Figure 5. IFNγ antagonizes cAMP but not Treg-derived IL-10 in Socs1^−/− BMDCs.

(a) Socs1^+/+ (5×10⁵ cells per ml; black bars) and Socs1^−/− BMDCs (5×10⁵ cells per ml; grey bars) were stimulated with LPS+IFNγ (10 ng ml⁻¹ each) for 12 h in the presence or absence of graded concentrations of membrane-permeable cAMP analogue, 8-Br-cAMP. The levels of IL-12p70 and TNFα in the culture supernatant were measured by ELISA. (b) Socs1^+/+ (5×10⁵ cells per ml; left half of each panel) and Socs1^−/− BMDCs (5×10⁵ cells per ml; right half of each panel) were stimulated with LPS (10 ng ml⁻¹) for 12 h with (white bars) or without (black bars) 100 μM of 8-Br-cAMP with graded concentrations of murine recombinant IFNγ. The levels of IL-12p70 and TNFα in the culture supernatant were measured by ELISA. IFNγ dose-dependently antagonized cAMP-mediated immunosuppression, which was enhanced by SOCS1 deficiency. (c) Stat1^+/+ (5×10⁵ cells per ml; white bars) and Stat1^−/− (5×10⁵ cells per ml; grey bars) BMDCs were stimulated with LPS (10 ng ml⁻¹) for 24 h in the absence or presence of PGE2 and graded concentrations of murine recombinant IFNγ. The level of TNFα in the culture supernatant was measured by ELISA. (d) Socs1^+/+ and Socs1^−/− BMDCs (5×10⁵ cells per ml) were co-cultured with 2.5×10⁵ cells per ml of CD4⁺CD25^high regulatory T cells in the presence of soluble anti-CD3 Ab (1 μg ml⁻¹). Cells were stimulated with 10 ng ml⁻¹ of LPS+IFNγ (10 ng ml⁻¹, each) for 12 h with or without anti-IL-10 neutralizing Ab (10 μg ml⁻¹) in the presence or absence of membrane-permeable cAMP. The levels of IL-12p70 and TNFα in the culture supernatant were measured by ELISA. Data are representative of two (b–d) to three (a) independent experiments. Error bars represent +s.d. Cells were stimulated in triplicate wells for each condition and s.d. was calculated from the values determined by ELISA. **P<0.01.