Figure 6.

In vitro cytotoxicity assay to demonstrate the major histocompatibility complex class I presentation of ovalbumin (OVA) peptide in ID8/meso or TC-1/Meso tumor cells treated with Meso-scFv-Fc-RO. (a) Schematic diagram depicting antihuman mesothelin scFv fragment conjugated with Fc protein (Meso-scFv-Fc) containing OVA peptide SIINKFEL at the carboxy end with (Meso-scFv-Fc-RO) or without (Meso-scFv-Fc-O) flanking by the furin recognition sequence RVKR. (b) Representative luminescence imaging to demonstrate in vitro cytotoxicity by OVA-specific CD8⁺ T cells of ID8/meso or TC-1/Meso tumor cells treated with Meso-scFv-Fc-RO. Luciferase expressing ID8-meso and TC-1-meso cells were treated with 0.5 µg of purified chimeric proteins (PBS, Meso-scFv-Fc, Meso-scFv-Fc-O, or Meso-scFv-Fc-RO). After 18 hours, treated tumor cells were incubated with 2 × 10⁵ OVA-specific CD8⁺ T cells. The degree of cytotoxic CD8⁺ T lymphocytes-mediated killing of the tumor cells was indicated by the decrease of luminescence activity using the IVIS luminescence imaging system series 2000. Bioluminescence signals were acquired for 1 minute. (c) Bar graph depicting viability of tumor cells treated with various chimeric protein and/or OVA-specific cytotoxic T cells (mean ± SE). Data shown are representative of two experiments performed. scFv, single-chain variable fragments; PBS, phosphate buffered saline.