Korean J Pediatr.  2006 Apr;49(4):439-445. 10.3345/kjp.2006.49.4.439.

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

• Cho JY
• Yoon YW
• Yoon HS
• Kim JD
• Choi DY

• KMID: 2140911
• DOI: http://doi.org/10.3345/kjp.2006.49.4.439

Abstract

PURPOSE: alpha-Galactosylceramide (alpha-GalCer)-stimulated human Valpha24 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/alphaGalCer 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-gamma, TNF-alpha. 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-alpha and anti-IFN-gamma neutralizing mAbs were used separately and it was completely abolished when the two mAbs were combined.
CONCLUSION
IFN-gamma and TNF-alpha produced by NKT cells could exert synergistically direct anti-tumor activity through apoptosis on some NB cell lines.

Keyword

alpha-Galactosylceramide (alpha-GalCer); Valpha24 NKT cells; INF-gamma; TNF-alpha; Neuroblastoma (NB)

MeSH Terms

Annexin A5
Apoptosis
Cell Line
Cell Survival
Cytokines*
Fluorescence
Humans*
Interleukin-2
Leukemia
Microscopy
Natural Killer T-Cells*
Neuroblastoma*
Tumor Necrosis Factor-alpha
Annexin A5
Cytokines
Interleukin-2
Tumor Necrosis Factor-alpha