Study Of Synergistic Effects Of Interleukin-18 Plus Interleukin-2 On Cytotoxicity And NKG2D Expression Of Human Peripheral Blood Derived NK Cells

Background and ObjectiveNatural killer (NK) cells are a major component of effector lymphocytes that play an important role in immune surveillance by directly killing tumor cells and preventing microbial invasion. Decline of NK cell number and activity in cancer patients is related to the occurrence and development of tumors and is a key factor in tumor immune escape. Thus, it may be a viable strategy to strengthen human’s anti-tumor responses by improving NK cell activities in vivo or adoptive transfer of exogenous activated NK cells. NK cell function is mediated by a large number of cytokines. Interleukin (IL)-18 is a pleiotropic cytokine that induces potent NK cell-dependent anti-tumor responses by enhancement of NK cell activation and IFN-y secretion. When administrated with other cytokines such as IL-12 or IL-15, IL-18 exerts intensive effects on the promotion of NK cell proliferation, cytotoxicity and IFN-y production. Interleukin (IL)-2 has been widely used for treatment of patients with cancer. However, a series of severe adverse effects related with high-dose IL-2 treatment have greatly limited its clinical application in tumor immunotherapy. Now, the research hotspot in the world is being directed at development combination regimens of IL-2 plus other cytokines with less toxicities. Clinical studies have demonstrated that IL-18 do not cause obvious side effects and can be given in widely active doses to patients with advanced cancer. The combination of IL-18 and IL-2 is considered as a viable strategy to induce NK cell-mediated anti-tumor responses. However, the exact regulatory mechanism of combining IL-18 and IL-2 on NK cell function is still not well understood.This study was to investigate the synergistic effects of IL-18 plus IL-2 on NKG2D expression and cytotoxicity of human NK cells against tumor cells, and analyze the regulatory mechanisms by which both cytokines regulate NK cell-mediated antitumor activity, in order to provide new ideas and experimental basis for cancer immunotherapy.MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood in sterile by Ficoll-Histopaque density gradient centrifugation. Freshly isolated human PBMCs were incubated for 48 h with various concentration combinations of recombinant human IL-18 (0,100,200 ng/ml) plus recombinant human IL-2 (0,100,200 U/ml) in vitro. The experiment was divided into:IL-18 group, IL-2 group, IL-18 combined with IL-2 group and control group. CD 107a expression on CD3-CD56+NK cells was determined by three-colour flow cytometry to evaluate the cytotoxicity of NK cells against human erythroleukemia K562 cells and human colon carcinoma HT29 cells. Flow cytometric analysis was also employed to determine NKG2D expression on human NK cells.Results1. The combined application of IL-18 and IL-2 resulted in a greater increase on CD 107a positive NK cells following stimulation, compared with using either cytokine (IL-18 or IL-2) alone (P<0.01), suggesting that the combination of these two cytokines exerted synergistic enhancement of NK cell cytotoxicity against K562 cells and HT29 cells.2. After different cytokine stimulations, we did not observe significant increases in the percentage of CD3-CD56+ NK cells among different groups (P> 0.05), suggesting that the combined use of IL-18 and IL-2 did not affect the proliferation of human NK cells.3. Isolated PBMCs were incubated for 48 h with IL-18 (200 ng/ml), IL-2 (100 U/ml) or a combination of both cytokines. Flow cytometric analysis showed that the combined administration of IL-18 and IL-2 significantly enhanced the expression of NKG2D on human CD3-CD56+ NK cells in comparison with using either IL-18 or IL-2 alone (P<0.05).4. After pre-incubation of stimulated cells with anti-NKG2D antibody for 1 h to block NKG2D signaling, the up-regulatory effects of combining IL-18 and IL-2 on NK cytolysis against K562 and HT29 cells were dramatically attenuated, suggesting that IL-18 synergized with IL-2 to improve the cytolytic activity of human NK cells at least partly via NKG2D pathway.ConclusionIL-18 acted synergistically with IL-2 to improve the cytolytic activity of human NK cells in vitro. Substantial up-regulation of NKG2D expression on the surface of NK cells may partly explain the synergistic enhancement of NK cytolysis by both cytokines. Our findings provide new ideas and important experimental basis for cancer immunotherapy. Appropriate administration of IL-18 plus IL-2 might be a viable approach to induce therapeutic NK cell-mediated antitumor responses.