Increased secretion of Th2-associated cytokines in SOCS2^−/− mice in vitro and in vivo. (A) CD4⁺CD25⁻ splenocytes purified from WT and SOCS2^−/− mice were cultured for 3 or 5 d with 2 µg/ml of plate-bound anti-CD3, 1 µg/ml of plate-bound anti-CD28, and 5 U/ml IL-2 in triplicate and cytokine production was evaluated by ELISA. Data are representative of at least three independent experiments. (B) Naive CD4⁺CD25⁻CD44⁻ splenocytes were cultured for 7 d under Th2 polarization conditions. Frequency of CD4⁺IL-4⁺ cells (Th2 cells) was evaluated at days 4 and 7 by intracellular staining. Data are representative of three independent experiments performed in triplicate. (C) SOCS2^−/− and WT mice (n = 5 per group) were i.p. injected with 10,000 S. mansoni eggs or PBS. 10 d later, splenocytes were restimulated with anti-CD3 and anti-CD28 or SEA and cytokine production was measured by ELISA. (D and E) The footpads of WT and SOCS2^−/− mice (n = 5 per group; D) or RAG-1^−/− mice reconstituted with CD4⁺ T cells sorted from WT (n = 4 per reconstituted group; E) or SOCS2^−/− mice (n = 7 per reconstituted group; E) were injected with 5,000 S. mansoni eggs or PBS. 10 d later, the amount of Th2 cells in popliteal LNs was determinate by IL-4 intracellular staining (D) or T1/ST2 (IL-33 receptor) staining (E). Data are representative of at least three experiments and presented as the mean and SEM of each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Increased allergic lung inflammation in SOCS2^−/− mice. (A) Methacholine-induced R_L in PBS or OVA-sensitized SOCS2^−/− and WT mice (n = 7 mice per group). (B) Representative image of lung sections from each group stained with periodic acid-Schiff stain (bars, 50 µm). (C) Levels of total and OVA-specific IgE in serum of mice from each group. (D) Cells from mediastinal LN were restimulated in triplicates with media or anti-CD3 and anti-CD28 and cytokine secretion was measured by ELISA. (E and F) RAG-1^−/− mice were reconstituted with PBS as control or with sorted CD4⁺ T cells isolated from OTII or SOCS2^−/− OTII mice. The number of mice per group of reconstituted RAG-1^−/− mice was between five and nine. The mice were then sensitized and challenged with OVA and methacholine-induced R_L was measured (E). The cytokine concentrations in the lungs were measured after homogenization (F). Data are from three independent experiments and are presented as the mean and SEM per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

SOCS2^−/− mice develop more severe AD than WT mice. (A) Calcipotriol (MC903) or ethanol was applied daily to the right ear of WT and SOCS2^−/− mice (n = 6 per group) and ear thickness was measured daily. (B) Representative pictures of H&E-stained ear sections from WT and SOCS2^−/− mice at the end of study (bars, 150 µm). (C) Quantitation of ear eosinophil infiltrates from 10 sections randomly selected out of one representative mouse per group. (D) Three mice per group were sacrificed at day 5, and auricular draining LN and spleen cells were isolated and restimulated in triplicate for 48 h with anti-CD3 and anti-CD28. Secreted cytokines were then measured by ELISA. Data are representative of three independent experiments and are presented as mean and SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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-β for the indicated times. Cells were then lysed and immunoblotted for the expression of SOCS2 and γ-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 immunoblotted for phospho-STAT6. (C) After 48 h and preactivation with anti-CD3, anti-CD28, 5 ng/ml TGF-β, and 5 U/ml IL-2, CD4⁺ splenocytes 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.