Function of Wdfy4 in cross-presentation and immunity

Wdfy4在交叉呈递和免疫中的功能

基本信息

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
项目类别：
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

项目摘要

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.

抽象的检查点封锁免疫疗法依赖于从正常抑制性对照中释放肿瘤特异性T细胞，并严重影响了几种类型癌症的治疗方法。但是，患者的反应率用检查点封锁处理仍需要改进。对检查点封锁的无反应性可能是由于许多原因所致，包括CD8 T细胞的树突状细胞启动失败。确实，检查站最近已显示封锁取决于树突状细胞的CDC1子集的有效性。树突状细胞的Cdc1谱系是primes 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。我们是第一个确定CDC1血统的人需要BATF3转录因子，对于肿瘤排斥而言至关重要。交叉呈现是中心的 CDC1能够促进肿瘤特异性DC8 T细胞的能力，但该过程仍然了解不足。我们已经对CDC1中交叉呈阳性机制进行了分子解剖。我们的ra- 蒂奥莱（Tionale CER患者通过将无反应性降低到检查点封锁。另外，这可能是用于改善癌症的树突状细胞疫苗接种方法。此应用程序建立在我们发现的基础上 CDC1交叉呈现体体内绝对必需的第一个基因，对于抗肿瘤重新至关重要赞助。我们在主要CDC1中的CRISPR/CAS9屏幕中确定了WDFY4的跨性别我们已经开发了WDFY4 - / - 鼠标模型，该模型是当前应用程序的基础。我们的初步数据表明，WDFY4 - / - 小鼠在交叉呈递方面具有深刻的缺陷，缺乏能力针对病毒和肿瘤的主要CD8 T细胞。我们建议使用此模型来确定 WDFY4在体内的功能，并确定WDFY4支持交叉分类的机制 CDC1。此外，我们将确定WDFY4蛋白的这种功能的分子和细胞机制。首先，我们将通过使用免疫荧光显微镜来确定DC中WDFY4的细胞内位置通过使用APEX2融合蛋白和电子微型Co- PY，并通过将内源性WDFY4定位在原代人和小鼠CDC1中。第二，我们将确定WDFY4 通过测试CLEC9A或DEC205相互作用的互动合作伙伴在CDC1中合作的合作伙伴使用WDFY4，通过免疫沉淀和质谱分析来鉴定WDFY4相互作用蛋白 SIS，以及通过接近标记，以及测试是否需要这些相互作用的蛋白质才能交叉呈递。