A Treg cell-intrinsic CTLA4-PKC-eta signaling pathway mediating contact-dependent suppression of tumor immunity: A novel target for cancer immunotherapy

摘要 调节性T细胞积累并与许多癌症的预后不良相关，以及Treg的消融 与其他疗法一起在临床上进行探索。然而，这一方法的成功需要， 阐明Treg的调节和作用机制，以提供新的、更具选择性的靶标 合理的免疫疗法这一建议源于我们发现了一种新的Treg-内在信号传导 肿瘤免疫的接触依赖性抑制所需的途径。在这条通路中，Treg表达 CTLA 4与蛋白激酶C-eta（PKCη）物理结合，并且在CTLA 4接合时，募集PAK-1。 局部粘连分离所需的PIX-GIT复合体。PKCη缺失或CTLA 4非表达 相互作用的PKCη突变体损害Treg抑制肿瘤特异性免疫的能力，但不损害自身免疫性结肠炎。 我们假设这种新的CTLA 4信号传导轴是接触依赖性抑制肿瘤的必需品。 Foxp 3 + Treg免疫。我们的首要目标是进一步探索在机械和动物水平， 信号通路及其对肿瘤免疫的功能意义。在目标1中，我们将机械地分析 CTLA 4和PKCη用于募集和激活GIT 2-PAK 2-αPIX复合物的分子途径， 介导小鼠和人类Treg中的接触依赖性抑制，并确定这种干扰是如何发生的。 信号通路影响Treg抑制。我们还将使用双光子显微镜来研究肿瘤内的Treg- 华盛顿动力局。考虑到CD 8 + T细胞作为肿瘤杀伤性CTL的关键作用，我们还将确定CD 8-T细胞如何在肿瘤细胞中发挥作用。 特异性PKCη缺失影响它们的活化和功能。在目标2中，我们将使用具有组成型Cre- Foxp 3 + Treg中PKCη（和GIT 2）介导的缺失，以分析体内如何破坏CTLA 4-PKCη 信号传导影响肿瘤免疫和肿瘤微环境。我们将使用临床前肿瘤模型， 包括携带BrafV 600 E突变的黑色素瘤和基因工程小鼠肝细胞 癌模型我们将重点关注肿瘤浸润性Treg，CD 8+细胞和DC，并探索其在肿瘤发生发展中的作用。 Treg介导的共刺激性CD 80/CD 86配体从抗原- 呈递细胞，其在Prkch-/- Treg中受损。如果Aim 1研究揭示了PKCη在CD 8+细胞中的重要作用， T细胞，我们将另外分析肿瘤生长和肿瘤微环境中的小鼠与CD 8特异性 Prkch缺失。在目标3中，我们将使用具有他莫昔芬（Tam）诱导的、时间控制的Treg特异性表达的小鼠。 PKCη缺失，单独或与辅助治疗联合，以研究这些治疗的疗效。 操作，使用目标2中的肿瘤模型。这些研究将受益于所有工具和模型的可用性 和专家合作者。我们希望这个项目提供一个新的途径的机制的理解 Treg用来抑制肿瘤特异性免疫并促进肿瘤生长。这种理解可能会导致， 反过来，基于CTLA 4-PKCη信号传导的选择性失活的新型癌症免疫疗法 在Treg中的免疫调节通路，导致以最小的自身免疫相关副作用抑制促癌Treg。

ABSTRACT Regulatory T cells accumulate and correlate with poor prognosis in many cancers, and ablation of Treg together with other therapies is being explored in the clinic. The success of this approach requires, however, elucidation of the mechanisms of regulation and action of Treg in order to provide new, more selective targets for rational immunotherapy. This proposal derives from our discovery of a novel Treg-intrinsic signaling pathway required for contact-dependent suppression of tumor immunity. In this pathway, Treg-expressed CTLA4 physically associates with protein kinase C-eta (PKCη) and, upon CTLA4 engagement, recruits a PAK- PIX-GIT complex required for focal adhesion disassembly. PKCη deletion or expression of a CTLA4 non- interacting PKCη mutant impaired Treg ability to inhibit tumor-specific immunity, but not autoimmune colitis. We hypothesize that this novel CTLA4 signaling axis is obligatory for contact-dependent suppression of tumor immunity by Foxp3+ Treg. Our overarching goal is to further explore at the mechanistic and animal levels this signaling pathway and its functional implications for tumor immunity. In Aim 1, we will mechanistically analyze the molecular pathways utilized by CTLA4 and PKCη to recruit and activate the GIT2-PAK2-αPIX complex and mediate contact-dependent suppression in mouse and human Treg, and determine how disruption of this signaling pathway affects Treg suppression. We will also use 2-photon microscopy to study intratumoral Treg- DC dynamics. Given the critical role of CD8+ T cells as tumor-killing CTL, we will also determine how CD8- specific PKCη deletion affects their activation and functions. In Aim 2, we will use mice with constitutive, Cre- mediated deletion of PKCη (and GIT2) in Foxp3+ Treg to analyze in vivo how disrupting CTLA4-PKCη signaling affects tumor immunity and the tumor microenvironment. We will use preclinical tumor models, including a melanoma carrying the BrafV600E mutation and a genetically engineered mouse hepatocellular carcinoma model. We will focus on tumor-infiltrating Treg, CD8+ cells and DC, and explore the transendocytosis pathway of Treg-mediated depletion of costimulatory CD80/CD86 ligands from antigen- presenting cells, which is impaired in Prkch–/– Treg. If Aim 1 studies reveal an important role for PKCη in CD8+ T cells, we will additionally analyze tumor growth and the tumor microenvironment in mice with CD8-specific Prkch deletion. In Aim 3, we will use mice with a tamoxifen (Tam)-induced, time-controlled Treg-specific PKCη deletion, either alone or in combination with adjunct therapies, to study the therapeutic effects of these manipulations, using the tumor models in Aim 2. The studies will benefit from availability of all tools and models and from expert collaborators. We expect this project to provide mechanistic understanding of a novel pathway that Treg utilize to suppress tumor-specific immunity and promote tumor growth. This understanding could lead, in turn, to novel cancer immunotherapies based on selective inactivation of the CTLA4-PKCη signaling pathway in Treg, resulting in inhibition of cancer-promoting Treg with minimal autoimmune-related side effects.