Synthetic rescue of antigen-driven T cells and alloimmunity

在移植物抗宿主病（GVHD）中，供体T细胞需要组蛋白 用于产生和维持介导宿主组织的效应T细胞的甲基转移酶Ezh 2 损伤我们最近确定Ezh 2作为产生Ezh 2的分子看门人。 GVHD中的CD 8记忆T细胞前体，对于应答中效应T细胞的产生至关重要 持久性抗原（Nat Commun 2017）。然而，我们开发新方法的努力， 选择性靶向同种异体反应性效应T细胞受到缺乏 理解为什么Ezh 2缺失导致抗原激活的T细胞的细胞死亡。基质相互作用 刺激分子（Stim）蛋白，Stim 1和Stim 2，是关键的动态内质网（ER）， Ca 2+信号的Ca 2+传感器和调节器。在T细胞受体（TCR）连接后， Stim 1激活导致其向质膜移位，在那里它激活细胞膜。 Ca 2+通道Orai 1，促进Ca 2+进入并驱动T细胞活化。条件刺激1 由于效应分化受损，缺失抑制小鼠中的GVHD。值得注意的是， Stim 1缺失拯救抗原激活的Ezh 2-null T细胞，导致恢复 小鼠中同种异体反应性效应T细胞的产生和严重的GVHD。因此，我们假设 A）Ezh 2和Stim 1协调运作以调节细胞的活力和功能， 抗原驱动的T细胞;和B）Ezh 2/Slim 1调节的分子途径对于 控制同种异体反应性T细胞介导的GVHD。我们进一步确定刺激1的作用 在Ezh 2介导的细胞死亡中，驱动线粒体Ca 2+（mitoCa 2+）超载，因为条件 线粒体钙单向转运体（MCU）的缺失，导致抗原激活的 Ezh 2-无效T细胞。为了确定这些发现的治疗潜力，我们进行了一项 初步筛选了800种化合物库，发现36种化合物阻断T细胞 增殖其中包括青蒿琥酯（ART），青蒿素的水溶性衍生物 临床上被批准用于治疗疟疾，并且已知靶向Sarco/Endoplasmic 网状细胞Ca 2 + ATP酶（SERCA），将Ca 2+从细胞质泵入内质网腔。SERCA 抑制导致Stim 1/Orai 1激活和mitoCa 2+摄取。初步 研究显示ART治疗减少了接受同种异体C57 BL/6 T的BALB/C小鼠中的GVHD 细胞综合考虑，这些发现表明，Ezh 2调节抗原特异性 通过调节胞质Ca 2+进入，从而限制效应T细胞存活 线粒体Ca 2+负载和保护细胞免于死亡。这一假设将得到检验 通过三个具体目标。在目标-1中，我们将定义调节存活的机制。 和抗原驱动的Ezh 2/Stim 1-null T细胞的分化。Aim-2将确定 Ezh 2缺乏导致细胞质和线粒体失调的分子机制 活化T细胞中的Ca 2+摄取。最后，目标-3将审查加强 T细胞Ca 2+负荷调节小鼠中的GVHD和GVL活性。这些实验的完成将 为T细胞生物学、T细胞介导的炎症性疾病， GVHD和自身免疫性疾病，并导致开发新的方法来改善免疫系统。 免疫疗法对慢性感染和癌症的疗效。

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.