Project Summary Immune checkpoint inhibitors, particularly anti-PD-1/PD-L1 (programmed cell death-1/ligand), represents an emerging and novel therapeutic class with clinical efficacy in many cancers. As such, characterizing features predictive of response, immune changes on therapy, and long-term clinical outcomes are critical objectives for a large and growing population. Currently, we remain largely unable to predict individual patient benefit from these agents. Using pre-treatment samples from patients who received anti-PD-1/PD-L1, we identified that expression of major histocompatibility class II (MHC-II) strongly correlated with response to therapy, as well as CD4 and CD8 T cell infiltration. Cell line studies also suggested that MHC-II expression also correlates with pro-immune gene signatures, including pathways such as “T cell receptor signaling”, “PD-1 reactome”, and “allograft rejection.” In a larger cohort, we will use qualitative and quantitative immunohistochemistry methods to verify and establish the predictive value of MHC-II expression on benefit to anti-PD-1/PD-L1 (Aim 1). We will also assess whether MHC-II may be induced by common melanoma therapeutics in vitro. Next, we will leverage cutting edge multiplexed mass cytometry to characterize the evolving anti-tumor immune response unleashed by anti-PD-1. Available data suggests that in anti-PD-1/PD-L1 responsive tumors, a pre-existing immune response exists that is released by the blockade of PD-1/PD-L1. As such, we hypothesize that peripheral and tumor infiltrating immune cells may be distinct prior to, and following treatment among treatment responders. To achieve unprecedented longitudinal characterization of multiple evolving immune subsets, we will perform high-dimensional multiplexed mass cytometry analysis on peripheral blood (obtained at baseline, 3 weeks, 12 weeks, 6 months after treatment) and tumor biopsies (baseline and 3 weeks) (Aim 2). Finally, while many patients respond to anti-PD-1/PD-L1, the molecular basis, incidence, timing, and clinical characteristics of acquired resistance are totally uncharacterized. Further, the incidence of chronic and delayed toxicities has not been explored. We plan to collect retrospective clinical data and tumor samples on a large population of patients who respond and then progress, and explore toxicities experienced by long-term survivors treated with anti-PD-1/PD-L1 (Aim 3). These studies, in conjunction with internationally recognized mentors and advisors, and a rigorous training plan, will provide the optimal conditions from which to develop an independent research career performing high-impact patient-oriented research.