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.

摘要 T细胞无反应性是一种对自身抗原无反应性的细胞内在程序，并且是T细胞免疫的重要机制。 外围公差的组成部分。由于无反应性的诱导和维持对于预防 耐受性的破坏和自身免疫的启动，重要的是要了解机制， 调节无反应性的分子最初，T细胞无反应性是由TCR刺激诱导的， 共刺激和多种刺激物已在体外用于诱导无反应性。早期的研究表明， Ras/Map激酶通路在TCR/CD 28刺激T细胞后激活的重要性 活化、IL-2产生和增殖。然而，该机制并不清楚Ras/Map 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CD 73和FR 4的高表达为特征。我们发现， 来自WT小鼠的体内天然无反应性FR 4 + CD 73 + CD 4 + T细胞表达较低水平的Runx 1蛋白， 与其他CD 4 + T细胞亚群相比。TCR/CD 28诱导的条件性CD 4 + T细胞活化 与WT激活相比，Runx 1的缺失导致IL-2表达降低和增殖减少。 CD 4 + T淋巴细胞。此外，在CD 4-T细胞中，天然无反应性FR 4 + CD 73 + CD 4 + T细胞的频率更高。 cre Runx 1条件性敲除（cKO）小鼠或他莫昔芬处理的ER-cre Runx 1 cKO小鼠混合骨 骨髓嵌合体因此，在成熟的CD 4 + T细胞中Runx 1的缺失使它们倾向于T细胞的发展。 细胞无能了解Runx 1缺陷型T细胞低反应性的机制 在TCR/CD 28刺激后，对WT和Runx 1缺陷型CD 4 + T细胞进行RNAseq分析。 Runx 1缺陷型CD 4 + T细胞上调Dab 2 IP的表达，Dab 2 IP是一种衔接分子， 非造血细胞是Ras信号传导的负调节因子。Dab 2 IP含有Ras-GAP结构域， 其通过加速GTP水解为GDP而导致Ras失活。以确定是否 增加Dab 2 IP表达可以抑制T细胞活化并促进无反应性诱导，我们产生了一种新的免疫抑制剂。 新型cre依赖性dox可调控的Dab 2 IP转基因小鼠。我们的初步结果表明，类似的 对于Runx 1条件性缺失的CD 4 + T细胞，Dab 2 IP Tg CD 4 + T细胞具有增加的 在它们的记忆细胞库中天然无反应性的FR 4 + CD 73 + CD 4 + T细胞。我们的假设是 Dab 2 IP的表达干扰正常TCR/CD 28信号传导，阻止T细胞活化，和 使T细胞倾向于无反应性的发展。