Defining an appropriate source of tumor antigen and a strategy for engineering DCs in vaccines to induce efficacious T-cell immunity are two of the major challenges currently facing modem tumor vaccine design. Recent evidence suggests that use of tumor cells as a source of antigen has several theoretical advantages over immunization schemes based on delivery of class I restricted epitopes, and that strategies to induce Th1 type T-cell immunity may result in improved objective clinical response rates. Here we propose, through innovative clinical trials and preclinical models, to evaluate and develop improved therapeutic melanoma vaccines by exploiting these approaches. In SPECIFIC AIM 1 we will test the hypothesis that potent anti-tumor immune responses can be induced by immunization with dendritic cells loaded simultaneously with multiple tumor antigens derived from the autologous melanoma. We will conduct 3 concurrent coordinate phase I/II evaluations of immunization against tumor in patients with metastatic melanoma using autologous dendritic ceils. Qualitative and quantitative vaccine-associated changes in immune responses will be correlated with clinical outcome. In SPECIFIC AIM 2, we will directly evaluate and compare the antigen presentation function of DCs in the vaccines prepared, and will use defined murine and human in vitro models to more precisely address issues of antigen internalization, processing and ligand formation. Our expectation is that the comparison and elucidation of key features of antigen uptake and presentation will lead to the development of more efficient strategies for antigen delivery that utilize tumor cells as a source of antigen. In addition, our in vitro analysis of the antigen presentation function of these DC vaccines may enable us to define preclinical assays, and characteristics of a DC vaccine, which are predictive of in vivo immunogenicity. The goal of SPECIFIC AIM 3 is to determine effective means to enhance Th1-type, tumor-reactive CD4+ T cell responses in order to promote and maintain "clinically-important" anti-tumor immunity. We will test the hypothesis that Th2-type anti-tumor T cell responses can be repolarized (corrected) to IFN- gamma associated Th1 T cell responses and be maintained as Th1-biased responses thereafter by "vaccines" containing dendritic cells conditioned to produce Th1-biasing cytokines. Together, these studies will serve as a foundation for designing innovative prospective preclinical models and clinical vaccine trials allowing for greater therapeutic efficacy and durable anti-tumor immunity.