GENERATION OF DENDRITIC CELLS IN-VITRO FROM PERIPHERAL-BLOOD MONONUCLEAR-CELLS WITH GRANULOCYTE-MACROPHAGE-COLONY-STIMULATING FACTOR, INTERLEUKIN-4, AND TUMOR-NECROSIS-FACTOR-ALPHA FOR USE IN CANCER-IMMUNOTHERAPY

Objective The purpose of the study was to characterize the requirement s in terms of precursors, developmental pathways, and media for the ge neration of large numbers of mature dendritic cells (DC) under conditi ons acceptable for use in adjuvant, active immunotherapy strategies fo r surgically treated malignancies. Summary Background Data Although li mited previously by the small numbers accessible, DC-based immunothera pies for malignancy have become more realistic with the development of methods for efficiently generating larger numbers of DC from peripher al blood mononuclear cells (PBMC) in vitro, but these methods rely on clinically unacceptable culture conditions (such as inclusion of fetal bovine serum), necessitating the development of methods for generatin g functionally equivalent DC in serum-free conditions. Methods Plastic -adherent PBMC (from healthy donors and patients with cancer) were inc ubated for 7 days with granulocyte-macrophage-colony-stimulating facto r (GM-CSF) and interleukin-4 (IL-4) with and without tumor necrosis fa ctor-alpha (TNF-alpha) in fetal bovine serum-containing and serum-free media and were analyzed by Wright's stain for morphology, flow cytome try for phenotype, and mixed lymphocyte reaction for allostimulatory f unction. Results Growth in either serum-containing or serum-free media supplemented with GM-CSF and IL-4 yielded a similarly heterogeneous p opulation of cells, 6% to 10% of which had the morphology (large cells with thin projections), immunophenotype (including CD83+), and functi on of mature DC. Tumor necrosis factor-ct significantly augmented the number of these mature DC, whereas preculture depletion of CD14+ PBMC virtually eliminated them. Conclusions Generation of mature DC in the authors' serum-free clinically applicable conditions is similar to ser um-containing conditions and requires CD14+ precursors, differentiatio n through a CD14-CD83- immature stage under the influence of GM-CSF an d IL-4, and maturation into a CD83+ DC under the influence of TNF-alph a.