Stimulation plates were made in 96-well flat bottom culture plate. One-hundred µl of PBS supplemented with LEAF purified anti-CD3 (OKT3, Biolegend, Cat# 317326, RRID:AB_11150592 ) at 0.25 µg/ml was added to each well and incubated at least 12 hr at 4°C. The plate was then emptied, and wells were given 100 µl of cytokine free T cell media. After cells were edited, expanded for 7 days, and rested 24 hr in cytokine free media, 100 µl of cells at 2 million/ml were added to each well. Plates were incubated at 37°C for up to 48 hr.
For Th1 skewing of edited T cells, cells that had been edited and rested were transferred to 96-well flat bottom culture plates, coated with anti-CD3 as discussed above. These cells were cultured for 3 days in media containing 20 ng/ml IL-12 (Peprotech), 50 ng/ml IL-2, 40 μg/ml anti-IL-4 (MP4-25D2, eBioscience Cat# 14-7048-81, RRID:AB_468414 ), and 500 ng/ml soluble anti-CD28 (CD28.2, BioXcell Cat# BE0291, RRID:AB_2687814 ) to promote a Th1 phenotype. Non-skewed controls were cultured in media that contained IL-2, anti-CD3, and anti-CD28 only.
For Th1 skewing of edited T cells, cells that had been edited and rested were transferred to 96-well flat bottom culture plates, coated with anti-CD3 as discussed above. These cells were cultured for 3 days in media containing 20 ng/ml IL-12 (Peprotech), 50 ng/ml IL-2, 40 μg/ml anti-IL-4 (MP4-25D2, eBioscience Cat# 14-7048-81, RRID: