α-Galactosylceramide (α-GalCer)-stimulated human Vα24 natural killer T (NKT) cells exert antitumor activity against some leukemia in a CD1d dependent and TCR-mediated manner, but could not kill CD1d - negative neuroblastoma (NB) cells. There are few reports about the direct antitumor effect of highly secreted cytokines by these cells on activation. In this study, using a cell-free supernatant (SPN) collected from plate bound hCD1d/αGalCer tetramers-stimulated NKT cells, we examined whether they could be helpful in the immunotherapeutic treatment of NB.

Cells were cultured in IMDM. The cytokines produced by NKT cells were measured with Cytometric Bead Array (CBA) analysis. Cell viability was evaluated by calcein-AM fluorescence with digital image microscopy scanning (DIMSCAN). The percentage of specific apoptosis was calculated by flow cytometric detection of apoptosis using annexin V and 7-AAD.

The activated NKT cells secreted high levels of IL-2, INF-γ, TNF-α. The SPN was significantly cytotoxic against four out of eight tested NB cell lines, through mainly apoptosis as evidenced by annexin-V staining and inhibition with the pretreatment of pancaspase blocker. This apoptosis was significantly inhibited when anti-TNF-α and anti-IFN-γ neutralizing mAbs were used separately and it was completely abolished when the two mAbs were combined.

IFN-γ and TNF-α produced by NKT cells could exert synergistically direct antitumor activity through apoptosis on some NB cell lines.