ATAK Cells Require Intact Cytochrome Oxidase Activity to Kill Fungi (38.18)

Abstract Invasive fungal infections cause high mortality rates in neutropenic patients. We developing an immunotherapy based on activation of HL-60 cells into Activated Targeted Killer (ATAK) cells. We have shown that ATAK cells phagocytize and kill C. albicans in vitro, and protect neutropenic mice from invasive candidiasis and aspergillosis. However, the mechanism by which ATAK cells kill fungi were unclear. Because we have recently found that gp91phox deficient mice were hypersusceptible to invasive candidiasis, we used siRNA to suppress gp91-phox expression in ATAK cells to define impact of suppression of NADPH-superoxide formation on fungal killing. We use a lentivirus to stably transfect a gp91-phox-targeting siRNA construct into HL-60 cells. Stable transfectants had significantly suppressed mRNA level (by RT-PCR) and protein expression (by western blot) of gp91-phox. To confirm functional suppression, we compared the superoxide production in wild-type ATAK cells and gp91-phox-/- cells by DHR123 staining and flow cytometry. We found the gp91-phox -/-cells had markedly reduced superoxide generation. In in vitro killing assays, gp91-phox-/- ATAK cells had marked reductions in their killing of C. albicans relative to wild type cells. Finally, gp91-phox deficient ATAK cells were unable to protect neutropenic mice from invasive candidiasis or invasive aspergillosis. Thus, ATAK cell fungal killing and in vivo efficacy requires intact superoxide production by the NADPH-oxidase system.