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: 10053328
• 负责人: Michael Croft
• 金额: $ 52.31万
• 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/10053328
• 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; Immune response; Immune system; Immunity; Immunotherapy; Impairment; In Vitro; Lead; Ligands; Malignant Neoplasms; Mediating; Membrane Proteins; Methodology; Modeling; Molecular; Mus; Mutation; Pathway interactions; Phosphotransferases; Primary carcinoma of the liver cells; 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; 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细胞在许多癌症中积累并与预后不良相关，而Treg的消融 在诊所正在探索其他疗法。但是，这种方法的成功需要 阐明Treg的调节机制和作用机制，以提供新的，更有选择性的目标 用于理性免疫疗法。该提议源于我们发现了一种新颖的Treg Intrinsic信号传导 接触依赖性抑制肿瘤免疫所需的途径。在此途径中，表达Treg CTLA4与蛋白激酶C-ETA（PKCη）物理相关，并在CTLA4参与后招募PAK- 局灶性粘附拆卸所需的PIX-GIT复合物。 ctla4 non-的pkcη删除或表达 相互作用的PKCη突变体损害了Treg抑制肿瘤特异性免疫史的能力，但没有自身免疫性结肠炎。 我们假设这个新型的CTLA4信号轴是对肿瘤的接触依赖性抑制的必要性 Foxp3+ Treg的免疫力。我们的总体目标是在机械和动物层面上进一步探索 信号通路及其对肿瘤免疫的功能意义。在AIM 1中，我们将机械地分析 CTLA4和PKCη用于募集和激活Git2-Pak2-αpix络合物的分子途径， 介导小鼠和人treg中的接触依赖性抑制，并确定这是如何破坏的 信号通路会影响Treg抑制。我们还将使用2光子显微镜研究肿瘤内Treg- DC动力学。鉴于CD8+ T细胞作为肿瘤CTL的关键作用，我们还将确定CD8-如何 特定的PKCη缺失会影响其激活和功能。在AIM 2中，我们将使用构成型的小鼠 - Foxp3+ Treg中PKCη（和Git2）的介导的缺失，以分析体内如何破坏CTLA4-PKCη 信号会影响肿瘤免疫学和肿瘤微环境。我们将使用临床前肿瘤模型， 包括携带BRAFV600E突变和基因工程小鼠肝细胞的黑色素瘤 癌模型。我们将专注于肿瘤浸润的Treg，CD8+细胞和DC，并探索 从抗原 - 呈现细胞，在prkch - / - treg中受到损害。如果AIM 1研究揭示了PKCη在CD8+中的重要作用 T细胞，我们将另外分析具有CD8特异性小鼠的肿瘤生长和肿瘤微环境 prkch删除。在AIM 3中，我们将使用小鼠与他莫昔芬（TAM）诱导的，特定的Treg特异性 PKCη缺失，无论是单独还是与辅助疗法，都可以研究这些治疗作用 操作，使用AIM 2中的肿瘤模型。研究将受益于所有工具和模型的可用性 以及专家合作者。我们希望这个项目能够提供对新途径的机械理解 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CTLA4-PKCη信号的选择性失活的新型癌症免疫疗法 Treg的途径，导致抑制癌症的Treg，自身免疫相关的副作用最少。