Skip to main content
. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: Nat Biomed Eng. 2022 Aug 8;6(11):1284–1297. doi: 10.1038/s41551-022-00915-0

Extended Data Fig. 4. CD8αβ single positive CAR+ iT cell development.

Extended Data Fig. 4.

WT-TiPS were differentiated on OP9-DLL4 and transduced to express the 1928z CAR at D35 utilizing γRV SFG-1928z-P2A-LNGFR. Cells were expanded for 7 days in expansion media supplemented with IL-2. a, CD4/CD8αβ expression prior to transduction (D35) and on D42 in LNGFR+ cells, LNGFR− cells and untransduced control cells which remained in differentiation on OP9-DLL4. Gated on live CD45+ cells. b, Cytotoxic activity of CAR+ iT cells in a 18 h bioluminescence assay, using FFLuc- NALM6 as target cells (n = 3 technical replicates, data are mean ± s.d). c, CRISPR/Cas9-targeted integration of CAR transgene into the TRAC locus. Top, TRAC locus; middle, plasmid containing the CAR transgene cassette flanked by homology arms; bottom, edited TRAC locus. d, f, PCR validation of CAR integration into the TRAC locus of TRAC-1928z-TiPS (d) and TRAC-1XX-TiPS (f) clones. e, g, Pluripotency marker expression on TRAC-1928z-TiPS (e) and TRAC-1XX-TiPS (g), gated on live cells.