Modulation of Teff/Treg cells by novel PKC-theta allosteric inhibitory strategies

描述(申请人提供)：蛋白激酶C Theta(PKCθ)是T细胞中含量最丰富的PKC家族成员，在T细胞激活中起关键作用。PKCθ通过与CD28的物理结合唯一地定位于T细胞免疫突触(IS)的中心，这种定位是下游PKCθ介导的信号所必需的。对PKCθ缺陷小鼠的体内分析表明，PKCθ在某些免疫反应(Th2型和Th17型炎症、移植物排斥反应和移植物抗宿主疾病)中具有选择性，但在其他免疫应答(Th1/CTL抗病毒和移植物抗白血病)中不需要。有趣的是，最近发现PKCθ被排除在诱导的Treg细胞(ITregs)的IS之外，并抑制其抑制功能，包括在人类中。我们的初步研究表明，干扰PKCθ-CD28相互作用并将PKCθ从IS中取代的策略，包括以前未知的PKCθ变构抑制剂(AI)，可以增强小鼠iTregs的体外分化和抑制功能。我们新的初步数据表明，同样的策略对人类T细胞也有类似的影响。这些发现表明，PKCθ很有希望成为一种选择性药物靶点，用于抑制有害的T细胞介导的自身免疫、炎症和移植物抗宿主病，其中抑制策略被预测为协同作用，抑制致病T细胞和促进Treg功能，而不干扰有益的抗病毒保护性免疫。在此，我们将详细研究在Th2细胞介导的变态反应性肺部炎症小鼠模型中阻断PKCθ-CD28相互作用的潜在治疗价值，以及PKCθ在人T细胞和T细胞体外分化和功能中的选择性和似乎相反的作用。这些研究将解决一个工作假设，即阻断PKCθ-CD28相互作用抑制致病T细胞的分化和功能，并促进Tregs在小鼠和人类中的功能，从而实现有益的协同作用，对抗有害的T细胞介导的炎症反应。在目标1中，我们将使用一个显性负的PKCθ突变体和新的PKCθAI来研究在完整小鼠、骨髓嵌合小鼠或通过过继T细胞转移(包括使用TCR转基因和FoxP3eGFP报告小鼠)对Th2介导的过敏性肺部炎症的发生和表现的影响。在目标2中，我们将首次使用与目标1相同的阻断策略来阻断TcR/CD28诱导的人θ+T细胞中PKCCD28的相互作用，并评估它们对人血源性T细胞的体外分化和功能的影响。这个项目将扩大我们对我们实验室发现的PKCθ-CD28相互作用的重要性的理解，以及它与T细胞分化和功能的生物学相关性，重要的是将这些发现扩展到人类T细胞。它还将代表对新的PKCθAI对T细胞反应的首次详细分析，并可能为开发新的、高度选择性的治疗策略铺平道路，以治疗T细胞介导的过敏、自身免疫、疾病和移植物抗宿主病。

DESCRIPTION (provided by applicant): Protein kinase C theta (PKCθ) is a PKC family member most abundant in T cells, where it plays critical roles in effector T (Teff) cell activation PKCθ uniquely localizes to the center of the T cell immunological synapse (IS) via physical association with CD28, and this localization is required for downstream PKCθ-mediated signaling. In vivo analysis of PKCθ-deficient mice revealed a selective requirement of PKCθ in some (Th2- and Th17- mediated inflammation, allograft rejection and graft vs. host disease) but not other (Th1/CTL antiviral and graft vs. leukemia) immune responses. Intriguingly, PKCθ was recently found to be excluded from the IS of induced Treg cells (iTregs) and to inhibit their suppressive function, including in humans. Our preliminary studies show that strategies, which disrupt the PKCθ-CD28 interaction and displace PKCθ from the IS, including novel, previously uncharacterized PKCθ allosteric inhibitors (AI), enhance the in vitro differentiation and suppressive function of mouse iTregs. Our new preliminary data suggest that the same strategies have similar effects on human T cells. These findings point to the high promise of PKCθ as a selective drug target for immunosuppression of harmful T cell-mediated autoimmunity, inflammation, and GvHD, where inhibitory strategies are predicted to act synergistically to inhibit pathogenic T cells and promote Treg function, without interfering with beneficial antiviral protective immunity. Here, we will investigate in detail the potential therapeutic value of blocking the PKCθ-CD28 interaction in a mouse model of Th2 cell-mediated allergic lung inflammation, and the selective and seemingly opposite roles of PKCθ in the in vitro differentiation and function of human Teffs and Tregs, respectively. These studies will address the working hypothesis that blockade of the PKCθ-CD28 interaction inhibits the differentiation and function of pathogenic T cells and promotes the function of Tregs in both mice and humans, thereby achieving a beneficial synergetic effect against harmful T cell- mediated inflammatory responses. In Aim 1, we will employ a dominant negative PKCθ mutant and novel PKCθ AI to study the resulting effects on the development and manifestation of Th2-mediated allergic lung inflammation in intact mice, bone marrow chimeric mice, or by using adoptive T cell transfers, including the use of TCR-transgenic and FoxP3eGFP reporter mice. In Aim 2, we will use, for the first time, the same blocking strategies of Aim 1 to disrupt the TCR/CD28-induced PKCθ-CD28 interaction in human CD4+ T cells, and evaluate their effect on the in vitro differentiation and function of human blood-derived Teff and Treg cells. This project will extend our understanding of the importance of the PKCθ-CD28 interaction, which was discovered in our laboratory, and its biological relevance for the differentiation and function of both Teff and Treg cells, importantly extending these findings to human T cells. It will also represent the first detailed analysis of novel PKCθ AI on T cell responses, and may pave the way to the development of new, highly selective therapeutic strategies to treat T cell-mediated allergies, autoimmune, diseases, and GvHD.