Cross-talk Between Antigen-presenting Cells And Natural Killer Cells Enhances NK-mediated Anti-tumor Activity

ObjectMalignant tumor, as a frequently occurring and common disease, has a great threat to human health; the death of it accounts for a quarter of total deaths in the world, and shows an increasing trend year by year. There are many treatment methods for malignant tumor, including surgery, radiotherapy, chemotherapy, endocrine therapy, Chinese medicine treatment, hyperthermia and radiofrequency ablation therapy, but most of them are lack of specificity and targeting, as they not only can kill tumor cells but also destroy normal cells, leading to a serious decline in immune function of patients. In recent years, immunotherapy based the cutting-edge science of modern immunology, cell biology and molecular biology, has become the latest anti-cancer biological science and technology in the21st century. Regarding immune effector cells as main body's, such as lymphocytes, macrophages, dendritic cells (DC), natural killer (NK) cells, cytotoxic T lymphocytes (CTL), or the combination of them, immunotherapy is attempting to stimulate the immune system to reject and destroy tumors. In some cases, the immune effector cells need active agents referred to as immunomodulators, which are a diverse array of recombinant, synthetic and natural preparations, such as cytokines, chemokines, oligodeoxynucleotides, Imiquimod, or CpG.NK cells are integral components of the innate immune system and are characterized by their strong cytolytic activity against tumors and virus-infected cells. NK cells also regulate innate and adaptive immune responses through secretion of immunoregulatory cytokines and cell-to-cell contact. Activated NK cells promote DC mature and cytokines production through production of gamma interferon (IFN-y) and tumor necrosis factor-α (TNF-α), leading to the induction and regulation of immune responses. In addition to DC, NK cells can strongly prompt the generation of inducible nitric oxide synthase (iNOS) from macrophages, resulting in the enhancement the antimicrobial effect of macrophages that also belong to antigen-presenting cells (APC). Moreover, NK cells induce the fusion of lysosomal in macrophages by secreting interleukin-22(IL-22), to inhibit the growth of intracellular Mycobacterium tuberculosis. Usually, the actions of NK cells are thought to be mediated by the complex interactions between inhibitory and activating signals sent by cell surface receptors following ligation. In many cases, cytokines, such as IL-2, IL-15, IL-12, IL-18and IL-21, produced by other immune cells, and particularly activated APC, can also play important roles in the regulation of NK cell activity. Therefore, a great deal of interest and information has emerged with respect to the crosstalk between APC and NK cell, in contrast to the interactions between NK cells and other innate immune cells.Toll-like receptors (TLRs) are receptors of the innate immune system that directly recognize conserved pathogen structures, and they also conserved receptors that recognize a variety of pathogen-break down products. TLRs are widely expressed on many cells, including epithelial cells, endothelial cells, mesothelial cells, fibroblasts, neutrophils, T lymphocytes, monocytes, NK cells, macrophages, and DC. Among the family of TLRs, TLR3, TLR7, TLR8and TLR9which belong to the TLR9subfamily that expressed in endosomal compartments, not only can directly activate a variety of immune cells, particularly APC and NK cells, but also induce tumor cell apoptosis and enhance their sensitive to effector cells. Therefore, TLR9subfamily can serve as the functional bridges and links of the cross-talk between APC and NK cells, and the agonists of TLR9subfamily are the best immunomodulators for the cells-based immunotherapy.In this study, TLR3agonist Poly Ⅰ:C and TLR7/TLR8agonists, Imiquimod, Gardiquimod, or ssRNA40were used to observe the APC-increased NK cells cytotoxicity against tumor cells. We also explored the cross-talk between APC and NK cells