Characterization of the Natural Killer Cell Cytokine Response to Antibody-Coated Tumor Cells

Cells from normal breast tissue have on their surface a specific protein known as HER2. Breast cancercells over-express this protein around twenty thousand times that of normal tissue. Targeting of HER2 over-expressing tumor cells can be achieved using an agent called a monoclonal antibody (mAb) that specifically binds to over expressed HER2 on the surface of the breast cancer cells. Binding of this antibody to HER2 can potentially inhibit tumor cell growth or lead to tumor cell destruction by the immune system. Unfortunately, only 20-40% of patients with HER2 over-expressing breast cancers currently respond to this treatment. We show here that natural killer (NK) cells secrete a distinct profile of potent immune stimulatory cytokines and T cell-recruiting chemokines in response to dual stimulation with mAb-coated tumor cells and interleukin (IL)-12. Cytokine production was dependent upon synergistic signals mediated by the activating receptor for the Fc portion of IgG (Fc-gamma-RIIIa) and the IL-12 receptor expressed on NK cells. The importance of this NK cell cytokine profile was confirmed in a phase I trial of breast cancer patients receiving IL-12 in combination with the anti-HER2 mAb, trastuzumab. Upon examination of the intracellular signaling pathways within primary human NK cells responsible for the elevated cytokine production, we found that levels of activated Syk and extracellular signal-related kinase (Erk) were synergistically elevated in Fc-gamma-RIIIa and IL-12 co-stimuated NK cells as compared to either stimulation alone. Moreover, inhibitor studies suggested that these molecules were critical for synergistic produciton of IFN-gamma. Interestingly, inhibition of lipid rafts within NK cell membranes abroaged the enhanced Erk activation and subsequent IFN-gamma production, suggesting the importance of lipid raft signaling in mediating cytokine production. Confocal microscopy of NK cells confirmed co-localization of both Fc-gamma-RIIIa and the IL-12R within areas of lipid raft micro-domains. Our studies also reveal a role for the inositol phosphatase SHIP-1 in the down-regulation of Fc-gamma-RIIIa-mediated NK cell cytokine produciton. Taken together, these results enhance our overall understanding of the NK cell response to mAb-coated tumor cells and provide a rationale for our attempts to enhance NK cells function for therapeutic gain.