Figure 2.
Figure 2. The glycosylation and RNase activity of omega-1 are essential for conditioning human DCs to prime Th2 responses. (A) Human moDCs were pulsed for 48 h with increasing concentrations of the mutant variants of recombinant omega-1 (ω-1) in combination with 100 ng/ml LPS as a maturation factor, and surface expression of CD86 was determined by FACS analysis. The expression levels, based on geometric mean fluorescence, are shown relative to the DCs stimulated with LPS alone, which is set to 1. Data are based on two independent experiments and shown as mean ± SD. (B) Conditioned DCs were co-cultured for 24 h with a CD40-L–expressing cell line to mimic the interaction with T cells. IL-12p70 cytokine expression levels are shown relative to the DCs stimulated with LPS alone, which is set to 1. Data are representative of triplicate wells from one of two independent experiments and shown as mean ± SD. (C) DCs conditioned as described in A were cultured with allogeneic naive CD4+ T cells for 12 d in the presence of staphylococcal enterotoxin B and IL-2. Intracellular cytokine production was assayed by FACS 6 h after the stimulation of primed T cells with PMA and ionomycin. Based on intracellular cytokine staining, the ratio of T cells single-positive for either IL-4 or IFN-γ was calculated relative to the control condition. Data are based on two independent experiments and shown as mean ± SD. (D) An example of T cell polarization assay as described in C induced by the different recombinant omega-1 variants. The frequencies of each population are indicated as percentages in the plot. One representative result from five independent experiments is shown. (E) T cell polarization assay as described in C but in the absence of LPS. Data are representative of three independent experiments. Bars represent mean ± SD. * and #, P < 0.05, for significant differences compared with control conditions (*) or between test conditions (#) based on paired analysis (two-sided paired Student’s t test). H58F, RNase mutant; N71/176Q, glycosylation mutant.

The glycosylation and RNase activity of omega-1 are essential for conditioning human DCs to prime Th2 responses. (A) Human moDCs were pulsed for 48 h with increasing concentrations of the mutant variants of recombinant omega-1 (ω-1) in combination with 100 ng/ml LPS as a maturation factor, and surface expression of CD86 was determined by FACS analysis. The expression levels, based on geometric mean fluorescence, are shown relative to the DCs stimulated with LPS alone, which is set to 1. Data are based on two independent experiments and shown as mean ± SD. (B) Conditioned DCs were co-cultured for 24 h with a CD40-L–expressing cell line to mimic the interaction with T cells. IL-12p70 cytokine expression levels are shown relative to the DCs stimulated with LPS alone, which is set to 1. Data are representative of triplicate wells from one of two independent experiments and shown as mean ± SD. (C) DCs conditioned as described in A were cultured with allogeneic naive CD4+ T cells for 12 d in the presence of staphylococcal enterotoxin B and IL-2. Intracellular cytokine production was assayed by FACS 6 h after the stimulation of primed T cells with PMA and ionomycin. Based on intracellular cytokine staining, the ratio of T cells single-positive for either IL-4 or IFN-γ was calculated relative to the control condition. Data are based on two independent experiments and shown as mean ± SD. (D) An example of T cell polarization assay as described in C induced by the different recombinant omega-1 variants. The frequencies of each population are indicated as percentages in the plot. One representative result from five independent experiments is shown. (E) T cell polarization assay as described in C but in the absence of LPS. Data are representative of three independent experiments. Bars represent mean ± SD. * and #, P < 0.05, for significant differences compared with control conditions (*) or between test conditions (#) based on paired analysis (two-sided paired Student’s t test). H58F, RNase mutant; N71/176Q, glycosylation mutant.

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