Figure 6.

Functional analysis of IFIT4-primed DCLCs. Mature and activated dendritic cell-like cells (DCLCs) were generated from THP-1 cells transfected with pEGFP-IFIT4 or pEGFP-C1 and harvested after 6 days of culture with granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-4 plus 2 days of stimulation with lipopolysaccharide (LPS; 100 ng/ml). (a) Effect of IFIT4 on modulating the ability of DCLCs to present antigens to T cells was examined by allogeneic mixed leukocyte responses (MLRs). Allogeneic CD4⁺ T cells were cultured for 3 days with γ-irradiated mature DCLCs primed with pEGFP-C1 or pEGFP-ITFIT4 transfection at different ratios of DCLCs to T cells (1:10, 1:20 and 1:40). The cells were harvested and the incorporated radioactivity was measured using a beta counter. Responses are reported as the mean of triplicate samples (counts/minute [cpm] ± standard deviation). (b) Effect of IFIT4 on IL-12 production by DCLCs. Mature and activated DCLCs were generated as described above. Supernatant from the DCLCs (1 × 10⁵ cells/ml) primed with pEGFP-C1 or pEGFP-ITFIT4 transfection was analyzed for IL-12p70 by ELISA. (c) Analysis of the cytokine-production in T cells primed with co-stimulation by DCLCs. Mature and activated DCLCs primed with pEGFP-C1 or pEGFP-ITFIT4 transfection were generated as described above. Then these mature and activated DCLCs were γ-irradiated and cultured with T cells at a ratio of DCLCs to T cells of 1:10 for 6 days. Phorbol-12-myristate-13-acetate (PMA) (10 ng/ml) was added for another day. Finally, the culture medium was measured to assess IFN-γ and IL-4 produced by T cells by ELISA (T cells: 2 × 10⁵ cells/ml; DCLC to T cell ratio = 1:10). DCLCs primed with pEGFP-IFIT4 (shaded histogram, test group) or pEGFP-C1 (open histogram, control group) were compared with each other. The results are expressed as the mean ± standard deviation. Data represent the mean of triplicate experiments. The asterisk indicates a highly significant difference between the test group and the control (P < 0.05). IFIT4, interferon-induced protein with tetratricopeptide repeats 4.