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细胞无能是一种对自身抗原无反应的细胞固有程序，是一种重要的 周长公差的组成部分。因为无能的诱导和维持是预防 耐受性的崩溃和自身免疫的启动，重要的是要了解其机制和 调节无能的分子。最初，T细胞无能是在缺乏TCR刺激的情况下诱导的 在体外，共刺激和各种刺激已经被用来诱导无能。这项早期工作证明了 激活RAS/MAPK通路在TCR/CD28刺激T细胞中的重要作用 活化、IL-2的产生和增殖。然而，RAS/MAP的作用机制尚不清楚 在无能T细胞中，激酶途径被下调。近年来，WT小鼠内源性无能T细胞 以CD73和FR4的高表达为特征。我们发现这些 WT小鼠体内天然无能FR4+CD73+CD4+T细胞表达较低水平的RUNX1蛋白 与其他CD4+T细胞亚群比较。条件性TCR/CD28诱导的CD4+T细胞活化 与WT的激活相比，RUNX1的缺失导致IL-2的表达降低和增殖减少 CD4+T细胞。此外，自然无能的FR4+CD73+CD4+T细胞在CD4-T细胞中的比例较高。 Cre RUNX1条件性基因敲除(CKO)小鼠或他莫昔芬治疗的ER-cre RUNX1 CKO混合骨小鼠 骨髓嵌合体。因此，成熟的CD4+T细胞中RUNX1的缺失使其易于T细胞的发育 细胞无能。了解RUNX1缺陷T细胞低应答的机制(S) 在TCR/CD28刺激下，对WT和RUNX1缺陷的CD4+T细胞进行RNAseq分析。 RUNX1缺陷的CD4+T细胞上调了Dab2IP的表达，Dab2IP是一种适配器分子，已在 非造血细胞可能是RAS信号的负调节因子。Dab2IP包含RAS-GAP域， 这导致了RAS的失活，加速了GTP对GDP的水解。以确定是否 Dab2IP表达增加可抑制T细胞活化，促进无能诱导，我们产生了 新型cre依赖、dox可调节的dab2ip转基因小鼠。我们的初步结果表明， 以RUNX1为条件缺失的CD4+T细胞，Dab2IP TG的CD4+T细胞出现频率升高 自然无能的FR4+CD73+CD4+T细胞在他们的记忆细胞池中。我们的假设是增加了 Dab2IP的表达干扰了正常的TCR/CD28信号，阻止了T细胞的激活 使T细胞容易发展为无能。