的充分治疗利用。 最近，我们利用条件遗传和骨髓嵌合体策略来保存 Treg动态平衡。出乎意料的是，同时含有WT和Nr4a1-/-Nr4a3-/-(DKO)骨髓的混合嵌合体 快速发展抗核自身抗体(ANA)和全身性炎症性疾病，尽管有充分的Treg 主要来源于WT的隔间。这种疾病与生殖系DKO小鼠的疾病有本质上的不同 Treg缺乏，是外源性B细胞。我们证明了DKO胸腺细胞的负性选择是深刻的 以细胞固有的方式受损。与自身反应性T细胞外周逃逸一致，DKO CD4 表达无能的表型和转录标记的Tconv细胞在这些嵌合体中积累。 然而，这些具有自我反应性的DKO细胞仍然表现出过度的增殖和IL-2的产生， 这表明功能性无能是有缺陷的。因此，我们假设NR4A家族发挥细胞固有的作用， 但这是多余的，在中枢和外周CD4T细胞耐受性中都起到了作用。要检验这一假设，请执行以下操作： 在我们的第一个目标中，我们建议定义NR4A家族在面对两者时胸腺负选择中的作用 无处不在的和组织限制性的抗原，并定义有助于这一功能的转录靶标。 在我们的第二个目标中，我们建议分离NR4A对外周T细胞耐受的贡献，并规范 成熟的CD4T细胞无能的特征--包括信号转导、细胞因子的产生和 扩散。我们将使用散装和单细胞基因组方法来定义 NR4A家族调节幼稚和耐受的CD4T细胞的转录组和表观遗传学特征。

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 T cell responses in immune-mediated diseases. NR4A 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 and others have shown that NR4A TF expression scales with the intensity of Ag stimulation, and are highly upregulated in thymocytes undergoing negative selection, in regulatory T cells (Tregs), as well as in self-reactive, anergic, or exhausted T cells. Due to an overlapping expression pattern and considerable structural homology in their DNA-binding domain, the NR4A TFs exhibit profound functional redundancy; thymic deletion of multiple - but not individual - NR4A family members (Nr4a1 and Nr4a3 >> Nr4a2, which is minimally expressed) results in severe Treg deficiency and a “scurfy-like” disease that phenocopies Foxp3-/- mice. Consequently, it has not been possible previously to unmask additional redundant functions of this family during thymic selection and in conventional mature CD4 T cells (Tconv). This represents a major gap in our knowledge that limits full therapeutic exploitation of these factors. Recently, we took advantage of both conditional genetic and bone marrow chimera strategies in order to preserve Treg homeostasis. Unexpectedly, mixed chimeras harboring both WT and Nr4a1-/- Nr4a3-/- (DKO) bone marrow rapidly develop anti-nuclear autoantibodies (ANAs) and a systemic inflammatory disease despite a replete Treg compartment of largely WT origin. This disease differs qualitatively from that seen in germline DKO mice with Treg-deficiency, and is B cell-extrinsic. We show that negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO CD4 Tconv cells expressing phenotypic and transcriptional markers of anergy accumulate in these chimeras. However, these self-reactive DKO cells nevertheless exhibit exaggerated proliferation and IL-2 production, suggesting that functional anergy is defective. We therefore hypothesize that the NR4A family play cell-intrinsic, but redundant, roles in both central and peripheral CD4 T cell tolerance. To test this hypothesis: In our first aim, we propose to define the role of the NR4A family in thymic negative selection in the face of both ubiquitous and tissue-restricted antigens, and to define the transcriptional targets that contribute to this function. In our second aim, we propose to isolate NR4A contributions to peripheral T cell tolerance, and to canonical features of mature CD4 T cell anergy - including impaired signal transduction, cytokine production, and proliferation. We will use both bulk and single cell genomic approaches to define the mechanism by which the NR4A family regulates the transcriptome and epigenetic profile of naïve and tolerant CD4 T cells.