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Overexpression of interleukin-12 enables dendritic cells to activate NK cells and confer systemic antitumor immunity¹

Hepatobiliary Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

²Correspondence: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. E-mail: dematter{at}mskcc.org

SPECIFIC AIMS

Dendritc cells (DC) are known initiators of T cell-mediated immunity. However, the relationship between DC and natural killer (NK) cells is less well studied, and direct evidence for a significant DC–NK relationship in vivo is lacking. We postulated that DC secretion of interleukin-12 (IL-12) would enable them to activate NK cells. Specific goals of this study were to determine whether murine bone marrow-derived DC could stimulate NK cells in vitro by their production of IL-12, determine whether IL-12 secretion by DC enables them to activate NK cells in vivo, and explore whether IL-12-producing DC can generate systemic, NK cell-mediated tumor protection.

PRINCIPAL FINDINGS

1. DC secretion of IL-12 and its autocrine effects on DC phenotype
We first assayed the amount of IL-12 produced by bone marrow-derived DC. Mock-infected DC did not secrete any detectable IL-12. However, DC infected with an adenoviral vector encoding IL-12 (AdIL-12) produced nearly 700 ng IL-12 per 5 x 10⁵ DC by 48 h. A small part of this effect was a result of the adenoviral vector alone, as DC infected with an adenovirus encoding the marker gene green fluorescent protein (AdGFP) produced 100 pg IL-12 per 5 x 10⁵ cells over the same time period.

To determine the effect of autocrine IL-12 on DC phenotype, we tested DC surface-maker expression by flow cytometry. DC transduced with AdIL-12 were phenotypically mature by 24 h after infection. Major histocompatibility complex class I and II proteins and costimulatory molecules CD40, CD80, and CD86 were up-regulated by up to 400% compared with mock-infected DC. To determine the relative contributions of adenoviral infection and IL-12 expression to these phenotypic changes, we tested each alone. DC cultured with IL-12 protein alone (100 ng/ml) were not mature. However, DC infected with AdGFP were matured in a similar manner to AdIL-12 infection. In addition, the presence of IL-12 protein (100 ng/ml) did not further alter the phenotype of AdGFP-infected DC. These findings indicated that DC maturation was completely dependent on adenovirus and independent of IL-12.

2. Mature DC expressing high IL-12 activate NK cells in vitro
To determine whether DC can activate NK cells in vitro, we cocultured AdIL-12-transduced DC or controls with purified splenic NK for 18 h and then tested NK cell lytic activity and interferon- (IFN-) production. NK cells cocultured with mock-infected DC showed no signs of activation; they did not lyse Yac-1 lymphoma cells and did not secrete IFN-. In contrast, DC infected with AdIL-12 induced nearly 50% Yac-1 lysis and produced 600 pg/ml IFN- (Fig. 1 A, B). Notably, AdGFP-infected DC also activated NK cells but to a lesser extent (35% Yac-1 lysis and 56 pg/ml IFN-). We postulated that AdGFP DC activated NK cells as a result of their small amount of IL-12 secretion. To test this, we added a blocking IL-12 antibody to the AdGFP DC–NK coculture wells. This reduced NK cell lytic activity (Fig. 1C ) and IFN- production by 50%, suggesting that IL-12 secretion was largely responsible for the enhanced ability of AdGFP DC to activate NK cells.

View larger version (17K): [in this window] [in a new window]   Figure 1. NK activation by DC is dependent on direct cellular contact and IL-12 secretion. NK cells were cocultured with DC, and NK activation was assessed by (A) Yac-1 lysis at effector-to-target ratios of 12:1 or 100:1 and (B) IFN- production. A) Mock-infected DC did not induce NK lytic activity, while AdIL-12 DC and even AdGFP DC induced considerable lysis. B) NK cells only expressed IFN- when cultured with AdIL-12 DC or to a lesser extent, with AdGFP DC. Prevention of direct cellular contact between DC and NK cells dramatically lowered (AdIL-12 DC) or eliminated (AdGFP DC) IFN- production. Notably, AdIL-12 DC did not secrete any IFN- when cultured alone, indicating that all detectable IFN- was NK cell-derived. C) IL-12 blockade lowered the lytic activity of NK cells cultured with AdGFP DC by 50%, suggesting that NK lytic activity after culture with AdGFP DC was contingent on IL-12 secretion.

After showing that IL-12 expression was critical for DC activation of NK cells in vitro, we wanted to determine whether the production of IL-12 alone was sufficient or if direct cellular interaction between the DC and NK cells was necessary. To test this, we separated the AdIL-12 DC from NK cells with a 0.4-µm insert, which permits free diffusion of proteins but prevents cellular contact. Under these conditions, the effect of AdIL-12 DC on NK cell Yac-1 lysis was reduced by more than 50%. A dramatic reduction was also seen in NK cell IFN- production (Fig. 1B ). Overall, these data indicate that in addition to IL-12 secretion, direct cellular contact between DC and NK cells is vital for maximal NK activation.

