Activation of FE11 γδTCR–transduced T cells is dependent on the presence of a specific HLA-A*24:02-restricted peptide. (A) Activation of TEG011 by HLA-A*24:02-positive or -negative target cells. (B) The differences between HLA-A*02:01 and HLA-A*24:02 mapped on the structure of HLA-A*24:02 (Protein Data Bank: 3wl9), the 2 nonhomologous amino acids between HLA-A*24:02 and HLA-A*24:03 are show in the red circle (top). Alignment of HLA-A*24:02, 02:01, 24:03, and 25:01 with the 2 nonhomologous amino acids in red (bottom). (C) Activation of T cells, transduced with γδTCR-FE11, by HLA-A*24:02-transduced, TAP-deficient T2 cells not loaded or loaded with the A*24-restricted viral peptides NEF_134-144 or CMV_341-349 (pp65 341-349). (D) WT1_126-134 tetramer, NY-ESO1_157-165 pentamer, and CMV_341-349 pentamer binding to WT1_126-134-specific TCR, NY-ESO1_157-165-specific TCR, and FE11 TCR–transduced T cells. (E) The effect of bortezomib treatment of HLA-A*24:02–transduced target cells COS-7 (left) and K562 (right) on the activation of FE11 γδTCR–transduced T cells. (F) Homodimerization was assessed on HLA-A*24:02⁺ cells, recognized and not recognized by flow cytometry FRET. (G) Activation of TEGs (left) or T cells transduced with the WT1_126-134-specific αβTCR (control) (right), by HLA-A*24:02-transduced COS-7 and K562 cells or HLA-A*02:01 (control). *Out of range. (H) Proximity Ligation Assay (PLA) was performed on HLA-A*24:02⁺ fibroblasts and the SW480 cell line. Cells were stained for 4′,6-diamidino-2-phenylindole and PLA signal. Where indicated, target cells were fixed before coincubation; target cells were coincubated with WT1_126-134. Error bars represent the SD (n ≥ 1).