Differential Desensitization to Type I Interferons phosphorylation in the USP18-expressing clone HU13 and in the parental HLLR1-1.4 cells. Cells were stimulated as indicated with 100 pM of IFN a2 or IFN b. Lysates (30 mg) were immunoblotted with the indicated Abs. (C) Kinetics of Tyk2 and Stat1/2 phosphorylation in parental HLLR1-1.4 cells and USP18-expressing HU13 cells. Cells were stimulated as indicated with 30 pM of IFN l1. (D) Luciferase activity induced by IFN a2 (closed circles) or IFN b (open circles) in HP1 control clone and in HU13 clone constitutively expressing USP18. (E) Ratio of the EC50 values determined for luciferase activity on the control clone HP1 and clone HU13. Cells were stimulated with the indicated IFN subtypes for 6 hr. Bars represent support limits of the ratio from 95% confidence intervals of the individual EC50. doi:10.1371/journal.pone.0022200.g006 have obtained preliminary evidence that, indeed, a catalytically inactive USP18 impairs IFN signaling when highly and stably expressed in na¨ıve cells. In this context, however, desensitization is severe and not differential, as cells become refractory to IFN a and b subtypes. At present, we cannot exclude the possibility that the isopeptidase activity of USP18 could be required in certain physiological contexts, for instance when USP18 is below a given threshold or when the level of ISG15 conjugates is maximal. In Figure 7. Cells expressing USP18 are defective in IFN a2 binding. (A) Cell surface levels of IFNAR1 (left) and IFNAR2 (right) in parental HLLR1- 1.4 cells, USP18-expressing clone HU13 and control clone HP1was determined by FACS. Binding of 125I labelled IFN a2 (B), or IFN a2-HEQ (C) at 37uC for 1 hr on HLLR1-1.4 cells (closed circles) and clone HU13 (triangles). doi:10.1371/journal.pone.0022200.g007 PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e22200