The laboratory has been involved in studies of two proteins that are encoded by genes that are selectively expressed by immature murine skin-derived dendritic cells. One of the proteins is a C-type lectin termed """"""""Langerin"""""""" because of its restricted expression in Langerhans cells, a population of dendritic cells that are found exclusively in stratified squamous epithelia. Although Langerin is known to be expressed on cell surfaces and localizes to unique endocytotic vesicles termed Birbeck granules, its physiologic ligands are only beginning to be identified and its function is incompletely characterized. We generated soluble forms of recombinant Langerin that retained mannan-binding activity and are utilized these probes to identify and characterize Langerin ligands and to further define Langerin's role in immunophysiology. One area of investigation focuses on identification of Langerin ligands that are associated with microbial pathogens and definition of the role of this protein as an important pathogen recognition receptor. Another area of investigation focused on characterization of endogenous Langerin ligands. Our results indicate that Langerin binds selectively to several extracellular matrix molecules, including the alpha chain of type I procollagen and cell-associated fibronectin. These results implicate Langerin in interactions beween Langerhans cells and extracellular matrix; interactions that may be important for Langerhans cell development or trafficking. MFG-E8 is a second protein that is under active investigation. In accordance with our prediction and consistent with its alpha-v beta-3 integrin- and phosphatidyl serine binding activity, others have demonstrated that MFG-E8 can mediate the uptake of apoptotic cells by macrophages. We have generated several MFG-E8 mouse mutants to determine if MFG-E8 has a non-redundant role in apoptotic cell uptake in vivo and, further, to determine if MFG-E8 is involved in cross presentation of antigens that are not expressed by antigen presenting cells. Because MFG-E8 is homologous to del1, a protein expressed exclusively by endothelial cells in embryos and in tumors, we are also defining the role that MFG-E8 may play in angiogeneis and tumorigenesis. Results, to date, indicate that MFG-E8 promotes tumorigenesis in both orthotopic and transgenic models of cancer in mice (melanoma, non-melanoma skin cancer and pancreatic beta-cell cancer). In addition, ongoing experiments suggest that MFG-E8 may be a relevant therapeutic target in cancer. The final project area in the laboratory involves testing the feasibility of using T cell receptor proteins expressed by clonal T cell malignancies as tumor antigens in vaccines. DNA-based (genetic vaccination) strategies are being emphasized because of concerns regarding the practicality of patient-specific therapies. Both alpha virus-based (self-replicating) as well as conventional eukaryotic expression vectors are being utilized. A reproducible model involving subcutaneous growth of T cell lymphomas in mice has been established in the laboratory. cDNAs encoding T cell receptor alpha and beta chains from this murine T cell lymphoma have been cloned, sequenced and introduced into relevant vectors, and testing of vaccination regimens has begun. We have determined that at least some of the constructs that we have generated have immunogenicity, and are in the process of optimizing regimens and identifying adjuvant strategies that will improve the therapeutic potential of the vaccine.
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