OBJECTIVES: We investigated whether umbilical cord blood (UCB) T cells could be ex vivo expanded and activated in short-term culture for potential utilization as adoptive cellular immunotherapy post-umbilical cord blood transplantation (UCBT). METHODS: Fresh UCB mononuclear cells (MNCs) were isolated by Ficoll density centrifugation. Cryopreserved UCB mononuclear cells were thawed and washed with 2.5% human serum albumin and 5% dextrose in isotonic saline. The nonadherent MNC fraction were then plated in a serum-free cocktail of IL-2, IL-12, and anti-CD3 with and without IL-7 for 48 hours. Proliferation, cytotoxicity, TH1 (IFN-gamma), CD25, and CD45RO assays were performed. RESULTS: Proliferation studies demonstrated a significant increase in the proliferative ability of the UCB MNCs incubated in anti-CD3, IL-2, IL-12, and IL-7 (fresh--p < 0.005, and thawed--p < 0.001). The combination of all four agonists significantly induced expression of CD45 RO (fresh--p < 0.05, and thawed--p < 0.001) in both the CD4(+) and CD8(+) T cells expressing CD25 (fresh UCB--p < 0.01 [CD4] and p < 0.005 [CD8], respectively; thawed UCB--p < 0.001 [CD4] and p < 0.001 [CD8]). Intracellular cytokine profiles also revealed a significant increase in the production of IFN-gamma (TH1 cells) (fresh UCB--p < 0.005, and thawed UCB--p < 0.001). The combination also significantly increased the killing of K562-labeled target cells (fresh--p < 0.0001, and thawed--0.731 +/- 0.03 vs 0.16 +/- 0.01) (p < 0.001). CONCLUSIONS: These data suggest that the ex vivo combination of IL-2, IL-12, anti-CD3, and IL-7 significantly enhances the proliferation, activation, maturation, and cytotoxic potential of UCB T cells of both fresh and thawed UCB MNC. Further studies, however, are required to determine whether these ex vivo--expanded MNC could also potentially exacerbate acute or chronic graft-vs-host disease and/or other toxicities if utilized post-UCBT.