Function of Wdfy4 in cross-presentation and immunity

Wdfy4在交叉呈递和免疫中的功能

• 批准号: 10203752
• 负责人: Kenneth M Murphy
• 金额: $ 51.36万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10203752
• 关键词: Alloantigen; Antibody Response; Antigen-Presenting Cells; Antigens; Area; Autoimmune; B-Lymphocytes; Bacteria; Biology; CD8-Positive T-Lymphocytes; CRISPR screen; Cancer Patient; Cells; Chimeric Proteins; Clinical; Clinical assessments; Cross Presentation; Data; Defect; Dendritic Cells; Development; Diagnostic; Dissection; Effectiveness; Electron Microscopy; Electrons; Epitopes; Failure; Genes; Genetic; Genetic Transcription; Goals; Human; Immune response; Immunity; Immunofluorescence Microscopy; Immunoprecipitation; Immunotherapy; In Vitro; Knockout Mice; Label; Lead; Listeria monocytogenes; Literature; Location; Lupus; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Modeling; Molecular; Mus; Parasites; Pathway interactions; Patients; Physiological; Process; Property; Proteins; Role; T cell response; T-Lymphocyte; Testing; Therapeutic; Toxoplasma gondii; Translations; Tumor Antigens; Tumor Immunity; Tweens; Virus; Virus Diseases; Work; adaptive immune response; antigen-specific T cells; cancer immunotherapy; cancer therapy; cancer type; checkpoint inhibition; dendritic cell vaccination; genome wide association study; immune checkpoint blockade; immunogenic; immunogenicity; improved; in vivo; inhibitor/antagonist; innovation; insight; monocyte; mouse model; neoantigens; novel; response; transcription factor; tumor; virtual

ABSTRACT Checkpoint blockade immunotherapy relies on releasing tumor-specific T cells from normal inhibitory control, and has significantly impacted treatment for several types of cancer. However, response rates in patients treated with checkpoint blockade still need to be improved. Non-responsiveness to checkpoint blockade could result from many causes, including failure of dendritic cell priming of CD8 T cells. Indeed, checkpoint blockade has recently been shown to be dependent on the cDC1 subset of dendritic cells for its effectiveness. The cDC1 lineage of dendritic cells is the major cross-presenting cell that primes CD8 T cells and has been the subject of our recent molecular and developmental analysis. We were the first to identify that the cDC1 lineage requires the BATF3 transcription factor and is critical in vivo for tumor rejection. Cross-presentation is central to the ability of cDC1 to prime tumor-specific DC8 T cells, but this process has remained poorly understood. We have undertaken a molecular dissection of the mechanisms of cross-presentation in cDC1. Our ra- tionale is that understanding cross-presentation at a basic level could be used to improve treatment of can- cer patients by reducing non-responsiveness to checkpoint blockade. In addition, this could potentially be used to improve dendritic cell vaccination approaches in cancer. This application builds on our discovery of the first gene that is absolutely required for cDC1 cross-presentation in vivo and is critical for anti-tumor re- sponses. We identified Wdfy4 in a CRISPR/Cas9 screen in primary cDC1 as required for cross-presentation and we have already developed the Wdfy4-/- mouse model which is the basis for the current application. Our preliminary data show that Wdfy4-/- mice have a profound defect in cross-presentation, lacking the ability to prime CD8 T cells against viruses and tumors. We propose to use this model to determine the full scope of Wdfy4's function in vivo and to determine the mechanism by which WDFY4 supports cross-presentation in cDC1. Further, we will determine the molecular and cellular mechanism for this function of the WDFY4 protein. First, we will determine the intracellular location of WDFY4 in DCs, by using immunofluorescence microscopy of epitope tagged WDFY4 proteins, by localizing WDFY4 using APEX2 fusion proteins and electron microsco- py, and by localizing endogenous WDFY4 in primary human and mouse cDC1. Second we will identify WDFY4 interacting partners that cooperate in cross-presentation in cDC1 by testing whether Clec9a or Dec205 interact with WDFY4, by identifying WDFY4 interacting proteins by immunoprecipitation and mass-spectrometry analy- sis, and by proximity labeling, and by testing if these interacting proteins are required for cross-presentation.

抽象的 检查点阻断免疫疗法依赖于从正常抑制控制中释放肿瘤特异性 T 细胞， 并对多种癌症的治疗产生了重大影响。然而，患者的反应率 检查站封锁治疗仍需改进。对检查站封锁无反应 可能由多种原因引起，包括 CD8 T 细胞的树突状细胞启动失败。确实，检查站 最近已证明封锁的有效性依赖于树突状细胞的 cDC1 子集。 树突状细胞的 cDC1 谱系是启动 CD8 T 细胞的主要交叉呈递细胞，并且一直是 我们最近的分子和发育分析的主题。我们是第一个发现 cDC1 谱系的人 需要 BATF3 转录因子，并且对于体内肿瘤排斥至关重要。交叉展示是核心 CDC1 启动肿瘤特异性 DC8 T 细胞的能力，但这一过程仍然知之甚少。 我们对 cDC1 中交叉呈递的机制进行了分子剖析。我们的RA- 重要的是，在基本层面上理解交叉表现可以用来改善对可能的治疗 通过减少对检查点封锁的无反应来治疗 cer 患者。此外，这可能是 用于改善癌症中的树突状细胞疫苗接种方法。该应用程序建立在我们的发现之上 第一个基因是 CDC1 体内交叉呈递所绝对必需的，并且对于抗肿瘤重新表达至关重要 响应。我们在原代 cDC1 的 CRISPR/Cas9 筛选中根据交叉呈递的需要鉴定了 Wdfy4 我们已经开发了 Wdfy4-/- 小鼠模型，这是当前应用的基础。我们的 初步数据表明，Wdfy4-/-小鼠在交叉呈递方面存在严重缺陷，缺乏 启动 CD8 T 细胞对抗病毒和肿瘤。我们建议使用该模型来确定 Wdfy4 的体内功能并确定 WDFY4 支持体内交叉呈递的机制 cDC1。此外，我们将确定 WDFY4 蛋白此功能的分子和细胞机制。 首先，我们将通过免疫荧光显微镜确定 WDFY4 在 DC 中的细胞内位置 表位标记的 WDFY4 蛋白，通过使用 APEX2 融合蛋白和电子显微镜定位 WDFY4 py，并通过在原代人和小鼠 cDC1 中定位内源性 WDFY4。其次我们将识别 WDFY4 通过测试 Clec9a 或 Dec205 是否交互，在 cDC1 中进行交叉展示的交互伙伴 与 WDFY4 一起，通过免疫沉淀和质谱分析鉴定 WDFY4 相互作用蛋白 sis，并通过邻近标记，并通过测试这些相互作用的蛋白质是否需要交叉呈递。