Mirzip 21 lentiviral vector

To antagonize miR-21, we used the miRZip-21 lentiviral vector expressing anti-sense miR-21 (Systems Biosciences). miRZip-scrambled hairpin vector was used as a control (System Biosciences). The vector additionally contained a GFP reporter. Lentivirus was produced by transfection of a lentiviral vector, along with psPAX2 (Plasmid #12260; Addgene) and pMD2.G (Plasmid #12259; Add gene) expression vectors into HEK293T cells by using FuGENE (Promega). Lentiviral particles were collected 48 and 72 hours after transfection, filtered through a 0.45-mm syringe filter (Millipore), concentrated using Peg-it solution (System Biosciences) and titered on HEK293T cells. For lentiviral transduction, naive CD4⁺ or CD8⁺ T cells labeled with CellTrace Violet (Thermo Fisher Scientific) were activated with anti-CD3/anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control RNA or anti-sense miR-21 at a multiplicity of infection of 10 in the presence of 8 mg/ml polybrene (Sigma) and 10 U/ml human IL-2 (Peprotech). After 36 hours, activated cells were washed and cultured on plates coated with 1 mg/ml anti-CD3 (CD3–2) plus 2 mg/ml soluble anti-CD28 Ab (CD28.2) and 10 U/ml human IL-2 (Peprotech).