All issues > Volume 49(4); 2006

Original Article

Korean J Pediatr. 2006;49(4):439-445. Published online April 15, 2006.

The study on cytotoxicity of cytokines produced by the activated human NKT cells on neuroblastoma

Jin Young JY Cho¹, Young Wook YW Yoon¹, Hyang Suk HS Yoon¹, Jong Duk JD Kim¹, Du Young DY Choi¹

¹Department of Pediatrics, School of Medicine, Wonkwang University, Iksan, Korea

Correspondence Du Young DY Choi ,Email: cdy8118@wonkwang.ac.kr

Abstract

Purpose
: 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.
Methods
: 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.
Results
: 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.
Conclusion
: IFN-γ and TNF-α produced by NKT cells could exert synergistically direct anti- tumor activity through apoptosis on some NB cell lines.

Keywords :α-Galactosylceramide (α-GalCer) , Vα24 NKT cells , INF-γ , TNF-α , Neuroblastoma (NB)