The laboratory is involved in studies of two proteins that are encoded by genes that are selectively expressed in 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 surface and localized to unique endocytotic vesicles termed Birbeck granules, its physiologic ligands are only beginning to be identified and its function is incompletely characterized. We have generated soluble forms of recombinant Langerin that retain mannan-binding activity in bacteria and plan to utilize these probes to identify and characterize Langerin ligands and thus further define Langerin's role in immunophysiology. MFG-E8 is a second protein that is under active investigation. In accordance with our prediction and consistent with its avb3 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. Studies of the immunogenicity of HIV TAT protein transduction domain (PTD)-containing antigens also continue. Having demonstrated that TAT PTD-antigens have enhanced abilities to elicit cytotoxic lymphocyte responses when used as a component of dendritic cell-based vaccines in proof of principle experiments, we are trying to assess the general utility of the approach and to simplify vaccine delivery. Recent efforts have been largely devoted to developing methodology that will improve the yield and solubility of bacteria-derived candidate tumor antigens encoded by eukaryotic cDNAs (eg. tyrosinase-related protein 2). The final project area involves determining if T cell receptors expressed by clonal T cell malignancies can be used as tumor antigens in vaccines. Both DNA- and protein-based strategies will be tested. The former involves alpha virus-based as well as conventional eukaryotic expression vectors, while the latter takes advantage of our experience with TAT PTD-containing protein antigens. cDNAs encoding alpha and beta chains from a murine T cell lymphoma of interest have been cloned and sequenced and preliminary experiments are underway.
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