EX-VIVO GENERATION OF FUNCTIONAL DENDRITIC CELLS FROM MOBILIZED CD34(-CELLS() HEMATOPOIETIC STEM)

The ability to generate dendritic cells (DCs) in sizeable numbers has enormous implications for the development of clinically-effective anti gen presentation procedures for cancer immunotherapy. We evaluated the generation of immunostimulatory DCs from peripheral blood CD34(+) cel ls collected from healthy donors. CD34(+) cells purified from leukaphe resis product were seeded at 1 x 10(4) cells/mL in complete medium sup plemented with GM-CSF, TNFa; IL-4, c-kit ligand, and flt3 ligand (FL). By day 14 of culture in the presence of GM-CFS + TNFa, the total cell number increased by 23.4 +/- 5.4-fold compared to the starting number of CD34(+) cells. When the c-kit and FL were added to GM-CSF and TNFa , the cell number increased by 109.8 +/- 11.2-fold without affecting t he immnophenotype of recovered cells. Flow cytometric analysis indicat ed that cells with the markers of mature dendritic cells, ie, CD1a(+)C D14(-)HLA-DR+, and CD80(+)CD86(+)HLA-DR+, constituted 49.0% +/- 7.5%, and 38.9% +/- 6.5%, respectively. This pattern of expression of surfac e antigen was unchanged whether the c-kit ligand and/or FL was added. The irradiated CD1a(+)HLA-DR+ cells recovered from in vitro cultures e licit a vigorous proliferation of allogeneic peripheral blood T-cells, irrespecitve of cytokine combinations. These findings provide advanta geous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in pati ents undergoing cancer treatment.