α-Fetoprotein impairs activation of natural killer cells by inhibiting the function of dendritic cells

Abstract

α-Fetoprotein (AFP) is a tumour-associated antigen in hepatocellular carcinoma (HCC). The biological properties of AFP have been identified in its regulatory effects on immune responses of T cells and B cells. However, AFP effects on natural killer (NK) cells are still unclear. In this study, we examined the immunoregulation of AFP on NK activity. The cytolytic activity against K562 cells and Huh7 cells of NK cells co-cultured with AFP-treated dendritic cells (DCs) (AFP-DCs) was lower than that with albumin-treated DCs (Alb-DCs). Direct addition of AFP to NK cells did not alter the cytolytic activity of NK cells. Adding AFP inhibited the interleukin (IL)-12 production of DCs after stimulation with lipopolysaccharide (LPS) [Toll-like receptor (TLR)-4 ligand], or Poly(I:C) (TLR-3 ligand), but not IL-18 production. The mRNAs of IL-12p35 and IL-12p40 were significantly inhibited in AFP-DCs compared with Alb-DCs, but those of TLR-4 or TLR-3 were not. Transwell experiments revealed that soluble factors derived from DCs played roles in inhibition of the ability of activating NK cells by AFP-DCs. Adding the neutralizing antibody of IL-12 to NK cells co-cultured with Alb-DCs resulted in a decrease of cytolytic activity to the levels of NK cells co-cultured with AFP-DCs. Adding IL-12 to NK cells co-cultured with AFP-DCs resulted in an increase of cytolytic activity to the levels of NK cells co-cultured with Alb-DCs. These demonstrated that the impairment of IL-12 production from AFP-DCs resulted in inhibition of the ability of the activation of NK cells by DCs, and thus suggests a role of AFP in HCC development.

Fig. 1

The cytolytic activity and interferon (IFN)-γ production of natural killer (NK) cells co-cultured with α-fetoprotein-dendritic cells (AFP-DCs) were impaired. (a,b) NK cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic cell sorting using CD56 MicroBeads according to the manufacturer's instructions. Enriched NK cells were co-cultured with AFP (25 µg/ml, AFP-DCs) or albumin (Alb) (25 µg/ml, Alb-DCs) pretreated DCs for 24 h. The cytolytic activities of NK cells co-cultured with AFP-DCs () or Alb-DCs (♦) against K562 cells (a) or Huh7 cells (b) were evaluated by ⁵¹Cr-releasing assay. *P < 0·05 versus the cytolytic activity of NK cells cultured with Alb-DCs. (c) NK cells were isolated from PBMCs by magnetic cell sorting using CD56 MicroBeads, according to the manufacturer's instructions. Enriched NK cells were co-cultured with AFP (25 µg/ml, AFP-DCs) or Alb (25 µg/ml, Alb-DCs) pretreated DCs for 24 h. The IFN-γ productions from NK cells were analysed by specific enzyme-linked immunosorbent assay (ELISA). To evaluate the IFN-γ production from NK cells, we also evaluated the IFN-γ production from AFP-DCs or Alb-DCs cultured without NK cells, and these values were subtracted from all experimental determinations to determine specific IFN-γ productions of NK cells (results in pg/ml; mean ± standard deviation of triplicate samples). We analysed statistically the production of IFN-γ between AFP-DCs and Alb-DCs. *P < 0·05.

Fig. 2

α-Fetoprotein (AFP) did not directly affect the cytolytic activity of natural killer (NK) cells and soluble factor from dendritic cells (DCs) played a role in the inhibition of NK activity. (a) NK cells were cultured with AFP (25 µg/ml, □, AFP-NK cells) or albumin (Alb) (25 µg/ml, ◊, Alb-NK cells) or cultured with AFP-DCs () or Alb-DCs (♦) for 24 h. We evaluated the cytolytic activity of AFP-NK cells and Alb-NK cells or NK cells stimulated by AFP-DCs or Alb-DCs using K562 cells as target cells by ⁵¹Cr-releasing assay. We analysed statistically between the cytolytic activity of NK cells co-cultured with AFP-DCs () and Alb-DCs (♦) or between that of AFP-NK cells (□) and Alb-NK cells (◊). *P < 0·05 versus the cytolytic activity of NK cells cultured with Alb-DCs, #P < 0·05 versus the cytolytic activity of Alb-NK cells. (b) Enriched NK cells were co-cultured with AFP-DCs (□) or Alb-DCs (◊) for 24 h in the presence of 0·4 µm of inserting membrane (Transwell). NK cells were harvested and subjected to examine the cytolytic activity against K562 cells by ⁵¹Cr-releasing assay. *P < 0·05 versus the cytolytic activity of NK cells cultured with Alb-DCs. Representative results are shown. Similar results were obtained from three independent experiments in all experiments.

