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Abstract

Natural killer (NK) cells form the first line of defense against viral infections and malignant transformation and therefore represent promising targets for cancer immunotherapy. However, NK cell anti-tumor efficacy in the patient is often impaired through inhibitory signals mediated by the interaction of inhibitory killer immunoglobulin-like receptors (KIRs) with self–HLA-I expressed on autologous tumor cells. Therefore, it is crucial to circumvent KIR-mediated self-inhibition in immunotherapy to unleash autologous NK cell potency. Although the molecular mechanisms of de novo KIR expression during NK development are well established, little is known about regulation of their expression on mature NK cells. We here present a new strategy of transiently and safely releasing NK cells from KIR-mediated inhibition to improve autologous NK cells transfer against HLA-I–expressing tumors. Stimulation of mature peripheral blood NK cells with a combination of the cytokines interleukin (IL)-12, IL-15 and IL-18 resulted in decreased surface expression of the major inhibitory KIR2DL2/L3, KIR2DL1 and KIR3DL1 molecules. In contrast, other NK receptors remained unchanged or even got upregulated such as the IL-2 receptor alpha-chain (CD25). Most pronounced KIR downregulation was observed two days after cytokine exposure and was attributed to decreased KIR mRNA levels. Downregulation of KIR expression was transient and KIR mRNA and surface expression could be re-induced upon culture in IL-2 or IL-15. Reduced KIR2DL2/L3 expression on IL-12/15/18–activated NK cells was associated with less KIR-mediated inhibition and increased CD16-dependent cytotoxicity in redirected lysis assays. Importantly, downregulation of KIR2DL2/L3 expression enabled improved cytotoxicity of IL-12/15/18–stimulated NK cells against cognate HLA-I–expressing tumor targets. Additionally, we observed downregulation of inhibitory KIR molecules on NK cells after 3 days in a human cytomegalovirus (HCMV) infection model, representing a physiological setting with high concentrations of pro-inflammatory cytokines such as IL-12. Taken together, our study reports a novel mechanism of KIR downregulation on mature peripheral blood NK cells by pro-inflammatory cytokines. Decreased KIR expression of IL-12/15/18–activated NK cells translated into reduced inhibition by self–HLA-I, generating potent effectors cells for the treatment of HLA-I–expressing tumors. These results imply that the transient resistance to self-inhibition might greatly improve immunotherapy protocols especially for autologous NK cell infusions.