The mRNA profiles of the macrophages with or without Trastuzumab-dependent tumor phagocytosis

Therapeutic antibodies trigger antibody-dependent cellular cytotoxicity (ADCC) of cancer cells and also their antibody-dependent cellular phagocytosis (ADCP) by tumor-associated macrophages (TAMs). We report here our unexpected finding that TAMs that have undergone ADCP of tumor cells induced by therapeutic antibodies display immunosuppressive characteristics and inhibit the proliferation and tumoricidal effects of NK and tumor-specific CD8+ T cells in breast cancers and lymphomas. Mechanistically, we show that DNA released from the phagocytosed tumor cells after ADCP activates caspase 1 inflammasome, leading to IL-1β-mediated PD-L1 and IDO upregulation in these cells. Combined treatment with anti- HER2 antibody and inhibitors of PD-L1 and IDO increased the infiltration of NK and CD8+ T cells and enhanced anti- HER2 therapeutic efficacy in mouse models of HER2+ breast cancers. Furthermore, neo-adjuvant Trastuzumab therapy significantly increased PD-L1 and IDO expression in the TAMs of HER2+ breast cancer patients, which correlate with poor Trastuzumab response and reduced NK and CD8+ T cells in the tumors. Collectively, our findings unveil an unexpected role of ADCP induced by therapeutic antibodies in cancer immunosuppression, and suggest that antibody plus immune checkpoint blockade may provide synergistic therapeutic effects in cancer patients. HER2+ breast cancer cells pre-dyed by CellTtracker Deep Red Dye (Cat# C34565, Thermo Fisher) were co-cultured with macrophages pre-dyed by CellTracker CMFDA Dye (Cat# C7025, Thermo Fisher) with or without Trastuzumab (Roche). The ratio between tumor cells and macrophages was 1:5. After 24 hr, the cells were rigorously washed by PBS, digested by 10×TrypLE Selected Enzyme (Cat# A1217701, Gibco) diluted 5× in PBS with 1mM EDTA and sorted by CD14 Isolation Kit (Cat# 130-050-201, Miltenyi Biotec) to collect macrophages followed the instructions. we named untreated macrophages as Mɸ and macrophages pre-incubated with HER2+ tumor cells in the presence or absence of Trastuzumab as ADCP-Mɸs or TC-Mɸs respectively. Total RNA was extracted from macrophages with indicated treatments (5 x 106 cells) using the TRIzol Reagent (Cat#A33251, Thermo Fisher). The quality of extracted RNA was assessed using the NanoDrop ND-2000 spectrophotometer.