based on cytokine stimulation and cell-cell contact.Methods(1) Magnetic bead sorting (MACS) was used to isolate NK cells from spleen in mice;(2) PBMCs were isolated with Ficoll by density gradient centrifugation from human peripheral blood of healthy donor;(3) Dendritic cells were induced from human PBMCs with recombinant cytokines in vitro;(4) The separation efficiency of murine NK cells and purification efficiency of macrophages, the expression of activating molecules, lysis-associated molecules, receptors and intracellular cytokines of murine NK cells, human NKL cells or PBMCs were tested by flow cytometry;(5) The gene expression of activating molecules, lysis-associated molecules, receptors or intracellular cytokines of murine NK cells, NKL cells and PBMCs were tested by real-time quantitative PCR;(6) The cytotoxicity of murine NK cells, NKL cells and PBMCs was assayed by Cr release assay or MTT assay;(7) The level of cytokines in cell culture supernatants were determined by ELISA;(8) Conventional reverse transcription PCR was used to detect the mRNA expression of TLR7and TLR8in NKL cells and PBMCs;(9) RNA interference was used to knockdown the expression of TLR3and Qa-1in macrophages;(10) The antibody neutralization assay was used to block the expression of NKG2D, and the production of IL-12and IFN;(11) The activation of NF-κB signaling pathway was analyzed by Western Blotting;(12) The HepG2nude mice xenograft was established and treated intraperitoneally;(13) The cell to cell contact between NK cells and macrophages, or between NKL cells and DC was blocked by Transwell assay.ResultsPart1:Poly I:C-treated macrophages enhance mouse NK cells anti-tumor activity1. Poly Ⅰ:C-treated macrophages increase NK cell-mediated cytotoxicity to target tumor cells, but not to macrophages. NK cell cytotoxicity against YAC-1cells, Hepal-6cells, MCA38cells or H22cells, but not macrophages, was dramatically increased when NK cells were pre-incubated with Poly Ⅰ:C-treated macrophages.2. NK cell-mediated target cell lysis is NKG2D-dependent. Blockade of NKG2D alleviated the activation and cytotoxicity of NK cells, reduced the expression of perforin, FasL and TRAIL, as well as the secretion of IFN-γ by NK cells.3. Poly Ⅰ:C up-regulates expression of NKG2D ligands on macrophages. The NKG2D ligands RAE-1, H60and MULT-1on macrophages were significantly elevated in a dose-dependent manner following stimulation with Poly Ⅰ:C.4. Poly Ⅰ:C-induced up-regulation of NKG2D ligands is dependent on TLR3. When TLR3expression on macrophage was silenced by TLR3specific siRNA, Poly Ⅰ:C-mediated up-regulation of the three NKG2D ligands was abrogated significantly.5. Qa-1contributes to the protection of macrophages from NK cell-mediated cy to lysis. Qa-1in poly Ⅰ:C-treated or untreated primary macrophages are much higher than that in YAC-1cells, and anti-Qa-lb blockade or Qa-1-siRNA silence significantly increased the NK cell-mediated cytolysis against Poly Ⅰ:C-treated macrophage.6. Poly Ⅰ:C increases many cytokines production by macrophages. Poly I:C stimulation markedly increased the secretion of IL-15, IFN-β, IL-18and IL-12in both RAW264.7cells and BALB/c peritoneal macrophages supernatants, in particular IL-15and IFN-p.7. Activation of NK cells is dependent on IL-15and IFN-β produced by Poly Ⅰ:C-stimulated macrophages. Blocking either IL-15or IFN-P in Poly Ⅰ:C-stimulated macrophages supernatants decreased NK cells function.8. Cell-to-cell contact was involved in the crosstalk between NK cells and macrophages. In Transwell system, Poly Ⅰ:C-stimulated macrophages still promote the expression of CD69and NKG2D, as well as the production of IFN-γ by NK cells, but the up-regulation effect was not as strong as that in direct contact co-culture.Part2:Role of NK-DC cross-talk mediated by TLR7/TLR8agonist in human HCC immunotherapy1. TLR7/TLR8agonists activate human NK cells directly. R837, GDQ and ssRNA40could activate NKL cells and primary NK cells, and enhanced cytotoxicity to HepG2cells, particularly GDQ.2. TLR7/TLR8agonists induce the production of IFN-γ and TNF-α by NKL cells. The protein Levels of IFN-γ and TNF-α in NK cells were promoted by R837, GDQ and ssRNA40. 