Separation, transfection and differentiation of naive CD4+ T cells
Murine CD4+ T cells were obtained from the splenocytes of C57BL/6 mice with a mouse naive CD4+ T cell isolation kit, according to the manufacturer’s instructions (Miltenyi Biotec, Gladbach, Germany). Cells were stimulated in vitro with anti-CD3 and anti-CD28 antibodies (BD Pharmingen, San Diego, CA) (both for 2 μg/ml) in RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin/streptomycin. ShRNA/lentivirus were purchased from Genepharma (Shanghai, P.R. China) to achieve the overexpression or knockdown of miR-143-3p. Under the stimulation by anti-CD3 and anti-CD28 antibody, the naïve CD4+T cells were infected by lentiviral-conditioned media following the manufacturer’s instructions. To induce the differentiation of Treg cells, naive CD4+ T cells were supplemented with 5 ng/mL TGF-β (R&D Systems, Minneapolis, USA) and 80 ng/mL IL-2 (R&D Systems, Minneapolis, USA) after 12 h transfection[18]. Cells were analyzed by RT–PCR and flow cytometry.