FIGURE 4.

TTP controls effector function of T_H17 cells by destabilizing IL-17 mRNA stability. (A) Splenic CD4⁺ T cells of TTP^{– /–} mice and wild-type (WT) littermates aged 6–8 months were stimulated under T_H17 polarizing conditions for 4 days and then actinomycin D (ActD) (5 μg/mL) and DRB (10 μg/mL) were added. Total RNAs were extracted at 30, 60, 90, and 120 min after adding ActD and DRB. cDNAs were reverse-transcribed and residual cytokine as well as GAPDH mRNA were measured by real-time quantitative PCR (qPCR). The levels of residual cytokine mRNAs were normalized to GAPDH mRNA at each time point and half-life of the mRNA determined by comparing to the levels of mRNA before adding ActD and DRB. (B) Jurkat cells were infected with control adenovirus (Ctrl/Ad) or TTP-expressing adenovirus (TTP/Ad) at MOI = 5 and 20 for 24 h, followed by adding ConA (5 ug/mL) and then measuring IL-17 mRNA by qPCR and TTP protein by immunoblotting. (C) HEK293 cells were transiently cotransfected with IL-17A 3′ UTR–driven luciferase construct along with CMV–TTP vector, as well as empty vector (Ctrl), followed by measurement of luciferase activity in cell lysates after 40 h. (D) HEK293 cells were transiently cotransfected with IL-17A 3′ UTR–driven luciferase construct along with WT TTP, as well as two TTP zinc-finger mutant constructs (C124R and C147R), followed by measurement of luciferase activity in cell lysates after 40 h. Data shown as relative levels compared to luciferase activity in cells transfected with the empty vector (Ctrl) from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 between groups.