Differential Desensitization to Type I Interferons Figure 2. Differential desensitization studies in HLLR1-1.4 cells. (A) Level of phosphorylation of Stat1, Stat2 and Stat3 induced at 30 min by the indicated doses of IFN in na¨ıve cells and in primed cells. Priming was for 8 hr and resting was for 16 hr. Cell lysates (30 mg) were fractionated on a 7% SDS polyacrylamide gel and immunoblotted with the indicated Abs. (B) Level of tyrosine phosphorylation of immunoprecipitated Tyk2 and Jak1 induced at 30 min by IFN a2 (100 pM), IFN b (100 pM) or IFN l1 (50 pM) in na¨ıve and primed cells. Priming was for 8 hr and resting was for 16 hr. Lysates (2 mg) were immunoprecipitated with Tyk2 Abs (top) or Jak1 Abs (bottom). The top membrane was incubated with phospho-tyrosine 4G10 mAb (P-Tyr) and the bottom membrane with phospho-Jak1 Abs. Protein content was assessed by re-blotting with Tyk2 or Jak1 specific Abs. (C) Kinetics of Stat1, Stat2 and Stat3 phosphorylation in na¨ıve and primed cells. Cells were stimulated with 50 pM of IFN a2 or IFN b, as indicated. Priming was for 8 hr and resting was for 16 hr. Lysates (30 mg) were fractionated on a 7% SDS polyacrylamide gel and immunoblotted with the indicated Abs. (D) Level of phosphorylation of Stat2 induced at 30 min by 100 pM of IFN in na¨ıve cells and in primed cells. Priming was for 8 hr and the resting phase varied from 24 hr to 72 hr. Cell lysates (30 mg) were fractionated on a 7% SDS polyacrylamide gel and immunoblotted with the indicated Abs. doi:10.1371/journal.pone.0022200.g002 USP18 expression vector or an empty vector. In the 10 positive clones analysed, the level of ectopic USP18 was higher (4 to 50 fold) than endogenous USP18 present in 8 hr-primed cells. Clone HU13 was chosen as it expressed the least USP18 (Fig. 6A). We checked the integrity of the deISGylase activity of USP18 expressed in clone HU13 by comparing the steady state level of ISGylated conjugates in IFN b-treated parental and HU13 cells (data not shown). The response to IFN a2, IFN b and IFN l1 was measured. Compared to na¨ıve HLLR1-1.4 cells, clone HU13 was severely impaired in its phosphorylation response to IFN a2. Conversely, responses to IFN b and IFN l1 were preserved or slightly reduced (Fig. 6A–C). By directly comparing luciferase induction in a control clone (HP1) and in HU13 clone, it appeared that constitutive USP18 in HU13 shifted the dose response relationship so that higher concentration of IFN a2 was required to trigger a response of the same magnitude (Fig. 6D). All IFN a subtypes tested and IFN v were found to be between 25 to 85-fold less potent on HU13 cells than on control HP1 cells, whereas the activities of IFN b and IFN l1 were marginally reduced (Fig. 6E). The specific activity of IFN a2-HEQ, engineered for higher binding towards IFNAR1 (see above), was reduced only by a factor of 10 on HU13 cells (Fig. 6E), in accordance with the level of activity of this mutant on cells desensitized by IFN priming (Fig. 4B). On the other hand, the specific activity of IFN a2-a8tail, an IFN a2 mutant engineered for higher IFNAR2 binding [7], was decreased by a factor of 45 on HU13 cells as compared to HP1 cells (Fig. 6E). These results indicate that the affinity of a given IFN a subtype towards IFNAR1 determines the degree of USP18- dependent desensitization to that subtype. As shown for desensitization caused by IFN priming (Fig. 4A), desensitization caused by expression of USP18 was not dependent on a change of IFNAR1 or IFNAR2 cell surface expression (Fig. 7A). In studies analogous to those performed on IFN-primed cells, we measured binding of radiolabeled ligands on HU13 and PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e22200