University of Virginia Health System

Recent studies have demonstrated that many patients make immune responses against their tumors, though they usually ultimately fail to control tumor outgrowth. Extensive investigations have identified many of the proteins that are the targets of these immune responses, raising the possibility of designing vaccines that enhance anti-tumor immune responses. The focus of our work is on the use of dendritic cells (DC) as immunotherapeutic vaccines against melanoma. DC are extremely potent antigen presenting cells that express MHC class I and class II molecules and an array of costimulatory molecules, which are required for the activation of naïve T cells. Thus, DC make ideal candidates as vaccines for the generation of immune responses against tumors. We are studying immune responses against tyrosinase, a melanoma associated antigen that is frequently the target of patient immune responses. Our approach is two fold. First, as CD4+ T cells are generally required for the activation immune responses, we are interested in defining MHC class II-restricted peptide epitopes that CD4+ T cells recognize. In collaboration with Dr Donald Hunt in the Department of Chemistry, we are using mass spectrometry to directly identify the HLA-DR4-restricted peptides presented at the surface of DC after DC have internalized tyrosinase via a variety of techniques. These studies are complemented by the use of computer assisted algorithms that select candidate peptides by their predicted ability to bind to MHC molecules. Identified peptides are then analyzed in a murine model system for their ability to activate CD4+ T cells, and whether the resulting T cells can either directly respond to tyrosinase-expressing tumors, or can enhance CD8+ T cell responses against the tumor. We are generating a transgenic mouse model that either expresses tyrosinase or not, in order to study the impact of tolerance on the CD4+ T cell response to this protein. Our second approach is to examine the efficiency that DC process and present MHC class I and class II-restricted peptides derived from tyrosinase. We hypothesize that DC will present MHC class I and class II-restricted peptides for a greater duration than achieved by exogenously pulsing DC with defined synthetic peptides if DC acquire an endogenous reservoir of antigen. Currently, we are targeting tyrosinase to DC via several different methodologies (e.g. apoptotic tumor cells, Fc-fusion proteins and recombinant viruses) and determining the relative efficiency of presentation of tyrosinase-derived peptides.

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