Human natural killer cells exhibit potent antifungal activity against azole-resistant Aspergillus fumigatus and diverse filamentous fungi.

Invasive aspergillosis in immunocompromised individuals is associated with a poor prognosis. Recently, the global emergence of azole-resistant Aspergillus fumigatus has underscored the urgent need for novel therapeutic strategies beyond conventional small-molecule antifungal agents. Natural killer (NK) cells, which are characterized by the absence of CD3 and presence of CD56 surface markers, play a pivotal role in the immune response against fungal pathogens and in the elimination of malignant cells and virus-infected host cells. Although the antifungal activity of NK cells against Aspergillus spp. and other fungi has been demonstrated, the efficacy of ex vivo-expanded human NK cells against azole-resistant A. fumigatus has not been investigated. NK cells, which were expanded from the CD3- peripheral blood mononuclear cells obtained from healthy donors, were co-cultured with fungi. Antifungal activity was assessed using the 2,3-(bis2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide reduction assay. NK cells demonstrated antifungal activity against azole-resistant A. fumigatus and other filamentous fungi. These findings suggest that a direct interaction between NK cells and hyphae is critical for exerting antifungal effects, with degranulation likely contributing to the underlying mechanism. However, contrary to previous reports, we failed to observe the direct antifungal activity of interferon-γ secreted by NK cells under our experimental conditions. Cellular immunotherapy using NK cells may represent a promising therapeutic approach for the treatment of azole-resistant aspergillosis.IMPORTANCEAspergillus fumigatus is a ubiquitous fungus that causes invasive aspergillosis (IA) in immunocompromised patients, with poor prognosis. Furthermore, azole-resistant A. fumigatus has spread globally in recent years. The present antifungal drugs pose challenges such as drug resistance and adverse events; thus, the development of novel therapies for IA is needed beyond the antifungal drugs. In cases of IA in immunocompromised patients with poor response to antifungal drugs, cellular immunotherapy is a reasonable approach. This study demonstrated that human natural killer (NK) cells derived from peripheral blood exhibit antifungal activity against azole-resistant A. fumigatus comparable to that of azole-susceptible strain. This indicates that cellular immunotherapy using NK cells could become an effective therapy for IA caused by azole-resistant A. fumigatus. Investigating the mechanism by which NK cells exhibit antifungal activity against A. fumigatus could lead to the development of novel therapies for IA.