Differential Desensitization to Type I Interferons Figure 1. Differential desensitization studies in HLLR1-1.4 cells. (A) Protocol used to measure desensitization. Unless otherwise indicated, cells were primed with IFN a2 or IFN b (500 pM) or IFN l1 (50 pM). The priming phase varied between 8 and 24 hr and the resting phase between 16 and 24 hr. Cells were then challenged with IFN for different times depending of the read out. (B) Graphic representation of the EC50 (pM) as determined by the luciferase activity induced by IFN a2, IFN b or IFN l1 in na¨ıve or primed cells. EC50 were calculated from the non-linear regression fits of the luciferase activity induced by IFN in a concentration range covering 2.4 log. Priming and resting times lasted 24 hr each. Bars represent the 95% confidence limits. (C) Level of OAS-69K mRNA induced by IFN a2 (10 pM), IFN b (10 pM) or IFN l1 (50 pM) in na¨ıve and primed cells as determined by RT-qPCR. Data are expressed as ratios to GAPDH levels. Priming and resting times lasted 24 hr each. Bars represent the 95% confidence limits (Student’s t-test). (D) Dose response induction profile of OAS-69K mRNA in na¨ıve (closed symbols) and IFN a2 primed cells (open symbols) stimulated for 4 hr with different doses of IFN a2 (circles) or IFN b (squares) as determined by RT-qPCR. Priming and resting times lasted 24 hr each. Data are expressed as ratios to GAPDH levels. Bars represent the 95% confidence limits (Student’s t-test). doi:10.1371/journal.pone.0022200.g001 with either type I or type III IFNs (i.e. desensitized) are unable to assemble a functional IFN a2 receptor complex. Expression of USP18 is necessary and sufficient to cause differential desensitization USP18 has been shown to downmodulate type I IFN activity through binding to IFNAR2 [13], and we therefore tested its role in differential desensitization. In HLLR1-1.4 cells USP18 mRNA was induced by a2, b and l1 IFNs, but not by other cytokines such as IFN c or IL-6 (Fig. 5C). Accordingly, cell priming with IFN c or IL-6 did not induce USP18 nor lead to a desensitized state (Fig. 5A and 5B). USP18 protein accumulated with similar kinetics in cells treated with IFN b or IFN l1, reaching maximum level between 8 and 16 hr of stimulation (Fig. 5D). As found in primary hepatocytes (Fig. 3C), two USP18- specific bands of comparable intensity were consistently detected. To study the involvement of USP18, its expression was silenced in HLLR1-1.4 cells (Fig. 5E). Remarkably, cells wherein USP18 was efficiently silenced were not desensitized to IFN a2. Of note, the level of Stat1/2 phosphorylation was higher in USP18- silenced/primed cells (lanes 14, 15 and 17, 18) than in USP18- silenced/naive cells (lanes 11, 12) and most likely this is consequence of the higher content of these Stats in primed cells. To determine if USP18 was sufficient to establish differential desensitization, HLLR1-1.4 cells were stably transfected with a PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22200