Two-step maturation of immature DCs with proinflammatory cytokine cocktail and poly(I:C) enhances migratory and T cell stimulatory capacity

Abstract

Effective induction of cell-mediated immune responses strongly depends on the ability of dendritic cells (DCs) to produce Th1-polarizing cytokines, migrate to lymph nodes and stimulate T cells through antigen-presenting complex and costimulatory molecules. While various protocols for optimizing DC maturation with single or multiple stimuli mimicking infections or inflammatory milieu have been proposed for the generation of DCs with features desired for clinical application, stepwise maturation of DCs by these multiple stimuli has not been systemically assessed. Among the combinations of several immune-modulating factors with known effects on DC maturation, we found that stepwise DC maturation with cytokine cocktail (TNF-alpha+IL-6+IL-1 beta+PGE(2)) followed by poly(I:C) stimulation enhanced the production of IL-12 with strong allostimulatory capacity. While there were no significant differences between DC matured by simultaneous or sequential activation by cytokine cocktail and poly(I:C) in expression of markers and costimulatory molecules of mature DCs, the delivery of inflammatory signal prior to poly(I:C) results in sustained interleukin-12 expression with reduced IL-10 than DC matured by simultaneous stimulation. This sequential stimulation significantly increased migratory capacity in response to CCL21 and CXCL12 compared to DC matured with cytokine cocktail. Furthermore, these DCs retained their responsiveness to CD40L stimulation in secondary IL-12 production and efficiently generated autologous antigen-specific effector T cells as evidenced by ELISPOT assay. Thus, we propose a novel DC maturation protocol in which stimulation of DCs with cytokine cocktail and subsequently with poly(I:C) generates DCs with a high migratory capacity with a preferential Th1 inducing capacity.