Soluble anti cd3

CD4⁺ T cells were isolated from the spleen and lymph nodes using the EasySep Mouse CD4 Positive Selection Kit II (Stemcell Technologies). Naive (CD25⁻CD44^hiCD62L^lo) CD4⁺ T cells were sorted by flow cytometry from the bead purified CD4⁺ T cells. The naive CD4⁺ T cells were resuspended in Click’s medium (Irvine Scientific) at 1 million cells per ml, and then plated on day 0 in 24 well plates coated with goat anti-hamster IgG antibody (200 ng ml⁻¹; MP Biomedicals) with the addition of soluble anti-CD3 (1 μg ml⁻¹; 145-2C11) and anti-CD28 (1 μg ml⁻¹; 37.51) from Bio X Cell. Polarizing conditions for different T helper subsets are as following: T_H1: human IL-2 (100 U ml⁻¹; PeproTech), mouse IL-12 (20 ng ml⁻¹; PeproTech) and anti-IL-4 (5 μg ml⁻¹; Bio X Cell); T_H2: human IL-2 (100 U ml⁻¹; PeproTech), mouse IL-4 (20 ng ml⁻¹; Biolegend), anti-IFN-γ and anti-IL-12 (5 μg ml⁻¹; Bio X Cell); T_H17: mouse IL-6 (20 ng ml⁻¹; Biolegend), human TGF-β (2 ng ml⁻¹; PeproTech), anti-IFN-γ and anti-IL-12 (5 μg ml⁻¹; Bio X Cell).