Function of Wdfy4 in cross-presentation and immunity

摘要 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， 并且显著影响了几种癌症的治疗。然而，患者的反应率 检查站封锁治疗仍需改进。对检查点封锁无反应 可能由许多原因引起，包括树突状细胞引发CD 8 T细胞的失败。事实上，检查站 最近显示阻断的有效性依赖于树突细胞的cDC 1亚群。 树突状细胞的cDC 1谱系是引发CD 8 T细胞的主要交叉呈递细胞，并且一直是CD 8 T细胞的主要来源。 我们最近的分子和发育分析的主题。我们是第一个发现cDC 1谱系 需要BATF 3转录因子，并且在体内对于肿瘤排斥至关重要。交叉呈现是核心 cDC 1启动肿瘤特异性DC 8 T细胞的能力，但这一过程仍然知之甚少。 我们对cDC 1中交叉呈递的机制进行了分子解剖。我们的- 在基本水平上理解交叉呈现可以用来改善对can的治疗。 通过降低对检查点阻断的无反应性来治疗患者。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。这个应用程序建立在我们发现的 cDC 1在体内交叉呈递绝对需要的第一个基因，并且对于抗肿瘤再表达至关重要。 海绵。我们在原发性cDC 1的CRISPR/Cas9筛选中鉴定了Wdfy 4作为交叉呈递所需的基因。 并且我们已经开发了Wdfy 4-/-小鼠模型，这是当前应用的基础。我们 初步数据显示，Wdfy 4-/-小鼠在交叉呈递方面存在严重缺陷，缺乏 使CD 8 T细胞对抗病毒和肿瘤。我们建议使用该模型来确定 Wdfy 4在体内的功能，并确定Wdfy 4支持交叉呈递的机制。 cDC1。此外，我们将确定WDFY 4蛋白这种功能的分子和细胞机制。 首先，我们将使用免疫荧光显微镜确定WDFY 4在DC中的细胞内定位 表位标记的WDFY 4蛋白，通过使用APEX 2融合蛋白和电子显微镜定位WDFY 4， py，并通过在原代人和小鼠cDC 1中定位内源性WDFY 4。其次，我们将确定WDFY 4 通过测试Clec 9a或Dec 205是否相互作用， 通过免疫沉淀和质谱分析鉴定WDFY 4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。

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.