Murine natural killer cells are fungicidal to Cryptococcus neoformans

Murine natural killer (NK) cells have been shown to bind to and inhibit the growth of Cryptococcus neoformans in vitro and to contribute to clearance of the organism in vivo. However, it is unclear whether NK cells actually kill cryptococci or simply inhibit proliferation of the fungal target. Therefore, the studies presented here were designed to determine whether NK cells are fungicidal to C. neoformans targets. C. neoformans viability was determined on the basis of the metabolic function of two different enzyme systems, as measured by the two vital stains MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and fluorescein diacetate. Cryptococcal viability, as determined by vital stains, was compared with cryptococcal proliferation, as measured by microcolony formation in agarose at the individual cell level and by CFU counts or extinction dilution analysis in the total cell suspension. Initial comparisons of the vital stains and proliferation assays indicated that these methods effectively distinguished between live and heat-killed cryptococci at the individual cell level and in the total cell suspensions. After cryptococci were incubated with murine NK cells for 18 h, vital stains demonstrated that at the single conjugate level and in the total cell suspension, NK cells kill bound C. neoformans target cells. In addition, the numbers of dead cryptococci in the NK cell-C. neoformans suspensions as determined by the vital stains were comparable to the numbers of cryptococci that were unable to proliferate. Kinetics of NK cell-mediated C. neoformans binding and killing at the single conjugate level and in the total cell suspension were assessed by MTT staining at 2-h intervals after mixing effector and target cells, and the data support the concept that NK cell-C. neoformans binding precedes cryptococcal death. Furthermore, unbound, dead fungal cells were observed in the NK cell-C. neoformans suspensions after 18 h, suggesting that NK cell-C. neoformans interactions may involve both effector cell recycling and killing of unbound cryptococci by soluble cytotoxic factors. In conclusion, the results of these studies firmly establish that NK cells kill C. neoformans.