Phoenix eco packaging cells

Phoenix-ECO packaging cells (catalog number CRL-3214) purchased from ATCC and 293 galv9 packaging cells (courtesy of the Sadelain lab) were maintained in DMEM supplemented with 10% heat-inactivated FBS nonessential amino acids, 2 mM L-glutamine, 1% penicillin/streptomycin. The B16F10 cell line, a kind gift from Dr. Jedd Wolchok in 2016, and A375 and SK-Mel5 human melanoma lines, a kind gift from Dr. David Scheinberg in 2018, were modified to express GFP-firefly luciferase for killing assays. Cell lines were kept in culture for up to two months. All tumor cell lines were maintained in RPMI-1640 supplemented with 10% heat-inactivated FBS, nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, 1% penicillin/streptomycin, 11 mM glucose, and 2 μM 2-mercaptoethanol. Cell lines were not re-authenticated in the past year but were routinely tested for potential mycoplasma contamination with Lonza MycoAlert Detection kit (LT07–318).

Mouse CD19scFv-CD28-CD3ζ CAR (m1928z) construct with GFP in SFG retroviral vector was described before and provided by M. Davila at the Moffitt Cancer Center³⁴ . The isolation, activation and transduction of mouse T cells followed the procedure described before^{34 ,35} . Briefly, the spleens were collected from female C57Bl/6 mice and T cells were enriched from splenocytes by passage over a nylon wool column (Polysciences). Mouse T cells were then activated with CD3/CD28 Dynabeads (Thermo Fisher) following the manufacturer’s instructions and cultured in the presence of human IL-2 at 30 IU ml⁻¹ (R&D Systems). Retrovirus was produced by transfecting Phoenix-Eco packaging cells (ATCC) and spinoculations were done twice with retroviral supernatant. mCART19 cells were expanded for 10–14 days as described³⁵ . UT-T were produced following the same procedure without viral transduction.