Generation of CAR.GD2 NKT cells. (A) Schematic representation of recombinant retroviral vectors encoding CAR.GD2 constructs. (B) NKT cells were isolated from human PBMCs, stimulated with αGalCer, and transduced with the indicated CAR.GD2 constructs followed by expansion in culture with IL-2. The expanded cells were analyzed by FACS for the frequency of NKT cells using 6B11 and anti-CD3 mAbs (day 21 after transduction, upper panel) and for the frequency of CAR.GD2 NKT cells using anti-idiotype 1A7 mAb on days 7, 14, and 21. Results are from a representative of 5 experiments. (C) Absolute cell count of CAR.GD2-expressing NKT cells at day 21 of expansion. Data are mean ± standard deviation (SD) from 5 donors.