Project Summary Despite recent successes, many melanoma patients still fail to respond to immunotherapy and there is a pressing need for identifying novel therapeutic targets. The primary goal of immunotherapy is to activate tumor- specific immune responses, in which tumor-specific T cells play a crucial role. Although cytotoxic CD8T cells have long been the main focus of research on tumor-specific immunity, growing evidence shows the contribution of CD4T cells to tumor rejection, especially in tumors treated with immunogenic therapy. However, the mechanism of CD4T cell activation in melanoma is less well understood compared to that of CD8T cells. Type 2 conventional dendritic cells (cDC2s) have high antigen presenting capacity to activate antigen-specific CD4T cells, but their role in tumor immunity has not been fully understood due partially to the phenotypic heterogeneity within this cell type. We previously identified CD301b+ DCs as the major cDC2 population in the mouse skin and showed that they transport antigens from the skin to the draining lymph node and efficiently prime antigen-specific CD4T cells. This proposal focuses on understanding their role in host protection and CD4T cell activation in experimental melanoma models in mice. To understand how cDC2 cells respond to the tumor microenvironment at the molecular level, we employ a novel technique of single-cell RNA sequencing to identify subset-specific gene expression changes in cDC2 subsets during melanoma progression. In parallel, we use targeted cell depletion approaches to identify the cellular mechanisms of cDC2-depedent host protection in both untreated melanoma as well as in those treated with immunogenic therapy. These experiments will collectively provide comprehensive understanding on the role of cDC2 subsets in melanoma and potentially reveal new cellular pathways that lead to activation of melanoma-specific CD4T cells.