We have previously demonstrated that dendritic cells (DCs) fused with malignant cells stimulate anti-tumor immunity by presenting a braod array of tumor antigens in the context of DC mediated costimulation. DCs differentiated from leukemia cells (LDCs) are also being explored as cancer vaccines in which leukemia associated antigens are presented. We examined the phenotypic and functional characteristics of DC/Leukemia fusions and LDCs to assess their potential as tumor vaccines. Leukemia blasts were isolated from peripheral blood of patients with AML. CD34 selection was performed on a subset of samples by magnetic bead separation. LDCs were generated by culturing blasts in the presence of GM-CSF, IL-4 and TNFα for 7 days. Alternatively, leukemia cells were fused with DC by coculture in the presence of polyethylene glycol. Differentiation of leukemic blasts into LDCs resulted in increased expression of HLA-DR and CD 11c. Unlike normal peripheral blood mononuclear cells, differentiation of leukemic blasts resulted in only modest expression of the costimulatory molecules, CD80 and CD86 (mean expression 12% and 30%) and no increase in expression of the maturation marker, CD83 (mean expression 4%). In addition, expression of the leukemia associated antigen c-kit (CD117) was lower on LDCs than on blasts (mean expression 34% on blasts, 15% on LDCs). To assess the capacity of the primitive leukemia clonal population to differentiate into DCs, CD34+ cells were isolated from the blast population and assessed after cytokine differentiation. Cytokine differentiation did not result in upregulation of CD80, CD83, or CD86 expression in the CD34+ population (mean expression 5%, 2%, 17%). In contrast, differentiation of the CD34- population resulted in moderate expression of CD80, CD83 and CD86 (mean expression 15%, 14%, 48%). In contrast to LDCs which do not strongly express co-stimulatory molecules and lose expression of leukemia associated antigens, fusion cells expressed both DC and tumor associated antigens (mean fusion efficiency 27%). The functional characteristics of DC derived from leukemic blasts were examined. Allogeneic T cell proliferation in response to stimulation by LDCs was not significantly higher than after stimulation with undifferentiated blasts (ratio 10:1, mean SI 17% with LDCs vs 9% with undifferentiated blasts, p=0.19). Neither stimulation with blasts nor with LDCs induced T cell production of interferon gamma. In contrast, interferon gamma production by T cells in response to stimulation with fusion cells was higher than after stimulation with undifferentiated blasts. In summary, LDCs do not demonstrate normal upregulation of costimulatory molecules, and lose expression of tumor antigens. In contrast, DC/leukemia fusions coexpress tumor and DC associated markers. While LDCs stimulate interferon gamma production by T cells poorly, fusion cells more potently stimulate interferon gamma production by allogeneic T cells than do undifferentiated blasts. This suggests that LDC may be ineffective as a tumor vaccine in AML, and that fusion cells may be superior to LDC in generating effective anti-tumor immune responses. Strategies to enhance the ability of both LDC and of fusion cells to stimulate anti-tumor immunity are being explored.