GENERATION AND FUNCTIONAL-CHARACTERIZATION OF HUMAN DENDRITIC CELLS DERIVED FROM CD34(-BLOOD - COMPARISON WITH BONE-MARROW CD34(+) CELLS() CELLS MOBILIZED INTO PERIPHERAL)

Dendritic cells (DCs) are the most powerful professional antigen-prese nting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34(+) cells from 14 indivi duals undergoing granulocyte colony-stimulating factor (G-CSP) treatme nt for peripheral blood stem cells (PBSC) mobilization and transplanta tion. Clonogenic assays in methylcellulose showed an increased frequen cy and proliferation of colony-forming unit-dendritic cells (CFU-DC) i n circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM-CSF and TNF-or with or without SCF and FLT-3L. Moreover , peripheral blood (PB) CD34(+) cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed l ymphocyte reactions (MLR), than their BM counterparts upon different c ulture conditions. DCs derived from mobilized stem cells were also cap able of processing and presenting soluble antigens to autologous T cel ls for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of cultu re of PB CD34(+) cells enhanced both the percentage of total CD1a(+) c ells and CD1a(+)CD14(-) cells and the yield of DCs after 14d of incuba tion. In addition, the alloreactivity of IL-4-stimulated DCs was signi ficantly higher than those generated in the absence of IL-4.Furthermor e, autologous serum collected during G-CSF treatment was more efficien t than fetal calf serum (FCS) or two different serum-free media for la rge-scale production of DCs. Thus, our comparative studies indicate th at G-CSF mobilizes CD34(+) DC precursors into PB and circulating CD34( +) cells represent the optimal source for the massive generation of DC s. The sequential use of early-acting and intermediatelate-acting colo ny-stimulating factors (CSFs) as well as the use of autologous serum g reatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.