Priamry T cell expansion and ell culture

For expansion, cells were either cultivated in ImmunoCult™-XF T Cell Expansion Medium or in Thera- PEAK® T-VIVO® medium, IL-2 supplemented and activated according to suppliers’ specifications. In detail, ImmunoCult™-XF T Cell Expansion Medium was supplemented with 218 IU/mL IL-2 (PeproTech, London, UK), and T cells were activated by 25 µL/mL ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator reagent at a cell density of 1 × 10^6 cells/mL (StemCell Technologies, Vancouver, Canada) (referred to as protocol A). TheraPEAK® T-VIVO® was supplemented with 100 IU/mL IL-2 (Novartis, Basel, Switzerland), and T cells were activated by 10 µL/mL T Cell TransAct™ reagent at a cell density of 1 × 10^6 cells/mL (Miltenyi Biotech, Bergisch Gladbach, Germany) (referred to as protocol B). For both protocols, T cells were activated on day 0 and incubated at 37 °C and 5% CO2 for up to 8 days. From days 3 to 8, T cells were monitored regarding cell number using cell counting chambers, cell viability by trypan blue staining, and T cell subsets by flow cytometry. The cell density was adjusted to 1.0–2.5 × 10^5 cells/mL by adding fresh medium.

Transient transfection and CAR expression analysis

On day 7, expanded T cells were transiently transfected with anti-CD123-CAR ± GFP mRNA using the 4D-Nucleofector ™ X Unit and P3 Primary Cell 4D X Kit L (Lonza, Walkersville, USA). For optimization of electroporation parameters, commercially available EGFP-mRNA and anti-CD123-CAR + GFP were used, while for media testing,
anti-CD123-CAR without GFP was used. A total of 3 × 10^6 T cells were resuspended in 90 µL 4D-Nucleofector ™ Solution, combined with 6 µg of mRNA, transferred to Nucleocuvette™ vessels, electroporated at a defined pulse code (see Supplemental Figs. S4, S6), resuspended in 500 µL from the respective media and cultured in a
24-well plate at 37 °C and 5% CO2.