MASSIVE EX-VIVO GENERATION OF FUNCTIONAL DENDRITIC CELLS FROM MOBILIZED CD34(+) BLOOD PROGENITORS FOR ANTICANCER THERAPY

We report that blood cell autografts, collected by single leukapheresi s in cancer patients (n=11) at the time of mobilization of hematopoiet ic progenitors into peripheral blood following anticancer therapy with high-dose cyclophosphamide (HD-CTX) plus interleukin-3 (IL-3) and gra nulocyte colony-stimulating factor (G-CSF/filgrastim), comprise 1.98 /- 0.39x10(5)/kg (mean +/- SE) CD34(+) progenitors of dendritic cells (DCs). This number corresponds to 140-fold more progenitors than in a control autograft collected in the steady state. DCs derived from mobi lized CD34(+) cells, morphologically and immunophenotypically undistin guishable from skin Langerhans cells and DCs from bone marrow and cord blood CD34(+) cells, are shown to be powerful stimulators of allogene ic T cell proliferation in primary MLR and of autologous HLA-DR-restri cted CD4(+) T cell proliferation in response to presentation of xenoge nic antigens. We show that the GM-CSF-plus-TNF-alpha-dependent ex vivo generation of DCs from mobilized CD34(+) cells is 2.5-fold enhanced b y flk-2/flt-3 ligand or c-kit ligand (stem cell factor) and five-fold enhanced by a combination of these growth factors. In addition, the op timal serum for the generation of DCs is autologous HD-CTX recovery-ph ase serum rather than fetal calf serum (FCS) or steady-state human ser um, which are clinically inadequate and ineffective, respectively. In practice, the stimulation of CD34(+) cells in a blood cell autograft ( 15.75 +/- 2.46x10(6)/kg) provided by the above four growth factors sho uld permit ex vivo generation of approximately 40x10(9) DCs in an adul t patient. These new findings provide advantageous tools for the large -scale generation of DCs that are potentially usable for clinical prot ocols of immunotherapy or vaccination in patients undergoing cancer tr eatment.