Altered TCR signaling in anergy

无能状态下 TCR 信号传导的改变

• 批准号: 10750486
• 负责人: Virginia Smith Shapiro
• 金额: $ 24.21万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-06 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750486
• 关键词: Acceleration; Autoantigens; Autoimmune Diseases; Autoimmunity; Biochemical Pathway; Bone Marrow; CD28 gene; CD4 Positive T Lymphocytes; Cells; Chimera organism; Defect; Development; Diabetes Mellitus; Frequencies; Generations; Guanosine Triphosphate; Hydrolysis; Immune response; In Vitro; Interleukin-2; Knockout Mice; Maintenance; Memory; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; PIK3CG gene; Pathway interactions; Peptides; Peripheral; Production; Proliferating; Regulation; Repression; Role; Signal Pathway; Signal Transduction; Stimulus; Structure; T cell anergy; T cell regulation; T-Cell Activation; T-Cell Development; T-Lymphocyte; T-Lymphocyte Subsets; Tamoxifen; Transgenic Mice; Transgenic Organisms; Up-Regulation; Work; acute myeloid leukemia 1 protein; anergy; autoreactive T cell; conditional knockout; in vivo; in vivo Model; insight; novel; novel therapeutics; overexpression; peripheral tolerance; prevent; programs; ras GTPase-Activating Proteins; transcription factor; transcriptome sequencing

Abstract T cell anergy is a cell-intrinsic program of non-responsiveness to self-antigens, and is an important component of peripheral tolerance. As the induction and maintenance of anergy is critical to preventing breakdown of tolerance and initiation of autoimmunity, it is important to understand the mechanisms and molecules that regulate anergy. Initially, T cell anergy was induced by TCR stimulation in the absence of costimulation and a variety of stimuli have been used in vitro to induce anergy. This early work demonstrated the critical importance of activation of the Ras/Map kinase pathway upon TCR/CD28 stimulation for T cell activation, IL-2 production and proliferation. However, the mechanism is not clear as to how the Ras/Map kinase pathway is downregulated in anergic T cells. Recently, endogenously anergic T cells in WT mice have been identified and are characterized by high expression of CD73 and FR4. We have found that these naturally anergic FR4+CD73+ CD4+ T cells in vivo from WT mice express lower levels of Runx1 protein as compared to other CD4+ T cell subsets. TCR/CD28-induced activation of CD4+ T cells with conditional deletion of Runx1 resulted in lower IL-2 expression and reduced proliferation as compared to activation of WT CD4+ T cells. In addition, there was a higher frequency of naturally anergic FR4+CD73+ CD4+ T cells in CD4- cre Runx1 conditional knockout (cKO) mice or tamoxifen-treated ER-cre Runx1 cKO mice in mixed bone marrow chimeras. Thus, deletion of Runx1 in mature CD4+ T cells predisposes them to the development of T cell anergy. To understand the mechanism(s) responsible for hypo-responsiveness of Runx1-deficient T cells upon TCR/CD28 stimulation, RNAseq analysis was performed on WT and Runx1-deficient CD4+ T cells. Runx1-deficient CD4+ T cells upregulated expression of Dab2IP, an adaptor molecule that has been shown in non-hematopoietic cells to be a negative regulator of Ras signaling. Dab2IP contains a Ras-GAP domain, which leads to the inactivation of Ras through accelerating hydrolysis of GTP to GDP. To determine whether increased Dab2IP expression could inhibit T cell activation and promote anergy induction, we generated a novel cre-dependent, dox-regulatable Dab2IP transgenic mouse. Our initial results demonstrate that similar to CD4+ T cells conditionally deleted for Runx1, Dab2IP Tg CD4+ T cells have increased frequencies of naturally anergic FR4+CD73+ CD4+ T cells in their memory cell pool. Our hypothesis is that increased expression of Dab2IP interferes with normal TCR/CD28 signaling, preventing T cell activation, and predisposing T cells to the development of anergy.

抽象的 T细胞无反应性是一种对自身抗原无反应的细胞固有程序，是一种重要的免疫反应。 外周耐受性的组成部分。由于无反应性的诱导和维持对于预防至关重要 耐受性的崩溃和自身免疫的启动，了解其机制和机制非常重要 调节无能的分子。最初，在缺乏 TCR 的情况下，T 细胞无反应性是由 TCR 刺激诱导的。 共刺激和多种刺激已在体外用于诱导无反应性。这项早期工作表明 TCR/CD28 刺激 T 细胞后 Ras/Map 激酶通路激活的至关重要性 激活、IL-2 产生和增殖。然而，Ras/Map 的机制尚不清楚。 激酶途径在无反应性 T 细胞中下调。最近，WT 小鼠中的内源性无反应性 T 细胞 已被鉴定并以 CD73 和 FR4 高表达为特征。我们发现这些 WT 小鼠体内天然无反应的 FR4+CD73+ CD4+ T 细胞表达较低水平的 Runx1 蛋白， 与其他 CD4+ T 细胞亚群相比。 TCR/CD28 诱导 CD4+ T 细胞的条件激活 与 WT 的激活相比，Runx1 的缺失导致 IL-2 表达降低并减少增殖 CD4+ T 细胞。此外，CD4- 中自然无反应的 FR4+CD73+ CD4+ T 细胞的频率较高。 cre Runx1 条件敲除 (cKO) 小鼠或他莫昔芬治疗的混合骨中的 ER-cre Runx1 cKO 小鼠 骨髓嵌合体。因此，成熟 CD4+ T 细胞中 Runx1 的缺失使它们易于发育为 T 细胞。 细胞无能。了解 Runx1 缺陷型 T 细胞反应低下的机制 TCR/CD28 刺激后，对 WT 和 Runx1 缺陷型 CD4+ T 细胞进行 RNAseq 分析。 Runx1 缺陷的 CD4+ T 细胞上调 Dab2IP 的表达，Dab2IP 是一种接头分子，已在 非造血细胞成为 Ras 信号传导的负调节因子。 Dab2IP 包含 Ras-GAP 域， 这通过加速 GTP 水解为 GDP 导致 Ras 失活。判断是否 增加的 Dab2IP 表达可以抑制 T 细胞活化并促进无反应性诱导，我们生成了 新型 cre 依赖性、dox 可调节 Dab2IP 转基因小鼠。我们的初步结果表明，类似 与针对 Runx1 有条件删除的 CD4+ T 细胞相比，Dab2IP Tg CD4+ T 细胞的频率增加 其记忆细胞库中存在天然无能的 FR4+CD73+ CD4+ T 细胞。我们的假设是增加 Dab2IP 的表达会干扰正常的 TCR/CD28 信号传导，阻止 T 细胞激活，并且 使 T 细胞容易产生无反应性。