title: The role of tumor necrosis factor alpha in modulating the quantity of peripheral blood-derived, cytokine-driven human dendritic cells and its role in enhancing the quality of dendritic cell function in presenting soluble antigens to CD4+ T cells in vitro.
abstract: Because dendritic cells (DC) are critically involved in both initiating primary and boosting secondary host immune responses, attention has focused on the use of DC in vaccine strategies to enhance reactivity to tumor-associated antigens. We have reported previously the induction of major histocompatibility complex class II-specific T-cell responses after stimulation with tumor antigen-pulsed DC in vitro. The identification of in vitro conditions that would generate large numbers of DC with more potent antigen-presenting cell (APC) capacity would be an important step in the further development of clinical cancer vaccine approaches in humans. We have focused attention on identifying certain exogenous cytokines added to DC cultures that would lead to augmented human DC number and function. DC progenitors from peripheral blood mononuclear cells (PBMC) were enriched by adherence to plastic, and the adherent cells were then cultured in serum-free XVIVO-15 medium (SFM) for 7 days with added granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). At day 7, cultures contained cells that displayed the typical phenotypic and morphologic characteristics of DC. Importantly, we have found that the further addition of tumor necrosis factor alpha (TNFalpha) at day 7 resulted in a twofold higher yield of DC compared with non-TNFalpha-containing DC cultures at day 14. Moreover, 14-day cultured DC generated in the presence of TNFalpha (when added at day 7) demonstrated marked enhancement in their capacity to stimulate a primary allogeneic mixed leukocyte reaction (8-fold increase in stimulation index [SI]) as well as to present soluble tetanus toxoid and candida albicans (10- to 100-fold increases in SI) to purified CD4+ T cells. These defined conditions allowed for significantly fewer DC and lower concentrations of soluble antigen to be used for the pulsing of DC to efficiently trigger specific T-cell proliferative responses in vitro. When compared with non-TNFalpha-supplemented cultures, these DC also displayed an increased surface expression of CD83 as well as the costimulatory molecules, CD80 and CD86. Removal of TNFalpha from the DC cultures after 2 or 4 days reduced its enhancing effect on DC yield, phenotype, and function. Thus, the continuous presence of TNFalpha over a 7-day period was necessary to achieve the maximum enhancing effect observed. Collectively, our findings point out the importance of exogenous TNFalpha added to cultures of cytokine-driven human DC under serum-free conditions, which resulted in an enhanced number and function of these APC. On the basis of these results, we plan to initiate clinical vaccine trials in patients that use tumor-pulsed DC generated under these defined conditions.
authors.affiliation: Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109-0666, USA.
authors.forename: B
authors.initials: B
authors.lastname: Chen
authors.forename: Y
authors.initials: Y
authors.lastname: Shi
authors.forename: J D
authors.initials: JD
authors.lastname: Smith
authors.forename: D
authors.initials: D
authors.lastname: Choi
authors.forename: J D
authors.initials: JD
authors.lastname: Geiger
authors.forename: J J
authors.initials: JJ
authors.lastname: Mulé
mesh_terms: D017951:Antigen Presentation; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002450:Cell Communication / Q000187:drug effects*; D002452:Cell Count / Q000187:drug effects; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology*; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D015847:Interleukin-4 / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor