Interleukin 2 (il 2)

Cytokine-induced killer (CIK) cells were generated from PBMCs obtained from volunteers using various animal PBMC separation liquid kits (Solarbio Life Sciences, Beijing, China) according to the manufacturer’s protocols and as previously described. To generate CIK cells, non-adherent PBMCs were prepared in complete RPMI 1640 medium with 1000 U/mL IFN-γ (Solarbio Life Sciences). After 24 hours of incubation, 50 ng/mL mouse anti-human CD3 monoclonal antibody (ACROBiosystems, Newark, Delaware, USA) and 1000 U/mL IL-2 (Solarbio Life Sciences) were added. The CIK cells were incubated at 37°C in a humidified atmosphere of 5% CO₂ and were subcultured every 3 days with cytokine replenishment.

This description was the same as in our previous study. In brief, first, the CD3-positive T cells were enriched from the PBMCs that were separated by density gradient centrifugation (TBD). Next, purified T cells were cultured in X–VIVO15 (Lonza), activated by CD3 and CD28 mAb (PeproTech), and supplied with 10%FBS, 100U/mL penicillin, 100ug/mL streptomycin(solarbio), and 200U/mL IL-2 (Peprotech). Then, 24h later, RetroNectin-coated plates (TaKaRa) were loaded with virus that carrying different CAR genes (CD19 Nb CAR, CD20 Nb CAR, Bispecific Nb CAR, or CD19 CAR) by centrifuging the plates at 2000g for 2h. Once activated, T cells were collected and added to the plates and centrifuged at 1000g for 1h, polybrene(solarbio), IL-2 and antibiotic were also added. Tomorrow, gently replenish constant volume T cell complete medium, and T cells were expanded for 7 days before in vitro and vivo analyze.