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

异基因造血干细胞移植是一种治疗危及生命的治疗方法 包括高危白血病和骨髓衰竭在内的疾病。然而，allHSCT的好处仍然存在。 受限于急性移植物抗宿主病(GVHD)，其中同种异体反应性供者T细胞破坏宿主组织中的 皮肤、肝脏和胃肠道。区分致病T细胞和介导性T细胞的新疗法 有益的免疫反应对于提高异基因造血干细胞移植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 这种选择性的干预。具体地说，我们的数据表明，在供体细胞中删除AMPK可以缓解 GVHD，但仍保留了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)效应。从机制上讲，我们的数据进一步表明，移植物抗宿主病的发生率较低是由于 AMPK基因敲除(KO)细胞对促炎细胞因子的敏感性。由此，我们形成了 遵循中心假说：AMPK在移植后早期以组织特异性的方式激活，增加 局部T细胞对促炎细胞因子的敏感性。在没有AMPK的情况下，炎症信号是 钝化、稳定调节性T细胞(Treg)的发育，降低效应者的反应。这些变化 缓解GVHD，而GVT反应不受影响，因为增加细胞因子敏感性对于 诱导白血病导向的细胞毒性，因为白血病清除发生在AMPK 激活则不那么明显。我们将通过三个具体目标来检验这一假设。在目标1中，我们将 通过在术后一定时间消除T细胞中的AMPK来确定AMPK的位置和时间必要性 移植并定量检测从多个组织中同时恢复的细胞中的AMPK活性。我们会 通过检测细胞因子来确定AMPK激活和细胞因子敏感性之间的关系 用一系列AMPK激动剂刺激后的反应。在目标2中，我们将阐明连接机制 AMPK缺乏改善GVHD和降低细胞因子敏感性 单个KO细胞，到缺乏AMPK和IL-6受体的细胞。此外，我们将使用质谱学来 测定细胞因子刺激和同种异体反应T细胞中新的AMPK靶蛋白的磷酸化。在AIM 3我们将测定AMPK缺陷的人T细胞在降低AMPK后的GVHD和GVT潜能 使用CRISPR/Cas9基因编辑和短发夹状RNA转导的水平，然后移植 将细胞修饰成异种GVHD模型和免疫缺陷GVT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 这些发现可以转化为人类细胞。如果成功，我们的研究将定义一种新的机制 将能量感知与T细胞效应器功能联系起来，这可能会扩展到GVHD之外，包括健壮和 任何T细胞的持续激活，包括在自身免疫期间和实体器官移植后。

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.