Article Figures & Data
Figures
- Figure 1.
TCR modification and function. A, schematic representation of the cysteine-modified TCR chains. The numbers indicate amino acid position according to the Immunogenetics database ( 18). EX, extracellular region; TM, transmembrane region; CY, cytoplasmic regions. Hα or Hβ, sequence of the wild-type TCR constant region; Hα-Cys or Hβ-Cys show where the cysteine residue was inserted. B, human PBLs were electroporated with 1G4 or 1G4-Cys and cocultured with HLA-A2+/NY-ESO-1+ cell lines (A375, 397/A2, 624.38, 1359/A2, and 1300) and with the control cell lines 526 (HLA-A2+/NY-ESO-1−) and 1359 (HLA-A2−/NY-ESO-1+). Twenty-four hours after the beginning of the coculture, the concentration of cytokine secreted in the medium (IFN-γ), normalized to the amount of mRNA used, was measured using an ELISA procedure. The difference between 1G4 and 1G4-Cys, based on seven independent experiments, was found to be statistically significant (P = 0.01).
- 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.
- Figure 3.
Enhanced properties of different cysteine-modified TCRs. A, purified CD8+ or CD4+ human PBLs (>98% purity) were electroporated with F5 or F5-Cys. Twenty-four hours after electroporation, we assessed MART-1 tetramer binding. Percentage of positive cells and the relative MFI (in brackets). B, electroporated cells were cocultured with melanoma cell lines (HLA-A2+: 526 and 624 and control HLA-A2−: 888 and 938). Twenty-four hours after the beginning of the coculture, the concentration of IFN-γ secreted in the medium was measured using an ELISA procedure. The difference between F5 and F5-Cys, based on 10 independent experiments, was found to be statistically significant (P < 0.001). C, human PBLs were electroporated with different p53-specific TCR: the humanized p53-H, cysteine-modified humanized p53-Hcys, wild-type (fully murine) p53-M, and cysteine-modified murine p53-Mcys. Twenty-four hours after electroporation, we assessed p53 pentamer binding. Percentage of positive cells and the relative MFI (in brackets). D, electroporated cells were cocultured with the tumors p53+/HLA-A2+ (H2087, MDA-MB-231, and Saos-2/*143) and the p53−/HLA-A2+ (Saos-2) cell lines ( 3). Twenty-four hours after the beginning of the coculture, the concentration of IFN-γ secreted in the medium was determined by ELISA. The difference between p53-H and p53-HCys, based on five independent experiments, was found to be statistically significant (P < 0.001).
- Figure 4.
Efficient and preferential pairing of cysteine-modified TCRs. A, TCR titration. OKT3-stimulated PBLs, electroporated with different amount of mRNA (0.2, 0.6, 2, and 6 μg total) encoding either the wild-type receptor F5 or its cysteine-modified version F5-Cys, were cocultured with the melanoma tumor 526. Twenty-four hours after the beginning of the coculture, the concentration of IFN-γ secreted in the medium was measured using an ELISA procedure. B, TCR competition assay. TCR-deficient Jurkat/RT3-T3.5 cells were electroporated with 1 μg of each chain of F4 (white) or F4-Cys (black) along with 1 μg of each chain of the competitor TCR: 1G4, TE-8 (an NY-ESO-1 class I–restricted TCR), and p53-H [for p53-H, we also used an additional amount (i.e., 0.5 μg) indicated as p53-H (1:0.5)]. Twenty-four hours after electroporation, the cells were stained with MART-1 tetramer, and the percentage of MART-1 TCR relative expression was calculated by dividing percentage of tetramer-positive cells of a given sample by that of the control sample (without competitor TCR). All the differences were found to be statistically significant based on Student's t test (P < 0.001).
Volume 67, Issue 8
