||Adoptive T cell transfer involves the administration of tumor-specific cytotoxic T lymphocytes (CTLs) into the patient to kill cancer cells. However, some limitations have restricted the generation of activated CTLs for clinical application. To overcome these limitations and generate CTLs with higher activation level for use in adoptive T cell transfer, it is necessary to understand how the activation of CTLs is regulated by the cytokines. Interleukin-18 (IL-18) is an interferon-γ (IFN-γ) inducing factor that has an important functional role in regulating CTLs. To generate tumor-specific CTLs, MAGE-A3 is used as a tumor marker, because it is an immunogenic melanoma antigen which belongs to the family of cancer testis antigens, and is widely expressed in many tumor cells. Dendritic cells (DCs), which are the most efficient antigen presenting cells, can present these antigens to naïve T cells. DCs possess the unique ability to activate naïve T cells, causing their differentiation into CTLs, which can kill tumor cells directly. Here, we attempt to use a DC-mediated system supplemented with a recombinant adenovirus (serotype 5) encoding IL-18 (rAd/IL-18) to produce DCs which can secrete HA-tagged IL-18 protein, so as to enhance the generation of activated MAGE-A3-specific CTLs in vitro. A human IL-18 gene was fused with an immunoglobulin lambda light chain leading sequence which leads to the secretion of IL-18 protein from mammalian cells. The recombinant adenovirus encoding IL-18 (rAd/IL-18) was produced by transfecting AD-293 cells with pAd/IL-18. To generate CTLs from naïve T cells, peripheral blood lymphocytes (PBLs) isolated from PBMC were primed with mature DCs, which were engineered to secret HA-tagged IL-18 protein by infection with rAd/IL-18. In Delfia EuTDA cytotoxicity assay, the cytotoxicity of MAGE-A3-specific CTLs was increased in the group in which rAd/IL-18 was loaded into the DC culture compared with the control group in which empty rAd was added. Furthermore, results of FACS analysis showed that the proportion of activated CTLs (as indicated by IFN-γ/CD8+ double-positive cells) in the total number of T cells was increased in the group in which rAd/IL-18 was loaded compared with the empty rAd control group. In conclusion, my data suggests that IL-18 significantly increases the cytotoxic efficacy of CTLs against MAGE-A3-expressing cancer cells, by inducing the activation of MAGE-A3-specific CTLs in a DC-mediated in vitro system, and it also enhances the proliferation of PBLs, which can be used in adoptive T cell transfer for cancer therapy.