Lentiviral transduction and shRNA knockdown

Plasmids containing the sequences of non-specific shRNA and mouse HIF-1α shRNA (Open Biosystems) were used to prepare lentivirus according to manufacturer’s protocol (Sigma-Aldrich). CD4+ T cells were activated on immobilized anti-CD3 and anti-CD28 antibodies for 18 h prior to transduction with lentivirus as follows. The lentivirus and polybrene (6 μg/ml) were added to the T cell culture and centrifuged at 1000 × g at 30 °C for 1 h. The transduced cells were grown for 2 d followed by puromycin (2.5 μg/ml) selection.