More than 95% of the cells were CD56+CD3- lymphocytes. 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 cytolytic activity of NK cells co-cultured with AFP-DCs or Alb-DCs against target cells (K562, NK sensitive cells, or Huh7, human HCC cells) was assessed by 4-h 51Cr-releasing assay with or without the presence of neutralizing antibody of IL-12 (BD Pharmingen) or recombinant IL-12p70 protein (PeproTech), as described previously [14]. In some experiments,

a Transwell insert was also used to prevent direct contact of NK cells and DCs in co-culture systems, as described previously [14]. The statistical significance of differences between the two groups was determined by applying the Mann–Whitney U-test. We defined statistical significance as P < 0·05. We investigated the activity of NK cells co-cultured with Selleck BYL719 AFP-DCs or Alb-DCs. NK cells from the same healthy volunteers were co-cultured with AFP-DCs or Alb-DCs for 24 h, and we evaluated the cytolytic activity of NK cells co-cultured with DCs against K562 cells as target cells with the 51Cr-releasing assay. The cytotoxicity of NK cells Protein Tyrosine Kinase inhibitor co-cultured with AFP-DCs against K562 cells was significantly lower than those with Alb-DCs (Fig. 1a). Similarly, the cytotoxicity of NK cells co-cultured with AFP-DCs against Huh7 cells was significantly lower than

that with Alb-DCs (Fig. 1b). We also evaluated the IFN-γ production from NK cells co-cultured with AFP-DCs or Alb-DCs by specific ELISA. IFN-γ production from NK cells co-cultured with AFP-DCs was significantly lower than that from NK cells co-cultured

with Alb-DCs (Fig. 1c). These results demonstrated that NK activity co-cultured with AFP-DCS was lower than that Buspirone HCl with Alb-DCs. Next, NK cells were cultured with AFP (AFP-NK cells) or Alb (Alb-NK cells) for 24 h, and we evaluated the cytolytic activity of AFP-NK and Alb-NK against K562 cells with the 51Cr-releasing assay. The cytotoxicity of AFP-NK cells was almost similar to that of Alb-NK cells, and the presence of DCs could enhance the cytotoxicity of NK cells (Fig. 2a). These results suggested that AFP does not directly impair NK cell function and that DCs play a critical role in activating NK cells. To examine whether this attenuation of NK cells was caused by the cytokine from DCs or by direct contact with DCs, NK cells were co-cultured with AFP-DCs or Alb-DCs in Transwell culture for 24 h. The cytotoxicity of NK cells co-cultured with AFP-DCs was lower than that with Alb-DCs, which was similar to the results without Transwell membrane (Fig. 2b). These results suggested that soluble factors derived from DCs played a role in activating NK cells. We next examined the function of AFP-DCs. We obtained DCs from eight healthy volunteers and cultured the DCs for 7 days in RPMI-1640 with AFP (AFP-DCs) or Alb (Alb-DCs). On day 6, we added LPS to induce DC maturation.