in the clinic as in chronic HCV infection and several forms of cancer, whereas IFN b is approved for treatment of multiple sclerosis, considered an autoimmune disease [1]. The pleiotropic activities of IFNs must be tightly down regulated in time and space and several mechanisms have been shown to co-exist in order to attenuate IFN-initiated Jak/Stat signaling (reviewed in [9]). In an in vivo model, Sarasin et al showed that liver cells from mice repeatedly injected with murine IFN a become refractory to further IFN a stimulation [10]. The ISG- encoded isopeptidase USP18/Ubp43 was found to be essential for the establishment of the desensitized state [10,11]. USP18 can remove the ubiquitin-like ISG15 from target proteins [12] and was found to inhibit IFN-induced Jak/Stat signaling when constitu- tively expressed in cultured cells [13]. Interestingly, USP18 expression was recently identified as a bad prognostic marker of the success of IFN a therapy in patients with chronic hepatitis C [14,15]. Here, we have studied how IFN induced desensitization integrates into the network built by the multiple type I and type III IFNs. We found that both type I and type III IFNs can induce a long lasting desensitization state in cells of different lineages, including human primary hepatocytes. Remarkably, the refractory state is targeted to the IFN a and v subtypes, leaving nearly intact the cells’ responsiveness to b and l IFNs. We show that USP18 is necessary and sufficient to differentially desensitize cells by disturbing the assembly of a IFNs with the receptor complex. Altogether, our findings emphasize the existence of differential activities within the type I IFN family and underline a novel type I/III IFN cross-talk acting at the receptor level that could have important consequences in the set up of clinical protocols, especially for the treatment of HCV-infected patients who are resistant to conventional pegylated IFN a2 therapy. Results Type I and type III IFNs induce desensitization to IFN a In a first set of experiments, we established to what extent cells that had responded to IFN a2, IFN b or IFN l1 could mount a response to a second stimulation. For this, we used HLLR1-1.4 cells, a clone derived from human fibrosarcoma HT-1080 cells stably expressing the IFNLR1 receptor chain and the luciferase reporter gene controlled by an ISGF3-dependent promoter [16]. Thus, HLLR1-1.4 cells are responsive to type I IFNs as well as to type III IFNs (Fig. 1B). HLLR1-1.4 cells were left untreated (na¨ıve) or stimulated (primed) with IFN a2, IFN b or IFN l1 for 24 hr, thoroughly washed and kept in fresh medium for another 24 hr (scheme in Fig. 1A). Following this resting period, the levels of Jak/Stat phosphorylation, luciferase activity and 29–59 oligo-adenylate-synthe- tase (OAS 69K) mRNA in primed cells had nearly returned to basal levels. Na¨ıve and primed HLLR1-1.4 cells were challenged with IFN a2, IFN b or IFN l1 for 6 hr (Fig. 1A) and luciferase activity was quantified (Fig. 1B). The potency of IFN a2 in luciferase induction (expressed as EC50) decreased 14.5–68.9 fold in primed cells as compared to na¨ıve cells, whereas the potency of IFN b decreased only 2.1–3.2 fold (Fig. 1B). A similar trend was observed when na¨ıve and primed cells were monitored by RT-qPCR for induction of OAS 69K mRNA in response to 10 pM of each IFN (Fig. 1C). As shown in Fig. 1D, upon desensitization the dose response relationship for IFN a2 had shifted down by a factor of 50-100. Interestingly, the activity of all the a/v subtypes assayed was decreased in type I and in type III IFN-primed. Desensitization was also evident at the level of early signaling events. Fig. 2A shows that cells primed with IFN b or with IFN l1 Differential Desensitization to Type I Interferons were refractory to low doses (10 and 100 pM) of IFN a2 in terms of tyrosine phosphorylation of Stat1, Stat2 and Stat3 (compare lanes 2–3 with lanes 9–10 or 16–17). Conversely, in primed cells Stat1, Stat2 and Stat3 were still phosphorylated upon treatment with IFN b and IFN l1. Moreover, in primed cells the activation of Jak1 and Tyk2, the earliest effectors of the pathway, was abrogated in the case of IFN a2, but still detectable in the case of IFN b and IFN l1 (Fig. 2B). To better characterize the differential desensitization state, we asked if primed cells resumed their response to IFNa2 when stimulated for longer than 30 min. Na¨ıve and primed cells were thus stimulated with IFN a2 and IFN b for up to 8 hr. As shown in Fig. 2C, the desensitization of primed cells to IFN a2 persisted, independently of treatment duration. To define for how long primed cells remained in a refractory state, we extended the interval between priming and restimulation (washing of 24, 48 and 72 hr). As shown in Fig. 2D, primed cells had regained IFN a2 sensitivity after 72 hr in the absence of cytokine. In conclusion, the differential desensitized state of the cell persists even at 8 hr of stimulation, but is reversible as seen when cells are kept in the absence of cytokine for 3 days. Priming with type I IFN induced a similar a2/b differential desensitized state in cell lines, such as bronchial epithelial BEAS- 2B and uroepithelial Hs 789.T cells, and in foreskin fibroblasts and T cell blasts (Fig. 3A and 3B). Human primary hepatocytes respond to type I IFNs and more weakly to IFN l1 (Fig. 3C, compare level of P-Stat2, lanes 1–8). After 24 hr of priming with either IFN a2 or IFN l1, hepatocytes expressed higher Stat2 protein and detectable levels of USP18, both proteins being encoded by ISG (Fig. 3C, lanes 9–24). Importantly, primed hepatocytes were desensitized to IFN a2 and only marginally to IFN b (Fig. 3C, compare P-Stat2 in lanes 3, 11 and 19) and the extent of desensitization was related to the level of sensitivity to the priming cytokine. Of note, the basal phosphorylation level of Stat3 was reduced in IFN l1-primed cells with respect to na¨ıve or IFN a2-primed cells (compare P-Stat3 in lanes 1, 9 and 17). Overall, these results demonstrate a previously unrecognized inhibitory cross-talk between the type I and type III IFN systems. Desensitized cells are impaired in their ability to bind IFN a2 As shown above, cell desensitization to IFN a2 is manifest at the level of Janus kinase activation and thus may result from reduced surface level of the receptor chains or impaired binding of IFN a2. FACS analysis clearly showed that na¨ıve and primed (i.e. desensitized) cells expressed equivalent levels of IFNAR1 and IFNAR2 (Fig. 4A). Therefore, we tested whether the ligand binding property of the receptor differed between na¨ıve and primed cells. For this, we iodinated IFN a2 and, in place of IFN b which cannot be iodinated without loss of bioactivity, we made use of an engineered mutant of IFN a2 (IFN a2-HEQ) whose affinity for IFNAR1 is similar to that of IFN b and which recapitulates IFN b unique biological activities [6]. Accordingly, in primed HLLR1-1.4 cells, IFN a2-HEQ was as potent as IFN b in inducing Stat phosphorylation (Fig. 4B). On this basis, we compared the binding of 125I-IFN a2 and 125I-IFN a2-HEQ to na¨ıve and to primed HLLR1-1.4 cells. Fig. 4C shows that the binding of 125I-IFN a2 was reduced in both IFN b-primed cells and IFN l1-primed cells with respect to na¨ıve cells. The reduction was most apparent for low 125I-IFN a2 concentrations, matching the decrease in specific biological activity (Fig. 1D). In contrast, the binding of 125I-IFN a2-HEQ was only marginally reduced in primed cells with respect to na¨ıve cells (Fig. 4D). In conclusion, despite unaltered levels of IFNAR1 and IFNAR2, cells primed PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22200