Retroviral transduction

Platinum-E ecotropic packaging cells (Cell Biolabs) were plated one day prior to transfections on poly-D-lysine coated 10cm plates (Corning) at a concentration of 6x10⁶ cells per plate. Packaging cells were transfected with 20µg of retroviral plasmid DNA encoding MSGV-Thy1.1, MSGV-Kcna3-Thy1.1, MSGV-Kcna3-Thy1.1 (W389F) (referred to as Kcna3_PD) ^{25, 40}, MSGV-Ppp2r1a(K416E) -Thy1.1 (referred to as PP2A_DN^41,42), MSCV-IRES-Thy1.1 (pMIT), or pMIT Akt1-CA⁴³ where indicated along with 6µg pCL-Eco plasmid DNA using 60µL Lipofectamine 2000 in OptiMEM (Invitrogen) for 8 hours in antibiotic-free media. Media was replaced 8h after transfection and cells were incubated for a further 48 hours. Retroviral supernatants were then collected and spun at 2000g for 2 h at 32°C onto 24 well non-tissue culture treated plates coated overnight in Retronectin (Takara Bio). For in vivo experiments live cells were isolated via ficoll density separation (Cedarlane) and subjected to positive-selection via CD90.1-microbead column enrichment according to the manufacturer’s protocol (Miltenyi) prior to transfer, yielding a CD8⁺ T cell population 70-95% Thy1.1⁺ prior to transfer. For retroviral transduction of human PBL with the NY-ESO-1 TCR, patients with metastatic melanoma on the clinical protocol NCI-13-C-0214 were subjected to leukopheresis and their PBL were transduced with a MSGV1 backbone encoding the NY-ESO-1 murine derived TCR as previously described¹⁷. After primary transduction and culture (10 days), these cells were then further expanded via a REP as described above (days 10-23) and subsequently assayed for target-specific effector function in the indicated conditions.