MECHANISMS OF BACILLUS CALMETTE-GUERIN MEDIATED NATURAL KILLER CELL ACTIVATION

Abstract

Natural killer (NK) cells are of crucial importance for bacillus Calmette-Guerin (BCG) mediated antitumor effects. We defined the mechanisms of BCG mediated NK cell activation in vitro. A standard Cr release assay was used to measure the cytotoxicity of BCG activated NK cells. Using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany) we depleted various immune cell subpopulations from BCG stimulated peripheral blood mononuclear cells to phenotype activated NK cells. During the stimulation process anticytokine antibodies and recombinant cytokines were added to define their role in NK cell activation. For costimulation studies peripheral blood mononuclear cells were separated into lymphocytes and monocytes by counterflow-centrifugation (elutriation). Inhibitory NK cell receptor expression on activated NK cells was measured by flow cytometry by antiCD3, antiCD56 and anti-inhibitory NK cell receptor triple staining. The accessory function of monocytes was indispensable for BCG mediated NK cell activation. However, the stimulatory potential of monocytes did not require direct cell-cell contact to NK cells or major histocompatibility complex dependent antigen presentation to T cells. Monocyte derived interleukin (IL)-12 and to a lesser extent interferon (IFN)-alpha were key mediators for stimulating BCG induced NK cell cytotoxicity and IFN-gamma production. In contrast, IL-10 inhibited NK cell cytotoxicity and IL-18 did not show any effect. Exogenous recombinant IFN-alpha and IL-12 enhanced BCG mediated secretion of IFN-gamma and yet BCG induced NK cell cytotoxicity remained unchanged. While the CD158a and CD158b subsets did not have a significant role, NKG2A cells represented the predominant cytolytic subset in BCG activated NK cells. Following BCG stimulation the monocyte derived TH1 cytokines IL-12 and IFN-alpha activate tumor cytotoxic CD3/CD56/NKG2A NK cells. Our results elucidate NK activating mechanisms that are operative during BCG immunotherapy for bladder cancer and are relevant for an early, innate antimycobacterial immune response.