Natural Killer cells exhibit direct activity against hyphae, but not against conidia of Aspergillus fumigatus

Background: Natural Killer (NK) cells play an important role in the elimination of malignant and virus-infected cells. Unfortunately, little is known about the antifungal activity of human NK cells against Aspergillus fumigatus. Methods: Human NK cells from healthy donors were purified using magnetic cell sorting (Miltenyi Biotech, Germany). Analyses were performed using either unstimulated or IL2 (1000 U/ml, 7–10d) stimulated human NK cells. Antifungal activity against Aspergillus hyphae was assessed by the XTT assay and activity against conidia by determination of colony forming units (CFUs). Perforin and interferon (IFN)-γ levels were determined using ELISA (Diaclone, France). Activation of unstimulated NK cells by A. fumigatus was assessed by means of flow cytometry analyzing the expression of CD69 (Beckman Coulter). Perforin isolated from human YT cells was used in a concentration of 100ng/ml, recombinant human granulysin in a concentration of 1µM. Results: We observed an E:T ratio dependent antifungal activity of unstimulated and IL2 stimulated NK cells against A. fumigatus hyphae. In contrast, Aspergillus conidia were not damaged by NK cells. The extent of the hyphal damage correlated with the concentration of perforin in the supernatant. Unstimulated NK cells did not release IFN-γ, neither with or without co incubation with Aspergillus hyphae. In contrast, high levels of IFN-γ were detected in the supernatants of IL2 stimulated NK cells not coincubated with Aspergillus. Interestingly, IFN-γ levels decreased when the NK cells were coincubated with Aspergillus hyphae, suggesting an immunosuppressive effect of the fungus. Administration of perforin and granulysin resulted in a significant damage of A. fumigatus hyphae, but not of conidia. In addition, Aspergillus hyphae led to an increase of the expression of CD69, which was not seen when NK cells were coincubated with conidia. Summary and perspectives: Whereas NK cells demonstrate a significant antifungal activity against Aspergillus hyphae, they do not damage Aspergillus conidia. Importantly, unstimulated NK cells are no activated by conidia. These results may have an important implication in the potential strategy of NK cells as adoptive immunotherapy (e.g., prophylaxis versus treatment).