FIGURE 2.

A, Cross-reactivity of CTL derived by IVS using wild-type EGFR_{853 – 861} peptide. CD8⁺ T cells from HLA-A*0201⁺ HD or SCCHN patients were stimulated using IVS with DC loaded with EGFR_{853 – 861}. Reactivity of CTL against peptide-pulsed target cells was tested using IFN-γ ELISPOT assays. This recognition was blocked by HLA class 1 and HLA-A–specific mAb, but not an HLA class 2-specific mAb. Cross-reactivity of EGFR_854L peptide and EGFR_{853 – 861} peptide-specific CTL was observed. Each bar represents the mean spot number of triplicate experiments ± SD with 10⁴ CTL per well. Background IFN-γ secretion was measured in response to T2 cells pulsed with HIV peptide or T2 cells alone. Background IFN-γ release was quantified using T2 plus HIV-1–derived peptide. Each data point represents the mean spot number of triplicate determinations ± SD with 10⁴ CTL per well. B, Comparison of EGFR_{853 – 861}-specific tetramer⁺ cells in PBMC from SCCHN patients or HD after 1-week IVS. IVS was performed using autologous DC from 5 HLA-A*0201⁺ SCCHN patients or 5 HLA-A*0201⁺ HD loaded with wt EGFR_{853 – 861} (10 μg/mL at 37°C for 4 h). Autologous CD8⁺ T cells were negatively isolated from PBMC with immunomagnetic beads (Miltenyi Biotech, Germany) and added at 1 × 10⁶ cells/ml to 1 × 10⁵ peptide-pulsed DC in a final volume of 2 mL of culture medium (24-well tissue culture plate). The cells were cultured for 7 days at 37°C with IL-2 (20 U/mL) and IL-7 (5 ng/mL). On day 7 lymphocytes were harvested and tested for the specificity of PE-labeled HLA-A*0201-EGFR_{853 – 861} tetramer. Tetramer staining of EGFR_{853 – 861}-specific CD8⁺ T cells was performed before and after IVS and statistically compared. Higher EGFR_{853 – 861} tetramer⁺ T cells were observed at baseline (pre-IVS) in the 5 SCCHN patient PBMC versus the 5 HD (P<0.05). IVS induced significantly higher levels of EGFR_{853 – 861} tetramer⁺ T cells in both groups (*P = 0.013 and **P = 0.0002, respectively). Interestingly, a stronger induction of EGFR_{853 – 861} tetramer⁺ T cells was observed during the 1-week IVS in HD versus SCCHN PBMC (***P = 0.014). C, EGFR_{853 – 861}-specific CTL recognition of EGFR_{853 – 861} and EGFR_854L peptides. Reactivity of EGFR_{853 – 861} peptide-specific CTL from a HD was tested against T2 cells incubated with decreasing concentrations of EGFR_{853 – 861} or EGFR_854L (ranging from 10 to 0.01 μM). Each data point represents the mean spot number of triplicate experiments ± SD with 1 × 10⁴ CTL per well. CTL indicates cytotoxic T lymphocyte; EGFR, epidermal growth factor receptor; ELISPOT, enzyme-linked immunosorbent spot; HD, healthy donors; IVS, in vitro stimulation; SCCHN, head and neck squamous cell carcinoma.