T-cells expressing the yfi T-cell receptor (TCR yo) comprise a small subpopulation of lymphocytes in the blood and lymphoid tissues. It is believed that y8 T-cells contribute to immune defense not only against infectious organisms, but also malignanj cells. To better understand y8 T-cell biology and the cellular/molecular basis by which y8 T-cells are activated, and to develop a y8 T-cell-mediated immunolherapeutic approach, we attempted to expand yo T-cells in vitro using dendritic cells (DC) and autologous CD4' helper T-cells. CD4 Tcells and y8 T-cells were purified from normal donor peripheral blood using cell sorting. Dendritic cells were generated with cytokines from autologous marrow CD34' cells or allogeneic CD34' leukemic cells from a patient with AML. Dendritic cells were purified using the CD 1 lc-mediated Miltenyi immunomagnetic separation system. Sorted y8 T-cells were co-cultured with DC ± CD4' T-cells in medium containing 20 U/ml rhIL-2. Expansion (-150 fold) of yS T-cells occurred after weekly stimulation with autologous DC alone or CD4 T-cells alone. A significantly larger expansion (-1500 fold) of y5 T-cells was achieved in cultures with weekly stimulation of both DC and CD4' cells. The proportion of yS T-cells was 86 ±1!)% (n=15) in cultures harvested at day 21 Phenotypic analysis showed that expanded yö T-cells were predominantly (>80%) oligoclonal for TCR 01 or fi2 chain expression, but with little y9 chain coexpression, yfi T-cells could also be expanded by stimulation of allogeneic AMLderived DC and autologous CD4' T-cells. Generated y& T-cells are cytotoxic against leukemic cell lines: K562, Molt-4, Raji, but not normal PHA-induced lymphoid blasts Cytotoxicity of yfi T-cells against multiple myeloid leukemic cell lines (e.g. K562, KBM5 and KGla), and primary leukemia could be significantly up-regulated by incubation with anti-TCRyfi antibody. Although the mechanism of y? T-cell activation and cytotoxic activity remains unclear, yfi T-cells are functionally different from NK cells and are MHC class I-independent. In summary, human yfi T-cells can be successfully expanded and functionally activated thereby providing a potentially new cell component for immunotherapy.