A contribution of mouse dendritic cell-derived IL-2 for NK cell activation

Abstract

Dendritic cells (DCs) play a predominant role in activation of natural killer (NK) cells that exert their functions against pathogen-infected and tumor cells. Here, we used a murine model to investigate the molecular mechanisms responsible for this process. Two soluble molecules produced by bacterially activated myeloid DCs are required for optimal priming of NK cells. Type I interferons (IFNs) promote the cytotoxic functions of NK cells. IL-2 is necessary both in vitro and in vivo for the efficient production of IFNgamma, which has an important antimetastatic and antibacterial function. These findings provide new information about the mechanisms that mediate DC-NK cell interactions and define a novel and fundamental role for IL-2 in innate immunity.

Figure 1.

DC-derived IL-2 is a key molecule for DC-mediated NK cell activation in vitro. Immature or E. coli–activated wtBMDCs and IL-2^−/−BMDCs were cultured together with either (A) syngeneic or (B) allogeneic NK cells for 18 h. Levels of IFNγ in the supernatant were then quantified by ELISA. The insets in A represent the intracellular staining performed in the mixed DC-NK populations after 4 h of coculture. NK cells were identified as DX5 positive. In B, IL-2 was also blocked using the S4B6 anti–IL-2 antibody. (C) Cell contact–dependent activation of NK cells by wtBMDCs. Unstimulated or E. coli–activated wtBMDCs and allogeneic NK cells were cocultured in the same wells or separated by a porous membrane. NK cells alone were also cultured in the presence of rIL-2. IFNγ in the supernatant was measured by ELISA after 18 h of coculture. The experiments were repeated four times with similar results (A, B, and C). (D) IL-2 produced by activated DCs directly activates NK cells. Unstimulated or 5-h bacterially activated IL-2^−/−BMDCs were fixed and incubated with allogeneic NK cells. 18 h later, IFNγ was measured in the supernatant by ELISA. The experiment was repeated twice with similar results.

Figure 2.

IL-2 is required in vivo for NK cell activation early after bacterial infection. (A) Frequency of IFNγ–producing NK cells in the spleen of untreated or E. coli–injected IL-2–deficient or wild-type mice measured by intracellular cytokine staining. The percentage of IFNγ-producing NK cells was also determined in wild-type mice pretreated with anti–IL-2, anti-Thy1, or isotype control antibodies before bacterial infection. The experiment was repeated three times with similar results. (B) Triple staining, 3 h after bacterial infection, of spleen cells with FITC-conjugated anti-CD11c antibody, PE-conjugated anti–IL-2 antibody, and biotinylated anti-CD19 and anti-TCRβ antibodies. In the left panels, the indicated fractions of IL-2–positive DCs have been calculated as the percentage of CD11c-positive cells (100%) before and after bacterial infection. Analogously, in right the panels the fraction of IL-2–positive non-DC leukocytes has been calculated as the percentage of CD19- or TCRβ-positive cells (100%) before and after bacterial challenge.

Figure 3.

IL-2 produced by DCs is required in vivo for NK cell activation. (A) Frequency of IFNγ-producing NK cells in the spleen of untreated or E. coli–injected RAG2^−/− or wild-type mice. The experiment was repeated three times with similar results. (B) Frequency of IFNγ-positive NK cells in the spleen of RAG2^−/− mice injected intraspleen with wtBMDCs, IL-2^−/−BMDCs or not injected (-), and then treated or not with 10⁷ E. coli i.v. Data are means and SDs from four mice analyzed in two independent experiments.

Figure 4.

DC-derived IL-2 is required in vivo to elicit antibacterial and antitumor NK cell activity. (A) Double staining with anti-NK1.1 and anti-IFNγ antibodies of spleen cells from RAG2^−/− mice injected (E. coli) or not (untreated) with 10⁷ bacteria. Before bacterial injection, mice were reconstituted either with wtBMDCs (wt) or IL2^−/−BMDCs (IL2^−/−), and the intracellular staining was performed 2 h after bacterial treatment. Percentages of cells in each quadrant are indicated. (B) Titers of free bacteria 2 h after i.v. injection of 10⁷ E. coli in the spleens of wild-type or RAG2^−/− mice reconstituted with either wtBMDCs or IL2^−/−BMDCs. The experiment was repeated twice with similar results. (C) The number of lung metastases in mice 14 d after i.v. injection of B16 melanoma alone (−), B16 melanoma and bacterially activated IL-2^−/−BMDCs, or B16 melanoma and bacterially activated wtBMDCs. Data represent means and SDs from three independent experiments.

Figure 5.

NK cell cytotoxicity is regulated by DC-derived type I IFNs. (A) DC-derived IL-2 is not required for the induction of NK cell cytotoxicity. Untreated or E. coli–activated wtBMDCs and IL-2^−/−BMDCs were cocultured with syngeneic NK cells for 48 h. NK cell cytotoxicity presented as the percentage of lysis of YAC targets was measured by a standard ⁵¹Cr release assay. (B) Bacterially activated BMDCs secrete type I IFNs. The amount of IFNαβ in the supernatant of wtBMDCs and NK cell cultures was measured after exposure to E. coli by a standard viral protection assay. (C) Type I IFNs control the induction of NK cell cytotoxicity after bacterial infection. Untreated or E. coli–activated wtBMDCs were cocultured with syngeneic NK cells for 24 h in the presence or absence of IFNαβ-blocking antibodies (P < 0.0001 when the percentage of lysis is compared between wtBMDC + E. coli + NK + isotype and wtBMDC + E. coli + NK + anti-IFNαβ at an E:T ratio of 7.5:1). The data are representative of three independent experiments conducted with triplicate samples.