with OVA and AHR evaluated. RAG-1/ animals that re- ceived the SOCS2/xOT/II cells had significantly higher RL responses than RAG-1/ mice reconstituted with OT/II CD4+ T cells (Fig. 3 E). Moreover, this was associated with enhanced peribronchial cell infiltration and goblet cell hyperplasia in recipients of CD4+ from SOCS2/xOT/II mice relative to cells from OT/II mice (Fig. S2 A) and in- creased bronchoalveolar cell infiltration (Fig. S2 B). Recipi- ents of CD4+ from SOCS2/xOT/II mice had significantly increased secretion of Th2-associated cytokines, lower IL-17, and unaffected IFN- both in the lungs (Fig. 3 F) and in BAL fluid (Fig. S2 C). Collectively, the data demonstrate that SOCS2/ mice are more susceptible to OVA-induced air- way inflammation and this can be a direct result of increased Th2 phenotype in SOCS2-deficient CD4+ T cells. Enhanced STAT5 and STAT6 phosphorylation and SOCS1 and SOCS3 expression after T cell activation in the absence of SOCS2 In the different allergic models used in this study, SOCS2/ mice consistently had enhanced IL-4, IL-5, and IL-13 and re- duced IL-17 expression. It was important, therefore, to deter- mine which cytokines involved in T helper cell polarization actually induced SOCS2 expression. Consequently, we next investigated which cytokine signals induced SOCS2 in purified WT CD4+ T cells. Both IL-4 and IL-6 strongly induced SOCS2 with expression being clearly up-regulated after 48 h (Fig. 4 A). JEM Vol. 208, No. 7 Figure 4. SOCS2 deficiency results in constitutive STAT6 activation, enhanced IL-2–mediated STAT5 activation, and reduced IL-6–mediated STAT3 activation. (A) CD4+ splenocytes from WT mice were cultured with anti-CD3 and anti-CD28 in the presence of 20 ng/ml IL-4, 200 U/ml IL-6, 20 ng/ml IL-12, or 5 ng/ml TGF- for the indi- cated times. Cells were then lysed and immuno- blotted for the expression of SOCS2 and -tubulin. (B) CD4+ splenocytes were cultured with anti-CD3 and anti-CD28 for 48 h. 200 ng/ml IL-4 was then added to the culture for times indicated. Cell were lysed and immuno- blotted for phospho-STAT6. (C) After 48 h and preactivation with anti-CD3, anti-CD28, 5 ng/ml TGF-, and 5 U/ml IL-2, CD4+ spleno- cytes were incubated for 2 h in serum-free media, restimulated with 100 U/ml IL-2 for the indicated times, and immunoblotted for phospho-STAT5. (D and E) CD4+ splenocytes were preactivated for 48 h with anti-CD3 and anti-CD28, rested 2 h in serum-free media, and restimulated with 50 ng/ml IL-12 (D) or 50 U/ml IL-6 (E) for the indicated times, lysed, and immunoblotted for phospho-STAT4 (D) or phospho-STAT3 (E). Total STAT6, STAT5, STAT4, or STAT3 expression was also assessed. Right panels show densitometry of phospho-STAT relative to total STAT. Data are mean and SEM from triplicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The data are representative of three independent experiments. Moreover, IL-4 was the most potent in- ducer of SOCS2, suggesting a possible role for SOCS2 in lim- iting Th2 differentiation. Interestingly, TGF- induced no detectable SOCS2 expression and IL-12 induced limited amounts in CD4+ T cells (Fig. 4 A). SOCSs are known to in- hibit STAT activation, and STATs, in turn, play a key role in the differentiation and commitment of helper T cell subsets. STAT6 is associated with Th2 development, whereas STAT4 and STAT3 reciprocally control Th1 and Th17 development, respectively (O’Shea et al., 2011). To examine the mechanism of preferential Th2 differentiation in SOCS2/ mice, we investigated the profile of STAT activation in T cells. SOCS2/ CD4+ T cells displayed constitutive and signifi- cantly enhanced STAT6 phosphorylation compared with WT CD4+ T cells (Fig. 4 B and Fig. S3 A), suggesting that the SOCS2-deficient CD4+ T cells are hyper-responsive to IL-4 in vivo. Moreover, IL-2–induced STAT5 activation is thought to promote early Th2 cell differentiation (Zhu et al., 2003; Cote- Sierra et al., 2004; Paul and Zhu, 2010) and we found that IL-2–induced STAT5 phosphorylation is enhanced in SOCS2/ CD4+ T cells. (Fig. 4 C and Fig. S3 B). Impor- tantly, IL-2-mediated STAT5 activation was shown to induce and maintain IL-4R expression (Liao et al., 2008). How- ever, we found that IL-4R expression was unchanged (Fig. S3 D). Finally, although IL-12–mediated STAT4 acti- vation was unaffected by the absence of SOCS2 (Fig. 4 D), A r t i c l e 1527