Recombinant mil 6

LN CD4⁺ T cells from WT and Crtam^−/− mice were enriched using a CD4⁺ T cell isolation kit (Miltenyi Biotec). 2 × 10⁵ cells were activated with plate-bound anti-CD3 (10 µg/ml) and anti-CD28 (2 µg/ml) under polarizing TH1 conditions, i.e., recombinant IL-2 (PeproTech), mIL-12 (PeproTech), and anti–mouse IL-4 mAb (11B11), or TH17 conditions, i.e., recombinant mIL-6 (PeproTech), hTGFb, mIL-23, mAb anti-IL-4 (11B11), and anti–IFN-γ (RUGA2) for 5 d (primary stimulation). For the secondary stimulation, T cells were washed, counted, and 2 × 10⁵ cells were restimulated with plate-bound anti-CD3 (10 µg/ml) and anti-CD28 (2 µg/ml) without added cytokines or antibodies for 2 more days. Cells were analyzed for cytokine production by intracellular staining either after 5-d stimulation or 2-d restimulation. Supernatants were also collected at these times, and IFN-γ and IL-17 concentrations determined by cytometric bead array (BD).