The long term objective of this proposal is to gain insight into the mechanisms involved in Toll-like receptor (TLR) enhancement of anti-tumor immune therapy and to translate these findings towards practical immunotherapies for patients with metastatic tumors.  Melanoma is a common cancer and a leading cause of loss of productive years.  Many active trials for metastatic melanoma utilize tumor antigen targeted immunotherapies.  Preliminary data suggests that the TLR7 agonist, imiquimod, is an effective adjuvant to immunization with recombinant Listeria monocytogenes (rLM) expressing melanoma antigens.  Adjuvant use of imiquimod with the rLM vaccine leads to profound protection from melanoma and to development of localized vitiligo.  We hypothesize that stimulation of TLR7 leads to activation, maturation, and migration of dendritic cells (DCs) to the tumor site and to the draining lymph nodes.  This DC activity leads to enhanced presentation of both vaccine and non-vaccine melanoma associated antigens to cytolytic CD8+ T-cells and potentially enhanced determinant spreading.  Aim 1 of this proposal uses a mouse melanoma model to determine the molecular mechanism involved in TLR7/8 enhancement of Listeria based anti-melanoma vaccines in both prophylactic and therapeutic modalities.  Aim 2 will determine the effects of TLR7/8 on mouse and human DCs in vitro and optimize a combinatorial approach to melanoma immunotherapy using both DCs and rLM vaccines.  Aim 3 will study the direct effects of TLR7/8 activation on the immune response in patients with high risk melanoma and will serve as a stepping stone to futre combinatorial therapies in humans.  In this aim, we will determine the non-specific cellular immune response in the skin and sentinel lymph node as well as the melanoma-antigen-specific immune response in the peripheral blood and sentinel lymph node.  TLRs are a critical part of the immune system's ability to generate host defense to pathogens.  This proposal is aimed at understanding the relationship between host immunity and self tolerance as it relates to tumor immunotherapy.  The team of investigators is multidisciplinary and includes co-investigators from Microbiology and Immunology, Hematology-Oncology, Surgical-Oncology, Neurosurgery, and Dermatology.  Several of these investigators are already involved in clinical trials of melanoma immunotherapies in humans.  These studies will lead to a better understanding of the mechanisms involved in breaking self tolerance and will enhance the design of future immunotherapies for melanoma and other, less immunogenic cancers.  This proposal is designed to provide a mechanistic foundation for the natural translation of our preliminary data--the development of GMP grade vaccines for clinical trials in humans.  Development of GMP vaccines is outside the scope of this proposal and will be pursued under another grant mechanism.  Project Narrative: Melanoma is a cancer that is rapidly increasing in prevalence in the U.S. The studies proposed in this application will lead to a better understanding of the mechanisms involved in cancer immune therapies and will enhance the design of future immunotherapies for melanoma and other cancers that can evade the immune system.  This proposal is designed to provide a mechanistic foundation for the natural translation of our preliminary data -- the development of safe vaccines for realistic and practical clinical trials in humans with metastatic cancers.