Generation of dendritic cells from peripheral blood of patients at different stages of chronic myeloid leukemia

We report a method to generate dendritic cells (DC) from frozen leukapheres is products of patients with chronic myeloid leukemia (CML), using sterile culture bags and serum-free culture medium, ie conditions feasible for re-i nfusion into the patient as part of immunotherapeutic protocols. Leukaphere sis products were stored from harvests performed either at diagnosis(13 pat ients) or after chemotherapy with subsequent granulocyte colony stimulating factor (G-CSF) administration (9 patients), for Peripheral Blood Stem Cell (PBSC) collections. In the presence of optimal concentrations of GM-CSF (5 0 ng/ml) and IL-4 (40 ng/ml) CML progenitors differentiated on day 7 and 14 of culture to DC, expressing CD1a, HLA-DR and CD86 surface antigens. Matur e DCs exhibited on average 12-fold higher allo-stimulatory capacity for CD4 (+) and CD8(+) cells compared to non-cultured PBMC in mixed lymphocyte reac tion (MLR). Only DCs obtained from CML patients at diagnosis exhibited bcr/ abl fusion gene when tested by fluorescent in situ hybridization (FISH). CD 34-selection on leukapheresis products from diagnosis (7 patients) resulted in later maturation of DCs (after 14-15 d), compared to the non-selected P BMC. CD34-selection significantly increased the DC growth, and improved the allostimulatory capacity in MLR (on average on day 14, 3.5- and 2.3-fold, respectively). Large differences were observed between individual patients and different leukapheresis products from the same patient. Our report demo nstrates the possibility to generate ex vivo autologous functionally active DC in CML in a way that allows their clinical application as immunotherape utic agents.