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细胞在许多癌症中积聚并与不良预后相关，以及Treg的消融 与其他疗法一起在临床上正在探索中。然而，这种方法的成功需要， 阐明Treg的调节和作用机制，以提供新的、更具选择性的靶点 进行合理的免疫治疗。这一建议源于我们发现了一种新的Treg-内在信号 依赖接触抑制肿瘤免疫所需的途径。在此途径中，Treg-表达 CTLA4在物理上与蛋白激酶C-etA(PKCη)结合，并在CTLA4参与时招募一名PAK- 局部粘连分解所需的PIX-GIT复合体。PKCη缺失或表达CTLA4Non 相互作用的PKCη突变会削弱Treg抑制肿瘤特异性免疫的能力，但不会影响自身免疫性结肠炎。 我们假设这个新的CTLA4信号轴对于接触性抑制肿瘤是必需的。 被Foxp3 Treg豁免。我们的首要目标是在机械和动物层面上进一步探索 信号通路及其在肿瘤免疫中的功能意义。在目标1中，我们将机械地分析 CTLA4和PKCη募集和激活GIT2-PAK2-αPIX复合体的分子途径 在小鼠和人类Treg中介导接触依赖性抑制，并确定如何破坏这种抑制 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- DC动力学。鉴于CD8 T细胞作为肿瘤杀伤CTL的关键作用，我们还将确定CD8- 特定的PKCη缺失会影响其激活和功能。在目标2中，我们将使用具有结构性Cre- Foxp3Treg介导的PKCη(和GIT2)缺失对CTLA4-PKCη的体内干扰作用 信号转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， 包括一个携带BRAFV600E突变的黑色素瘤和一个基因工程小鼠肝细胞 肿瘤模型。我们将重点关注肿瘤浸润性Treg、CD8细胞和DC，并探讨 Treg介导的共刺激CD80/CD86配体耗尽抗原的跨内吞途径 呈现细胞，在Prkch-/-Treg中受损。If Aim 1研究揭示了pkcη在CD8中的重要作用 T细胞，我们还将分析CD8特异性小鼠的肿瘤生长和肿瘤微环境 删除Prkch。在目标3中，我们将使用三苯氧胺()诱导的、时间控制的Treg特异性的小鼠 PKCη缺失，无论是单独或联合辅助治疗，以研究这些治疗效果 操作，使用AIM 2中的肿瘤模型。所有工具和模型的可用性将使研究受益 以及专家合作者。我们希望这个项目能提供对一种新途径的机械性理解 Treg用来抑制肿瘤特异性免疫和促进肿瘤生长。这种理解可能会导致， 反过来，基于选择性失活CTLA4-PKCη信号的新型癌症免疫疗法 在Treg中的途径，导致抑制促癌Treg与最小的自身免疫相关的副作用。

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.