Abstract 787: The role of monocytes in promoting natural killer cell activation in response to antibody-coated tumor cells and IL-12
Authors: Bhave Neela S. , Parihar Robin , Lewis Adrian , Karpa Volodymyr , Carson William E. 
Published: June 2012

Abstract
Introduction: Previous studies from our group has demonstrated that co-stimulation of NK cells via the interleukin-12 receptor (IL-12R) and the FcgRIIIa activates the extracellular signal-regulated kinase (ERK) signaling pathway in vitro, in murine tumor models and in phase I clinical trials. The ERK pathway in turn promotes the secretion of interferon-gamma (IFN-g) thereby mediating potent anti-tumor effects.
Hypothesis: NK cell cytokine secretion would be significantly enhanced following simultaneous stimulation of the NK cells via the activating receptor, NKG2D and their ligands (such as MICA/B) expressed on monocytes.
Methods: Purified NK cells, Monocytes and peripheral blood mononuclear cells (PBMC) were co-cultured with antibody (Ab)-coated tumors cells in the presence of IL-12. Cell free supernatants were analyzed for the presence of inflammatory cytokines.
Results: Co-stimulation of purified NK cells with trastuzumab-coated HER2+ SKBR3 breast cancer cells and IL-12 (10 ng/mL) resulted in synergistic production of IFN-g (&gt;20,000 pg/mL) as compared to the single conditions (&lt;3000 pg/mL). Cytokine production in response to antibody-coated tumor cells and IL-12 was significantly enhanced in the presence of autologous monocytes (2-3-fold, P&lt;0.05) but not T cells or B cells. Maximum cytokine secretion was observed at 48 hours, and preceded by a 6-fold increase in IFN-g transcript. This enhancement of cytokine response was dependent on cell-cell contact as determined by a Transwell assay. NK cells co-cultured with monocytes (1:1=200,000 cells per well) also secreted significantly higher amounts of TNF-a (&gt;2 fold) and MIP1 alpha (&gt;4 fold) as compared to un-supplemented NK cells exposed to Ab and IL-12. A dose-response effect was observed with increasing numbers of added monocytes. Pre-treatment of monocytes with LPS and/or IFN-alpha led to increased expression of NKG2D ligands (MICA and MICB) and increased the ability of monocytes to act as co-stimulators of NK cell cytokine secretion. The stimulatory effects of MICA/B+ monocytes were duplicated by the use of a MICA over-expressing cell line (C1R-MICA) but not the parental MICA-negative cell line (2-3.2 fold increase). Incubation of C1R MICA cells with a MICA neutralizing antibody prior to co-culture with NK cells led to a significant reduction in IFN-g secretion. The stimulatory effects of monocytes were also observed in whole PBMC in that depletion of monocytes from PBMC markedly inhibited the production of IFN-g by the NK cell compartment whereas supplementation of PBMC with additional monocytes led to dose-dependent increases in cytokine production.
Conclusions: These data suggest that stimulation of NKG2D by monocyte ligands can enhance the NK cell cytokine response to Ab-coated targets. Enhancement of NK monocyte interactions could increase the efficacy of Ab-based anti-cancer therapies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 787. doi:10.1158/1538-7445.AM2011-787