Fig 4. PGE2 signals through cAMP, but not EPAC or PKA, to inhibit IL-27 mediated Tr1 differentiation.

(A) il10^gfp naïve CD4⁺CD62L⁺ cells were pretreated with PI3K inhibitor LY294002 (LY) for 30 min prior to stimulation in the presence of IL-27 and PGE2. (B) Naïve CD4⁺CD62L⁺ cells were stimulated as described in the presence of dbcAMP. (C) Cells were stimulated in the presence of a cell-permeable EPAC activator 8-pCPT-2′-O-Me-cAMP (8-CPT). Cells were collected on day 3 and analyzed by FACS for Tr1 markers. (D, E) Naïve CD4⁺CD62L⁺ cells were pretreated with PKA inhibitors PKI 6–22 and PKI 5–24 (2x10^-5M) for 30 minutes prior to stimulation with IL-27 and PGE2 and analyzed on day 3 for Tr1 markers (D) and IL-10 within Tr1 cell populations (E). Data are representative of three independent experiments. Each sample was tested in duplicate and results represent means ± SD. Significance was evaluated by one-way ANOVA and * symbolizes significance of experimental condition versus IL-27 control (B); *P<0.05, **P<0.01, ***P<0.001.