Rv bound retronectin

Murine CAR-T cells were produced as previously described [15 (link)]. Briefly, the Rv packaging CAR gene was produced by transfecting Plat-E cells with pMXs-Puro/CAR. Murine T cells were activated by using anti-CD3ε mAb (clone 145-2C11, Bioxcell, West Lebanon, NH) and an anti-CD28 mAb (clone 37.51, Bioxcell), and then transduced with Rv-bound Retronectin (Takara Bio, Kusatsu, Japan) under anti-CD3ε/CD28 mAbs stimulation. After Rv-transduction, CAR-T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 50 μM 2-mercaptoethanol, MEM Non-essential Amino Acids Solution (FUJIFILM Wako Pure Chemical, Osaka, Japan), 10 U/mL interleukin-2 (Peprotech, Rocky Hill, NJ), and 5 μg/mL puromycin.
Human CAR-T cells were produced as previously described [13 (link)]. Briefly, CAR mRNA was transcribed using the mMESSAGE mMACHINE T7 Ultra kit (Thermo Fisher Scientific) and linearized pcDNA3.1-Zeo/CAR. Human T cells were activated by using an anti-CD3 mAb (Janssen Pharmaceutica, Beerse, Belgium) for 13 days, and CAR mRNA was introduced by electroporation.

Murine CAR-T cells were produced as previously described [22 (link)]. Briefly, a murine leukaemia virus-derived pMXs-Puro Retroviral Vector (Cell Biolabs, San Diego, CA, USA) was used as a plasmid for Rv production. The Rv packaging CAR gene was produced by transfecting Plat-E cells with the pMXs-Puro/CAR. The culture supernatant of Plat-E cells obtained 48 h later was filtered through a 0.45 μm filter and used as an Rv solution for gene transfer. Murine T cells were activated by incubation with an anti-CD3ε mAb (clone 145-2C11, Bioxcell, West Lebanon, NH, USA) and anti-CD28 mAb (clone 37.51, Bioxcell, West Lebanon, NH, USA) and then transduced with Rv-bound Retronectin (Takara Bio, Kusatsu, Japan) under anti-CD3ε/CD28 mAbs stimulation. The gene-transduced cells were cultured in cRPMI medium, supplemented with 5 μg/mL puromycin. We defined the end of the 24 h culture as the end of the gene transfer operation on the 0th day (day 0).

Murine CAR-T cells were produced as previously described [42 (link)]. Briefly, the Rv packaging CAR gene was produced by transfecting Plat-E cells with pMXs-Puro/CAR. Murine T cells were activated by using anti-CD3ε mAb (clone 145-2C11, Bioxcell, West Lebanon, NH, USA) and an anti-CD28 mAb (clone 37.51, Bioxcell), and then transduced with Rv-bound Retronectin (Takara Bio) under anti-CD3ε/CD28 mAbs stimulation. After Rv-transduction, CAR-T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 50 μM 2-mercaptoethanol, MEM Non-essential Amino Acids Solution (FUJIFILM Wako Pure Chemical, Osaka, Japan), 10 U/mL interleukin-2 (Peprotech, Rocky Hill, NJ, USA), and 5 μg/mL puromycin.