title: NK cytotoxicity and alloreactivity against neuroblastoma cell lines in vitro: comparison of Europium fluorometry assay and quantification by RT-PCR.
abstract: New therapies for children with high risk neuroblastoma are needed, and haploidentical stem cell transplantation with NK post-graft injections is a potential option. To develop this strategy, we compared and correlated two methods of NK cytotoxicity assay. The aim of this work is to optimize in vitro NK cytotoxicity assays, investigate the effect of interleukin stimulation on NK cells and use of antiGD2 antibodies against tumor target cells and finally establish an in vitro model for haploidentical stem cell transplantation.
  
  
  METHODS
  We evaluated NK cell cytotoxicity in vitro against NB cell lines (IMR-32 and SK-NSH) in different culture conditions using a Europium BATDA fluorescence test, and correlated the results with quantification of TH, Phox2B, and DCX transcripts evaluated by RT-PCR.
  
  
  RESULTS
  Both IMR-32 and SK-N-SH neuroblastoma cell lines were sensitive to NK cells and particularly when NK cells were stimulated by interleukin IL-2 and IL-15 or when using anti-GD2 antibodies against tumor target cells. All these results were observed either with Europium fluorometry assay or with RT-PCR quantification. There is a clear correlation between the two methods, for the three transcripts at the ratio effector/target 50/1 (TH r=0.75, Phox2B r=0.79 and DCX r=0.8), for all the values whatever the cell line. Besides for all three transcripts, the correlations were significantly independent of the cell line and the ratio E/T (all p values non-significant) even if the best correlation was observed for the ratio 50/1. After prolonged incubation times of effector and target cells (24 h), which could be evaluated only by RT-PCR, all the transcripts clearly decreased, confirming the haploidentical effect of NK against the two neuroblastoma cell lines in our two in vitro haploidentical models but no advantage of mismatch.
  
  
  CONCLUSIONS
  NK cytotoxicity against neuroblastoma cell lines can be evaluated by Europium assay and by RT-PCR with clear correlation for the three transcripts TH, Phox2B and DCX whatever the ratio E/T and cell line used. This new method of RT-PCR is simple and suitable for large-scale conditions like study of adherent tumor cells or prolonged incubations of target/effector cells which allowed us to observe haploidentical effect.
authors.affiliation: CHU de Clermont-Ferrand, Centre Régional de Cancérologie et Thérapie Cellulaire Pédiatrique, Hôpital Estaing, 63001 Clermont-Ferrand, France. cpaillard@chu-clermontferrand.fr
authors.forename: C
authors.initials: C
authors.lastname: Paillard
authors.forename: P
authors.initials: P
authors.lastname: Halle
authors.forename: A
authors.initials: A
authors.lastname: Tchirkov
authors.forename: C
authors.initials: C
authors.lastname: Confland
authors.forename: R
authors.initials: R
authors.lastname: Veyrat-Masson
authors.forename: F
authors.initials: F
authors.lastname: Quainon
authors.forename: B
authors.initials: B
authors.lastname: Perreira
authors.forename: E
authors.initials: E
authors.lastname: Rochette
authors.forename: M
authors.initials: M
authors.lastname: Pfeiffer
authors.forename: P
authors.initials: P
authors.lastname: Lang
authors.forename: F
authors.initials: F
authors.lastname: Deméocq
authors.forename: J
authors.initials: J
authors.lastname: Kanold
mesh_terms: D000328:Adult; D000368:Aged; D045744:Cell Line, Tumor; D003601:Cytotoxicity Tests, Immunologic; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D005063:Europium / Q000032:analysis*; D005260:Female; D005470:Fluorometry / Q000379:methods*; D018398:Homeodomain Proteins / Q000096:biosynthesis; D006801:Humans; D018664:Interleukin-12 / Q000276:immunology; D019409:Interleukin-15 / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology*; D008297:Male; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis; D008875:Middle Aged; D009447:Neuroblastoma / Q000276:immunology*; D009479:Neuropeptides / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014157:Transcription Factors / Q000096:biosynthesis; D055815:Young Adult
chemical_list: C000714328:DCX protein, human; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D018398:Homeodomain Proteins; C569274:IL15 protein, human; D019409:Interleukin-15; D008869:Microtubule-Associated Proteins; C109220:NBPhox protein; D009479:Neuropeptides; D014157:Transcription Factors; D018664:Interleukin-12; D005063:Europium