PROJECT SUMMARY Our broad, long term objective is to understand the molecular details of Fcγ receptor (FcγR) function, with the goal of improving monoclonal antibody therapy for cancer. The antitumor effects of antibody therapy are largely mediated by FcγR, which become clustered and activated upon binding to the Fc portion of antibodies. Monocytes and macrophages are essential for antibody therapy to be effective, and FcγR activation in these cells can lead to pleiotropic responses. These include phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), production of inflammatory cytokines, or a combination of these. Amongst these activities, the mechanisms by which monocytes / macrophages carry out ADCC remain the least understood. We have recently found that FcγR clustering can induce Granzyme B production by human monocytes and that ligands for Toll-like receptors (TLR) 4 and 8 can significantly enhance this response, as well as induce Granzyme B themselves. Granzyme B is a serine protease expressed predominantly by natural killer (NK) cells and CD8+ T cells, and is required for their cytotoxic functions. Given the central role of Granzyme B in NK cell- mediated ADCC, our finding that monocytes can also produce Granzyme B is remarkable as it provides an as- yet undiscovered anti-tumor function of monoclonal antibodies. Most notably we have also found that nurse- like cells (NLCs) generated from CLL patient blood also produce Granzyme B following FcγR activation and TLR8 ligand treatment, and they can engage in Granzyme-dependent ADCC of CLL cells. This latter observation is of particular interest, as NLCs typically promote CLL-cell survival and proliferation. We therefore hypothesize that Granzyme B production by monocytes and NLCs represents a critical aspect of the effector response to antibody-coated target cells. We propose to explore the significance of these findings specifically related to CLL immunotherapy in the following 3 specific aims: 1) Elucidate the mechanisms of Granzyme B induction by FcγR in monocytes/macrophages and NLCs, 2) Determine the mechanism of augmentation of FcγR-induced Granzyme B production by immune modulators and 3) Analyze the expression and function of Granzyme B by monocytes and monocyte-lineage cells in patient samples and mouse models of CLL. Upon completion of this study we will have fully explored an entirely new mechanism of antibody-mediated destruction of tumors by monocytes/macrophages and NLCs. These mechanistic studies will significantly enhance our understanding of ADCC mediated by these cells, and will likely provide information that can lead to the further enhancement of antibody therapy.