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

描述（由申请人提供）：蛋白激酶Cθ (PKCθ)是在T细胞中最丰富的PKC家族成员，它在效应T （Teff）细胞激活中起关键作用PKCθ通过与CD28的物理关联独特地定位于T细胞免疫突触（is）的中心，并且这种定位是下游PKCθ介导的信号传导所必需的。对PKCθ缺陷小鼠的体内分析显示，PKCθ在某些（Th2-和Th17-介导的炎症、同种异体移植排斥和移植物抗宿主病）免疫应答中有选择性需求，但在其他（Th1/CTL抗病毒和移植物抗白血病）免疫应答中没有选择性需求。有趣的是，PKCθ最近被发现在诱导Treg细胞（iTregs）的IS中被排除，并抑制其抑制功能，包括在人类中。我们的初步研究表明，破坏PKCθ- cd28相互作用并将PKCθ从IS中置换的策略，包括新颖的，以前未表征的PKCθ变构抑制剂（AI），增强了小鼠iTregs的体外分化和抑制功能。我们新的初步数据表明，相同的策略对人类T细胞有相似的影响。这些发现表明PKCθ作为有害T细胞介导的自身免疫、炎症和GvHD的免疫抑制的选择性药物靶点具有很高的前景，其中抑制策略被预测为协同作用以抑制致病性T细胞和促进Treg功能，而不会干扰有益的抗病毒保护性免疫。在这里，我们将详细研究阻断PKCθ- cd28相互作用在Th2细胞介导的小鼠变过敏性肺炎症模型中的潜在治疗价值，以及PKCθ在人Teffs和Tregs的体外分化和功能中分别具有选择性和似乎相反的作用。这些研究将解决这样一个假说，即阻断PKCθ-CD28相互作用可抑制致病性T细胞的分化和功能，并促进小鼠和人类Tregs的功能，从而实现对有害T细胞介导的炎症反应的有益协同作用。在Aim 1中，我们将采用显性阴性PKCθ突变体和新型PKCθ AI来研究其对完整小鼠、骨髓嵌合小鼠或过继T细胞转移（包括使用tcr转基因小鼠和FoxP3eGFP报告小鼠）中th2介导的变应性肺部炎症的发展和表现的影响。在Aim 2中，我们将首次使用与Aim 1相同的阻断策略来破坏人CD4+ T细胞中TCR/ cd28诱导的PKCθ-CD28相互作用，并评估其对人血源性Teff和Treg细胞体外分化和功能的影响。该项目将扩展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Teff和Treg细胞的分化和功能的生物学相关性，重要地将这些发现扩展到人类T细胞。它也将首次详细分析新的PKCθ AI对T细胞反应的影响，并可能为开发新的高选择性治疗策略铺平道路，以治疗T细胞介导的过敏、自身免疫、疾病和GvHD。

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.