We selected cell lines representing bona fide non-professional APCs constitutively expressing MHCII molecules. Because constitutive expression (i.e., IFNγ-independent) of MHCII genes has been described in melanoma [ 41] and glioma cell lines [ 42] and because both non-bone marrow derived cell types possess the MHCII-mediated ability to present antigens to CD4+ T lymphocytes [ 43, 44], we chose four already well-characterized cell lines that could be used for these experiments: three melanoma cell lines (SK MEL-23, Me10538 and M14) and one glioma cell line (U-87). All of them showed a strong IFNγ-dependent upregulation of MHCII

expression (data not shown). To begin, we established selleckchem the baseline level of MHCII expression in our cultures of SK MEL-23, Me10538, M14 and U-87 cells (see Fig. 1, no IFNα data). Flow cytometry analysis of SK MEL-23 confirmed the lack of expression of MHCII on the cell surface, matching the absence of HLA-DRA and HLA-DQA1 specific mRNA, as tested by quantitative

RT-PCR qRT-PCR assay. Unlike SK MEL-23 cells, the other two melanoma cell lines Me10538 and M14, and the glioma cells U87 showed a significant level of HLA-DR and -DQ antigens, both at the protein and at the RNA level. For IFNα stimulation, cells were cultured with Ruxolitinib or without IFNα (250 U/ml) for

48 h. Because the ability of IFNα to upregulate the expression of MHCI molecules in melanoma cell lines is well established [45], the HLA-A,B,C immunophenotype was specifically measured as a positive control of the effects of this cytokine on gene expression in the cells used in the study. As shown in Fig. 1A, 48 h of incubation of each of the four cell lines with IFNα resulted in the expected significant increase in the density of MHCI molecules on the cell surface measured as MFI ( p < 0.01). Liothyronine Sodium On average, IFNα-treated MHCII-positive tumor cells showed a 1.5–2 fold increase of HLA-A,B,C molecules on the cell surface compared to untreated cells. In all MHCII-positive cells used as models of non-professional APCs, the flow cytometry analysis showed a consistent significant decrease of the level of MHCII molecules on IFNα-treated cells compared to untreated cells. The density of HLA-DR and HLA-DQ heterodimers on the surface of IFNα-treated cells was reduced to 40% and 50%, respectively, of the density of the corresponding molecules on untreated cells ( p < 0.05, for both), as seen in Fig. 1A. Incidentally, at no time did IFNα induce the expression of either HLA-DR or HLA-DQ on the cell surface of SK MEL-23.