3. IL-12 enables DC to activate NK cells in vivo
We next determined if IL-12-secreting DC activate NK cells in vivo. Animals were treated intraperitoneally (i.p.) with 5 x 10⁵ AdIL-12-transduced DC, and 24 h later, their splenic or nodal NK cells were isolated and tested for lytic activity and IFN- production. NK cells from animals treated with exogenous AdIL-12 DC had a greater than 2-fold higher lysis of Yac-1 targets than controls and produced 170 pg IFN- per 10⁶ cells. In contrast, purified NK cells from mice treated with saline, mock DC, or AdGFP DC did not produce detectable IFN-. To determine whether maximal activation of NK cells in vivo required DC as well as IL-12, as seen in the in vitro experiments, we inoculated animals with MC57 fibroblasts or B16 melanoma cells that had been infected with AdIL-12 or a comparable dose of IL-12 protein (0.2 µg). Splenic NK cells were then harvested 24 h later and were compared with those from AdIL-12 DC-treated mice. NK-specific lysis of Yac-1 cells was 2-fold higher for animals treated with AdIL-12 DC compared with controls. Similarly, although NK cells from mice treated with AdIL-12 DC overexpressed IFN-, splenic NK cells from any of the control groups did not produce detectable IFN-.

4. DC expressing IL-12 confer NK-mediated tumor protection
We next tested the ability of DC overexpressing IL-12 to induce NK-mediated tumor protection in a hepatic metastases model. Mice were inoculated i.p. with saline, mock DC, AdGFP DC, or AdIL-12 DC and were then challenged 2 h later with 2.5 x 10⁴ B16 murine melanoma cells by intrasplenic inoculation. Animals were then killed on day 21 and staged, based on the number and size of liver nodules. Animals treated with AdIL-12-infected DC had a significantly lower tumor burden than controls (P=0.001; Fig. 2 A). We next determined if this translated into a survival advantage. Mice treated with AdIL-12 DC lived significantly longer than saline, mock DC, or AdGFP DC-treated controls (P<0.0001; Fig. 2B ). Moreover, 33% (6 of 18) of experimental mice lived beyond day 75 compared with no long-term survivors in any of the control groups (0 of 26). To elucidate the cellular requirements for AdIL-12 DC-induced antitumor immunity, we depleted animals of T cells or NK cells. Animals depleted of CD4⁺ or CD8⁺ T cells or both had no significant change in median survival. In contrast, NK cell depletion abrogated the advantage of AdIL-12 DC treatment.

View larger version (11K): [in this window] [in a new window]   Figure 2. Treatment with AdIL-12-transduced DC delays or prevents tumor development. A) Animals from each treatment group were killed 21 days after tumor inoculation and were assessed for hepatic tumors using the staging system: 0 = no tumor; 1 = 1–3 small (<3 mm) or 1 large (>3 mm) nodule(s); 2 = 4–10 small nodules; 3 = >10 small or >1 large nodule(s); 4 = >1/4 liver replaced by tumor; 5 = dead by day 21. Horizontal lines indicate the mean stage. B) Survival of animals treated with saline (n=6), mock DC (n=15), AdGFP DC (n=5), or AdIL-12 DC (n=18). Animals treated with AdIL-12 DC have a 33% long-term survival rate, and control groups had no survivors (P<0.0001).

To determine whether the NK-mediated antitumor effects were DC-specific, we infected B16 melanoma cells in vitro with AdIL-12 (at a dose that produced similar levels of IL-12 to infected DC) before inoculation into the spleen. This cohort of animals (n=5) had no long-term survivors and had a median survival similar to saline-treated mice, suggesting that simply producing low levels of IL-12 in the vicinity of the tumor is not sufficient to delay tumor development. To test this further, we injected AdIL-12-transduced MC57 fibroblast cells before tumor challenge. Although this provided slight protection, it was far inferior to DC expressing IL-12 (P=0.02). Collectively, these data demonstrate that DC are critical for the NK-mediated antitumor effects observed.

CONCLUSIONS AND SIGNIFICANCE

Our data show that bone marrow-derived DC are enabled, through secretion of IL-12, to activate NK cells. We have shown this important relationship between IL-12-secreting DC and NK cells in 3 ways: by coculture of DC with purified NK cells in vitro, by analysis of NK cells isolated from animals treated with exogenous DC, and by depleting NK cells in tumor survival experiments (Fig. 3 ). The fact that our results are DC-specific and are not seen when fibroblast or tumor cells secrete IL-12 or when DC–NK cellular contact is blocked implies a fundamental DC–NK relationship. In the current experiments, we expressed IL-12 in DC using an adenoviral vector. However, our findings have broader biological relevance, as many DC subsets secrete IL-12 on exposure to a variety of physiological stimuli and pathogens. Finally, our study may have important implications to the design of DC vaccines for cancer immunotherapy. Engineering mature DC to overexpress IL-12 may be used to induce NK cell-mediated tumor lysis while avoiding the toxicity from high, systemic cytokine levels that are otherwise necessary to achieve tumor protection.

View larger version (45K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0900fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0900fje

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