Modulating T Cell Differentiation via miRNA-374b-5p

Naïve CD4⁺ T cells (5 × 10⁵ cells per well) were grown in a 96-well plate containing T cell expansion medium (Thermo-Fisher) at 37 °C and 5% CO₂, and then transfected with miRNA-374b-5p inhibitor (200 nmol/mL; Qiagen) and miRNA-374b-5p mimic (50 nmol/mL; Qiagen) using the Hiperfect Transfection Reagent (Qiagen). MiScript NC siRNA (Qiagen) was employed as a control. After 4 h, T cell differentiation was performed in using 48-well plates different cytokine regimens according to a previous method [23 (link)]. For Th1, the transfected cells were incubated with complete RPMI, plate-bound 1 μg/mL anti-CD3 and 1 μg/mL soluble CD28 antibodies, 20 ng/mL IL-2, 10 ng/mL anti-IL-4 and 50 ng/mL IL-12 antibodies (BD Biosciences) for 96 h. For Th2, the cells were exposed to 1 μg/mL plate-bound anti-CD3 and 1 μg/mL anti-CD28 antibodies, 10 ng/mL IL-4, 20 ng/mL IL-2 and 10 ng/mL anti-IFN-γ antibodies (BD Biosciences) for 96 h. For Th17 cell differentiation, the transfected cells were incubated with complete RPMI, 1 μg/mL plate-bound anti-CD3 and 0.2 μg/mL soluble CD28 antibodies, 5 ng/mL TGF-β, 100 ng/mL IL-6, 50 ng/mL IL-23, 10 ng/mL anti-IL-4, and 10 ng/mL anti-IFN-γ antibodies (BD Biosciences) for 96 h.