DHD-K12 transfected cells (2 × 104/well) LXH254 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. Discussion The development of sensitive assays to assess specific T cell responses against cancer represents a key tool for both experimental and clinical immunology as well as in the pre-clinical and clinical settings [9, 22, 23]. In recent years, the increase in the understanding the biology of tumor cells and the identification of tumor antigens capable to elicit potent and effective T cell immune responses, opened an avenue of possibilities for the design of specific vaccination strategies based on the use of peptide antigens [24, 25]. Is therefore of utmost relevance the development of assays that can Alisertib mw provide qualitative and quantitative measurement of the anti-tumour immune responses. Several techniques for immune monitoring of specific T-cell responses are now available including assays

which provide information about the specific T cell recognition of cancer antigens, irrespective SB273005 price of their functional Urease activity, such as those based on the use of tetramers [26], assays aimed at detecting T-cell precursors by amplifying cells that proliferate in response to the antigenic stimulation [27], as well as assays that measure the secretion of a particular cytokine [28] All these test do not provide information about the anti-tumour lytic activity of the immune cells [9, 28]. On the other hand,

the assessment of cytotoxicity, is generally measured on the basis of the Chromium or Europium release assay, Such cytotoxicity assays measure the percentage of targets lysed by a bulk population of effectors, but they do not provide any information about the frequency of cyotoxic T cells. The biologic relevance of these methods is therefore limited to the specific information about cytokine secretion, extent of cell-mediated cytotoxicity and/or proliferation in response to tumour antigens. Nevertheless, antigen-activated T cells do not necessarily secrete the same set of cytokines, neither cytotoxicity always correlates with cytokine secretion in a bulk T cell population [12, 14, 29]. It is well recognised that activated CD8+ T cells mediate their functions by secretion of different cytokines, including IFN-γ, that initiate a “”lytic program”" ending with a direct perforin-mediated transfer of lytic enzymes (granzyme) capable of inducing apoptosis in target cells [10, 30–32].