Function of Wdfy4 in cross-presentation and immunity

Wdfy4在交叉呈递和免疫中的功能

• 批准号: 10430144
• 负责人: Kenneth M Murphy
• 金额: $ 50.73万
• 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/10430144
• 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; 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.

摘要 检查点阻断免疫疗法依赖于从正常的抑制性对照中释放肿瘤特异性T细胞， 并对几种癌症的治疗产生了重大影响。然而，患者的应答率 对检查站封锁的处理仍需改进。对检查站封锁无反应 可能由多种原因引起，包括CD8T细胞的树突状细胞启动失败。事实上，检查站 最近研究表明，BLOKADE的疗效依赖于树突状细胞的cDC1亚群。 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，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血统的人 需要BATF3转录因子，在体内对肿瘤排斥反应至关重要。交叉演示是核心 对于cDC1启动肿瘤特异性DC8T细胞的能力，但这一过程仍然知之甚少。 我们已经对cDC1中的交叉呈现机制进行了分子解剖。我们的RA- 建议在基础水平上理解交叉陈述可以用来改善对CAN-2的治疗。 CER患者通过减少对检查站封锁的无反应性。此外，这可能是潜在的 用于改进癌症中的树突状细胞疫苗接种方法。此应用程序构建在我们发现的 第一个基因是cDC1在体内交叉呈递所必需的，并且对抗肿瘤复发至关重要。 海绵。我们在主cDC1的CRISPR/Cas9屏幕中确定了交叉演示所需的Wdfy4 我们已经开发了Wdfy4-/-鼠标模型，这是当前应用的基础。我们的 初步数据显示，Wdfy4-/-小鼠在交叉呈现方面存在严重缺陷，缺乏 激发CD8 T细胞对抗病毒和肿瘤。我们建议使用此模型来确定 WDFY4‘S在体内的作用，并确定WDFY4支持体内交叉呈现的机制 CDC1.此外，我们将确定WDFY4蛋白这一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 利用APEX2融合蛋白和电子显微镜对WDFY4进行定位。 Py，并将内源性WDFY4定位在原代人和小鼠的cDC1中。第二，我们将确定WDFY4 通过测试Clec9a或Dec205是否交互，在cDC1中进行交叉演示时合作的交互合作伙伴 对于WDFY4，通过免疫沉淀和质谱分析鉴定WDFY4相互作用的蛋白质。 SIS，通过邻近标记，以及通过测试这些相互作用的蛋白质是否需要交叉呈现。