Differential Desensitization to Type I Interferons indicated IFN doses. Cell lysates (50 mg) were analysed with the indicated Abs to evaluate tyrosine phosphorylation and content of Jak1 and Stats. The arrow points to the band corresponding to phosphorylated Jak1. The level of USP18 (bottom panel) was assessed in a 10% SDS PAGE. Of the two USP18 bands (apparent MW of 38 and 35 kDa), the faster migrating one results from proteolytic processing [46]. This latter comigrates with a non specific cross-reacting band detected in na¨ıve cells and indicated by the asterisk (bottom panel). doi:10.1371/journal.pone.0022200.g003 na¨ıve HLLR1-1.4 cells. Binding of 125I-IFN a2 on HU13 cells was clearly reduced (Fig. 7B) and this to the same extent as for IFN- primed HLLR1-1.4 cells (see Fig. 4C). As expected, the binding of 125I-IFN a2-HEQ was similar for the two clones (Fig. 7C). In conclusion, these data demonstrate that the sole expression of USP18 recapitulates the binding alteration observed in IFN- primed (i.e. desensitized) cells. Discussion The major findings of our study are hereafter summarized: i) type I IFN and type III IFN desensitize cells to several a IFNs but only marginally to IFN b; ii) cells of different lineages - including primary hepatocytes - undergo differential desensitization; iii) the extent of desensitization is controlled by the level of an ISG, USP18; vi) forced expression of USP18 in naive cells blunts IFN a response at the level of its assembly to the receptor complex. USP18 is a cysteine protease specialized in the removal of ISG15 from ISGylated proteins. However, the phenotypic alterations caused by USP18 deletion in the mouse have been dissociated from ISG15-dependent mechanisms [17,18,19]. One group has proposed that USP18 attenuates IFN a signaling regardless of the isopeptidase activity of the protein by compet- itively displacing Jak1 from its interaction with IFNAR2 [13]. We Figure 4. Analysis of the type I IFN receptor in na¨ıve and primed HLLR1-1.4 cells. (A) Surface level of IFNAR1 and IFNAR2 in na¨ıve cells and in IFN b or IFN l1-primed cells as determined by FACS. Cells were primed for 24 hr, washed and maintained in medium without IFN for 24 hr. Cells were then stained with AA3 mAb (IFNAR1) or CD118 mAb (IFNAR2) followed by biotinylated rat anti-mouse Ab and streptavidin-PE. (B) Level of phosphorylation of Stat1 in na¨ıve and primed cells stimulated for 30 min with 100 pM of IFN a2, IFN b or IFN a2-HEQ. Lysates (30 mg) were immunoblotted with the indicated antibodies. Priming was for 8 hr followed by 16 hr resting in medium without IFN. (C and D) Binding of 125I labelled IFN a2 (C) or IFN a2-HEQ (D) at 37uC for 1 hr to na¨ıve (closed circles), IFN b-primed cells (triangles) or IFN l1-primed cells (open circles). Cells were primed for 8 hr and maintained without IFN for 16 hr. doi:10.1371/journal.pone.0022200.g004 PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22200