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      Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes

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          Abstract

          We developed a new 2-day protocol for the generation of dendritic cells (DCs) from human monocytes in vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of total culture period, expression of activation markers was more pronounced in cells generated by the 2-step differentiation and activation method. Our new protocol for 2-day DC differentiation reduces labor, cost and time and also reliably renders high numbers of mature and viable DCs.

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          Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells.

          M. Cella, A. Engering, V Pinet … (1997)
          Dendritic cells have the remarkable property of presenting any incoming antigen. To do so they must not only capture antigens with high efficiency and broad specificity, but must also maximize their capacity to load class II molecules of the major histocompatibility complex (MHC) with antigenic peptides in order to present a large array of epitopes from different proteins, each at a sufficient copy number. Here we show that formation of peptide-MHC class II complexes is boosted by inflammatory stimuli that induce maturation of dendritic cells. In immature dendritic cells, class II molecules are rapidly internalized and recycled, turning over with a half-life of about 10 hours. Inflammatory stimuli induce a rapid and transient boost of class II synthesis, while the half-life of class II molecules increases to over 100 hours. These coordinated changes result in the rapid accumulation of a large number of long-lived peptide-loaded MHC class II molecules capable of stimulating T cells even after several days. The capacity of dendritic cells to load many antigenic peptides over a short period of initial exposure to inflammatory stimuli could favour presentation of infectious antigens.
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            Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking.

            S Beaulieu, Adrienne G Randolph, R Steinman … (1998)
            Essential to the dendritic cell system of antigen-presenting cells are the veiled dendritic cells that traverse afferent lymph to enter lymph nodes, where they initiate immune responses. The origin of veiled cells, which were discovered 20 years ago, is unclear. Monocytes cultured with endothelium differentiated into dendritic cells within 2 days, particularly after phagocytosing particles in subendothelial collagen. These nascent dendritic cells migrated across the endothelium in the ablumenal-to-lumenal direction, as would occur during entry into lymphatics. Monocytes that remained in the subendothelial matrix became macrophages. Therefore, monocytes have two potential fates associated with distinct patterns of migration.
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              Type I Interferon as a Powerful Adjuvant for Monocyte-Derived Dendritic Cell Development and Activity in Vitro and in Hu-Pbl-Scid Mice

              Stefano M. Santini, Caterina Lapenta, Mariantonia Logozzi … (2000)
              Type I interferons (IFNs) are cytokines exhibiting antiviral and antitumor effects, including multiple activities on immune cells. However, the importance of these cytokines in the early events leading to the generation of an immune response is still unclear. Here, we have investigated the effects of type I IFNs on freshly isolated granulocyte/macrophage colony-stimulating factor (GM-CSF)–treated human monocytes in terms of dendritic cell (DC) differentiation and activity in vitro and in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) mice. Type I IFNs induced a surprisingly rapid maturation of monocytes into short-lived tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)–expressing DCs endowed with potent functional activities, superior with respect to the interleukin (IL)-4/GM-CSF treatment, as shown by FACS® analyses, mixed leukocyte reaction assays with allogeneic PBLs, and lymphocyte proliferation responses to HIV-1–pulsed autologous DCs. Type I IFN induced IL-15 production and strongly promoted a T helper cell type 1 response. Notably, injection of IFN-treated HIV-1–pulsed DCs in SCID mice reconstituted with autologous PBLs resulted in the generation of a potent primary immune response, as evaluated by the detection of human antibodies to various HIV-1 antigens. These results provide a rationale for using type I IFNs as vaccine adjuvants and support the concept that a natural alliance between these cytokines and monocytes/DCs represents an important early mechanism for connecting innate and adaptive immunity.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Biol Proced Online
                Title: Biological Procedures Online
                Publisher: Biological Procedures Online
                ISSN (Electronic): 1480-9222
                Publication date Collection: 2003
                Publication date (Electronic): 24 October 2003
                Volume: 5
                Pages: 197-203
                Affiliations
                [1 ]Division of Clinical Pharmacology, Medizinische Klinik Innenstadt, University of Munich. Ziemssenstr. 1, 80336 Munich. Germany. B.O. and M.D. contributed equally to this manuscript.
                [2 ]Division of Clinical Pharmacology and Section of Gastroenterology, Medizinische Klinik Innenstadt, University of Munich. Ziemssenstr. 1, 80336 Munich. Germany.
                [3 ]Abteilung fuer Klinische Pharmakologie, Medizinische Klinik Innenstadt, University of Munich. Ziemssenstr.1, 80336 Munich. Germany.
                Author notes
                Andreas Eigler, Abteilung fuer Klinische Pharmakologie, Medizinische Klinik Innenstadt, University of Munich. Ziemssenstr.1, 80336 Munich. Germany. Phone: 49-89-51602291. Fax: 49-89-51604576. Andreas.Eigler@ 123456med.uni-muenchen.de
                Article
                Publisher ID: m62
                DOI: 10.1251/bpo62
                PMC ID: 248478
                PubMed ID: 14615816
                SO-VID: 3dc12146-c441-4d4b-af3b-4baf04d0531d
                Copyright © Copyright © October 10, 2003, B Obermaier et al. Published in Biological Procedures Online under license from the authors. Copying, printing, redistribution and storage permitted.
                History
                Date received : 18 August 2003
                Date revision received : 21 September 2003
                Date accepted : 22 September 2003
                Categories
                Subject: Research Article

                ScienceOpen disciplines: Life sciences
                Keywords: cell differentiation,vaccination,antigen presenting cells,dendritic cells,monocytes
                Data availability:
                ScienceOpen disciplines: Life sciences
                Keywords: cell differentiation, vaccination, antigen presenting cells, dendritic cells, monocytes

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