Figure 5

Klfl-mediated PD-L1 induction requires the activation of PI3K-mTOR signaling pathway. (a) Splenocytes from each Stat-deficient or WT mouse were transfected with a pMIG-Mock or pMIG-Klf1 vector. PD-L1 expression levels were assessed by FCM 48 h after the transfection. In the Stat1 KO mouse, anti-CD3ε/anti-CD28 stimulation was used whereas Concanavalin A (ConA) stimulation was used for the other KO mice. (b) Phosphorylation of Akt and S6K was analyzed by Western blotting. The indicated blots were derived from the same experiments. Full length blots are presented in Supplementary Figure S4. (c) Splenocytes from B6 mice were transfected with pMIG-Mock or pMIG-Klf1 and the indicated inhibitors were added 24 h later. Then, cells were cultured for 48 h and PD-L1 expression was evaluated by FCM. The proportion of cells expressing PD-L1 at high levels was compared between the GFP-positive and GFP-negative groups. (n = 3). Control: without inhibitor; SP 600125: JNK1/2/3 inhibitor (5 μM); SB 203580: MAPK-p38 inhibitor (20 μM); PD 98059: MEK/ERK inhibitor (50 μM); LY 294002: PI3K inhibitor 20 μM); Torin: mTOR inhibitor (200 nM). *p < 0.01 (Bonferroni’s multiple comparison test). Plots are representative of 3 independent experiments.