Different levels of control prevent interferon-gamma-inducible HLA-class II expression in human neuroblastoma cells

The HLA class II expression is controlled by the transcriptional activator CIITA. The transcription of CIITA is controlled by different promoters, among which promoter-IV is inducible by IFN-gamma. We analysed the regulation of HLA class II molecules by IFN-gamma in a large series of human neuroblastoma cell lines. No induction of surface or intracellular HLA class II molecules and of specific mRNA was observed, in all neuroblastomas, with the exception of a nonprototypic cell line, ACN. In a large subset of neuroblastomas IFN-gamma induced expression of CIITA mRNA, derived from promoter-IV, which was not methylated. In contrast, in another subset of neuroblastomas, CIITA was not inducible by IFN-gamma and CIITA promoter-IV was either completely or partially methylated. Interestingly, the use of DNA demethylating agents restored CIITA gene transcriptional activation by IFN-gamma, but not HLA class II expression. The defect of HLA class II was not related to alterations in RFX or NF-Y transcription factors, as suggested by EMSA or RFX gene transfection experiments. In addition, the transfection of a functional CIITA cDNA failed to induce HLA class II expression in typical neuroblastoma cells. Confocal microscopy and Western blot analysis suggested a defective nuclear translocation and/or reduced protein synthesis in CIITA-transfected NB cells. Altogether, these data point to multiple mechanisms preventing HLA class II expression in the neuroblastoma, either involving CIITA promoter-IV silencing, or acting at the CIITA post-transcriptional level.