Synthetic rescue of antigen-driven T cells and alloimmunity

抗原驱动T细胞和同种免疫的综合拯救

• 批准号: 10543445
• 负责人: Jonathan A Soboloff
• 金额: $ 63.76万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-12 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543445
• 关键词: Allogenic; Antigens; Artemisinins; Autoimmune Diseases; Automobile Driving; CD8B1 gene; Ca(2+)-Transporting ATPase; Calcineurin inhibitor; Cell Death; Cell Differentiation process; Cell Survival; Cell membrane; Cellular Assay; Cellular biology; Clinical; Cyclosporine; Cytosol; DNA Synthesis Inhibitors; Development; Differentiation Antigens; Disease; Endoplasmic Reticulum; Epigenetic Process; Gatekeeping; Generations; Goals; Homeostasis; Human; ITPR1 gene; Immunity; Immunotherapy; Impairment; Inbred BALB C Mice; Inflammatory; Investigation; Libraries; Ligation; Malaria; Malignant Neoplasms; Mediating; Methods; Mitochondria; Molecular; Mus; Na(+)-K(+)-Exchanging ATPase; Natural Compound; Outcome; Pathogenesis; Pathway interactions; Patients; Production; Proteins; Pump; Reaction; Regimen; Repression; Role; STIM1 gene; Signal Transduction; T cell response; T memory cell; T-Cell Activation; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Tacrolimus; Testing; Therapeutic; Toxic effect; Translating; Water; artesunate; calcium uniporter; chronic infection; disorder prevention; effector T cell; experimental study; graft vs host disease; graft vs leukemia effect; histone methyltransferase; improved; insight; interest; isoimmunity; mitochondrial metabolism; novel; novel strategies; nuclear factors of activated T-cells; pharmacologic; preservation; prophylactic; response; sensor; tissue injury; uptake

During graft-versus-host disease (GVHD), donor T cells require the histone methyltransferase Ezh2 for producing and sustaining effector T cells that mediate host tissue injury. We recently established that Ezh2 serves as a molecular gatekeeper for the generation of CD8 memory T cell precursors in GVHD, critical for the production of effector T cells in response to persistent antigen (Nat Commun 2017). However, our efforts to develop novel approaches to selectively target alloreactive effector T cells has been limited by the lack of understanding of why Ezh2 loss causes cell death of antigen-activated T cells. Stromal interaction molecule (Stim) proteins, Stim1 and Stim2, are crucial dynamic endoplasmic reticulum (ER) Ca2+ sensors and modulators of Ca2+ signals. Upon T cell receptor (TCR) ligation, Stim1 activation causes its translocation towards the plasma membrane, where it activates the Ca2+ channel Orai1, facilitating Ca2+ entry and driving T cell activation. Conditional Stim1 deletion inhibits GVHD in mice due to impaired effector differentiation. Remarkably, Stim1 deletion rescues antigen-activated Ezh2-null T cells, leading to restored production of alloreactive effector T cells in mice and severe GVHD. Therefore, we hypothesize that: A) Ezh2 and Stim1 operate coordinately to regulate the viability and function of antigen-driven T cells; and B) Ezh2/Stim1- regulated molecular pathway(s) are crucial for controlling alloreactive T cell-mediated GVHD. We further establish that the role of Stim1 in Ezh2-mediated cell death is to drive mitochondrial Ca2+ (mitoCa2+) overload since conditional deletion of the mitochondrial calcium uniporter (MCU), leads to rescue of antigen-activated Ezh2-null T cells. To establish the therapeutic potential of these findings, we performed a preliminary screen with an 800 compound library, finding 36 compounds that block T cell proliferation. Amongst them was artesunate (ART), a water-soluble derivative of artemisinin clinically approved for the treatment of malaria and known to target the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA), which pumps Ca2+ from the cytosol to the ER lumen. SERCA inhibition leads to Stim1/Orai1 activation and mitoCa2+ uptake. Preliminary investigations show that ART treatment reduces GVHD in BALB/C mice receiving allogeneic C57BL/6 T cells. Considered collectively, these findings suggest that Ezh2 regulates antigen-specific effector T cell survival through modulation cytosolic Ca2+ entry, thereby limiting mitochondrial Ca2+ loading and protecting against cell death. This hypothesis will be tested through three specific aims. In Aim-1, we will define the mechanisms that regulate the survival and differentiation of antigen-driven Ezh2/Stim1-null T cells. Aim-2 will determine the molecular mechanisms by which Ezh2 deficiency dysregulates cytosolic and mitochondria Ca2+ uptake in activated T cells. Finally, Aim-3 will examine the beneficial effect of enhancing T cell Ca2+ load to modulate GVHD and GVL activity in mice. Completion of these experiments will provide novel insights into T cell biology, T cell-mediated inflammatory disorders such as GVHD and autoimmune diseases, and lead to development of novel methods for improving the efficacy of immunotherapy for chronic infections and cancer.

