PROJECT SUMMARY: One in fifty Americans will be diagnosed with melanoma in their lifetime and skin cutaneous melanoma is the deadliest skin cancer. Cancer immunotherapy is a breakthrough approach to treat this disease and cytotoxic CD8+ T-cell tumor infiltration is a critical factor to immunotherapeutic success. As such, identifying effective strategies to increase the magnitude and functionality of the patient’s tumor-specific CD8+ T-cell response remains an important goal. Inhibitory molecules on CD8+ T cells are imperative to T-cell signaling and immune homeostasis. However, elevated expression of these molecules is correlated with dampened antitumor effector response as well as poorer patient survival. FcγRIIB is an inhibitory Fc receptor recently discovered on a subset of CD8+ T cells. FcγRIIB+ CD8+ T cells exhibit increased expression of activation markers, higher proliferative ability, and secrete more proinflammatory cytokines than their FcγRIIB- counterparts in mice and humans, making them imperative to the antitumor response. Recently, we discovered that an immunosuppressive cytokine, fibrinogen-like protein 2 (Fgl2), is a ligand that binds FcγRIIB on CD8+ T cells and induces FcγRIIB- mediated apoptosis of CD8+ T cells. The goal of this research is to interrogate the mechanism by which Fgl2 regulates tumor-specific FcγRIIB+ CD8+ T cells using syngeneic mouse models via the following aims. AIM 1: Determine the cellular source of Fgl2 that critically regulates tumor-specific CD8+ T cells. Our studies show that both Foxp3+ regulatory T cells and CD8+ T cells express Fgl2 at the tumors of mice and humans. Thus, we will determine if Fgl2 secreted by these cell types is necessary and/or sufficient for FcγRIIB-mediated CD8+ T-cell apoptosis, findings which would provide the impetus for subsequent therapeutic targeting of this cell type. AIM 2: Elucidate the mechanism by which the CD8+ T-cell isoform of FcγRIIB induces apoptosis upon ligand binding by Fgl2. Our studies show that CD8+ T cells express a distinct isoform of FcγRIIB that can be bound by Fgl2, but the immediate signaling events after Fgl2-FcγRIIB binding remain unknown. We will identify proteins associated with the intracellular domain of FcγRIIB, specifically after ligand binding by Fgl2. This project is in the context of melanoma as therapeutic success is heavily influenced by CD8+ T-cell infiltration, but the impact of these findings could be applied to other immunologically “hot” tumors where immunotherapies are gaining momentum. Our possession of several mouse models and unique expertise on the role of FcγRIIB in CD8+ T-cell responses uniquely qualify us to address the proposed aims. The impact of the proposed aims is considerable as they will identify novel targets, that when blocked, could rescue a population of memory CD8+ T cells that are crucial to the immune response to tumor. Then, these “rescued” cells would unleash their effector function to improve melanoma patient re...