Fig 4. Human IL-39 does not activate STAT3 and does not inhibit IL-23-induced signaling.

(A) HEK-Blue^TM STAT3 IL-6 cytokine reporter cells were transiently transfected with a plasmid encoding for the human IL-23 receptor and after 48 hrs, expression of the IL-39 receptor genes IL23r and Gp130 were quantified by quantitative RT-PCR. The Ct values for IL23r and Gp130 were 22 and 26, respectively. (B) HEK-Blue^TM STAT3 transfected cells from (A) were incubated with recombinant, human IL-6 (1 ng/ml), with IL-39 IgG1-Fc fusion or IgG1-Fc control protein (10 μg/ml) or with concentrated IL-39 containing supernatants originating from HEK293FT cells transfected with either p19, EBI3-His or with a combination of p19/EBI3-His. After 20 hrs, secretion of the STAT3-induced SEAP reporter was measured by QUANTI-Blue^TM. (C and D) IL-39 does not antagonize IL-23-mediated signal transduction. HEK-Blue^TM IL-23 cells stably expressing the human IL-23 receptor and a STAT3-inducible SEAP reporter were stimulated for 20 hrs with various concentrations of recombinant human IL-23 to induce the STAT3-mediated SEAP reporter. Alternatively, the cells were incubated with a cocktail containing IL-23 plus different concentrations of IL-39 IgG1-Fc fusion or IL-23 plus IgG1-Fc control protein. Secretion of the STAT3-induced SEAP reporter was measured by QUANTI-Blue^TM.