HLA-DR phenotype of parental THP-1 and of THP1-L. Indirect immunofluorescence analysis by flow cytometry by using FITC-conjugated anti-mouse IgG secondary antibody. Cell surface expression in untreated cells (mock) or after 48 h in presence of 150 U ml⁻¹ of IFN-γ. Cells stained with an isotype-matched antibody were used as negative control (thin line). Results are expressed in abscissa as log fluorescence value in arbitrary units. The results shown are from one representative experiment of four with similar results.

Quantitative real-time RT–PCR analysis of promoter-specific CIITA transcripts in various cells. THP1-L cells were treated with 5-aza for 5 days and then either left untreated or incubated with IFN-γ for 2 days. THP1-pc, untreated or similarly incubated with IFN-γ, iDCs and the B cell Raji were also analysed. For AIR-1 p-I, the housekeeping normalized PCR data value obtained in iDC was defined as the arbitrary unit (value 1). For AIR-1 p-III, the housekeeping normalized PCR data value obtained in Raji was defined as the arbitrary unit (value 1). For AIR-1 p-IV, the housekeeping normalized PCR data value obtained in THP1-pc treated with IFN-γ was defined as the arbitrary unit (value 1). The results shown are from one representative experiment of two with similar results.

Expression of HLA-DR and CIITA in U937 cells incubated with IFN-γ before and after treatment with 5-aza and analysis of the AIR-1 pIV methylation status. (A) HLA-DR cell surface phenotype of U937 cells as assessed by immunofluorescence and flow cytometry. Thin histograms represent the negative controls after each treatment (staining with irrelevant isotype-matched antibody); mock, U937 cells untreated; +IFN-γ, U937 cells treated with IFN-γ for 48 h; +5-aza + IFN-γ, U937 cells treated with 5-aza for 5 days and then with IFN-γ for 48 h. Results are expressed in abscissa as log fluorescence value in arbitrary units. The results shown are from one representative experiment of four with similar results. (B) Quantitative real-time RT–PCR analysis of CIITA and DRA transcripts. U937 cells were treated as in (A) and total RNA was extracted. The data values obtained for CIITA and DRA transcripts were normalized with respect to the value of the housekeeping gene transcript of choice in mock-treated U937 cells and defined as the arbitrary unit (value 1). All other normalized PCR data values were then expressed as fraction or multiple of the arbitrary unit. Bars represent the standard deviation of the mean of three distinct experiments. (C) Quantitative analysis of −86 and −77 CpG sites by pyrosequencing (see schematic representation of AIR-1 p-IV, Fig. 4A). U937 cells were treated as in (A) and analysed in duplicate. Values are expressed as mean percentage of methylation at the two sites.

Treatment with 5-aza restores CIITA and consequent HLA-DR inducibility by IFN-γ in THP1-L. (A) Quantitative analysis by real-time RT–PCR of CIITA and DRA transcript. THP1-L cells were treated with 5-aza for 5–7 days or with TSA for 2 days. Freshly prepared 5-aza was added to cultures every day. Thereafter, THP1-L and THP1-pc were treated with IFN-γ for 2 days and total RNA was extracted. The housekeeping normalized data value obtained in mock THP1-pc was defined as the arbitrary unit (value 1). All other normalized PCR data values were then expressed as fraction or multiple of the arbitrary unit (mRNA a.u.) in the ordinate in a log scale. Bars represent the standard deviation from the mean of three distinct experiments. (B) THP1-L cells treated with 5-aza for 5 days were then cultured in absence (mock) or in presence of 150 U ml⁻¹ IFN-γ (IFN-γ) for 2 days and analysed for HLA-DR surface expression by indirect immunofluorescence and flow cytometry. The results shown are from one representative experiment of three with similar results. (C) THP1-L cells were transfected with a CIITA expression vector under the control of CMVp. Stable bulk transfection was obtained under G418 selection and further subcloned. HLA-DR surface expression (DR) was evaluated by indirect immunofluorescence and flow cytometry. Results refer to a representative clone of 10 with similar DR phenotype.

Hyper-methylation of AIR-1 pIV as the major cause of lack of CIITA expression in response to IFN-γ. (A) Schematic representation of the AIR-1 p-IV region. Thick arrows indicate the positions of the primers used to analyse CpG sites by semi-quantitative restriction analysis; thin arrows indicate the positions of the primers used to analyse CpG sites by quantitative pyrosequencing. CpG sites under investigation are shown as vertical bars: sites −238 (HpaI MspI) and −77 (HhaI) were studied by restriction analysis; sites −86 and −77 were studied by quantitative pyrosequencing. (B) Semi-quantitative analysis of two CpG sites by restriction enzyme digestion. THP1-pc (pIV nMet), THP-1 parental cells with a non-methylated pIV; THP1-L (pIV Met), the THP1 variant with a fully methylated pIV; THP1-L 5-aza (pIV Met/nMet), the THP1-L variant treated with 5-aza showing a significantly reduced methylation of pIV. IMR5 and ACN are two neuroblastoma cell lines chosen as control of methylated (Met) and non-methylated (nMet) AIR-1 p-IV, respectively (15). The results shown are from one representative experiment of two with similar results. (C) Quantitative analysis of two CpG sites by pyrosequencing. ACN, IMR5 and THP1-pc were analysed in duplicates (mean value), THP1-L, THP1-L + aza and THP1-L TSA + aza were analysed in triplicate (mean value ± SD). Values are expressed as percentage of methylation at the two sites.

HLA-DR2-restricted, antigen-specific T cell activation assay using THP-1 as APC. THP1-L was treated with 5-aza for 4 days; after this period, both THP1-L and THP1-pc cells were treated with IFN-γ for 6 h and then extensively washed to remove traces of the cytokine. Cells were pulsed with antigen (either peptide pep11 or whole PPD protein) and then mixed with a pep11-specific CD4+ T cell clone in flat-bottomed microtiter plate at a 2:1 stimulator/responder ratio. Activation of T cell was quantitatively assessed after 20 h of co-culture by measuring the amount of IFN-γ secreted by ELISA.