The Role of AMP-activated Protein Kinase in GVHD-causing T Cells

AMP 激活的蛋白激酶在引起 GVHD 的 T 细胞中的作用

• 批准号: 10359032
• 负责人: Craig Alan Byersdorfer
• 金额: $ 48.82万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359032
• 关键词: 5' -AMP-activated protein kinase; Abate; Acute Graft Versus Host Disease; Adrenal Cortex Hormones; Agonist; Allogenic; Autoimmunity; Blocking Antibodies; Bone marrow failure; CRISPR/Cas technology; Cells; Cellular Metabolic Process; Data; Development; Disease; Gastrointestinal tract structure; Genes; Goals; Graft-Versus-Tumor Induction; Hematopoietic Stem Cell Transplantation; Human; Hyperglycemia; Hypertension; IL6ST gene; Immune System Diseases; Immune response; Immunologic Deficiency Syndromes; Infection Control; Inflammation; Inflammatory; Interleukin 6 Receptor; Interleukin-6; Intervention; Knock-out; Knockout Mice; Life; Link; Liver; Location; Lymphopenia; Mass Spectrum Analysis; Measures; Mediating; Metabolism; Modeling; Mus; Organ; Organ Transplantation; Pathogenicity; Pathway interactions; Peripheral; Pharmacology; Phosphorylation; Physiological; Property; Protein Kinase; Proteins; Publications; Reagent; Regulatory T-Lymphocyte; Research; Risk; Role; Safety; Severity of illness; Signal Transduction; Site; Skin; Solid; System; T cell response; T-Lymphocyte; Tamoxifen; Testing; Time; Tissues; Toxic effect; Translating; Transplantation; base; curative treatments; cytokine; cytotoxicity; design; effector T cell; experience; graft vs host disease; high risk; immune reconstitution; improved; infection rate; leukemia; mortality; novel; novel therapeutics; peripheral blood; post-transplant; preservation; prevent; response; sensor; side effect; small hairpin RNA

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative therapy for life-threatening disorders including high-risk leukemia and bone marrow failure. However, the benefits of alloHSCT remain limited by acute graft-versus-host disease (GVHD), where alloreactive donor T cells destroy host tissues in the skin, liver and gastrointestinal tract. New therapies to distinguish pathogenic T cells from T cells mediating beneficial immune responses are necessary to improve the safety and applicability of alloHSCT. Our previous publications and current preliminary data suggest that targeting alloreactive T cell metabolism may allow for this selective intervention. Specifically, our data demonstrate that deletion of AMPK in donor cells mitigates GVHD but still preserves lymphopenia-driven immune reconstitution and T cell-driven graft-versus-tumor (GVT) effects. Mechanistically, our data further suggest that lower rates of GVHD result from decreased sensitivity of AMPK knock-out (KO) cells to the effects of pro-inflammatory cytokines. From this, we form the following central hypothesis: AMPK is activated early post-transplant in a tissue-specific fashion, increasing local T cell sensitivity to pro-inflammatory cytokines. In the absence of AMPK, inflammatory signals are blunted, stabilizing regulatory T cell (Treg) development and decreasing effector responses. These changes mitigate GVHD, while GVT responses are unaffected because increased cytokine sensitivity is unnecessary for inducing leukemia-directed cytotoxicity and because leukemia clearance occurs at sites where AMPK activation is less pronounced. We will test this hypothesis through three specific aims. In Aim 1, we will determine the location and temporal necessity of AMPK by eliminating AMPK in T cells at defined times post- transplant and quantitating AMPK activation in cells recovered from multiple tissues simultaneously. We will also define the relationship between AMPK activation and cytokine sensitivity by measuring cytokine responses following stimulation with an array of AMPK agonists. In Aim 2, we will elucidate mechanisms linking AMPK deficiency to improved GVHD and decreased cytokine sensitivity by comparing the GVHD potential of single KO cells, to cells lacking both AMPK and the IL-6 receptor. In addition, we will use mass spectrometry to measure phosphorylation of novel AMPK target proteins in cytokine-stimulated and alloreactive T cells. In Aim 3 we will determine the GVHD and GVT potential of AMPK-deficient human T cells after decreasing AMPK levels using CRISPR/Cas9 gene editing and short hairpin RNA transduction, followed by transplantation of modified cells into xenogeneic models of GVHD and immunodeficient models of GVT. These studies will deepen our understanding of AMPK activation, how this activation impacts cytokine sensitivity, and whether these findings can be translated into human cells. If successful, our studies will define a novel mechanism linking energy sensing to T cell effector function that will likely extend beyond GVHD to include the robust and sustained activation of any T cell, including during autoimmunity and following solid organ transplantation.

异基因造血干细胞移植（alloHSCT）是一种治疗危及生命的造血干细胞移植的有效方法。 包括高危白血病和骨髓衰竭在内的疾病。然而，alloHSCT的益处仍然存在， 受到急性移植物抗宿主病（GVHD）的限制，其中同种异体反应性供体T细胞破坏体内的宿主组织 皮肤、肝脏和胃肠道。区分致病性T细胞与介导T细胞的新疗法 有益的免疫应答对于提高alloHSCT的安全性和适用性是必要的。我们以前的 出版物和目前的初步数据表明，靶向同种异体反应性T细胞代谢可能允许 这种选择性干预。具体来说，我们的数据表明，供体细胞中AMPK的缺失减轻了 GVHD，但仍保留淋巴细胞减少驱动的免疫重建和T细胞驱动的移植物抗肿瘤 (GVT)方面的影响.从机制上讲，我们的数据进一步表明，GVHD的发生率较低， AMPK敲除（KO）细胞对促炎细胞因子作用的敏感性。由此，我们形成了 以下中心假设：AMPK在移植后早期以组织特异性方式被激活， 局部T细胞对促炎细胞因子的敏感性。在缺乏AMPK的情况下，炎症信号是 钝化、稳定调节性T细胞（Treg）发育和降低效应子应答。这些变化 减轻GVHD，而GVT反应不受影响，因为增加的细胞因子敏感性是不必要的， 诱导白血病导向的细胞毒性，并且因为白血病清除发生在AMPK 激活不太明显。我们将通过三个具体目标来检验这一假设。在目标1中，我们 通过在术后的规定时间消除T细胞中的AMPK来确定AMPK的位置和时间必要性， 移植和同时定量从多个组织回收的细胞中AMPK活化。我们将 还通过测量细胞因子来确定AMPK激活与细胞因子敏感性之间的关系 在用一系列AMPK激动剂刺激后的反应。在目标2中，我们将阐明 AMPK缺乏改善GVHD和降低细胞因子敏感性的作用， 单KO细胞，转化为缺乏AMPK和IL-6受体的细胞。此外，我们还将使用质谱仪， 测量新的AMPK靶蛋白在奎宁刺激的和同种异体反应性T细胞中的磷酸化。在Aim中 我们将测定AMPK缺陷的人T细胞在降低AMPK后的GVHD和GVT潜能。 水平使用CRISPR/Cas9基因编辑和短发夹RNA转导，然后移植 将修饰的细胞转化为GVHD的异种模型和GVT的免疫缺陷模型。这些研究将 加深我们对AMPK激活的理解，这种激活如何影响细胞因子的敏感性，以及是否 这些发现可以转化为人类细胞。如果成功，我们的研究将确定一种新的机制， 将能量感测与T细胞效应器功能联系起来，这可能会超出GVHD，包括强大的 任何T细胞的持续活化，包括在自身免疫期间和实体器官移植后。