Introduction: Cytotoxic CD8+ T lymphocyte (CTL) cells are central in mediating anti-tumour immunity. Hence, CTLs are a major target for cancer immunotherapy, such as peptide-based T cell vaccines. The primary mechanism of CTL anti-tumour response is through cell-mediated cytotoxicity. Therefore, we developed an ex-vivo assay to evaluate the ability of novel therapeutics to induce CTL cytolytic activity on numerous target cell lines. We analysed the cytotoxic killing response of in-vivo generated antigen-specific CTLs, on fluorescently labelled target cells ex-vivo. Using this direct ex-vivo assay, we will be able to screen and identify potent and widely applicable vaccine formulations.
Method: Mice were immunised with a vaccine formulation (antigen + adjuvant) to generate in-vivo vaccine antigen-specific CTLs. On day 7, CTLs were harvested from lymph nodes and isolated via AutoMACS separation. Subsequently, CTLs were incubated (18 hours) ex-vivo with fluorescently labelled target (hi-FITC) and control (lo-FITC) cell lines at various effector: target cell ratios (0:1, 1.5:1, 3:1, 6:1, 12:1, 25:1, 50:1, 100:1). Cell samples were analysed via flow cytometry, and the CTL specific lysis was calculated based on the frequencies of the target and control cell lines.
Results: The level of target cell specific killing was found to correlate with an increasing effector: target cells ratio with observable killing occurring at 6:1 ratio and reaching a maximum CTL lysis of 81.67% at 100:1 ratio. Additionally, non-specific CTL response was absent.
Conclusion: We have developed an ex-vivo assay that allows analysis of CTL response on target cells. This assay will be instrumental in translation for high throughput screening of cancer cell lines. In addition, it will significantly reduce animal use in research, as opposed to conventional in-vivo testing. It will also overcome the limitation of testing a single vaccine candidate in a single animal model. Thus, this method will allow rapid identification of lead vaccine formulations against cancer targets.