A closed and single-use system for monocyte enrichment: potential for dendritic cell generation for clinical applications.
ABSTRACT This study evaluated the ability of a modified cell separator (Cobe Spectra Apheresis) system to isolate monocytes (MOs) by elutriation. The evaluation was performed in two independent international laboratories. The capacity of collected MOs to differentiate into dendritic cells (DCs) was also assessed.
MNCs from platelet apheresis residues were elutriated on a modified cell separator (Cobe Spectra Apheresis system) using a custom disposable set. Cells were separated according to their size and density. Recovery and purity of the collected cell product were evaluated by impedance counting and flow cytometry. DCs were differentiated in culture from the elutriated MOs and characterized by their surface markers and stimulatory capacity in a mixed WBC reaction assay.
Six apheresis mononuclear cell products were used by each laboratory. The separation was achieved in less than 1 hour. Collected MOs had the potential to differentiate into DCs.
The modified cell separator is an easy and fast device to obtain highly enriched MOs with a DC differentiation potential. The system is closed and employs a single-use disposable set and is more amenable to good tissue practice. This method could become a valuable tool for DC-based active immunotherapy.
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ABSTRACT: Sixteen patients with metastatic stage IV melanoma were treated with use of intravenous infusions of dendritic cells (DC) derived by incubation of plastic-adherent peripheral blood mononuclear cells (PBMC) with IL-4 and GM-CSF for 8 days in serumless AIM-V medium, followed by overnight pulsing with peptides. The tyrosinase368-376 (370D) and gp100(209-217 (210M)) peptides restricted to HLA class I A*0201 each differed from wild type by one amino acid modified to increase HLA binding. Median age was 49, with nine men and seven women. All patients, except one, had visceral disease. Patients received escalating doses of peptide-pulsed DCs at 10e7, 3 x 10e7, and 10e8 cells/dose twice at 2 weeks apart, with toxicity and clinical and immune responses as the principal endpoints. The first infusion of DCs was fresh, and frozen DCs were given for the second infusion of each cycle. Mean DC purity by flow cytometry was 49%, with a mean HLA-DR level of 57%, CD86 of 41%, CD58 of 46%, and mean CD14 cells of 0.9%. Toxicity was minimal, with two patients having transient grade III DC-related toxicity. Ten patients received one cycle of treatment and six patients received two cycles of treatment. One patient had a complete remission (CR) of lung and pleural disease after two cycles of DC therapy. Two additional patients had stable disease and two patients had mixed responses. Overall immunity was assessed by recall skin testing with peptides, gamma interferon ELISA assays of peptide specific cytolytic T cell (CTL) stimulated twice with peptide, IL-2, and IL-7 over 24 days, and peptide-specific tetramer assays performed before and after vaccination. Five of 16 patients had an immune response to gp100 or tyrosinase by gamma interferon ELISA assay; four of five were clinically stable or had tumor regression. These data suggest that melanoma antigen peptide-pulsed DC given intravenously are not toxic, and regression or stability of tumor appeared to correlate with the detection of a peptide-specific immune response in the peripheral blood.Journla of Immunotherapy 01/2001; 24(1):66-78. · 3.46 Impact Factor
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ABSTRACT: Dendritic cells (DCs) are professional antigen presenting cells (APCs) that are required for the initiation of the immune response. DCs have been shown to be generated from hematopoietic stem cells, but relatively little is known about the regulation underlying differentiation and activation of DCs. Here, we report that recombinant human (rh)IL-13 induces functional maturation of rhGM-CSF plus rhIL-4 generated monocyte-derived immature DCs. Incubation of these immature DCs with rhIL-13 or rhTNF-alpha for 2 days resulted in increased surface expression of CD1a, CD11c, CD86 and HLA-DR. The DCs treated with rhIL-13 or rhTNF-alpha, but not rhIL-4, for 2 days were more efficient than unstimulated DCs in the primary autologous/allogeneic T-cell response whereas the antigen (Ag)-specific T-cell response was suppressed. The treatment of DCs with rhIL-13 as well as rhTNF-alpha for 4 days down-modulated endocytic capacity for FITC-dextran (FITC-DX) and lucifer yellow (LY), and induced surface expression of CD83. Morphological, phenotypical, and functional analyses revealed that the monocytes cultured with rhGM-CSF plus rhIL-13 gave rise to a DC type more mature than rhGM-CSF plus rhIL-4-induced DCs. These findings revealed a new role for rhIL-13 in regulating both the maturation and activation of DCs.Experimental Hematology 03/1999; 27(2):326-36. · 2.91 Impact Factor
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ABSTRACT: CD34+ cells in human cord blood and marrow are known to give rise to dendritic cells (DC), as well as to other myeloid lineages. CD34+ cells are rare in adult blood, however, making it difficult to use CD34+ cells to ascertain if DC progenitors are present in the circulation and if blood can be a starting point to obtain large numbers of these immunostimulatory antigen-presenting cells for clinical studies. A systematic search for DC progenitors was therefore carried out in several contexts. In each case, we looked initially for the distinctive proliferating aggregates that were described previously in mice. In cord blood, it was only necessary to deplete erythroid progenitors, and add granulocyte/macrophage colony-stimulating factor (GM-CSF) together with tumor necrosis factor (TNF), to observe many aggregates and the production of typical DC progeny. In adult blood from patients receiving CSFs after chemotherapy for malignancy, GM-CSF and TNF likewise generated characteristic DCs from HLA-DR negative precursors. However, in adult blood from healthy donors, the above approaches only generated small DC aggregates which then seemed to become monocytes. When interleukin 4 was used to suppress monocyte development (Jansen, J. H., G.-J. H. M. Wientjens, W. E. Fibbe, R. Willemze, and H. C. Kluin-Nelemans. 1989. J. Exp. Med. 170:577.), the addition of GM-CSF led to the formation of large proliferating DC aggregates and within 5-7 d, many nonproliferating progeny, about 3-8 million cells per 40 ml of blood. The progeny had a characteristic morphology and surface composition (e.g., abundant HLA-DR and accessory molecules for cell-mediated immunity) and were potent stimulators of quiescent T cells. Therefore, large numbers of DCs can be mobilized by specific cytokines from progenitors in the blood stream. These relatively large numbers of DC progeny should facilitate future studies of their Fc epsilon RI and CD4 receptors, and their use in stimulating T cell-mediated resistance to viruses and tumors.Journal of Experimental Medicine 08/1994; 180(1):83-93. · 13.21 Impact Factor