An In Vitro Assay for Studying the Cytotoxicity of Pre-Activated CD8⁺ T Cells against Cancer Cells

Take two CD8⁺ T cell populations: effector  CD8⁺ T cells pre-activated through antibody-mediated engagement of T cell and co-stimulatory receptors and suppressed CD8⁺ T cells inhibited by myeloid-derived suppressor cells.

Add these T cells to basement-membrane-coated wells with cancer cells expressing a nuclear-restricted fluorescent protein.

Pipette a fluorogenic  caspase-3 substrate — a DNA-binding fluorogenic dye conjugated to a caspase-3 recognition site-containing peptide.

Incubate. Effector T cell receptors bind to the class I MHC-peptide complex on the cancer cells, triggering the release of cytotoxic granules containing perforins and granzymes.

Perforins create cancer cell membrane pores, allowing granzyme entry and caspase activation, initiating apoptosis.

Activated caspases additionally cleave the fluorogenic caspase substrate, releasing the dye that binds to DNA and fluoresces.

Under a fluorescence microscope, cancer cells co-cultured with effector T cells exhibit dual nuclear fluorescence, indicating cancer cell apoptosis.

In contrast, cancer cells co-cultured with suppressed T cells display only nuclear protein fluorescence, suggesting non-apoptotic cells.