Series 2

First, 1 × 105 CD4⁺ T cells were seeded in a 24-well plate added with RPMI 1640 media. Then, lentiviral particle solution was added, together with polybrene to obtain a final concentration of 8 μg/mL, followed by incubation for 2 hours in 5% CO₂ at 37°C and 100% humidity (SERIES II, Thermo Fisher Scientific Inc., MA). Afterwards, centrifugation was performed at 1000 rpm for 10 minutes (L-500, Xiangyi Centrifuge Co.,Ltd, Changsha, China). The precipitate was obtained, followed by addition of 10% fresh fetal bovine serum and incubation for another 72 hours.
Total RNA was extracted from the CD4⁺ T cells expressing STAT3 shRNAs and the control CD4⁺ T cells, respectively. Reverse transcription was carried out using a 10 μL system containing 2 μL 5 × PrimeScript RT Master Mix, 2 μL total RNA and RNase Free dH₂O added to the final 10 μL. Quantitative real-time PCR was performed in a 20 μL system containing 10 μL SYBR Green Master Mix (2×), 0.4 μL of forward primer and reverse primer each, and cDNA and RNase free dH₂O added to the final 20 μL on ViiA7 instrument (Thermo Fisher Scientific Inc., MA). The protocol consisted of an initial annealing at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 60 °C for 60 seconds, and final dissociation of 95°C for 15 seconds, 60°C for 60 seconds, and 95°C for 15 seconds.