Article Figures & Data
Figures
- FIG. 1.
ssRNA-DCs and LPS-DCs display a mature phenotype. Monocyte-derived DCs were differentiated in the presence or not (cDCs) of LPS (LPS-DCs) or ssRNA/Dotap (ssRNA-DCs). On day 5, DCs were kept for 24 h in medium (filled histograms) or stimulated with LPS (thick lines). Expression of surface HLA-DR, CD1a, CD14, CD40, CD80, CD83, and CD86 was determined by FACS analysis. The background signal of an Ig isotypic control is shown (dotted lines). The phenotype of DCs differentiated in the presence of Dotap did not differ from that of cDCs (not shown).
- FIG. 2.
ssRNA-DCs and LPS-DCs induce proliferation of allogeneic T cells. cDCs, LPS-DCs, and ssRNA-DCs (day 0 treatment) not receiving further stimuli or receiving a late LPS maturation stimulus (day 5) were cocultured with carboxyfluorescein diacetate succinimidyl ester-loaded naive CD4 T cells (ratio, 1:3) from one allogeneic donor. At day 6 of coculture, the different division cycles undergone by T cells were detected by FACS analysis and the number of stimulated cells on the initial sample was calculated. Proliferation induced by cells receiving a late LPS stimulus was significantly greater in all DC preparations (P < 0.05). Results are representative of experiments with at least six different donors. The frequency of responding autologous naive CD4 T cells was in all cases under 2% (not shown). Control peripheral blood mononuclear cells stimulated with PHA are also shown. DCs differentiated in the presence of Dotap induced T-cell proliferation which was not significantly different from that induced by cDCs (not shown).
- FIG. 3.
ssRNA-DCs and LPS-DCs have a reduced Th-polarizing capacity. Allogeneic naive CD4 T cells were stimulated for 6 days with the different DC preparations: cDCs, LPS-DCs, and ssRNA-DCs not receiving or receiving a late LPS maturation stimulus. Control cells were also stimulated with ssRNA. The phenotype of activated T cells was determined by FACS analysis after stimulation with PMA-ionomycin and intracellular staining of IFN-γ and IL-4. Results are shown as dot plots (A) or bar histograms (B) representative of experiments with different donors. Compared to cDCs, LPS-DCs and ssRNA-DCs receiving a late TLR4-mediated signal induced significantly fewer IFN-γ-producing cells (indicated with asterisks [P < 0.05]).
- FIG. 4.
Early ssRNA or LPS priming suppresses late IL-12p70 response of DCs to LPS but not to CD40LT. Freshly purified blood monocytes were cultured with GM-CSF and IL-4 to induce DC differentiation. (A) Medium (none), 10 ng/ml LPS, or 500 ng/ml ssRNA was added at the beginning of the culture (day 0). (B) On day 5 (d5), cells either were left in medium or were stimulated with 10 ng/ml LPS or 200 ng/ml CD40LT. (C) On day 5, cells either were left in medium or were stimulated with 200 ng/ml CD40LT. The amounts of IL-10, IL-12p70, and TNF-α released during the 24 h following stimulation were determined by ELISA: culture supernatants were collected on day 1 (A) or day 6 (B and C).
- FIG. 5.
Pretreatment of LPS-DCs and ssRNA-DCs with the p38 MAPK inhibitor SB203580 recovers the late IL-12p70 response but not a Th1-polarizing potential. For pretreatment, on day 0 (d0), monocytes cultured in GM-CSF and IL-4 to induce DC differentiation were treated for 30 min with 1 μM SB203580 (SB) or left untreated before medium, LPS, or ssRNA was added. For maturation stimulus, on day 5 (d5), DCs were stimulated with LPS for 24 h or left untreated. (A) Day 6 supernatants were tested for IL-12p70 by ELISAs. (B and C) DCs were cocultured with allogeneic carboxyfluorescein diacetate succinimidyl ester-labeled naive CD4 T cells. On day 6, T-cell proliferation (B) and intracellular IFN-γ accumulation (C) were determined by FACS analysis. In the proliferation assay (B), the frequency of responding autologous naive CD4 T cells was under 1.0% in all cases (not shown). Statistically significant differences (P < 0.05) between the control and a given test are indicated with an asterisk.
- FIG. 6.
Exogenous IL-12p70 recovers the Th1 polarization mediated by LPS-DCs and ssRNA-DCs. Allogeneic naive CD4 T cells were stimulated with the different DC preparations either without (black bars) or in the presence of (hatched bars) 5 ng/ml recombinant IL-12p70. After 6 days, cell were stimulated with PMA-ionomycin, stained for intracellular IFN-γ, and analyzed by FACS. Percentages of IFN-γ-producing cells significantly different (P < 0.05) between IL-12p70-treated and nontreated cultures are indicated by asterisks.
- FIG. 7.
Dynamic of IL-10 and IL-12p70 response induced by LPS or by ssRNA during the DC differentiation process. Freshly isolated blood monocytes were cultured in the presence of GM-CSF and IL-4 to allow DC differentiation. Cells cultured for different periods of time (x axis) were stimulated with 10 ng/ml LPS (A) or 500 ng/ml ssRNA (B). Twenty-four hours after stimulation, released IL-10 and IL-12p70 were quantified on culture supernatants by ELISAs. The IL-12p70 response by 5-day-differentiated DCs was significantly higher than the response by DCs in earlier differentiation states (indicated by an asterisk [P < 0.05]).
