The expression of Tyk2 in DCs is required for the optimal Th1-cell differentiation. (A) Splenic CD4⁺ T cells (1 × 10⁶ cells) from DO11.10⁺ mice were stimulated with OVA323–339 peptide (0.2 μg/mL) in the presence of WT or Tyk2^−/− CD11c⁺ DCs (2 × 10⁵ cells). Where indicated, IL-12 (10 ng/mL) or anti–IFN-γ antibody (15 μg/mL) was added to the culture. Five days later, intracellular staining for IL-4 and IFN-γ was performed and analyzed by FACS. Representative FACS profiles of IL-4 versus IFN-γ gated on CD4⁺ CD11c⁻ cells are shown from 5 independent experiments. (B) DO11.10⁺ CD4⁺ T cells were stimulated with OVA323–339 peptide in the presence of WT or Tyk2^−/− CD11c⁺ DCs with or without IL-12 (10 ng/mL). Five days later, the levels of IFN-γ, IL-4, and IL-5 in the culture supernatants were determined by ELISA. Data are means (± SD) from 5 independent experiments. *Significantly different from the corresponding mean values of WT DCs, *P < .05. (C) DO11.10⁺ CD4⁺ T cells (2 × 10⁵ cells) were stimulated with OVA323–339 peptide in the presence of WT or Tyk2^−/− CD11c⁺ DCs (4 × 10⁴ cells) with or without IL-12 in a 96-well microtiter plate for 72 hours, with 1 μCi (0.037 MBq) [³H] thymidine added for the final 12 hours. Data are means (± SD) of [³H] thymidine uptake from 4 independent experiments. (D) DO11.10⁺ CD4⁺ T cells were stimulated with OVA323–339 peptide in the presence of WT or Tyk2^−/− CD11c⁺ DCs. Where indicated, IL-12 or anti–IFN-γ antibody was added to the culture. Five days later, the levels of IL-17 in the culture supernatants were determined by ELISA. Data are means (± SD) from 5 independent experiments. *P < .05.