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.
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.
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