2.4. Lentiviral Vector Production and Cell Transduction

The siRNA-expression lentivirus vectors were constructed, packed, and purified by Bioyong Technologies Inc. (Beijing, China). The sequences used for Axl knockdown were sense, 5′-GAGAUGGACAGAUCCUAGA-3′, and antisense, 5′-UCUAGGAUCUGUCCAUCUC-3′. The sequences for control siRNA were sense, 5′-CCUACGCCACCAAUUUCGU-3′, and antisense, 5′-ACGAAAUUGGUGGCG-UAGG-3′, as described previously [21]. CD4⁺CD25⁺T cells were resuspended to 10⁶/ml in 4 ml of complete medium (RPMI 1640 with 10% fetal bovine serum) in 25 cm² dishes. After being cultured for 12 h, cells were washed, counted, and plated at 5 × 10⁵ in complete medium in 25 cm² dishes. Lentivirus was added to a final multiplicity of infection (MOI) of 30 colony-forming units (CFU) per cell. In initial experiments, polybrene was added to give a final concentration of 5 mg/ml. After 16 h, the cells were washed with PBS and resuspended to 10⁶/ml in fresh medium.