However, a modest increase of IRF transcript is evident in IFNα-treated cells after 24 h, becoming a little more substantial after 48 h of stimulation. In contrast with the slow and weak IFNα-driven

upregulation of IRF1 gene, IFNα-driven upregulation of SOCS1 in M14 was robust within 15 min, peaking at 24 h of treatment and still active after 48 h. In conclusion, we showed evidence that quantitative differences in the expression of SOCS1 and IRF1 are underlying the kinetics of a weak and transient activation of IFNα-dependent STAT1 activation as well as of CIITA-PII and CIITA–PIV activation. The unique effects of IFNα on CIITA-PIV expression observed in these studies suggested that targeting the expression of this isoform in non-professional APCs might be an effective means selleck screening library of manipulation of click here MHCII expression without critically affecting professional APCs. We therefore tested the feasibility of utilizing CIITA-PIV specific RNA interference to downregulate MHCII expression. The effects of the gene silencing mediated

by the specific interference with HLA-DRA, CIITA, and CIITA-PIV transcripts on the cell surface expression of HLA-DR and HLA-DQ molecules in Me10538, M14 and U87 cells are presented in Fig. 6. In summary, determination of cell surface expression of MHCII molecules was performed by direct immunofluorescence using anti-HLA-DR and -DQ Abs in all cells 72 h after transfection with 50 nM of the various siRNAs described in Section 2 (Material and methods). In all cell lines transfected with the control siRNA, the expression of either HLA-DR or HLA-DQ on

the cell surface was not significantly modified. Transfection with siRNAs directed against the HLA-DRA sequence (indicated as HLA-DRA_2 and HLA-DRA_3 in Fig. 6) were used as positive controls of specificity. These siRNAs significantly reduced cell surface expression of HLA-DR in all cell lines tested, without significantly affecting the expression of HLA-DQ. Transfection with two different siRNAs (indicated as CIITA_2 and CIITA_3 in Fig. 6) each targeting all known human CIITA isoforms significantly Histamine H2 receptor reduced the surface expression of both HLA-DR and -DQ in all the MHCII-positive tumor cell lines. Finally, a similar efficiency of knockdown of both the MHCII molecules was achieved by the transfection of two different siRNAs specifically directed against CIITA-PIV (indicated as CtPIV-a and CtPIV-b in Fig. 6). To confirm our data on the MHCII cell-surface expression, we measured the amount of HLA-DRA, HLA-DQA1, CIITA, CIITA-PIV, and CIITA-PIII transcripts in the total RNA from our set of samples both 16 and 48 h after transfection with the different siRNAs used in our study (including the control siRNA) as well as in the sham-transfected samples (data not shown).