CAR T cell generation

Peripheral blood was obtained from healthy donors after written informed consent to participate in a research study. The study was approved by the Institutional Review Board of the University of Würzburg. Mononuclear cells were isolated by density centrifugation with Biocoll separating solution (Biochrom), and CD4⁺ and CD8⁺ T cells were sorted by negative isolation with immuno-magnetic associated cell sorting (MACS, Miltenyi). CD8⁺ and CD4⁺ primary human T cells were activated by anti-CD3/CD28 bead stimulation (Thermo Fisher). For assays in Fig. 4, two days after activation, CD19 CAR_EGFRt-encoding transposon MC and hsSB protein were transfected consecutively using a 4D-Nucleofector according to the manufacturer’s instructions (Lonza). 2 x 10⁶ T cells were pulsed with 10 μg hsSB in a volume of 20 μl. LV transduction of controls was performed on day one by spinoculation. CAR T cells were propagated in RPMI-1640 with 10% (v/v) human serum, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin and 50 U/ml IL-2. CAR-modified (i.e. EGFRt-positive) T cells were enriched using biotin-conjugated anti-EGFR mAb and anti-biotin beads (Miltenyi) and expanded with irradiated CD19⁺ feeder cells for 7 days. Functional analysis of CAR T cells was performed as described elsewhere^67–69. For assays in Fig. 6, two days after activation, 1 x 10⁶ T cells were transfected with 0.5 μg CD19 CAR_EGFRt-encoding transposon MC in a volume of 20 μl using a 4D Nucleofector according to the manufacturer’s instructions (Lonza). Directly after electroporation, cells were incubated with 20 μg hsSB in a volume of 500 μl RPMI-1640 serum-free medium for 4 hours. Then, the medium was replaced with fresh serum-containing medium without hsSB. CD8⁺ T cells were cultured in RPMI-1640 with 10% (v/v) human serum, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin and 50 U/ml IL-2 for additional five days. Then, CD3/CD28 beads were removed and CAR-modified (i.e. EGFRt-positive) T cells (~6.3% of the initial population, as determined by flow cytometry) were enriched using biotin-conjugated anti-EGFR mAb and anti-biotin beads (Miltenyi) and expanded using irradiated CD19⁺ feeder cells.