In vitro Polarization of T Helper Cells

In vitro-polarized Th17 or Th1 cells or ex vivo CD4 T cells were incubated with only the indicated recombinant cytokines at the following concentrations: IL-1β (10 ng/ml), IL-6 (20 ng/ml), IL-18 (50 ng/ml), IL-23 (10 ng/ml), TGFβ (5 ng/ml) and IL-12 (10 ng/ml), all purchased from R&D Systems. Control cultures were left without additional cytokines or plated in wells coated with anti-CD3 and supplemented with soluble anti-CD28 (5 μg/ml). Supernatants were collected after 48 hours and the production of IL-17A, IL-17F and IFNγ were detected by color development (TM-Blue; Sigma) of HRP-avidin substrate (Vector) following capture by antibodies directed against mouse IL-17A, IL-17F or IFNγ and detection by biotinylated anti-mouse IL-17A, biotinylated anti-mouse IL-17F, or biotinylated anti-mouse IFNγ, respectively (all purchased from BD Biosciences, IL-17F R&D Systems). ELISA was performed as previously described [10 (link)]. IL-22 ELISA was performed according to the manufacture's instructions (R&D Systems). The amounts of cytokine were determined from standard curves established with serial dilutions of recombinant murine IL-17A, IL-17F, IL-22 or IFNγ (R&D Systems).