HS1 Function in Dendritic Cells

描述(申请人提供)：树突状细胞(DC)是一种强大的抗原提呈细胞(APC)，因为它能用抗原刺激NAIHAD T细胞。树突状细胞也是疫苗开发的一个很有前途的新来源。内吞和迁移是树突状细胞启动免疫反应的两个关键过程。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的肌动蛋白调节蛋白。其中一种蛋白是HS1，其在DC中的功能尚未被研究。我们发现HS1是DC高效提呈抗原所必需的，尤其是在受体介导的可溶性抗原的内吞作用中。此外，HS1-/-DC在跨孔迁移实验中存在向趋化物质的异常迁移，在足体的组织和定位上存在缺陷，富含肌动蛋白的结构参与迁移、黏附和基质降解。通过调节肌动蛋白的细胞骨架动力学，我们认为HS1在DC有效的抗原提呈中起着重要作用。通过免疫荧光通过内吞途径运输荧光标记的抗原将识别到达溶酶体隔室的缺陷。检测特定多肽-MHC复合体的抗体将用于免疫印迹和流式细胞术分析，以确定抗原处理、MHC负载或将多肽-MHC分子运输到细胞表面是否需要HS1。流式细胞术和Transwell分析将确定HS1-/-DC上表达的趋化因子受体，以及HS1缺乏对向特定趋化因子迁移的影响。通过荧光标记耳部皮肤中的树突状细胞和腹膜炎模型中的炎症部位，体内迁移将被监测到淋巴器官。我们将用视频显微镜来描述薄层和足体的动力学特征，以确定HS1在这些结构的形成和持续时间中的作用。HS1包含一个SH3结构域，它与参与内吞和迁移的蛋白质相互作用。通过SH3结构域点突变在HS1-/-DC中的表达，我们将研究该结构域在调节HS1功能中的作用。综上所述，这些研究将使我们能够开始确定DC有效发挥APC功能所需的蛋白质网络。对DC生物学的了解对于有效利用这些细胞进行疫苗免疫治疗是必要的。通过了解肌动蛋白调节蛋白(如HS1)在DC功能中的作用，这些蛋白可以针对DC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.