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. 2012 Nov 27;21(3):542–553. doi: 10.1038/mt.2012.233

Figure 7.

Figure 7

Characterization of the major histocompatibility complex class I presentation of E7 peptide to E7-specific CD8+ T cells by ID8-meso tumor cells treated with Meso-scFv-Fc-RE7. (a) Schematic diagram depicting antihuman mesothelin scFv fragment (Meso-scFv) conjugated to Fc protein-containing E7 peptide, RAHYNIVTE, at the carboxyl end proceeded by furin recognition sequence, RVKR. (b) Flow cytometry analysis to characterize the activation of E7-specific CD8+ T cells by ID8-meso tumor cells treated with the various Meso-scFv-Fc chimeric proteins. ID8-meso tumor cells (1 × 105 cells/well) were added to 48-well plates and incubated with 0.5 µg/ml of proteins. After 18 hours, treated tumor cells were incubated with 2 × 105 E7-specific cytotoxic CD8+ T lymphocytes (CTLs). Activated interferon γ (IFN-γ)-secreting E7-specific CD8+ T cells were determined by staining for surface CD8+ and intracellular IFN-γ before flow cytometry analysis. (c) Representative bar graph depicting the % of IFN-γ-secreting E7-specific CD8+ T cells out of total E7-specific T cells (mean ± SD). (d) Representative luminescence imaging to demonstrate in vitro cytotoxicity by E7-specific CD8+ T cells of ID8-meso tumor cells treated with Meso-scFv-Fc- RE7. Luciferase-expressing ID8-meso tumor cells were plated in a 24-well plate (1 × 105 cells/well) and incubated with 0.5 µg/ml of Meso-scFv-Fc chimeric proteins. After 18 hours, treated tumor cells were incubated with 2 × 105 ovalbumin (OVA)-specific CD8+ T cells. Luciferase-expressing ID8 tumor cells not incubated with chimeric protein and treated with 2 × 105 OVA-specific CTL alone were the control. The degree of CTL-mediated killing of tumor cells was seen as a reduction in luminescence activity and measured by IVIS luminescence imaging system series 2000. Bioluminescence signals were acquired for 1 minute. (e) Bar graph depicting viability of tumor cells treated with E7-specific cytotoxic T cells and/or protein (mean ± SD). Data shown are representative of two experiments performed. IFN, interferon.