Quantifying and Imaging NY-ESO-1/LAGE-1-Derived Epitopes on Tumor Cells Using High Affinity T Cell Receptors

Staining of NY-ESO-1_157–165 minigene expressing cells with high affinity TCR. A, Imaging of individual SLLMWITQC/HLA-A2 Ags on SLLMWITQC-minigene-transfected J82 bladder carcinoma cell line. Cells were stained with high affinity NYE(113) TCR-bio monomers and streptavidin-PE. Each dot on the fluorescent images represents a single streptavidin-PE/TCR/Ag-HLA complex. Specificity of TCR-bio binding was demonstrated by costaining with a 10- or 100-fold excess of cold (nonbiotinylated) NYE(113) TCR. Three-dimensional fluorescent images of cells were acquired, although only the lower, slide-proximal surface of each cell is shown. Cells stained with streptavidin-PE only are shown as control. Cells shown are representative of the brightest 10% found among each staining condition. Bar, 10 μm. Levels of cell surface SLLMWITQC/HLA-A2 (B), or HLA-A2 complexes (C) were determined for each SLLMWITQC-minigene-transfected cell line by FACS, using a high affinity NYE(113) TCR or anti-HLA-A2 mAb to stain with, respectively. Where indicated, cell lines were cultured in the presence of IFN-γ. D, NYE(113) TCR staining of snap-frozen SLLWITQC-minigene-transfected J82 and SK-Mel-37 cells by immunohistochemistry. MFI, Mean fluorescence intensity.

Inhibition of NY-ESO-1_157–165-specific CTL responses by high affinity TCR. A, IFN-γ response of NY-ESO-1_157–165-specific CTL (clone 1G4) to T2 cells pulsed with a titration of SLLMWITQV peptide. B, Inhibition of the CTL (clone 1G4) IFN-γ response to SLLMWITQV-pulsed T2 cells by a titration of high affinity NYE(113) TCR. Efficiency of TCR-mediated CTL inhibition was investigated at the various peptide concentrations indicated. C, Inhibition of the CTL (clone 1G4) IFN-γ response to SLLMWITQV-pulsed T2 cells by a titration of wild-type NYE TCR. Efficiency of TCR mediated CTL inhibition was investigated at the various peptide concentrations indicated. D, Inhibition of CTL-mediated lysis of T2 cells (pulsed with 10 nM SLLMWITQV) by a titration of high affinity NYE(113) TCR. Data shown are for NY-ESO-1_157–165-specific CTL line 1045-MTLC and CTL clone NW2549-CD8–2/2. Target cell lysis in the absence of TCR, in the presence of wild-type NYE TCR and in the presence of irrelevant TAX(134) TCR are shown as controls.

Number of NY-ESO-1_157–165 Ags presented by human cancer cells. A, NY-ESO-1 and LAGE-1 expression in melanoma cells. FACS plots of Mel-624, SK-Mel-37, and Mel-526 melanoma cells stained with NY-ESO-1-specific mAb E978 (bold line) or isotype control (shaded area). NY-ESO-1⁻ T2 cells were stained as negative control. NY-ESO-1 and LAGE-1 expression in SK-Mel-37, Mel-624, and Mel-526 cells was also determined by RT-PCR. B–E, Number of SLLMWITQC/HLA-A2 complexes presented by NY-ESO-1⁺ melanoma cells. B, Imaging of total NY-ESO-1_157–165 Ag presented on Mel-624 melanoma cells. Cells were stained with high affinity NYE(113) TCR-bio monomers and streptavidin-PE. The entire three-dimensional surface of the cell was imaged by fluorescent microscopy. The fluorescent image is a maximum projection of the three-dimensional image, thus depicting the total PE count per cell. Brightfield image shows position of cell cluster. Ten cells are shown in image. Bar, 10 μm. C–E, Ag levels on individual cells were determined from three-dimensional fluorescent images of SK-Mel-37 (C), Mel-624 (D), and Mel-526 (E) melanoma cells stained with TCR-bio/streptavidin-PE. Each experimental condition included: (i) staining with NYE(113) TCR-bio to determine cell surface Ag levels, (ii) costaining with 50-fold excess of cold (nonbiotinylated) NYE(113) TCR to demonstrate TCR specificity, and staining with (iii) the irrelevant high affinity TAX(134)-bio(26) or (iv) streptavidin-PE alone to determine background staining levels. Vertical bars indicate mean values of the individual counts. Anti-IFN-γ ELISPOT of CTL (clone 1G4) responses were investigated for all three melanoma call lines. Each experimental condition included: (v) target cells alone (T); (vi) effector cells alone (E); (vii) targets added to effectors (T+E); (viii) targets plus effectors in the presence of NYE(113) TCR (T+E+NYE(113)); and (ix) targets and effectors in the presence of high affinity TAX(134) TCR (T+E+TAX(134)). F, FACS analysis of HLA-A2 levels on Mel-526, Mel-624, and SK-Mel-37 cell lines. B–F, Where indicated, cell lines were cultured in the presence of IFN-γ before analysis.