Project Summary The killing of target cells by therapeutic antibodies is expanding the effective treatment options for a wide range of autoimmune diseases and cancers. Mounting evidence from mouse models, humanized mouse systems and the analysis of human tissues, indicates that macrophages are principal effectors of therapeutic antibodies, mediating the destruction of infected, malignant and immunologically aberrant cells. Fc? receptors (FcR) on the surface of macrophages bind target-cell associated monoclonal IgG class antibodies (mAbs) to initiate antibody-dependent cellular phagocytosis (ADCP) and killing of the target cell. Patient responses to mAb therapies can vary from complete remission to minimal therapeutic effect. One poorly explored possibility is that the variability of the ADCP response is its dependence on macrophage polarization under the influence of immune modulation. Specifically, immunosuppressive environments alter macrophage polarization leading to ineffective ADCP. Conversely, stimulators of interferon genes agonists (STINGa), acting through type 1 interferons (IFN-1) can dramatically potentiate ADCP and overcome immunosuppression. Our overarching goal is to elucidate the mechanistic pathways by which macrophage activation controls FcR function and ADCP using a systems biology approach across in vivo transcriptomics and whole genome CRISPR screens. Identified gene-function relationships for ADCP will be validated in a novel vivo model and translated to human macrophages and therapeutic antibodies. We hypothesize that ADCP is regulated across major axes of macrophage polarization (M0, M1(IFN?/LPS), M(IFN-1/STING), M2(IL4/13) and M(S)) by gene-expression changes of yet undefined genes that modulate the A:I ratios of FcRs, their signaling machinery and innate cellular recognition receptors. Our proposal has two innovative aims that will vastly expand understanding of the regulation FcR-dependent ADCP. In Aim 1, we will elucidate the macrophage genes contributing to differential FcR function and ADCP across M1, M(IFN-1/STING), M2 and M(S). This aim takes advantage of a new CRISPR-based whole genome screening strategy to identify genes that promote and inhibit ADCP in primary derived macrophages. Aim 2 will delineate macrophage gene regulation supporting FcR function and ADCP in vivo. Here we will translate findings from patient data and the CRISPR screen from Aim 1 to define regulators of ADCP in vivo. Both aims will focus on clinically relevant anti-B cell (Rituximab) and anti-T cell (CAMPATH) antibodies, and will generate findings that extend our understanding of Fc-dependent killing mechanism of ADCP.