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.
