Fig 1: AFP TCR1, 2, and 3 demonstrate comparable potent reactivity toward the target cells in vitro. (A) Alignment of amino acid sequence of CDR3 regions of the a (left) and ß (right) chains of the 7 AFP TCRs. (B) FACS analysis of the surface expression of TCR ß chain and AFP tetramer staining of T cells 8 days after transducing of indicated AFP TCRs. The percentage of TCR ß (top) or AFP tetramer (bottom) positive cells as indicated. (C) Cytokine production and degranulation of AFP TCR T cells upon encountering HepG2 target cells as revealed by intracellular staining of IL-2, IFN-? (top), TNF-a and CD107a (bottom). (D) IFN-? concentration in the supernatant of overnight, 1:1 ratio co-culture of AFP TCR T cells with HepG2 target cells or control Huh7 cells measured by ELISA. Data represents the mean + s.d. of quadruplicate co-culture samples. (E) Cytolytic capacity of AFP TCR T cells toward HepG2 target cells over a 4-day co-culture. Data represents the mean of triplicate samples derived from RTCA instrument, as compared to target only wells. (E:T): effect to target ratio. Colors are represented as in (D). (F) Time to eradicate 50% of target cells (KT50) of different AFP TCR T cells at the indicated effect to target (E:T) ratio. Colors are represented as in (D). T cells transduced with TCR 8 and TCR 11 did not eradicate 50% of the target by the end of the analysis at E:T = 0.25:1, thus were not plotted. Data shown in b-f is representative of at least 3 independent experiments on T cells isolated from 3 healthy donors. UT, untransduced T cells.