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

项目概要/摘要 这项提案的长期目标是利用孤儿核激素NR 4A家族的生物学 受体，以选择性地操纵免疫介导的疾病中的抗原特异性T细胞应答。 NR 4A成员（NR 477、NR 4 R1和NOR 1）由三个基因（分别为Nr 4a 1 -3）编码， 在淋巴细胞中通过抗原（Ag）刺激快速诱导。尽管它们被认为是配体- 独立的、组成型活性转录因子、小分子激动剂和拮抗剂配体已经被 使它们变得可药用。我们和其他人已经表明，NR 4A TF表达与肿瘤大小成比例。 Ag刺激的强度，并在经历负选择的胸腺细胞中高度上调，在调节 T细胞（Tcells），以及自身反应性、无反应性或耗竭的T细胞。由于重叠的表达模式 和相当大的结构同源性，在它们的DNA结合域，NR 4A转录因子表现出深刻的功能 冗余;多个-而非单个-NR 4A家族成员的胸腺缺失（Nr 4a 1和Nr 4a 3>> Nr 4a 2， 最低限度表达）导致严重的Treg缺乏和表型模仿的“皮屑样”疾病 Foxp 3-/-小鼠。因此，先前不可能揭露附加的冗余功能， 在胸腺选择和常规成熟CD 4 T细胞（Tconv）中，这是一个重大差距 这限制了对这些因素的充分利用。 最近，我们利用了条件遗传和骨髓嵌合体策略，以保持 Treg稳态。出乎意料的是，携带WT和Nr 4a 1-/-Nr 4a 3-/-（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Treg 大部分WT起源的区室。这种疾病在性质上不同于在生殖系DKO小鼠中观察到的， Treg缺陷，并且是B细胞外源性的。我们发现，DKO胸腺细胞的负选择是深刻的， 以细胞内在的方式受损。与自身反应性T细胞逃逸到外周一致，DKO CD 4 表达无反应性的表型和转录标记的Tconv细胞在这些嵌合体中积累。 然而，这些自身反应性DKO细胞仍然表现出过度的增殖和IL-2产生， 提示功能性无反应性是有缺陷的。因此，我们假设NR 4A家族发挥细胞内在作用， 但是多余的，在中枢和外周CD 4 T细胞耐受中的作用。为了检验这一假设： 在我们的第一个目标中，我们建议定义NR 4A家族在胸腺负选择中的作用， 普遍存在的和组织限制性的抗原，并确定有助于这一功能的转录靶点。 在我们的第二个目标中，我们建议分离NR 4A对外周T细胞耐受性的贡献，以及对典型的T细胞耐受性的贡献。 成熟CD 4 T细胞无反应性的特征-包括受损的信号转导，细胞因子产生， 增殖我们将使用散装和单细胞基因组方法来定义的机制， NR 4A家族调节幼稚和耐受性CD 4 T细胞的转录组和表观遗传谱。

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.