Figure 3

Immunological monitoring for peptide specific T-cell responses in melanoma patients using ELISPOT and MHC-Dextramer assay ELISPOT and the MHC-Dextramer assay are shown in the left (a, e, i, c, g and k) and right (b, f, j, d, h and l) side of each panel. The arrow indicates the timing of each vaccine injection. In ELISPOT assay, to evaluate the characteristics of peptide-specific CTLs, PBMCs of the patients were stimulated in vitro with gp100-in4, HIV or CMV peptide (data not shown) and examined for their ability to produce IFN-γ. The MHC-Dextramer assay was performed on blood samples identical to the ones used for ELISPOT assay to identify CD8⁺ T-cells recognizing the epitope peptide used. For flow cytometric analysis, PBMCs, which were stimulated in vitro, were stained with the MHC-Dextramer for 20 min in the dark at room temperature, followed by staining with FITC-conjugated anti-CD8 mAb and APC-conjugated anti-CD3 mAb and 7AAD (Beckton Dickinson Biosciences) at 4°C for 30 min. Flow cytometric analysis was performed using FACSCalibur and CellQuest software (BD Biosciences).