T2 binding assay identifies mesothelin protein-derived epitopes that bind to HLA-A2, A3, and A24 molecules. T2 cells were pulsed with 0–225 μg/ml of peptide overnight at room temperature before analysis by flow cytometry. (A) T2 cells expressing HLA-A2 and pulsed with either a mesothelin A1_(309–317) peptide (closed diamond), mesothelin A2_(20–28) (closed square), and mesothelin A2_(530–538) (closed triangle). (B) T2 cells genetically modified to express HLA-A3 and pulsed with either mesothelin A1_(309–317) peptide (closed diamond), mesothelin A3_(83–91) (closed square), and mesothelin A3_(225–233) (closed triangle). (C) T2 cells genetically modified to express HLA-A24 and pulsed with either mesothelin A1_(309–317) peptide (closed diamond), mesothelin A24_(435–443) (closed square), and mesothelin A24_(475–483) (closed triangle).

ELISPOT analysis of CD8⁺ T cells from PBMCs demonstrates similar pre- and postvaccination responses to the influenza matrix protein HLA-A2 binding epitope M1 (GILGFVFTL) in all HLA-A2⁺ patients This analysis was performed on the same PBL samples described in Fig. 2. The DTH responders are represented by dotted lines, and the DTH nonresponders are represented by solid lines. For the detection of nonspecific background, the number of IFN-γ spots for CD8⁺ T cells specific for the irrelevant control peptides were counted. The HLA-A2 binding HIV-gag protein-derived epitope (SLYNTVATL), the HLA-A3 binding HIV-NEF protein-derived epitope (QVPLRPMTYK), and the HLA-A24 binding melanoma tyrosinase protein-derived epitope (AFLPWHRLF) were used as negative control peptides in these assays. Data represent the mean of each condition assayed in triplicate, and standard deviations were <5%. The number of human IFN-γ spots per 10⁵ CD8⁺ T cells is plotted. Analysis of each patient's PBLs was performed at least twice, and all ELISPOT assays were performed in a blinded fashion.

A mesothelin-specific CTL line derived from patient 13 PBL lyses HLA-A3⁺, mesothelin-expressing cells. Patient-derived CD8⁺ T cells stimulated with an HLA-A3 mesothelin peptide (peptide 6293) were tested for their capability to recognize and kill mesothelin-expressing tumor and EBV cell lines shown in Fig. 4. ⁵¹Cr-labeled target cells (3 × 10³) were mixed with varying concentrations of patient 13 CD8⁺ T cell line (starting with 9 × 10⁴) in a total of 200 μl in a v-bottom 96-well plate. Percent lysis was calculated after 4 h at 37°C. Results are expressed as the percentage of specific lysis of triplicate samples.

ELISPOT analysis of CD8⁺ T cells from PBMCs demonstrates postvaccination induction of mesothelin-specific T cells in three DTH responders. (A) ELISPOT analysis of PBLs from two patients who were HLA-A3⁺. (B) ELISPOT analysis of PBLs from two patients who were HLA-A-2 and HLA-A3⁺. (C) ELISPOT analysis of PBLs from two patients who were HLA-A24⁺. (D) ELISPOT analysis of PBLs from all 14 patients who were treated with the vaccine (reference 24). ELISPOT analysis for IFN-γ–expressing cells was performed using PBMCs that were isolated on the day before vaccination or 28 d after the first vaccination. T2-A3 cells were pulsed with the two mesothelin-derived epitopes MesoA3_(83–91) (squares), MesoA3_(225–233) (X), and HIV-NEF_(94–102) (not shown). T2-A2 cells were pulsed with the two mesothelin-derived epitopes MesoA2_(20–28) (triangles), MesoA2_(530–538) (circles), and HIV-gag_(77–85) (not shown). T2-A24 cells were pulsed with the two mesothelin-derived epitopes MesoA24_(435–443) (asterisks), MesoA24 _(475–483) (diamonds), and tyrosinase A24_(206–214) (not shown). All DTH responders are represented by dotted lines and open symbols, and DTH nonresponders are represented by solid lines and closed symbols. For the detection of nonspecific background, the number of IFN-γ spots for CD8⁺ T cells specific for the irrelevant control peptides were counted. The HLA-A2 binding HIV-gag protein-derived epitope (SLYNTVATL), the HLA-A3 binding HIV-NEF protein-derived epitope (QVPLRPMTYK), and the HLA-A24 binding tyrosinase protein-derived epitope (AFLPWHRLF) were used as negative control peptides in these assays. Background was minimal to negative control peptides, ranging from zero to three spots total (data included in each graph). Data represent the mean of each condition assayed in triplicate, and standard deviations were <5%. The number of human IFN-γ spots per 10⁵ CD8⁺ T cells is plotted. Analysis of each patient's PBLs was performed at least twice.

Expression of surface mesothelin of both the Panc 6.03 and Panc 10.05 vaccine and on cell lines used as targets for CTL assays. Panc 6.03, Panc 10.05, autologous EBV, autologous EBV transduced with mesothelin, Panc 2.5 (HLA-A3⁺), Panc 3.014 (HLA-A3⁺), and Panc 3.11 (HAL-A3⁻) were analyzed by flow cytometry for their levels of surface mesothelin using the mesothelin-specific monoclonal antibody CAK1 as the primary antibody and goat anti–mouse IgG1 FITC as the secondary antibody. The dotted line represents the isotype control, and the solid line represents mesothelin staining.