LARGE SCALE PREPARATION OF DENDRITIC CELLS FROM CD34+ PROGENITOR CELLS FOR IMMUNOLOGICAL STUDIES.
Specialized antigen-presenting cells (APC), particularly Langerhans cells/dendritic cells (LC/DC) residing in the skin, mucosa, and lymphoid tissues play a key role in inducing immunological responses following exposure to antigens that induce danger signals to the host. However, difficulty in harvesting, short survival time, and variability in cytokine production capacity of cultured LC have prevented researchers from widespread use of these cells for in vitro studies. Recently, we have developed a new culture medium and method to generate large number of DC (DC-100) from CD34+ progenitor cells. The CD34+ progenitor cells, derived from approximately 60 ml of umbilical cord blood, can be expanded to produce greater than 200 million LC/DC. These cells express surface markers including CD1a, HLA-DR, CD11c, CD54, CD209 (DC SIGN), and CD206 (mannose receptor), all characteristic of LC/DC. Transmission electron microscopy showed the presence of Birbeck granules, a key ultrastructural marker of LC, starting from day 12 of culture of progenitor cells. The life span of these cells has been prolonged up to 41 days and the cells can be frozen and recovered with a viability of 70-80%. The generated cells contain both plasmacytoid and myeloid DC populations. Upon stimulation with lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA), the release of IL-12, MIP-1a, MIP-3a, RANTES, and IL-6 was significantly enhanced above non-stimulated DC. Functionally, these DC: 1) initiate allogeneic T cell proliferation, 2) induce primary and secondary T-cell responses to a strong allergen (fluorescein-5-isothiocyanate, FITC) but not for an irritant sodium dodecyl sulfate (SDS), 3) migrate in response to chemoattractant factors, and 4) are infectable with macrophage and T-cell tropic HIV-1 viruses. In conclusion, we have developed a method to harvest and culture long-lived, functional LC that should be useful for the study of: 1) allergenicity, 2) microbial infection, 3) neutralizing antibodies, 4) antigen presentation, 5) immuno-therapy, and 6) tissue engineering, amongst many others.
APC, Allergenicity, Allergic reactions, Allogeneic T cell, Antigen presentation, Antigen-presenting cells (APC), Birbeck granules, CD11c, CD123, CD1a, CD206, CD209, CD34+, CD40, CD54, CD80, CD86, Chemokine, Cytokine, Dendritic Cells (DC), FACS analysis, FBS, FITC, Fetal bovine serum (FBS), Fluroescein-5-isothiocyanate (FITC), HIV-1, HLA-DR, IL-12, IL-6, Immuno-therapy, Immunological studies, In Vitro, LPS, Langerhans Cells (LC), Lipopolysaccharide (LPS), MIP-1a, MIP-3a, MLR, Mannose receptor, Microbial infection, Mixed lymphocyte reaction (MLR), Myeloid, Neutralizing antibodies, PDC, Phorbol-12-myristate-13-acetate (PMA), Phosphate buffered Saline, Plasmacytoid, Plasmacytoid dendritic cells, Progenitor cells, RANTES, SDS, Sodium dodecyl sulfate (SDS), T-Cell proliferation, Tetanus toxoid, Tissue Engineering, Transmission electron microscopy, pDC
Fluroescein-5-isothiocyanate (FITC), HIV-1 , Lipopolysaccharide (LPS), Phorbol-12- myristate-13-acetate (PMA), Phorbol-12-myristate-13-acetate (PMA), Sodium dodecyl sulfate (SDS), Tetanus toxoid
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