Using a commercially available IFN-γ ELISpot assay, we confirmed an antigen-specific, dose-dependent, IFN-γ release by PBMC isolated from rats when primed with DHD-K12 cells. The dual-colour assay was developed by combining an IFN-γ ELISpot assay, a LysiSpot

assay, and β-gal transfection selleck inhibitor of the target cells. This assay allowed us to detect simultaneously the lysis of tumour target cells and the identification of CTLs producing IFN-γ. The use of a dual-colour software programme, allowed to count separately the spots of three different colours, thus overcoming the reported difficulty in discerning the difference in the colours of the spots previously described The LysiSpot was performed with a number of target cells high enough to virtually allow all CTLs present in the culture to find the target, however respecting the limit of an acceptable background level of positive spots. The assessment of effector/target cells ratio was determined in preliminary experiments (data not shown) to ensure that all the key parameters to assess PD-1/PD-L1 inhibitor cancer T cell cytotoxicity were optimized. 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. Those cells could represent an incomplete stage of differentiation toward

fully developed effector cells [42]. DHD-K12 cells naturally express a Unoprostone tumour-associated antigen that induces specific cytotoxic responses in immune competent syngeneic animals [16, 17]. The synthetic nonapeptide antigen, CSH-275, was previously used in a vaccination protocol and gave proof of the induction of an antitumour activity as elicited by the vaccination [17]. These data demonstrate that CSH-275 is full recognized by ex vivo lymphocytes from DHD-K12 primed rats and since CSH-275 is a major epitope identified on the TLP (Tumour Liberated

Proteins) isolated from human lung, colon and breast cancer [18–20] it is evident the importance of this antigen as a potential target for new diagnostic and/or therapeutic approaches to human cancer. Conclusions In this study we show a reproducible and easy technique capable of measuring even low frequencies of antigen-specific cytolytic cells against tumour, and provided further evidence of the multiple aspects of the different regulatory pathways governing the induction of cytolytic mechanisms. The proposed lysispot assay, and this rat colon carcinoma model, could be used to evaluate the specific cell mediated immunity and or cytochine production in preclinical study, pharmacological treatment and development of immune intervention. Acknowledgements This work was partially supported by MIUR Italy, PRIN 2008 n°20089E83YR_005 to Maria Pia Fuggetta. References 1. Kochenderfer JN, Gress RE: A comparison and critical analysis of preclinical anticancer vaccination strategies.