Identification of gene that drives Tcells to exhaustion may lead to more effective immunotherapies NatureComms
Generation of lentivirus
To generate lentivirus, 2.5 million low passage HEK293T cells were cultured in DMEM medium and seeded into a 15 cm tissue-culture treated dish. Three days later, 2nd generation LTR-containing donor plasmid, packaging plasmid pCMV-delta8.9 and the envelope plasmid VSV-G were mixed at a ratio of 4:2:1 ratio in unsupplemented Opti-MEM and sterile filtered. This solution was then mixed with polyethyleneimine 25 kDa , also diluted in Opti-MEM at a DNA:PEI ratio of 1:3.
. At each stimulation, 75% of the medium was replaced. The cells were expanded 1:2 on the day of the second stimulation with tumor cells and peptide. T, we only exchanged 75% of the medium every three days replenishing IL2. Acute stimulation controls were only cultured as Twith IL2 on days 0, 3, 6, and with tumor cells + peptide + IL2 on day 9 after plating. After 12 days , cells were stained and analyzed by flow cytometry.
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