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

Treg 细胞固有的 CTLA4-PKC-eta 信号通路介导接触依赖性肿瘤免疫抑制：癌症免疫治疗的新靶点

• 批准号: 10310411
• 负责人: Michael Croft
• 金额: $ 51.26万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10310411
• 关键词: Ablation; Address; Affect; Animals; Antigen-Presenting Cells; Autoimmune; Autoimmune Diseases; Autoimmunity; Biochemical; CD8-Positive T-Lymphocytes; CD80 gene; CD86 gene; CD8B1 gene; CTLA4 gene; Cancer Model; Cell model; Cells; Cellular biology; Clinic; Colitis; Collaborations; Complex; Enzymes; Eragrostis; FOXP3 gene; Family member; Focal Adhesions; GIT2 gene; Genetic; Genetic Models; Genetically Engineered Mouse; Goals; Growth; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Human; Immune response; Immune system; Immunity; Immunosuppression; Immunotherapy; Impairment; In Vitro; Lead; Ligands; Malignant Neoplasms; Mediating; Membrane Proteins; Methodology; Modeling; Molecular; Mus; Mutation; Pathway interactions; Phosphotransferases; Prognosis; Protein Kinase C; Reagent; Regulation; Regulatory T-Lymphocyte; Risk; Role; Signal Pathway; Signal Transduction; Solid; T-Cell Activation; T-Lymphocyte; Tamoxifen; Technical Expertise; Therapeutic Effect; Therapeutic Studies; Therapeutic Uses; Time; Transplantation; Tumor Immunity; Tumor Suppression; anti-CTLA4; base; cancer immunotherapy; effector T cell; high resolution imaging; immunological synapse; implantation; improved; in vivo; innovation; liver cancer model; melanoma; mouse model; mutant; novel; pre-clinical; prevent; recruit; side effect; success; tool; tumor; tumor growth; tumor microenvironment; tumor-immune system interactions; two photon microscopy

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.

抽象的 调节性 T 细胞的积累与许多癌症的不良预后相关，而 Treg 细胞的消融 然而，这种方法的成功需要与其他疗法一起在临床中进行探索。 阐明 Treg 的调节和作用机制，以提供新的、更具选择性的靶点 该提议源自我们对一种新型 Treg 内在信号传导的发现。 接触依赖性抑制肿瘤免疫所需的途径 在该途径中，Treg 表达。 CTLA4 与蛋白激酶 C-eta (PKCη) 物理结合，并且在 CTLA4 参与后，招募 PAK- 粘着斑分解所需的 PIX-GIT 复合物 PKCη 缺失或 CTLA4 非表达。 相互作用的 PKCη 突变体损害了 Treg 抑制肿瘤特异性免疫的能力，但不抑制自身免疫性结肠炎。 我们发现这种新型 CTLA4 信号轴对于接触依赖性肿瘤抑制是必需的 Foxp3+ Treg 的免疫作用我们的首要目标是在机制和动物水平上进一步探索这一点。 在目标1中，我们将从机制上分析信号通路及其对肿瘤免疫的功能影响。 CTLA4 和 PKCη 利用分子途径招募和激活 GIT2-PAK2-αPIX 复合物， 介导小鼠和人类 Treg 中的接触依赖性抑制，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 2 光子显微镜来研究肿瘤内 Treg- 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- 如何发挥作用。 特定的 PKCη 缺失会影响其激活和功能。在目标 2 中，我们将使用具有组成型 Cre- 的小鼠。 介导 Foxp3+ Treg 中 PKCη（和 GIT2）的删除，以分析体内如何破坏 CTLA4-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 BrafV600E 突变的黑色素瘤和基因工程小鼠肝细胞 我们将重点关注肿瘤浸润性Treg、CD8+细胞和DC，并探索其作用机制。 Treg 介导的抗原共刺激 CD80/CD86 配体消耗的转内吞途径 Prkch–/– Treg 中的呈递细胞受损，Aim 1 研究揭示了 PKCη 在 CD8+ 中的重要作用。 T 细胞，我们将另外分析 CD8 特异性小鼠的肿瘤生长和肿瘤微环境 在目标 3 中，我们将使用具有他莫昔芬 (Tam) 诱导的时间控制 Treg 特异性的小鼠。 PKCη 缺失，单独或与辅助疗法联合，以研究这些疗法的治疗效果 使用目标 2 中的肿瘤模型进行操作。研究将受益于所有工具和模型的可用性 我们希望这个项目能够提供对新途径的机械理解。 Treg 利用来抑制肿瘤特异性免疫并促进肿瘤生长。 反过来，基于 CTLA4-PKCη 信号选择性失活的新型癌症免疫疗法 Treg 通路，从而抑制促癌 Treg，同时将自身免疫相关副作用降至最低。