PROJECT SUMMARY In response to NCI’s Provocative Question #11, we propose to investigate the mechanisms of action of radiotherapy (RT) on the function of dendritic cells (DCs) and other myeloid cells, and how these mechanisms affect the efficacy of immunotherapies. Given their unique role in activating and modulating new antigen- specific T cell immune responses, our long-term goal is to harness DCs for immunotherapy. Currently, there is a fundamental mechanistic gap in our understanding of the effect of RT on DC subsets and other myeloid cells localized in the tumor microenvironment (TME). This gap represents an important problem for the rational combination of RT with immunotherapies. Recent data in tumor-free mice demonstrated that ionizing radiation (IR) differentially affects DC subsets, causing the rapid death of immunogenic but not tolerogenic DCs. Furthermore, IR changes the gene signature of DC subsets and, consequently, their capacity to promote antigen-specific regulatory T cells (Tregs). Therefore, exposure of skin to IR promotes the growth of tumors transplanted one-day post-IR exposure through a mechanism dependent on tolerogenic DC subsets and Tregs. Based on these findings, we hypothesize that tumor-localized RT will induce a shift in the proportion of DC subsets localized in the TME by promoting the survival and function of tolerogenic DCs, which in turn will induce T cell-mediated tolerance. We further hypothesize that this unstudied effect of IR on DC subsets and other myeloid cells will significantly impact the outcome of RT/immunotherapy combination strategies. In three specific aims, we propose to perform an unbiased characterization of myeloid cells localized in the TME following tumor-targeted RT and RT/immunotherapy combinations using newly available technology, CyTOF, and RNA-seq. We will use mouse models that resemble melanoma-driving mutations in patients, and image- guided RT that allows for fractionation and stereotactic delivery schemes similar to those used in clinical practice. We will correlate these characterizations with the generation of tumor-specific T cell responses. We anticipate that findings obtained from this proposal will enhance our current understanding of DC biology and function in response to RT, and positively impact the rational design of combination strategies.