T cell differentiation

Mice were injected subcutaneously with 2 × 10⁶ Flt3L-producing EL4 cells, and spleens were collected 10 days later. Flow-sorted splenic DCs were used as APCs. Alternatively, iCD103 BMDC was cultured as previously described with RPMI complete media supplemented with Flt3L (100 ng/ml) and GM-CSF (10 ng/ml; both from PeproTech, Rocky Hill, NJ) for 12 days before used as APCs in T cell differentiation assays. Naïve CD4⁺ T cells were enriched using MicroBeads (Miltenyi Biotec, Germany), and T cells were cocultured with DCs (DC:T = 1:4) in the presence of 1 μM OVA323-339 peptide (InvivoGen, San Diego, CA) in RPMI complete media supplemented with recombinant IL-2 (10 ng/ml) under various T cell differentiation conditions for 4 to 5 days. T_H1: recombinant IL-12 (10 ng/ml) and anti–IL-4 (10 μg/ml; clone 11B11); T_H2: recombinant IL-4 (10 ng/ml), anti–interferon-γ (IFN-γ) (10 μg/ml; clone XMG1.2), and anti–IL-12 (10 μg/ml; clone C17.8); T_H17: recombinant IL-6 (20 ng/ml), recombinant transforming growth factor–β (TGF-β) (1 ng/ml), and anti–IL-4/IFN-γ (10 μg/ml each); and T_reg: TGF-β (1 ng/ml) and anti–IL-4/IFN-γ/IL-12 (10 μg/ml each). All antibodies and recombinant proteins besides TGF-β (PeproTech) were purchased from BioLegend (San Diego, CA). In MPEG1 (macrophage-expressed gene 1) blocking experiments, anti-MPEG1 antibody (10 μg/ml) (Thermo Fisher Scientific, Waltham, MA) was added to the media. Equal amount of normal rabbit IgG (PeproTech) was used as control.