HS1 Function in Dendritic Cells

描述（由申请人提供）：树突状细胞（DC）是有效的抗原呈递细胞（APC），因为它能够用抗原刺激幼稚T细胞。DC也是疫苗开发的一个有前途的新来源。内吞作用和迁移是DC启动免疫应答能力的两个关键过程。肌动蛋白细胞骨架的调节对于这些过程是必不可少的，并且依赖于一组造血特异性肌动蛋白调节蛋白。这些蛋白质之一是HS 1，其在DC中的功能尚未研究。我们已经发现，HS 1是DC有效呈递抗原所必需的，特别是在可溶性抗原的受体介导的内吞作用中。此外，HS 1-/- DCs在transwell迁移实验中表现出向趋化因子的异常迁移，并在组织和定位上存在缺陷，富含肌动蛋白的结构参与迁移、粘附和基质降解。通过调节肌动蛋白细胞骨架动力学，我们认为HS 1在DC有效的抗原提呈中起重要作用。通过免疫荧光通过内吞途径运输荧光标记的抗原将鉴定到达溶酶体区室的缺陷。检测特异性肽-MHC复合物的抗体将用于蛋白质印迹和流式细胞术测定，以确定抗原加工、MHC加载或肽-MHC分子向细胞表面的运输是否需要HS 1。流式细胞术和transwell试验将鉴定HS 1-/-DC上表达的趋化因子受体以及HS 1缺陷对向特定趋化因子迁移的影响。将通过荧光标记耳皮肤中的DC来监测向淋巴器官的体内迁移，并监测向腹膜炎模型中的炎症部位的体内迁移。将通过视频显微镜来表征板状伪足和足体动力学，以确定HS 1在这些结构的形成和持续时间中的作用。HS 1含有SH 3结构域，其介导与参与内吞作用和迁移的蛋白质的相互作用。通过在HS 1-/-DC中表达SH 3结构域点突变体，我们将研究该结构域在介导HS 1功能中的作用。总之，这些研究将使我们能够开始鉴定DC作为APC有效发挥功能所必需的蛋白质网络。了解DC生物学对于有效地利用这些细胞用于疫苗免疫疗法是必要的。通过了解肌动蛋白调节蛋白（如HS 1）在DC功能中的作用，可以靶向这些蛋白质进行修饰，从而使DC能够更有效地用作疫苗。

DESCRIPTION (provided by applicant): Dendritic cells (DCs) are potent antigen presenting cells (APCs) due to the ability to stimulate nai've T cells with antigen. DCs are also a promising new source of vaccine development. Endocytosis and migration are two key processes in the ability of DCs to initiate an immune response. Regulation of the actin cytoskeleton is essential for these processes and depends on a set of hematopoietic-specific actin regulatory proteins. One of these proteins is HS1, for which a function in DCs has not been investigated. We have found that HS1 is required for efficient antigen presentation by DCs, specifically in receptor- mediated endocytosis of soluble antigen. Additionally, HS1-/- DCs have abnormal migration toward a chemoattractant in transwell migration assays and have defects in organization and localization podosomes, actin-rich structures involve in migration, adhesion and matrix degradation. Through regulating actin cytoskeletal dynamics, we believe that HS1 plays an important role in efficient antigen presentation by DCs. Trafficking fluorescently tagged antigen by immunofluorescence through the endocytic pathway will identify defects in reaching the lysosomal compartment. Antibodies that detect specific peptide-MHC complexes will be used in western blotting and flow cytometry assays to determine if antigen processing, MHC loading or trafficking of peptide-MHC molecules to the cell surface requires HS1. Flow cytometry and transwell assays will identify the chemokine receptors expressed on the HS1-/- DCs and the effects of HS1- deficiency on migration towards specific chemokines. In vivo migration will be monitored to lymphoid organs by fluorescently labeling DCs in ear skin and to sites of inflammation in a peritonitis model. Lamellipodial and podosome dynamics will be characterized by video microscopy to determine the role of HS1 in the formation and duration of these structures. HS1 contains an SH3 domain that mediates interactions with proteins involved in endocytosis and migration. Through expression of SH3 domain point mutants in HS1-/- DCs, we will investigate the role of this domain in mediating HS1 functions. Together, these studies will allow us to begin to identify the networks of proteins necessary for DCs to function efficiently as APCs. An understanding of DC biology is necessary to utilize these cells efficiently for vaccine immunotherapies. Through understanding the role of actin regulatory proteins, such as HS1, in DC function, these proteins can be targeted for modification that will allow DCs to be tailored to serve more effectively as vaccines.