3. TLR7/TLR8agonists enhance the expression of NKG2D and NKp80on NKL cells. GDQ enhanced the expression of NKG2D, and all three agonists strongly up-regulated NKp80expression.4. DCs strongly enhance NK cells function in the present of TLR7/TLR8agonists. Although NK cells could be directly activated by these three agonists, changes in the activation status of the co-cultured NK cells were more significantly, particularly NKL cells treated with DCs and GDQ.5. DCs plus GDQ improve the antitumor effects of NK cells in vivo. The human HepG2liver carcinoma xenografts were established and treated with NKL cells. NKL plus DC and GDQ exerted a significantly greater inhibitory effect and delayed the growth of tumor nodules compared to other groups.6. Increase of mature maker expression and cytokine secretion by the DCs exposed to NKL cells and TLR7/TLR8agonists. The expression of co-stimulatory molecules CD1a, CD11c, CD83and CD86on DCs were up-regulated. IFN-α, IFN-β, IL-12p35, IL-15, and IL-18, pro-inflammation cytokines IL-1(3and L-6, but not the Th2type cytokine IL-10, in the co-culture supernatant were increased by exposure to NKL cells and TLR7/TLR8agonists.7. DC induces NK cells activation in a Type ⅠIFN and IL-12-dependent manner. Blockade of type Ⅰ IFN pathway on DC and NKL cells in the co-culture system significantly attenuated CD69and CD25expression on NK cells which were also failed to maintain high levels of lysis-associated molecules. Recombinant IL-12plus agonists could increase CD69expression and IFN-y production.8. Cell-to-cell contact is critical for DC-mediated NK cells activation. DC in Transwell culture could still promote the expression of CD69and IFN-y secretion of NK cells, but the effect is not as strong as that in direct contact co-culture. Activating receptors NKG2D and NKp80and their ligands, co-stimulatory molecules CD40-CD40L and LFA-1-ICAM-1, and chemokines CCL2and CCL4and their receptors may involve in the NK/DC cross-talk.Conclusion In this study, we demonstrated that the cross-talk between APC and NK cells mediated by TLR agonists enhanced NK cells anti-tumor activity. On the one hand, macrophages treated by TLR3agonist, Poly I:C, enhanced mouse NK cell anti-tumor effect. Poly I:C induced the expression of NKG2D ligands on murine macrophages, and up-regulated the production of IL-15, IL-12, IFN-β and other cytokines, leading to the activation and IFN-y secretion of NK cells. However, macrophages escaped the attack from activated NK cells, because of the high expression of Qa-1, the ligand of inhibitory receptor NKG2A. On the other hand, the DC/NK cross-talk mediated by the TLR7/TLR8agonist improved the anti-tumor effect of human NK cells. Although the TLR7agonist R837, TLR8agonist ssRNA40, and TLR7/TLR8agonist GDQ could directly induce the activation of NK cells, the participation of DC further significantly promoted NK cell activation. And this phenomenon was further confirmed by the in vivo therapy of hepatoma in HepG2nude mice xenograft model. In addition, it was demonstrated that the promotions of TLR7/TLR8agonist-stimulated DC for NK cell activation were dependent on the secretion of Type Ⅰ IFN and IL-12, as well as the cell-cell contacts between NKG2D-NKG2DL, NKp80-AICL, CD40-CD40L, LFA-1-ICAM-1, CCR2-CCL2and CCR4-CCL4.This study strongly confirmed the enhancement function of NK cells by APC, and identified the need of cytokines and cell-cell contact between cells interaction. It provided theoretical foundations and new ideas for the further exploration of the molecular mechanisms in the APC/NK cross-talk. The present study suggests that Poly Ⅰ:C, R837, GDQ and ssRNA40can be used as vaccine adjuvant or combined with other anti-tumor agents for treatment of cancer. And the combination of TLR agonists, APC and NK cells may be served as a new form of cell-based immunotherapy used in the clinical application and investigation for anti-tumor, anti-viral and autoimmune diseases.