Differentiation of Naive CD4+ T Cells

Naive CD4⁺ T cells were obtained from spleen and peripheral lymph nodes of CRL and IF1-KO mice by negative selection using the MojoSort™ Mouse CD4 Naïve T Cell Isolation Kit (BioLegend). For in vitro activation, naive CD4⁺ T cells were incubated in the presence of 2 μg/ml plastic-bound purified anti-CD3, 1 μg/mL soluble anti-CD28, and 5 ng/mL recombinant mouse IL-2 during 48 h. For Th1 differentiation, culture medium was also supplemented with IL-12 (10 ng/mL) and anti-IL-4 (4 μg/mL). For Treg cell differentiation, TGF-β (10 ng/mL) was also added to culture medium. For Th17 differentiation, IL-2 was replaced and anti-IL-4 (5 μg/mL), anti-IFN-γ (5 μg/mL), TGF-β (5 ng/mL), IL-23 (20 ng/mL), and IL-6 (20 ng/mL) were incorporated in the culture medium.⁴³ (link) Cells were differentiated for 4 days in a humidified incubator at 37°C with a controlled atmosphere of ambient air 10% CO₂.