Figure 3

EVT-conditioned DCs show reduced capacity to prime naive T cell proliferation and activation, but enhanced capacity to induce Tregs generation.

(A) DCs and H-DCs were harvested and washed after treated with LPS (100 ng/ml, 48 h), then seeded with CFSE-labeled allogeneic naive CD4⁺T lymphocytes at a DC/lymphocyte ratio of 1:5 in the present of IL-2(50 U/ml). The dilution of CFSE staining was analyzed at day 5 by flow cytometry. T cells cultured alone with or without PMA (10 mg/ml) were used as positive and negative controls, respectively. Histograms from a representative experiment (n = 5) are shown. (B) Graphical representation of data in (A) is shown as mean ± SEM from 3 to 5 independent experiments with consistent results. (C) Mature DCs and H-DCs were incubated with allogeneic naive CD4⁺T cells using a DC/lymphocyte ratio of 1:2. At day 5, cells were harvested for surface staining of CD4 and CD69. For intracellular staining of IFN-γ, cells were resuspended in fresh medium, stimulated with PMA/ionomycin for 6 h and treated with brefeldin A for the last 4 h of cultures. The representative flow cytometry pictures are depicted from a representative experiment (n = 5). Gray filled histograms, isotype control Ab staining; Solid lines, anti-CD69 Ab staining. (D) Summary of the proportion of CD69 (upper) and IFN-γ (lower) on CD4⁺T cells. The results are shown as mean ± SEM from 5 independent experiments. (E) Mature DCs and H-DCs were recovered and then cultured for 5 days with isolated naive CD4⁺T cells in the present of IL-2(50U/ml). Cells were stained with anti-CD4, anti-CD25 and anti-Foxp3. Flow cytometry dot plots showing that CD25⁺Foxp3⁺ Tregs increased in the CD4⁺T cell population were shown. (F) The percentage of CD25⁺Foxp3⁺ cells in the CD4⁺T cell population was summarized in the bar graph(mean ± SEM, n = 5). *p < 0.05, **p < 0.01, ***p < 0.001.