HARVEST, PROLIFERATION, AND FUNCTIONAL TESTING OF HUMAN DENDRITIC CELLS.

Purpose: Difficulty in harvesting large numbers of cells, short survival time, and rapid phenotypic changes in culture have prevented the widespread use of human dendritic cells (DC) for many fundamental studies applicable to the initial stages of pharmaceutical discovery and development. The ability to overcome these problems would enable a broad variety of extremely useful in vitro assays. The purpose of this study was to develop an improved means of generating DC. Methods: DC precursors were harvested from umbilical cord blood samples and proliferated with a newly developed medium to produce non-stimulated, tissue-resident DC/Langerhans cells. The resultant DC were characterized phenotypically by flow cytometry (FACS), ultrastructurally by transmission electron microscopy (TEM), and functionally using the mixed lymphocyte reaction and RT-PCR. In addition, their ability to induce T-cell (TC) proliferation following exposure to allergens and their infectability with pathogenic viruses was investigated. Results: Increases of 200-300 fold in DC number were obtained. FACS analysis showed that the DC expressed CD1a, HLA-DR, CD11c, CD40, CD80, CD83, and CD86 for up to 26 days in culture; Birbeck granules were also observed over this culture period by TEM. Upon stimulation, the DC showed gene and protein responsiveness in terms of IL-12, MIP-1α, MIP-3α, IL-6, and TNF-α expression and DC were able to stimulate allogeneic TC. Finally, DC exposed to allergens initiated both primary and secondary autologous TC responses and were infectable with HIV. Note: Subsequent to submission of this abstract, additional data relating to the proliferation of autologous TC by antigen stimulated DC were obtained. These results are also included in this poster. Conclusions: Improved culture conditions have allowed the harvest and expansion of functional DC. The DC cells will be useful in: 1) allergenicity, 2) viral infection, 3) antigen presentation, 4) immuno-therapeutic, and numerous other studies related to the development of prophylactic and therapeutic pharmaceuticals.

Allergenicity, Allergens, Allergic/irritant response, Antigen presentation, Autologous, Birbeck granules, CD11c , CD1a, CD40, CD80 , CD83, CD86, Chemokines , Cytokine, DC, Dendritic cells, Flow cytometry(FACS), Gamma interferon (IFN-gamma), Gene transfection experiments, Green fluorescent protein (GFP), HIV, HIV-1 viruses, HIV-1ADA, HIV-1yu-2 virus, HLA-DR, Human dendritic cells (DC), IL-12, IL-12 (p40), IL-1b, IL-6, Immuno-therapeutic, Immunology, Immunotherapy, Infection with HIV, Interferon gamma, Keyhole limpet hemocyanin (KLH, 0.5 mg/ml), Langerhans cells, Lipopolysaccharide (LPS), Lymphocyte, MIP-1a, MIP-3a, Microbial infection, Mitomycin C, Neo-antigen, Phorbol 12-myristate 13-acetate (PMA), Prophylactic pharmaceuticals, RANTES, Recall-antigen, T-cell (TC), TNF-a, Tetanus toxoid, Therapeutic pharmaceuticals, Transmission electron microscopy (TEM), Viral infection