Modulation of M1/M2 markers and down-regulation of MHC-II molecules by DcR3. (A) Validation of microarray analysis. Quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) was used to determine gene expression. The mean fold change was calculated by comparing the expression level of each gene, relative to that of GAPDH, in hIgG1- or DcR3-treated MDMs. (B) Heat map of genes involved in MHC-II–mediated antigen presentation pathway. Down-regulated genes are shown in blue, while up-regulated genes are shown in red. (C) Expression of MHC-II molecules in MDMs. DcR3-treated or control MDMs were stained with FITC-conjugated anti–HLA-ABC mAb in conjugation with allophycocyanin-conjugated anti–HLA-DR mAb (top panel) or PE-conjugated anti–HLA-DR, -DP, and -DQ mAbs (bottom panel), followed by flow cytometric analysis. (D) Down-regulation of HLA-DR expression in MDMs after cocultivation with SW480 cells. At 24 hours after siRNA transfection, SW480 cells were cocultured with PKH67-labeled monocytes for 5 days, followed by gating PKH67-positive MDMs to determine surface expression of HLA-DR by FACS Calibur system (top panel). Open histograms correspond to isotype control and shaded histograms to specific staining. The concentrations of DcR3 in SW480 culture media were determined by ELISA (bottom panel). (E) Impaired T-cell stimulatory ability of DcR3-treated MDMs. Allogeneic CD4⁺ T cells were cocultured with γ-irradiated macrophages for 4 days at different ratios, followed by addition of 1 μCi (0.037 MBq)/well [³H]-thymidine for another 16 hours. The incorporation of [³H]-thymidine was measured using a β-counter. P values for the coupled differences, compared with control, were determined using the Student t test: *P < .05; **P < .01 (A,E). Error bars in panels A, D, and E represent SD.