Myeloid derived suppressor cells inhibit natural killer cells in patients with hepatocellular carcinoma via the NKp30 receptor

Abstract

Several immune suppressive mechanisms that evade the host immune response have been described in patients with hepatocellular carcinoma (HCC); one of these mechanisms is expansion of myeloid-derived suppressor cells (MDSCs). MDSCs have been shown to inhibit T cell responses in tumor-bearing mice, but little is known about these cells in humans. Here, we have analyzed and characterized the effect of MDSCs on the innate immune system, in particular, their interaction with natural killer (NK) cells in patients with HCC. MDSCs from patients with HCC inhibited autologous NK cell cytotoxicity and cytokine secretion when cultured together in vitro. This suppression was dependent on cell contact, but did not rely on the arginase activity of MDSCs, which is a hallmark function of these cells. However, MDSC-mediated inhibition of NK cell function was dependent mainly on the NKp30 on NK cells.

Conclusion: Our study suggests a new role for MDSCs in patients with HCC in disarming the innate immune system and further contributing to the immune suppressor network in these patients. These findings have important implications when designing immunotherapy protocols.

Fig. 1.

NK cell cytotoxicity is reduced in patients with HCC. NK cells were FACS-sorted from PBMCs of either healthy donors (n = 10) or patients with HCC (n = 10), or from tumor-infiltrating lymphocytes (n = 5) by gating on CD56⁺CD3⁻ population and used in a ⁵¹Cr-release assay against K562 target cells. Figure shown is average of four independent experiments (*P < 0.05).

Fig. 2.

CD14⁺HLA-DR^−/low cells suppress NK cell function. (A) Purified NK cells were cultured in the absence or presence of different ratios of CD14⁺HLA-DR⁺ or CD14⁺HLA-DR^−/low cells as indicated. After 12 hours, K562 cells were added at a ratio of 2.5:1 (E:T) and lysis was determined by standard ⁵¹Cr-release assay (P < 0.05). Shown are cumulative results from four independent experiments. (B) NK cells were stimulated with IL-2 and cultured in the presence or absence of CD14⁺HLA-DR^−/low or CD14⁺HLA-DR⁺ cells as indicated. IFN-γ release was determined after 48 hours by ELISA. Shown are cumulative results from five independent experiments (*P < 0.05, **P < 0.001). NK cells cultured without IL-2 were used as background (arrow).

Fig. 3.

Intracellular cytokine analysis of CD14⁺HLA-DR^−/low in the presence or absence of NK cells. (A) NK cells were stimulated with IL-2 and cultured either alone, with CD14⁺HLA-DR^−/low, or with CD14⁺HLA-DR⁺ cells. IFN-γ was analyzed after 48 hours by intracellular FACS as shown in representative dot plots. (B) Cumulative results of three independent experiments are shown (*P < 0.05; **P < 0.001). Cytokine secretion was analyzed gating on CD14⁺ cells (black bars) and CD56⁺ cells (white bars).

Fig. 4.

Phenotypic analysis of NK cells and CD14⁺HLA-DR^−/low cells upon coculture. Purified CD14⁺HLA-DR^−/low or CD14⁺HLA-DR⁺ cells were cultured alone or with FACS-sorted autologous NK cells as indicated, and the expression of different surface markers (black lines) or isotype control (filled histograms) was analyzed by FACS. Representative histograms for (A) CD14⁺HLA-DR⁺, CD14⁺HLA-DR^−/low or (B) NK cells are shown. Cumulative results of seven independent experiments for (C) HLA-DR and (D) NKp30 are shown (*P < 0.05; **P < 0.001, ***P < 0.0001).

Fig. 5.

Inhibition of IFN-γ production by MDSC is mainly cell contact–dependent, but independent of arginase and NO. (A) CD14⁺HLA-DR^−/low cells were cocultured with NK cells as described and transwell inserts were used as indicated (white bar). IFN-γ secretion was determined by ELISA. Shown are cumulative results from three independent experiments (*P < 0.05). (B) Purified NK cells and CD14⁺HLA-DR^−/low cells were cocultured in the absence or presence of N-omega-hydroxy-L-arginine (L-NOHA), N(G)-monomethyl-L-arginine (L-NMMA), 1-MT (10 μmol/L each), or media alone as indicated. IFN-γ production was measured by ELISA after 48 hours. Figure shown is a representative of three independent experiments. (C) Purified NK cells and CD14⁺HLA-DR^−/low cells were cocultured for 36 hours. NK cells were reisolated, and a ⁵¹Cr-release assay against K562 target cells was performed. Shown is representative data from three independent experiments.

Fig. 6.

MDSC-mediated suppressive mechanism is independent of anti-CD94, anti-NKG2D, anti-NKp44, anti-HLA-DR, and anti-MHC class I. Purified NK cells were cultured with IL-2 in the absence or presence of different blocking antibodies as indicated. (A-E) IFN-γ secretion was measured in supernatants after coincubation in the presence of the indicated antibodies by ELISA. Shown are cumulative results from two independent experiments.

Fig. 7.

MDSC-mediated suppressive mechanism is dependent on NKp30. (A) NK cells were cultured with IL-2, CD14⁺HLA-DR^−/low in the absence or presence of anti-NKp30 antibody or isotype control. IFN-γ secretion was measured by ELISA. Cumulative results ± SEM of three independent experiments are shown (*P < 0.05). (B) NK cells were cultured with CD14⁺HLA-DR^−/low in the presence or absence of anti-NKp30 antibody or isotype control. K562 cells were added at a ratio of 2.5:1 (E:T) and lysis was measured. Cumulative results ± SEM of three independent experiments are shown.