Compared with NK cells alone, NK cells incubated with iNKT cells and vehicle\pulsed moDC were partially activated. were upregulated, and the cytotoxicity of NK cells treated with anti\GD2 antibodies was increased. Not only cytokines produced by activated iNKT cells, but also NK\NKT cell contact or NK cell\dendritic cell contact contributed to the increase in NK cell cytotoxicity and further IFN production by iNKT cells and NK cells. In conclusion, iNKT cell\based immunotherapy could be an appropriate candidate for anti\GD2 antibody therapy for neuroblastoma. (((Hs00169473_m1) and (as a housekeeping gene. Cytokine measurement To determine the amount of cytokine secretion, a Bio\Plex assay was performed according to the manufacturer’s recommendations using the Bio\Plex 3D Suspension Array System and Bio\Plex Human Cytokine 17\plex Assay (Bio\Rad, Hercules, CA, USA). The cytokines that can be detected using this assay are: IL\1, IL\2, IL\4, IL\5, IL\6, IL\7, IL\8, IL\10, IL\12 (p70), IL\13, IL\17, G\CSF, GM\CSF, IFN\, MCP\1 (MCAF), MIP\1 and TNF\. The data were analyzed using the Bio\Plex Manager version 6.1 software program. DMH-1 Transwell system Transwell plates with two chambers per well separated by a 400\nm pore DMH-1 membrane (Corning) were used for the transwell assays. Statistical analysis The data are expressed as the means??SD. Statistical analyses were performed using Student’s cytotoxicity assay using NK cells against NB cell lines with various GD2 expression levels was performed. NK cells were cultured for 4?h at various E:T ratios with NB cell lines in the presence of anti\GD2 Abs (14.G2a). ADCC mediated by NK cells toward NMB (high GD2 expression, Fig.?1c) was highest and that toward NLF (low GD2 expression) was lowest. The cytotoxicity toward IMR\32, which had a heterogeneous expression of GD2, was not as high as that against NMB (Fig.?2b). iNKT cell\mediated cytotoxicity toward NMB was not increased by the addition of anti\GD2 Ab (Fig.?2c, right), whereas NK cell\mediated cytotoxicity was dramatically increased by the addition of anti\GD2 Ab (Fig.?2c, left). When iNKT cells are activated by APC, TMOD3 it is known that iNKT cells produce a substantial amount of IFN. Therefore, iNKT cells were cultured with NB cells in the presence of anti\GD2 Abs and the IFN production was measured. There was no increase of IFN production by iNKT cells with NB cells and antibodies (data not shown). Open in a separate window Figure 2 Natural killer (NK) cell\mediated antibody\dependent cellular cytotoxicity (ADCC) DMH-1 is related to the expression level of the tumor antigen, whereas invariant natural killer T (iNKT) cells themselves do not mediate ADCC. (a) The surface FcR (CD16) expression of freshly isolated NK cells and expanded iNKT cells is shown. The data are from one representative experiment of a total of five experiments. (b) NK cells were cultured for 4?h at various E:T ratios with NB cell lines DMH-1 with various intensities of the GD2 expression in the presence of anti\GD2 antibodies or isotype controls. (c) NK cells and iNKT cells were cultured for 4?h at various E:T ratios with NMB NB cells. Natural killer cell activation by invariant natural killer T cells It has been reported that the cytokines produced by activated iNKT cells can DMH-1 activate and induce the proliferation of NK cells and enhance tumor immunity;10, 11, 24 however, precisely which function of NK cells is enhanced remains unclear. To examine whether the expression of Fas ligand (FasL) or cytotoxic granules by NK cells was enhanced by activated iNKT cells, freshly isolated NK cells were incubated together with or without expanded iNKT cells and moDC without exogenous cytokines. The Fas expression of NB cell lines was examined before this experiment (Fig.?3a). Open in a separate window Figure 3 Activated invariant natural killer T (iNKT) cells have no effect on the.