Enhancing Treg Therapeutic Efficacy in GVHD

摘要：这次更新延续了我们对调节性T细胞(Treg)输注的优化，以创造移植 临床上预防移植物抗宿主病(GVHD)的耐受性。我们的首个人类第一阶段Treg试验显示 减少但不是消除GVHD。需要较高的Treg数，并且抑制是可变的。我们 建议通过注入高效力树突状细胞来解决这两个局限性。而蛋白激酶Cθ(PKC-θ) 近端免疫突触(IS)的定位是最佳效应器功能所必需的，它被排除在外 来自特雷格斯的IS，它位于一个远端极复合体(DPC)。小鼠或人的Tregs用一种 临床试验表明，PKC-θ抑制剂的抑制率提高了300%。磷酸化蛋白质组学显示下调 波形蛋白，一种在Treg中优先表达的细胞骨架细丝，以及与肌动蛋白有关的靶点 细胞骨架动力学和细胞运动性。波形蛋白物理隔离DPC中的PKC-θ，据信具有 负调节功能，并束缚线粒体。PKC-θ抑制剂和波形蛋白siRNA分别引起DPC 中断和更多的抑制，并在收养转移时提高了GVHD的效力 预防和减少宿主树突状细胞对同种抗原特异性T细胞的激活。这些数据指向 令人惊讶的结论是，DPC本身被破坏，而不是以PKC-θ激酶活性为靶点的抑制剂 负责Treg超级抑制器的功能。增殖树增加线粒体脂肪酸β- 氧化(粮农组织)，以满足能源需求。超级抑制因子Tregs上调脂肪酸(FA)摄取和 在不改变糖酵解的情况下增加氧化磷酸化和粮农组织。更多的镇压是 与mTOR成分mTORC2下调有关，mTORC2影响细胞骨架动力学和 线粒体锚定，没有mTORC1效应。DPC中断树的细胞内代谢组学表明 对FA代谢和从头合成FA的显著影响，导致了中心假说 细胞骨架结构通过调节线粒体功能和FAO来抑制Treg功能。因此，DPC 被破坏的Treg具有超抑制表型，具有协调调节的信号属性， 新陈代谢和功能。在目标1A中，我们假设线粒体融合是由Treg DPC中断引起的 而功能融合的得失将改变超抑制。在目标1B中，我们假设DPC中断 通过改变线粒体-细胞骨架来增加线粒体对IS的运动性以调节钙信号 系绳。在目标2A中，我们将确定Treg对FA摄取、从头合成或 线粒体新陈代谢排除了超抑制功能。在目标2B中，我们假设关键目标2A 被波形蛋白或肌动蛋白纤维隔离的代谢蛋白必须分散以驱动Treg的超抑制。 局限性结肠炎和全身性多器官系统炎性GVHD模型的能力。这些数据将 为将Treg PKC-θ抑制剂和相关治疗方法转化为临床提供重要的生物学见解。

Abstract: This renewal continues our optimization of regulatory T cell (Treg) infusion for creating transplant tolerance to prevent graft-vs-host disease (GVHD) in the clinic. Our first-in-human phase I Treg trials showed reduction but not elimination of GVHD. High Treg numbers were needed and suppression was variable. We propose to tackle both limitations by infusing high potency Tregs. Whereas protein kinase C theta (PKC-θ) localization to the proximal immunological synapse (IS) is required for optimal Teffector function it is excluded from the IS in Tregs where it is located in a distal pole complex (DPC). Mouse or human Tregs treated with a clinically tested PKC-θ inhibitor increased suppression by 300%. Phospho-proteomics show down-regulation of vimentin, a cytoskeletal filament preferentially expressed in Tregs, as well as targets involved in actin cytoskeletal dynamics and cell motility. Vimentin physically sequesters PKC-θ in the DPC, believed to have negative regulatory function, and tethers mitochondria. PKC-θ inhibitors and vimentin siRNA each caused DPC disruption and increased suppression and, upon adoptive transfer, had heightened potency in GVHD prevention and reduced host dendritic cell priming of alloantigen-specific Teffectors. These data point to the surprising conclusion that DPC disruption per se and not inhibitor targeting of PKC-θ kinase activity was responsible for Treg super-suppressor function. Proliferating Tregs increase mitochondrial fatty acid β- oxidation (FAO) to meet energy needs. Super-suppressor Tregs up-regulated fatty acid (FA) uptake and increased oxidative phosphorylation and FAO without altering glycolysis. Increased suppression was associated with down-regulation of the mTOR component, mTORC2, that affects cytoskeletal dynamics and mitochondrial anchoring, with no mTORC1 effects. Intracellular metabolomics of DPC disrupted Tregs indicate significant effects on FA metabolism and de novo FA biosynthesis, leading to the central hypothesis that the cytoskeleton structure restrains Treg function by regulating mitochondrial function and FAO. Thus the DPC disrupted Tregs have a super-suppressive phenotype with coordinately regulated attributes of signaling, metabolism and function. In aim 1A, we hypothesize mitochondrial fusion is induced by Treg DPC disruption and gain-or-loss-of function fusion will alter super-suppression. In aim 1B, we hypothesize that DPC disruption increases mitochondrial motility to the IS to modulate calcium signaling by altering mitochondria-cytoskeletal tethering. In aim 2A, we will determine whether Treg loss-of-function for FA uptake, de novo synthesis or mitochondrial metabolism precludes super-suppressor function. In aim 2B, we hypothesize that key aim 2A metabolic proteins sequestered by vimentin or actin fibers must be dispersed to drive Treg super-suppression. In aim 3, we hypothesize super-suppressor mechanisms identified above will translate into in vivo suppressor capacity in localized colitis and systemic multi-organ system inflammatory GVHD models. These data will provide important biological insights for translating Treg PKC-θ inhibitor and related treatments into the clinic.