The Role of Tumor Necrosis Factor α 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 α (TNFα) at day 7 resulted in a twofold higher yield of DC compared with non–TNFα-containing DC cultures at day 14. Moreover, 14-day cultured DC generated in the presence of TNFα (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–TNFα-supplemented cultures, these DC also displayed an increased surface expression of CD83 as well as the costimulatory molecules, CD80 and CD86. Removal of TNFα from the DC cultures after 2 or 4 days reduced its enhancing effect on DC yield, phenotype, and function. Thus, the continuous presence of TNFα over a 7-day period was necessary to achieve the maximum enhancing effect observed. Collectively, our findings point out the importance of exogenous TNFα 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.

Comparative Analysis of Genetically Modified Dendritic Cells and Tumor Cells as Therapeutic Cancer Vaccines

“…Our finding that the engineered expression of either of the three molecules tested led to significantly improved biological activity suggests that the optimal in vivo behavior of DCs may be quite dependent on a pattern of normal endogenous gene expression not easily maintained after ex vivo manipulation of the cells. This notion is consistent with a variety of studies by others that suggest that optimal DC function can be highly dependent on the precise methodology used for their generation and expansion (46,47). While the precise explanation for the enhanced immunogenicity of GM-CSF-transduced DCs (or cells transduced by TNF-␣ or CD40L viruses) remains to be determined, our dye-tracking experiments indicate that GM-CSF expression results in more transduced DCs reaching the LN.…”

Dendritic Cells Discriminate between Yeasts and Hyphae of the Fungus Candida albicans

“…Our study shows that, in doing that, murine DCs are exquisitely sensitive to the different forms of a pathogen, a finding in line with the increasingly recognized importance of pattern recognition receptors in host defense (79,80). Considering that human DCs also phagocytose (81) and activate T cell responses to C. albicans (82), our findings provide important and novel insights into the general mechanisms of immunoregulation in fungal infections. Moreover, as the morphogenesis of C. albicans is activated in vivo by a wide range of signals, including stress and metabolic signals (83), DCs may also act as key regulators of Th reactivity in saprophytism.…”

Activities of granulocyte‐macrophage colony‐stimulating factor and interleukin‐3 on monocytes

“…The potential of GM-CSF to support the differentiation of monocytes into DCs as a single factor may at least in part be involved in enhancement of the immunological effects induced by the vaccine. Although several approaches to generate DCs from PBMCs including monocytes in serum-free cultures for clinical vaccine trials have been investigated [32][33][34][35], IL-4 was used in combination with GM-CSF for these studies. Our serum-free culture method may provide another attractive mode to generate DCs for human DCsbased immunotherapy, because IL-4 is not required for the generation of DCs from monocytes.…”