A limitation of UCB as a source for adoptive cellular immunity (ACI) is the limited number of immunoeffector T cell subsets and/or UCB immaturity. We have previously demonstrated the ex vivo expansion, maturation, and activation of fresh UCB derived T lymphocytes with anti-CD3, IL-2, IL-12 and IL-7 (AB/CY) (Robinson/Cairo et al, Exp Hem 30:245, 2001). IL-18 has been shown to enhance T cell cytolytic activity and provide antitumor immunity (Osaki et al, J Immunol 1998, 160: 1742), in addition to working synergistically with IL-2 in NK cell proliferation (Son YI et al, J Immunother, 2003, 26:234). We have shown that IL-18 acts synergistically with IL-12 to induce NK and LAK cellular cytotoxicity in UCB (Cairo et al. J Pediatr Hematol/Oncol 2003; 25(4):8, p. S2.). For this study, we compared the expansion, maturation, activation, and cell survival of UCB in IL-18 with the AB/CY combination without IL-7. UCB MNCs were isolated by Ficoll density centrifugation and incubated overnight @ 37°C, 5% CO2 in serum free (SF) AIM-V media. The nonadherent MNC fraction was cultured in SF AIM-V media alone and with IL-2 (5 ng/ml), IL-12 (10 ng/ml), anti-CD3 (50 ng/ml) and IL-18 (10 ng/ml) for 48 hours @ 5% CO2, 37°C. Expression of T cell receptors was analyzed by flow cytometry using CD3, CD45RO, CD8, CD4, CD25, CD16, and CD56 monoclonal antibodies. Apoptosis markers were determined by measuring Annexin V and PI by flow cytometry. Expression of lymphocyte subsets of CD8+/25+, CD4+/25+ and CD16+/56+ was significantly increased in the AB/CY with IL-18 compared to media alone (CD8+/25+: 58.4±16.4 vs 28.8±13.9%, p < 0.05; CD4+/25+: 61.4 ± 4.4 vs 2.9 ± 0.9, p < 0.001; CD16+/56+: 57.9±13.0 vs 24.0±7.1%, p < 0.05, respectively). There was no significant increase in the CD3+/45RO+ activation when compared to media alone. To determine if the increases were due in part to increased cell survival, AB/CY with IL-18 expanded cultures demonstrated no significant difference in apoptotic markers compared to media alone. In comparison to historical data, there was no significant difference in AB/CY with IL-7 vs. AB/CY cocktail with IL-18. These data suggests that CD8+/25+, CD4+/25+ and CD16+/56+ subsets can be ex vivo expanded with AB/CY with IL-18 for possible use in ACI for DLI after UCBT. In vitro functional and in vivo xenotransplant studies are underway to further examine the cytolytic activity of these activated UCB cell subsets.