在移植物抗宿主病 (GVHD) 期间，供体 T 细胞需要组蛋白 甲基转移酶 Ezh2 用于产生和维持介导宿主组织的效应 T 细胞 受伤。我们最近确定 Ezh2 作为分子守门人，用于产生 GVHD 中的 CD8 记忆 T 细胞前体，对于反应性 T 细胞的产生至关重要 持久性抗原（Nat Commun 2017）。然而，我们努力开发新方法 选择性靶向同种异体反应性效应 T 细胞由于缺乏 了解为什么 Ezh2 缺失会导致抗原激活 T 细胞死亡。基质相互作用 分子 (Stim) 蛋白 Stim1 和 Stim2 对动态内质网 (ER) 至关重要 Ca2+ 传感器和 Ca2+ 信号调制器。 T 细胞受体 (TCR) 连接后， Stim1 激活导致其向质膜易位，从而激活 Ca2+ 通道 Orai1，促进 Ca2+ 进入并驱动 T 细胞激活。条件刺激1 由于效应器分化受损，缺失会抑制小鼠的 GVHD。值得注意的是， Stim1 缺失可拯救抗原激活的 Ezh2 缺失 T 细胞，从而恢复 小鼠体内同种反应性效应 T 细胞的产生和严重的 GVHD。因此，我们假设 A) Ezh2 和 Stim1 协调运作以调节 抗原驱动的T细胞； B) Ezh2/Stim1 调节的分子途径对于 控制同种反应性 T 细胞介导的 GVHD。我们进一步确定 Stim1 的作用 Ezh2 介导的细胞死亡是为了驱动线粒体 Ca2+ (mitoCa2+) 超载，因为有条件 线粒体钙单向转运蛋白（MCU）的缺失，导致抗原激活的拯救 Ezh2 缺失 T 细胞。为了确定这些发现的治疗潜力，我们进行了 使用 800 种化合物库进行初步筛选，发现 36 种阻断 T 细胞的化合物 增殖。其中包括青蒿素（ART），一种青蒿素的水溶性衍生物 临床批准用于治疗疟疾，已知可靶向肉瘤/内质 网状 Ca2+ ATP 酶 (SERCA)，将 Ca2+ 从细胞质泵入内质网腔。塞卡 抑制导致 Stim1/Orai1 激活和 mitoCa2+ 摄取。初步的 研究表明，ART 治疗可降低接受同种异体 C57BL/6 T 的 BALB/C 小鼠的 GVHD 细胞。综合考虑，这些发现表明 Ezh2 调节抗原特异性 通过调节胞质 Ca2+ 进入，从而限制效应 T 细胞的存活 线粒体 Ca2+ 负载并防止细胞死亡。这个假设将被检验 通过三个具体目标。在Aim-1中，我们将定义调节生存的机制 以及抗原驱动的 Ezh2/Stim1-null T 细胞的分化。 Aim-2 将确定 Ezh2 缺陷导致细胞质和线粒体失调的分子机制 活化 T 细胞中的 Ca2+ 摄取。最后，Aim-3 将检验增强的有益效果 T 细胞 Ca2+ 负载可调节小鼠 GVHD 和 GVL 活性。完成这些实验将 为 T 细胞生物学、T 细胞介导的炎症性疾病（例如 GVHD 和自身免疫性疾病，并导致开发改善 免疫疗法对慢性感染和癌症的疗效。