DNA-based vaccines activate innate and adaptive antitumor immunity by engaging the NKG2D receptor

Abstract

The interaction of NKG2D, a stimulatory receptor expressed on natural killer (NK) cells and activated CD8(+) T cells, and its ligands mediates stimulatory and costimulatory signals to these cells. Here, we demonstrate that DNA-based vaccines, encoding syngeneic or allogeneic NKG2D ligands together with tumor antigens such as survivin or carcinoembryonic antigen, markedly activate both innate and adaptive antitumor immunity. Such vaccines result in highly effective, NK- and CD8(+) T cell-mediated protection against either breast or colon carcinoma cells in prophylactic and therapeutic settings. Notably, this protection was irrespective of the NKG2D ligand expression level of the tumor cells. Hence, this strategy has the potential to lead to widely applicable and possibly clinically useful DNA-based cancer vaccines.

Fig. 1.

Construction and expression of vectors encoding murine NKG2D ligand-H60 and survivin. (A) Expression vectors were constructed based on the pBudCE4.1 plasmid backbone. (B) Expression of murine survivin was detected by Western blotting analyses of pSurv-transfected 293T cells, by using rabbit anti-mouse survivin antibody. (C) Expression of H60 was demonstrated by flow cytometry using NKG2D tetramer. 293T cells were transfected with either pH60 (thick black line) or pBud vectors (thin gray line). (D) Expression of NKG2D ligands in Peyer's patches in vivo. Mice were vaccinated with either pBud (Left) or pH60 (Right). Green, DC Ab; red, NKG2D/Fc chimera; arrow-head, colocalization; *, both markers expressed on the same cells without colocalization.

Fig. 2.

NKG2D ligand H60 enhances the antitumor efficacy of survivin-based DNA vaccines in a murine CT-26 colon carcinoma model. (A) Endogenous expression (thick blue lines) in CT-26 cells of survivin (Left) or NKG2D ligand (Right). Thin red lines, staining controls. (B) Prophylactic setting. Vaccinated mice (n = 8) were challenged i.v. with 1 × 10⁵ CT-26 colon carcinoma cells. (Upper) Representative lungs. (Lower) Average lung weight. Normal lung weight is ≈0.2 g. *, P < 0.00005, 0.001, 0.02, or 0.005 compared with PBS, pBud, pH60, or pSurv, respectively. Experiments were repeated 3 times with similar results. (C) In vivo depletion assays. Experiments were terminated 2 days earlier in CD8- and NK-depleted mice than in control and CD4-depleted animals. (D) Therapeutic setting. Mice were challenged i.v. with 1 × 10⁵ CT-26 cells and later vaccinated. The results presented are the average of two separate experiments (n = 12 and 4).

Fig. 3.

The pH60/Surv vaccine protects mice in a murine D2F2 breast carcinoma model. (A) Endogenous expression of survivin (Left) and NKG2D ligands (Center) by D2F2 cells (thick black lines), compared with NKG2D ligand positive control-Yac-1 cells (Right). Thin gray lines, staining controls. (B) Prophylactic setting. (C) Therapeutic setting. *, P < 0.01; *, P < 0.05 (compared with pBud); ***, P < 0.0002 and 0.05 (compared with pBud and pSurv, respectively).

Fig. 4.

The pH60/Surv enhanced NK and CD8⁺ T cell activity. Freshly isolated or in vitro-stimulated splenocytes were tested in a standard ⁵¹Cr-release assay against Yac-1 (A) or CT-26 (B) target cells. ⋄, pBud group; ▪, pH60 group; ▵, pSurv group; •, pH60/Surv group. Experiments were repeated twice with similar results. (C) Cytotoxicity assays against CT-26 target cells in the absence or presence of blocking Abs. In vitro-stimulated splenocytes were originally isolated from pH60/Surv-vaccinated mice. •, no blocking Ab; shaded symbols, specific lysis in the presence of anti-CD4 (○), anti-CD8 (▵), or anti MHC class I (□) Abs. Experiments were repeated once with similar results.

Fig. 5.

Another NKG2D ligand, RAE1, enhances the antitumor efficacy of survivin-based DNA vaccines. (A) Expression of RAE1 was demonstrated by flow cytometry using NKG2D tetramer. COS-7 cells were transfected with the pRAE (thick black line) or pBud control (thin gray line) vector. (B) In vivo CT-26 colon carcinoma model. Vaccinated mice (n = 8) were challenged i.v. with 1 × 10⁵ CT-26 cells. *, P < 0.02 compared with pBud control group. Freshly isolated (C)or in vitro-stimulated (D) splenocytes were tested in a standard ⁵¹Cr-release assay against Yac-1 (C) or CT-26 (D), respectively. ⋄, pBud group; □, pRAE group; •, pRAE/Surv group.

Fig. 6.

NKG2D ligand H60 enhances the antitumor efficacy of CEA-based DNA vaccines. (A) Expression vectors were constructed based on the pBudCE4.1 backbone. (B) Flow cytometric analyses of 293T cells transfected with pCEA (thick blue line) or the empty pBud (thin red line) vector by using anti-CEA Ab. (C) Western blotting analyses of 293T cells transfected with different expression vectors by using anti-CEA Ab. (D) In vivo expression of CEA (red) in the Peyer's patches from pBud-vaccinated (Upper) or pCEA-vaccinated (Lower) mice. Each panel indicates two representative CD11c⁺ (green) dendritic cells. (E) Vaccinated CEA-A2Kb double transgenic mice were challenged s.c. with 6 × 10⁵ MC-38-CEA-A2Kb cells and killed 28 days later. Tumor weights from each mouse are represented by individual symbols. Solid line, average tumor weights of each group. *, P < 0.0002 or 0.05 (compared with PBS or pBud control groups, respectively). Experiments were repeated three times with similar results.

Fig. 7.

The pH60/CEA vaccine enhanced NK- and CEA-specific CTL responses. (A) Standard ⁵¹Cr release assays were performed with freshly isolated splenocytes against Yac-1 NK target cells. In vitro-stimulated splenocytes were tested in cytotoxicity assays against MC-38-CEA-A2Kb (B) or MC-38 (C) target cells. (A–C) ⋄, pBud group; ▪, pH60 group; ▵, pCEA group; •, pH60/CEA group. (D) Cytotoxicity of in vitro-stimulated splenocytes from pBud (open symbols) or pH60/CEA (closed symbols) groups against unloaded (▵) or CEA₆₉₁-loaded (○) T2 target cells. (E) ELISPOT assays were performed with freshly isolated splenocytes either without (open bars) or in the presence of control peptide (kindly provided by J. Schlom of the National Institutes of Health; shaded bars) or CEA₆₉₁ peptides (striped bars) at 10 μg/ml.