Effects of leukapheresis protocol, cell processing and cryopreservation on the generation of monocyte-derived DC for immune therapy.

BACKGROUND: Many clinical trials of DC-based immunotherapy involve administration of monocyte-derived DCs (Mo-DC) on multiple occasion. We aimed to determine tbe optimal cell processing procedures and timing (leukapheresis, RBC depletion and cryopreservation) for generation of Mo-DC for clinical purposes. METHODS: Leukapheresis was undertaken using a COBE Spectra. Two instrument settings were compared - the standard semi-automated software (Version 4.7) (n = 10) and the fully automated software (Version 6.0) (N = 40). Density gradient centrifugation using Ficoll, Percoll, a combination of these methods or neither for RBC depletion were compared. Outcomes (including cell yield and purity) were compared for cryopreserved unmanipulated monocytes and cryopreserved Mo-DC. RESULTS: Software Version 6.0 provided significantly better enrichment for monocytes (P < 0.05) but 25% fewer total monocytes. Final Mo-DC purity was not influenced by leukapheresis or RBC depletion method, but was critically dependent on monocyte adherence. Version 6.0 produced significantly lower RBC and platelet contamination (P < 0.0005) but in vitro RBC depletion could not routinely be omitted. Only 5-6% of monocytes harvested resulted in Mo-DC (95% lost in cell processing or failing to differentiate). DISCUSSION: Cell losses remained significant despite attempts to minimise processing steps during Mo-DC generation. Reduction in RBC and platelets achieved with software version 6.0 was insufficient to offset the disadvantage of the lower monocyte yield. Substantial savings in materials and other costs can be achieved if Mo-DC for multiple treatments are generated from cryopreserved monocytes rather than from fresh monocytes.

Human peripheral blood Valpha24+ Vbeta11+ NKT cells expand following administration of alpha-galactosylceramide-pulsed dendritic cells.

BACKGROUND AND OBJECTIVES: We have undertaken the first clinical trial involving the administration of alpha-GalactosylCeramine (alpha-GalCer)-pulsed dendritic cells (DCs) to human subjects, to determine safety, optimal dose, optimal administration route and immunological effects. MATERIALS AND METHODS: Subjects (n = 4) with metastatic malignancy received two infusions of alpha-GalCer-pulsed DCs intravenously, and two infusions intradermally. The percentages of Valpha24 Vbeta11 NKT cells in peripheral blood (PB) were determined by three-colour flow cytometry and the PB NKT cell numbers were calculated using the total number of PB lymphocytes/ml determined by automated full-blood counts. RESULTS: No serious treatment related adverse events were observed during the study period. Administration of alpha-GalCer-pulsed DCs in vivo can significantly (P < 0.03) increase PB Valpha24+ Vbeta11+ NKT cell numbers above pretreatment baseline levels after the transient fall in the NKT numbers within 48 h. CONCLUSIONS: Administration of alpha-GalCer-pulsed DCs is well tolerated, modulates PB Valpha24+ Vbeta11+ NKT cells and may have a role in the therapy of malignancies sensitive to activities of Valpha24+ Vbeta11+ NKT cells, or for autoimmune diseases.