(A) IL-17A levels from anti-CD3/28 Ab-stimulated OT-II cells co-cultured with BM-APCs from WT mice treated with various cAMP agonists: CPT (50 μM), pertussis toxin (PTX, which inhibits Gαi, 100 ng/ml), prostaglandin E2 (PGE2, 1 μM), cholera toxin (CT, an activator of Gαs, 1 μg/ml), forskolin (Fsk, an activator of AC, 10 μM), rolipram (Rol, PDE4 inhibitor, 10 μM). (B) Levels of the indicated cytokines and (C) mRNA expression of lineage commitment factors in OT-II cells co-cultured with WT BM-APCs treated with CPT. (D) QPCR of TFs in the WT BM-APCs treated with the indicated cAMP agonists. Two-way ANOVA with Sidak’s multiple comparisons test; n = 3 in each group, different from untreated in the CPT-treated group; *p<0.05, **p<0.01, ***p<0.001. Effect of treatment; Irf4 (p<0.001), Klf4 (p<0.001), Irf8 (p<0.001) and Crem (p<0.001). (E, F) qPCR of Irf4 in the WT BM-APCs treated with PGE2 in the presence of inhibitors of PKA (Rp-cAMP, 50 μM), EPAC (CE3F5, 50 μM), or CREB (666–15, 1 μM). Two-way ANOVA with Sidak’s multiple comparisons test; n = 3 in each group, different from untreated; ***p<0.001. Data are representative of three independent experiments.