Anti cd3 antibody

Naïve CD4⁺ T cells were purified from splenocytes using mouse CD4⁺ T cell isolation kit (Stemcell Technologies), and further sorted by FACS Aria II (BD Biosciences). 24-well plates were coated with 40 μg/ml anti-hamster antibody (MP Biomedical) at 37°C for 4 hours. For iTreg differentiation, naïve CD4⁺ T cells were cultured in T cell media IMDM (Sigma-Aldrich) supplemented with soluble 0.25 μg/ml anti-mouse CD3 (145–2C11), 1 μg/ml anti-mouse CD28 (37.51), 2 μg/ml anti-mouse IL-4 (11B11), 2 μg/ml anti-mouse IFN-γ (XMG1.2) and 5 ng/ml TGF-β, with (Th17) or without (iTreg) 20 ng/ml IL-6, for 3.5 days. For human cells, naïve T cells were enriched using the human CD4⁺ T cell isolation kit (Stemcell) from human PBMC cells and further sorted (CD3⁺CD4⁺CD45RA⁺CD25⁻). For iTreg differentiation, the sorted naïve human CD4⁺ T cells were seeded into 48-well plates precoated with 1 μg/mL anti-CD3 antibody (clone HIT3a), and cultured in RPMI media (Sigma-Aldrich) supplemented with IL-2 (10 ng/ml), soluble anti-CD28 (1 μg/ml; clone CD28.2) and 5 ng/ml TGF-β for 6 days. In some experiments, FICZ was added at a concentration of 200 nM as indicated in the text.