Dendritic cells (DCs) are well characterized for their ability to induce adaptive immune responses. To mimic these properties, in vitro generated monocyte-derived DCs (moDCs) have been extensively studied and used as antigen presenting cells, initiating specific T cell immune responses against virus infected and malignant cells. There is increasing evidence that the immunoregulatory effect of DCs is exerted also towards innate immune effector cells, like NK cells. Recently, we have shown a significant anti-Aspergillus fumigatus impact of NK cells, mainly mediated by the release of IFN- γ, which demonstrated direct fungicidal properties. The interest for our study is driven by the high mortality characterizing invasive aspergillosis, an infection of immunosuppressed patients caused mainly by this fungus. To date, it has been shown that NK cells activated by DCs demonstrate increased IFN- γ release, proliferation and cytotoxicity against malignant cells. In our study, we have investigated the potential effect of moDCs, previously pulsed with fungal proteins and with cell wall fragments on NK cells and on their cytotoxicity against A. fumigatus.
For the generation of moDCs, we used CD14+ cells isolated from peripheral blood of healthy donors by positive selection with magnetic microbeads. Cells were cultured for 5 days in serum-free GMP grade medium supplemented with GM-CSF and IL-4. On the second day of culture, immature DCs were pulsed either with a β-(1, 3)- glucan–rich alkali-insoluble (AI) cell wall fraction extracted from fungal conidia or from mycelia, inactivated conidia and germ tubes or with the recombinant catalase Cat1 and transglycosidase Crf1. Three hours later, DCs were maturated with TNF- α. Before being used, DC properties were characterized by quantifying the expression of CD1a, CD14, CD40, CD80, CD83, CD86, HLA-I, HLA-DR, CCR7, Dectin-1, TLR-2, TLR-4, IL12p35, IL15, and IFN- γ. Autologous NK cells were obtained by magnetic negative selection of peripheral mononuclear cells and were kept frozen in human serum supplemented with 8% DMSO at −80°C. DC/ NK cell co-cultures were set in unconditioned GMP grade medium on different ratios (1:1, 1:5, 1:10). To evaluate proliferation, NK cells were stained with CFSE. Proliferation was measured at the 7th day of DC / NK co-culture. IFN- γ release by NK cells was quantified by FACS analysis after 16 h of DC / NK co-culture. Finally, the antifungal impact on A. fumigatus germ tubes was evaluated by 2, 3-bis[2-methoxy-4-nitro-5-sulpho-phenyl]2H-tetrazolium-5-carboxanilide (XTT) assays.
DCs pulsed with the different fungal stimuli demonstrated a ‘mature’ surface profile with negative CD1a & CD14 expression and high positivity of CD40, CD80, CD83, CD86, HLA-I, HLA-DR & CCR7. All DCs produced IL12p35 and IL15, but no IFN- γ. The expression of Dectin-1 was strongly down-regulated (p<.001) by the alkali-insoluble cell wall fraction and by germlings, moderately by conidia (p<.05) and it was unaffected after Cat1 and Crf1 stimulation. This observation suggests a major involvement of Dectin-1 and its ligand, β-(1, 3)-glucan in the interaction of moDCs with A. fumigatus. In contrast, TLR-2 and TLR-4 were not found to be regulated on DCs. Furthermore, DCs induced the proliferation of NK cells, higher DC / NK ratios elicited higher NK cell expansion. DCs stimulated with AI cell wall fractions or germlings provoked a significantly higher proliferation of NK cells in comparison to unstimulated DCs (p<.05). Similarly, significant up-regulation of IFN- γ expression was observed in NK cells co-cultured with DCs previously pulsed with germ tubes and with fungal cell wall fractions, respectively (p<.05). Finally, we revealed significant higher fungal damage in NK cell / DC co-cultures (especially in those primed with fungal cell wall fractions or germlings), compared to NK cell or DC cultures alone (p<.05).
To summarize, our study shows for the first time that moDCs pulsed with A. fumigatus cell wall components induce NK cell proliferation and potentiate IFN- γ release and cytotoxicity against this fungus. These results provide evidence for a role of moDCs as activators of NK cells towards an immune response against IA.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.