Fig. 3

The maturation of α-fetoprotein-dendritic cells (AFP-DCs) was inhibited more than that of Alb-DCs. Monocyte-derived DCs were generated from eight healthy volunteers. DCs were cultured for 7 days in RPMI-1640 with AFP (25 µg/ml) or albumin (Alb) (25 µg/ml). On day 6, lipopolysaccharide (LPS) was added to induce DC maturation. (a) We defined DCs with CD11c⁺ and human leucocyte antigen D-related (HLA-DR⁺) cells by flow cytometry. (b) We evaluated the expression of CD80, CD86, CD40 and CD83 on AFP-DCs (black line) and Alb-DCs (dotted line). Grey histogram indicates control immunoglobulin (Ig)G staining. The expression of each molecule on AFP-DCs and Alb-DCs from seven healthy volunteers was evaluated by the mean fluorescence intensity (MFI) ± standard deviation. All experiments were performed three times independently and representative results (upper panels) as well as the statistical analysis (lower panels) are shown as the MFI of the staining cells. *P < 0·05.

Fig. 4

The production of interleukin (IL)-12p70 from α-fetoprotein-dendritic cells (AFP-DCs) was lower than that from Alb-DCs, but that of interleukin (IL)-18 was not. We cultured DCs for 7 days in RPMI-1640 with AFP (25 µg/ml, 12·5 µg/ml, 6·25 µg/ml) or albumin (Alb) (25 µg/ml). On day 6, we added lipopolysaccharide (LPS) (10 µg/ml, a,b) or Poly(I:C) (10 µg/ml, c) to induce DC maturation. Twenty-four hours later, IL-12p70 (a,c) or IL-18 (b) production from LPS- or Poly(I:C)-treated DCs was measured by specific enzyme-linked immunosorbent assay (ELISA) (results in pg/ml; mean ± standard deviation of triplicate samples). (a, left panel; b,c) We analysed statistically the production of both cytokines between AFP-DCs and Alb-DCs. *P < 0·05. (a, right panel) We analysed statistically the production of IL-12p70 between Alb-DCs and AFP (6·25 µg/ml)-DCs, AFP (12·5 µg/ml)-DCs or AFP(25 µg/ml)-DCs. *P < 0·05 versus IL-12p70 production of Alb-DCs.

Fig. 5

The mRNAs of interleukin (IL)-12p35 and p40 from α-fetoprotein-dendritic cells (AFP-DCs) were lower than that from albumin (Alb)-DCs, but the mRNA of Toll-like receptor (TLR)-4 and TLRL-3 were not. We cultured DCs for 7 days in RPMI-1640 with AFP (25 µg/ml) or Alb (25 µg/ml). On day 6, we added lipopolysaccharide (LPS) (10 µg/ml) or Poly(I:C) (10 µg/ml) to induce DC maturation. Twenty-four hours later, total RNA was isolated from LPS or Poly(I:C)-treated AFP-DCs or Alb-DCs and was subjected to real-time polymerase chain reaction (PCR) to detect mRNA of IL-12 (a: IL-12p35 mRNA, IL-12p40 mRNA) or mRNA of TLRs (b, TLR-4 mRNA or TLR-3 mRNA). Similar results were obtained from three independent experiments. We analysed statistically the mRNA levels of IL-12p35, IL-12p40, TLR-4 and TLR-3 between AFP-DCs and Alb-DCs. *P < 0·05.

Fig. 6

Interleukin (IL)-12 derived from dendritic cells (DCs) played a critical role in natural killer (NK) cell activation. (a) Enriched NK cells were co-cultured with α-fetoprotein (AFP)-DCs or albumin (Alb)-DCs for 24 h with or without the presence of the neutralizing antibody of IL-12. The cytolytic activity of NK cells against K562 cells were evaluated by ⁵¹Cr-releasing assay. The cytolytic activity against K562 cells of NK cells co-cultured with AFP-DCs () or Alb-DCs (♦) without neutralizing antibody of IL-12 or AFP-DCs (□) or Alb-DCs (◊) with neutralizing antibody of IL-12. We analysed statistically between the antibody-adding and not-adding groups in both AFP-DC and Alb-DC cultures, respectively. *P < 0·05 versus the cytolytic activity of NK cells cultured with Alb-DCs. Significant difference was observed between the antibody-adding group and not-adding group in the cytolytic activity of NK cells cultured with Alb-DCs. In contrast, no significant difference was observed between the groups in the cytolytic activity of NK cells cultured with AFP-DCs. (b) Enriched NK cells were co-cultured with AFP-DCs or Alb-DCs for 24 h with or without recombinant IL-12p70 protein (150 pg/ml, 300 pg/ml). NK cells were harvested and subjected to examine the cytolytic activity against K562 cells by ⁵¹Cr releasing assay. The cytolytic activity co-cultured with AFP-DCs without IL-12 () or with IL-12 (150 pg/ml, •; 300 pg/ml, ▴) or Alb-DCs (♦). *P < 0·05 versus the cytolytic activity of NK cells cultured with Alb-DCs. Representative results are shown. Similar results were obtained from three independent experiments in all experiments.