CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells

Abstract

Toll-like receptors (TLRs) are pattern-recognition receptors responsible for triggering cells of innate immunity. In this study we investigated the expression and function of TLRs 3 and 9 in human natural killer (NK) cells. In the presence of IL-12, freshly isolated NK cells responded to double-stranded RNA or unmethylated CpG DNA and expressed CD69 and CD25 activation markers. Because both markers were expressed by virtually all NK cells, this would suggest that most of them can be triggered by TLRs. Remarkably, NK cell stimulation also resulted in the induction of their functional program as revealed by IFN-gamma and tumor necrosis factor-alpha release and by up-regulation of cytolytic activity against tumor cells. IL-8 could efficiently substitute IL-12 in supporting NK cell responses to TLR-mediated stimulation. Importantly, freshly isolated NK cells acquired the ability to lyse immature dendritic cells after stimulation with double-stranded RNA and IL-12. However, responses to these stimuli were not restricted to fresh NK cells, because significant responses were also detected in polyclonal NK cells cultured in the presence of exogenous IL-2 for several weeks. The analysis of NK cell clones revealed some degree of heterogeneity in the ability to respond to TLR stimulation also among NK clones derived from a single donor. These data suggest that stimuli acting on TLR not only activate immature dendritic cells to release IL-12 but also render NK cells capable of receiving triggering signals from pathogen-associated molecules, thus exerting a regulatory control on the early steps of innate immune responses against infectious agents.

Figures

Fig. 1.

ODN A, ODN B, or poly(I·C) induces the expression of CD69 and CD25 by peripheral blood NK cells. (a) Freshly isolated NK cells were cultured for 20 h in medium supplemented with suboptimal doses of IL-12 (1 ng/ml) in the absence or presence of ODN A (5 μg/ml), ODN B (5 μg/ml), or poly(I·C) (50 μg/ml), and were then analyzed by one-color immunofluorescence for the expression of CD69 and CD25. (b) Freshly isolated NK cells were cultured for 20 h in medium supplemented with suboptimal doses of IL-12 (1 ng/ml) or IL-8 (10 ng/ml) in the absence or presence of poly(I·C) (50 μg/ml), and were then analyzed by two-color immunofluorescence for the expression of CD69 and CD25 in combination with NKG2A or KIR molecules. This experiment is representative of >15 that gave similar results.

Fig. 2.

ODN A, ODN B, or poly(I·C) induces functional responses by freshly isolated human peripheral blood NK cells. (a) Peripheral blood NK cells were cultured in the presence of suboptimal doses of IL-12 (1 ng/ml) in either the absence (black boxes) or presence of the following stimuli: ODN A (white boxes), ODN B (dark-gray boxes), or poly(I·C) (light gray boxes). After 20 h of culture, supernatants were harvested and assessed for IFN-γ and TNF-α content by specific ELISA (n = 3, mean ± SD). (b) Freshly isolated peripheral blood NK cells were cultured for 20 h in the presence of suboptimal doses of IL-12 (1 ng/ml) or IL-8 (10 ng/ml) in either the absence (black boxes), or presence of the following stimuli: ODN A (black circles), ODN B (black triangles), or poly(I·C) (×) and were then analyzed for cytotoxicity against the K562 target or FO-1 melanoma cell line at two different E/T ratios. As a control, the cytolytic activity of cells cultured in medium without cytokines is shown (white boxes). (c) Peripheral blood NK cells were cultured for 20 h with the stimuli discussed above in the presence of IL-12 (2 ng/ml) and were then analyzed for cytotoxicity against iDCs at various E/T ratios. Each value represents the mean of triplicate experiments. The SD did not exceed 4% in the cytotoxicity assays. Results are representative of at least 10 different experiments.

Fig. 3.

Stimuli acting on TLRs promote functional responses by cultured NK cell populations. (a) RT-PCR analysis of TLR3 and TLR9 transcripts was performed on total RNA isolated from a representative NK bulk population and NK92 cell line. Molecular weights of Φx HaeIII principal bands are indicated on the left. CTR-, negative control. (b) A representative polyclonal NK cell population was cultured for 20 h with medium (black boxes), ODN A (black circles), ODN B (black triangles), or poly(I·C) (×) in the presence of IL-12 (1 ng/ml) or IL-2 (10 units/ml) and was then analyzed for cytotoxicity against two human HLA class I- target cell lines (LCL 721.221 EBV and FO-1 melanoma) at different E/T ratios. Each value represents the mean of triplicate experiments. The SD did not exceed 4% in the cytotoxicity assays. (c) A representative polyclonal NK cell population was cultured with medium (black boxes), ODN A (white boxes), ODN B (dark-gray boxes), or poly(I·C) (light-gray boxes) in the presence of IL-12 (1 ng/ml) or IL-2 (10 units/ml). After 20 h of culture, supernatants were harvested and assessed for IFN-γ, TNF-α, and GM-CSF content by specific ELISA (n = 3, mean ± SD). These data are representative of at least five different experiments.

Fig. 4.

TLR stimulation up-regulates cytotoxicity and cytokine release by human NK clones. (a) Two representative NK clones, characterized by the NKG2A+KIR- (50D) or NKG2A-KIR+ (10D) phenotype, were cultured in medium supplemented with IL-2 (10 units/ml) or IL-12 (1 ng/ml) in either the absence (black boxes) or presence of ODN A (black circles), ODN B (black triangles), or poly(I·C) (×) for 20 h and were then assessed for cytotoxicity against the FO-1 target cell line at different E/T ratios. Each value represents the mean of triplicate experiments. The SD did not exceed 4% in the cytotoxicity assays. (b) The same NK clones were cultured with medium (black boxes), ODN A (white boxes), ODN B (dark-gray boxes), or poly(I·C) (light-gray boxes) in the presence of IL-2 (10 units/ml) or IL-12 (1 ng/ml). After 20 h of culture, supernatants were harvested and assessed for IFN-γ, TNF-α, and GM-CSF content by specific ELISA (n = 3, mean ± SD). These data are representative of at least five different experiments.