Fig. 2.

5T4_p17-specific TCRs expression on CD8⁺ T-cells from healthy donors and redirected effector functions against peptide-pulsed T2 targets. a Vector structure of 5T4_p17-specific TCR assembly: TRB and TRA are transcribed under control of the MSCV promotor; P2A cleaves the primary transcripts to equal molar quantity of TRA and TRB in the cytosol. The enlarged graph indicates the gene segments and CDR3s, contributing to TRA and TRB sequence assembly. The relative location for the introduced transmembrane cysteines in TRAC and TRBC are indicated. b Cell surface expression of 5T4_p17-specific TCRs stained by 5T4_p17/HLA-A2 tetramer at day 7 post-transduction is shown in comparison to tetramer staining of the native T-cell clone expressing the corresponding TCR (upper panels). Cell surface expression of 5T4_p17-specific TCRs on healthy donor T-cells is shown after sorting for 5T4_p17/HLA-A2 tetramer⁺ cells and 12 days expansion (lower panels). CD8⁺ T-cells expressing 5T4_p17-specific TCRs were tested for recognition of c T2 cells pulsed with 10 nM of 5T4_p17 or control HLA-A2-binding peptides DDX3Y_428–436 or UTY_148–156 in a 4-h cytotoxicity assay. The effector: target ratio (E:T) was 10:1. d TNF-α release was measured by ELISA in culture supernatants harvested after 18 h co-culture of effector T-cells with T2 cells pulsed with 10 nM of 5T4_p17 or the control peptide DDX3Y_428–436 at 10:1 E:T. e Effector T-cells were tested for recognition of T2 cells pulsed with 5T4p17 peptide in a 4-h cytotoxicity assay at 10:1 E:T. f TNF-α release was measured by ELISA in culture supernatants harvested after 18 h co-culture of effector T-cells with T2 cells pulsed with 5T4_p17 peptide at 10:1 E:T