Induction of interferon-gamma from natural killer cells by immunostimulatory CpG DNA is mediated through plasmacytoid-dendritic-cell-produced interferon-alpha and tumour necrosis factor-alpha

Abstract

Immunostimulatory sequences (ISS) that contain CpG motifs have been demonstrated to exert antipathogen and antitumour immunity in animal models through several mechanisms, including the activation of natural killer (NK) cells to secrete interferon-gamma (IFN-gamma) and to exert lytic activity. Since NK cells lack the ISS receptor TLR9, the exact pathway by which NK cells are activated by ISS is unclear. We determined that ISS-induced IFN-gamma from NK cells is primarily dependent upon IFN-alpha release from plasmacytoid dendritic cells (PDCs), which directly activates the NK cell. However, further analysis indicated that other PDC-released soluble factor(s) may contribute to IFN-gamma induction. Indeed, tumour necrosis factor-alpha (TNF-alpha) was identified as a significant contributor to ISS-mediated activation of NK cells and was observed to act in an additive fashion with IFN-alpha in the induction of IFN-gamma from NK cells and to up-regulate CD69 expression on NK cells. This activity of TNF-alpha, however, was dependent upon the presence of PDC-derived factors such as type I interferon. These results illustrate an important function for type I interferon in innate immunity, which is not only to activate effectors like NK cells directly, but also to prime them for enhanced activation by other factors such as TNF-alpha.

Figure 1

A soluble factor produced by ISS-stimulated PDCs confers IFN-γ secretion to NK cells. Human PDCs were stimulated with CpG-B 1018, CpG-C C274, or a negative control ODN for 24 hr. Cell-free SNs were collected and used to stimulate freshly purified NK cells for another 24 hr. These NK SNs were then harvested and assayed for IFN-γ and IFN-α content via ELISA. Results are reported as the mean of two donors, representative of two experiments.

Figure 2

Blockade of type I interferon partially inhibits ISS-induced IFN-γ secretion. Human PDCs were stimulated with medium or CpG-C C274 for 24 hr. Cell-free SNs were collected and used to stimulate freshly purified NK cells for another 24 hr in the presence of cytokines, neutralizing antibodies, or no stimulation. These NK SNs were then harvested and assayed for IFN-γ and IFN-α content via ELISA. Results are reported as the mean ± SEM of 10 donors. **P < 0·01; ***P < 0·001.

Figure 3

NK-produced IFN-γ in response to cytokine stimulation. (a) Human NK cells were isolated and stimulated with cytokines for 24 hr. Cell-free SNs were harvested and assayed for IFN-γ content via ELISA. Data are reported as the mean of six donors ±SEM. Statistical significance against medium stimulation was computed.(b) Human PDCs were stimulated with medium or CpG-C C274 for 24 hr. Cell-free SNs were collected and used to stimulate freshly purified NK cells for another 24 hr in the presence of cytokines. These NK SNs were then harvested and assayed for IFN-γ content via ELISA. Results are reported as the mean ± SEM of five donors. Statistical significance against ISS-PDC SN was computed.(c) Human NK cells were isolated and stimulated with IFN-α⁺ cytokines for 24 hr. Cell-free SNs were harvested and assayed for IFN-γ content via ELISA. Results are reported as the mean ± SEM of four to 10 donors. *P < 0·05; **P < 0·01; ***P < 0·001.

Figure 4

Effect of cytokine neutralization on ISS-induced IFN-γ secretion. Human PDCs were stimulated with medium or CpG-C C274 for 24 hr. Cell-free SNs were collected and used to stimulate freshly purified NK cells for another 24 hr in the presence of neutralizing antibodies or cytokines. These NK-cell SNs were then harvested and assayed for IFN-γ content via ELISA. Results are reported as the mean ± SEM of five to seven donors. Statistical significance was computed against C274-PDC SNs alone. *P < 0·05; **P < 0·01.

Figure 5

Induction of TNF-α from PDCs by ISS. Human PDCs were stimulated with CpG-B 1018, CpG-C C274, or a negative control ODN for 24 hr. Cell-free SNs were collected and assayed for TNF-α content via ELISA. Results are reported as the mean ± SEM of 14 donors. **P < 0·01; ***P < 0·001.

Figure 6

Dependency of ISS-mediated NK activation on IFN-α/TNF-α. (a,b) Human NK cells were isolated and stimulated with medium (black) or cytokines (red) for 24 hr. Cells were stained for CD69 expression and analysed by FACs. Data are from one representative donor of four. (c–f) Human PDCs were stimulated with CpG-C C274 for 24 hr, and cell-free SNs were collected. Freshly purified NK cells were stimulated with medium (black), C274-PDC SN (red), or C274-PDC SN + neutralizing antiboies (green) for another 24 hr. Cells were stained for CD69 expression and analysed by FACs. Data are from one representative donor of four.