we observed consistent blunting of IL-6–induced STAT3 phosphorylation (Fig. 4 E and Fig. S3 C). Interestingly, SOCS2 expression was induced after IL-6 and TGF- stimulation (Fig. S3 E) and, when stimulated under Th17 polarization con- ditions, SOCS2-deficient T cells express less RORt (Fig. S3 F) and secrete less IL-17 (Fig. S3 G). These results are consistent with the decreased IL-17 levels observed in vivo (Fig. 1 C and Fig. 3 D), including in the CD4+ cell reconstitution experi- ments (Fig. 3 F and Fig. S2 C), and they suggest that SOCS2 is required for normal Th17 differentiation. STAT5 is known to inhibit Th17 differentiation (Laurence et al., 2007; Yang et al., 2011) and, therefore, our data suggest that the reduced STAT3 activation and increased IL-2–mediated STAT5 phos- phorylation in the SOCS2/ T cells primes Th2 development, probably at the expense of Th17. Interestingly, SOCS3 is a strong inhibitor of STAT3 signal- ing (Elliott and Johnston, 2004) and constitutive expression of SOCS3 in T cells was shown to favor Th2 polarization (Seki et al., 2003). Moreover, several studies suggest that SOCS2 can control the expression of both SOCS1 and SOCS3 (Tannahill et al., 2005; Piessevaux et al., 2006). We therefore hypothesized that SOCS1 and SOCS3 levels may be altered in SOCS2- deficient T cells. When splenocytes from SOCS2/ mice were stimulated with anti-CD3/CD28 and IL-2 for periods of up to 24 h, we consistently observed significantly elevated SOCS1 and SOCS3 expression (Fig. S4 A). Interestingly, in WT cells, SOCS2 expression after T cell activation correlated with loss of detectable SOCS3 protein (Fig. S4 A). To assess whether SOCS3 levels were elevated in Th2 cells from SOCS2/ mice, we purified and cultured CD4+ T cells for 7 d under Th2 conditions. Basal levels of SOCS3 were higher in SOCS2- deficient T cells relative to controls (Fig. S4 B), with the differ- ence between WT and SOCS2/ cells being amplified under polarizing conditions (Fig. S4 C). Finally, we also found that SOCS1 and SOCS3 mRNA levels are elevated in SOCS2- deficient CD4+ T cells stimulated for 3 and 5 d with anti-CD3/ CD28 antibodies (Fig. S4 D). Because SOCS1 and SOCS3 are important regulators of Th differentiation (Palmer and Restifo, 2009), the elevated SOCS3 expression in SOCS2/ CD4+ T cells would be consistent with the observation that SOCS3 blocks Th17 development by inhibiting IL-6 and IL-23 re- sponses (Chen et al., 2006) while favoring Th2 differentiation (Seki et al., 2003). It is therefore plausible that the increased levels of SOCS1 and SOCS3 may also affect polarization toward other subsets, indirectly favoring Th2 differentiation. DISCUSSION Collectively, our findings show that SOCS2/ mice are predisposed to AD and allergic lung inflammation and devel- oped enhanced type 2 immune responses to helminth anti- gen. Because the SOCS2 mice are ubiquitous knockouts, other immune cells also lacking SOCS2 could contribute to this phenotype. Of note, we made the observation that the SOCS2-deficient B cells do not spontaneously class-switch toward IgE (unpublished data), which could have explained the predisposition of the SOCS2/ mice for atopy. Of course, 1528 this should be further investigated, together with the effect of SOCS2 deficiency on APC, eosinophils, and mast cells in particular. However, as RAG-1/ mice reconstituted with SOCS2-deficient CD4+ T cells developed enhanced pathol- ogy compared with those reconstituted with WT (Fig. 1 E; and Fig. 3, E and F), these findings suggest that SOCS2 exerts an intrinsic inhibition on CD4+ T cells, which prevents po- larization toward Th2 both in vitro and in vivo. Interestingly, and consistent with the normal IFN- production we observe, SOCS2/ mice mount a normal Th1 response to L. major infection (Bullen et al., 2003). SOCS2 has primarily been studied in relation to the regu- lation of growth hormone and cytokine signaling and has not previously been thought to influence T cell lineage commit- ment. CD4+ T cell lineage maturation is governed by the ex- pression of master regulator transcription factors that drive differentiation, but recent evidence suggests that their expres- sion is not as stable as initially thought; indeed, there are now many examples of plasticity in Th cell subsets (Murphy and Stockinger, 2010). T cell plasticity and lineage fate are also governed by the cytokines they encounter after antigen recog- nition. Therefore it is plausible that the SOCS proteins might play a significant role in Th cell polarization. Indeed, constitu- tive expression of SOCS3 during CD4+ T cell differentiation facilitates Th2 expansion (Seki et al., 2003; Ozaki et al., 2005), whereas selective deletion of SOCS3 in T cells facilitated en- hanced STAT3 activation and elevated IL-17 production (Chen et al., 2006). Moreover, recent studies have shown that SOCS3 may play a role in controlling regulatory T cell re- sponses because SOCS3-deficient T cells demonstrate a Th3- like phenotype with increased TGF- and IL-10 and increased STAT3 activation (Kinjyo et al., 2006; Taleb et al., 2009). In conclusion, therefore, we propose that SOCS2 inhibits Th2 development, and that the enhanced STAT5 and STAT6 phosphorylation in the absence of SOCS2 favors Th2 polar- ization. Finally, we have defined new functions for SOCS2 in the regulation of allergic inflammation and, potentially, CD4+ T cell differentiation, providing new insights on how the in- fluence of SOCS proteins on T cell plasticity may be exploited for the development of therapies for atopic disorders. MATERIALS AND METHODS Mice. All experiments used 12–16-wk-old sex- and age-matched C57BL/6 and SOCS2/ mice. SOCS2/ mice were a gift from D.J. Hilton (Royal Mel- bourne Hospital, Victoria, Australia) and were generated on a C57BL/6 back- ground as previously described (Metcalf et al., 2000). C57BL/6 mice were either purchased from Harlan Laboratories or were WT littermates. RAG-1/ and OT/II strain mice on a C57BL/6 background were purchased from The Jackson Laboratory. OT/II mice were subsequently crossed to SOCS2/ mice in house to generate SOCS2/xOT/II mice. Mice were housed under specific patho- gen-free conditions. All animal experiments were performed in compliance with either the UK Home Office or the Irish Department of Health and Children reg- ulations and approved either by the Queen’s University Ethical Review Com- mittee or by the Trinity College Bioresources Ethical Review Board. T cell culture, purification, and differentiation. All in vitro assays were performed by using between two and three mice per group. After extraction, splenocytes and lymphocytes were depleted of erythrocytes by lysis with ammonium chloride solution. Cells were cultured at 106/ml in complete SOCS2 regulates Th2 responses | Knosp et al.