Abstract
BackgroundAntigen-specific CD8+ cytotoxic T lymphocytes represent potent effector cells of the adaptive immune response against viruses as well as tumours. Therefore assays capable at exploring the generation and function of cytotoxic T lymphocytes represent an important objective for both clinical and experimental settings.MethodsHere we show a simple and reproducible assay for the evaluation of antigen-specific CD8+ cytotoxic T lymphocytes based on a LysiSpot technique for the simultaneous determination of antigen-specific IFN-γ production and assessment of tumor cytolysis. The assay was developed within an experimental model of colorectal carcinoma, induced by the colorectal tumor cell line DHD-K12 that induces tumors in BDIX rats and, in turn, elicits a tumor- specific immune response.ResultsUsing DHD-K12 cells transfected to express Escherichia coli β-galactosidase as target cells, and by the fine setting of spot colours detection, we have developed an in vitro assay that allows the recognition of cytotoxic T lymphocytes induced in BDIX rats as well as the assessment of anti-tumour cytotoxicity. The method highlighted that in the present experimental model the tumour antigen-specific immune response was bound to killing target cells in the proportion of 55%, while 45% of activated cells were not cytotoxic but released IFN-γ. Moreover in this model by an ELISPOT assay we demonstrated the specific recognition of a nonapeptide epitope called CSH-275 constitutionally express in DHD-K12 cells.ConclusionsThe assay proved to be highly sensitive and specific, detecting even low frequencies of cytotoxic/activated cells and providing the evaluation of cytokine-expressing T cells as well as the extent of cytotoxicity against the target cells as independent functions. This assay may represent an important tool to be adopted in experimental settings including the development of vaccines or immune therapeutic strategies
Highlights
Antigen-specific CD8+ cytotoxic T lymphocytes represent potent effector cells of the adaptive immune response against viruses as well as tumours
Several putative anti-cancer vaccines have been studied in animal models through immunization with intact tumour cells, cancer-related peptides, Ag-loaded dendritic cells (DCs), different viral delivery systems as
The target tumour cells were transduced by an herpes simplex virus (HSV) amplicon vector to express Escherichia coli b-galactosidase (b-gal) as the marker protein
Summary
Antigen-specific CD8+ cytotoxic T lymphocytes represent potent effector cells of the adaptive immune response against viruses as well as tumours. Antigen-activated T cells may not always secrete the all set of their potential cytokine production [11] and cytotoxicity does not always correlate with IFN-g secretion in bulk PBMC populations [12,13,14]. For this reason, few years ago has been proposed a LysiSpot assay, which is capable to detect cytotoxic T cells, and to provide an evaluation of the target cell lysis by measuring the release of a foreign marker protein [15]. The target tumour cells were transduced by an herpes simplex virus (HSV) amplicon vector to express Escherichia coli b-galactosidase (b-gal) as the marker protein
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