Figure 3.

Lack of CIITA expression is responsible for the absence of HLA-II expression in HCC tumor cell lines.

(a) HLA class I (HLA-I) and HLA class II (HLA-DR) cell surface phenotype of Alex, Hep3B, HepG2 and HUH7 HCC cell lines were carried out by immunofluorescence and FACS analysis. The monocytic THP-1 cell line was used as a positive control for both baseline and IFNγ-induced HLA-I and HLA-DR expression. Histograms represent fluorescence profiles of the cells indicated on the top incubated with specific anti-HLA-I or HLA-DR mAbs followed by incubation with FITC-conjugated F(ab)2 anti mouse antibody as second reagent. Cells were either untreated (solid line) or treated with IFNγ (bold line). Controls (dashed line) are cells incubated with the second reagent only. Mean fluorescence (m.f.) values are expressed in the abscissa as arbitrary units (a.u.). A representative experiment is shown out of three independent experiments with very similar results. (b) CIITA mRNAs expression was assessed by qRT-PCR in cells treated or not with IFNγ. The results of a representative experiment, out of at least three experiments with similar results, performed in triplicates are shown. CIITA mRNA levels are expressed as values relative to those of untreated Jurkat T cells set to 1. THP-1 cells were used as a positive control for both basal and IFNγ-induced- CIITA mRNA expression. Error bars represent the standard deviation. (c) The stable expression of CIITA in Alex, Hep3B, HepG2 and HUH7 cells restores the HLA-DR expression. HLA-I and HLA-DR cell surface expression was carried out by immunofluorescence and FACS analysis. Histograms represent fluorescence profiles of the cells indicated on the top incubated with specific anti HLA-I or HLA-DR mAbs (solid line) followed by incubation with FITC-conjugated F(ab)2 anti mouse antibody as second reagent. Controls (dashed line) are cells incubated with the second reagent only. Mean fluorescence (m.f.) values are expressed in the abscissa as arbitrary unit (a.u). A representative experiment is shown out of three independent experiments with very similar results.