Figure 2.

Expression and function of the cysteine-modified anti-MART-1 TCR F4. A and B, comparison of the surface expression of the wild-type F4 and its cysteine-modified F4-Cys. OKT3-stimulated PBLs were electroporated with mRNA encoding different combinations of F4-TCR chains: F4, F4-Cys, the α chain of the wild-type F4 + the β chain of F4-Cys (noted as F4 α/β Cys), or the opposite noted as F4 α Cys /β. Twenty-four hours after electroporation, we assessed MART-1 tetramer (A) and Vβ12 (B) binding. Percentage of positive cells and the relative MFI (in brackets). The difference of TCR surface expression, based on 13 independent experiments done with 10 different donors, was found to be statistically significant (P < 0.001). C, recognition of tumor lines. Human PBLs were electroporated with the F4 (white), F4-Cys (black), the α chain of the wild-type F4 + the β chain of F4-Cys (noted as F4 α/β Cys; horizontal hatching), or the opposite noted as F4 α Cys/β (diagonal hatching). The electroporated cells (1 × 10⁵) were cocultured with the HLA-A2⁺ 526 and 624 and the HLA-A2⁻ 888 and 938 melanoma cell lines (1 × 10⁵). Twenty-four hours after the beginning of the coculture, the concentration of IFN-γ and GM-CSF secreted in the medium were measured using an ELISA procedure. This was observed in independent experiments for 10 different donors, and the difference between F4 and F4-Cys was found to be statistically significant (P < 0.001). D, specific killing of tumor cell lines. CD8⁺ purified human PBLs expressing F4 (◆), F4-Cys (■), or mock electroporated (○) were cocultured for 3 h with the indicated tumor cell lines previously labeled with ⁵¹Cr. Specific lysis was measured at the effector/target (E/T) ratios indicated: (specific release − spontaneous release) / (total release − spontaneous release). We used the following melanoma cell lines: HLA-A2⁺ (526 and 624) and HLA-A2⁻ (888 and 938) as control lines.