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. 2012 Feb 1;31(1):9.
doi: 10.1186/1756-9966-31-9.

Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model

Valentina Bordignon  1 , Paola Cordiali-Fei, Monica Rinaldi, Emanuela Signori, Andrea Cottarelli, Manuela Zonfrillo, Fabrizio Ensoli, Guido Rasi, Maria Pia Fuggetta
Affiliations

Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model

Valentina Bordignon et al. J Exp Clin Cancer Res. .

Abstract

Background: Antigen-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.

Methods: Here 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.

Results: Using 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.

Conclusions: The 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.

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Figures

Figure 1
Figure 1
DHD-K12 cells expressing β-gal. DHD-K12 cells were transiently transfected with a plasmid vector expressing LacZ gene. Twenty-four hours after transfection, cells were checked for expression of β-gal through the development of blue colour. Cells expressing β-gal (mark with an arrow-head) ranged between 50% and 60% without significant cell death. The images (20x) was captured using Spot RT software version 3.0 (Diagnostic Instruments, inc) using a conventional inverted microscope.
Figure 2
Figure 2
IFN-γ release. IFN-γ-ELISpot results from three different experiments, expressed as number of spots per well (mean ± SD), showed the immune-response of DHD-K12-inoculated rats (dark grey) against CSH-275 peptide. No effect was produced on PBMC from control rats (light grey). Increasing concentration of peptide yielded an increasing numbers of IFN-γ producing PBMC. Under each histogram there is the corresponding image illustrative of blue spots. As negative contros we showed the non stimulated PBMC (W/O).
Figure 3
Figure 3
Panel A - LysiSpot assay. LysiSpot assay results, expressed as net number of spots per well (spots from wells containing only target cells were subtracted), from four different experiments (mean ± SD). Increasing numbers of target cells were plated in short term cultures with effector cells (2 × 105/well PBMC). Spots were the imprint of β-gal, released by the transfected DHD-K12 target cells after lysis. Cytotoxic activity of PBMC from DHD-K12-inoculated rats or control rats are represented by dark and light grey respectively. Panel B - LDH-Cytotoxicity assay. Cytotoxic activity expressed as percent of specific lysis (mean ± SD) of DHD-K12 target cells from PBMC of intact (control) or DHD-K12-inoculated rats (Primed) evaluated by Promega CytoTox 96 kit. Concentration ratio of effector and target cells was 10:1 (light grey), 5:1 (dark grey), 2.5:1 (white), 1.25:1 (black) and corresponding respectively to 2 × 104,1 × 104, 5 × 103, 2.5 × 103 of DHD-K12 target cells.
Figure 4
Figure 4
Dual-colour LysiSpot assay. Dual-colour LysiSpot assay results from three different experiments (mean ± SD), expressed as net number of spots per well (spots from control wells containing only target cells or PBMC cultured alone were subtracted from the spots counted in the cocultures. DHD-K12 transfected cells (2 × 104/well) were cocultured with 2 × 105/well PBMC. The panel shows the image of the different spots left on the wells at the end of assay: 1) pure red spots indicate cell lysis by IFN-γ non-producing cells; 2) pure blue spots indicate IFN-γ secreting cells; 3) violet spots indicate cell lysis by IFN-γ producing cells. Dark and light grey bars represent number of spots from DHD-K12-inoculated rats or from control rats respectively.
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