Generation of DC-based vaccine for therapy of B-CLL patients.: Comparison of two methods for enriching monocytic precursors

Background The generation of Ag-loaded DC under good manufacturing practice ( GMP) conditions is logistically challenging and further compounded when the starting precursors need to be purified from B-CLL patients who have overwhelming numbers of circulating B-CLL cells and decreased numbers of monocytes. Methods We have previously demonstrated that DC with endocytosed B-CLL apoptotic bodies are powerful stimulators of anti-leukemic T cells. In this study we compared counterflow elutriation and immunomagnetic separation for enriching monocyte precursors, and evaluated the feasibility of generating DC from B-CLL patients and the effects of cryopreservation. Results Monocyte yield from a single leukapheresis product of a B-CLL patient varied from 1 x 10(8) to 10 x 10(8) total cells, from which 40 - 200 x 10(6) mature DC could be produced. Adequate numbers of monocytes could not be enriched from one patient with 0.2% monocytes in the leukapheresis product, and the target of 50 x 10(6) DC was barely achieved in another patient with 0.9% monocytes in the pheresed cells. These results suggested that successful production of DC is dependent on a minimum frequency of 1% CD14(+) monocytes in the leukapheresis product. Cryopreservation of tumor cell-loaded DC yielded a recovery rate of 86 +/- 4.4% upon thawing, with a total viability of 90 +/- 2.8%. Most importantly, cryopreserved Ag-loaded DC retained their morphology, phenotype and function. Discussion The results demonstrate that adequate numbers of functional DC required for clinical therapy can be generated from patients who have > 1% of CD14(+) monocytes in the leukapheresis product. Moreover, Ag-loaded DC can be cryopreserved and recovered without significant change in phenotype or function.