Project Summary Over 300,000 new global cases of invasive pulmonary aspergillosis (IPA) are reported each year, with a mortality rate as high as 90%. Therefore, the development of effective antifungal treatments is of high medical significance. The current antifungal arsenal consists of 3 core classes of drugs: the azoles, echinocandins and polyenes. However, increasing antifungal resistance within Aspergilli (azoles and echinocandins) and secondary cytotoxicity (polyenes) limits the treatment options. Consequently, there is a need for the development of alternative treatment strategies to control IPA progression. Granulocyte transfusion therapy (GTX) has been pursued as an alternative treatment strategy for microbial infections, but the short lifespan of primary human neutrophils and insufficient donor supply has limited the use of this approach. Induced human pluripotent stem cell (iPSC) derived neutrophils (iNeutrophils) can be produced in large quantities independent of a donor, are genetically malleable to permit engineering, and may serve as invaluable replacement to primary neutrophils in GTX. However, the efficacy of iNeutrophils in controlling fungal growth is currently unknown. Our lab and others have recently discovered that deletion of GATA1 in iPSCs, a transcription factor that is important in eosinophil and basophil differentiation, results in a highly homogenous culture of mature iNeutrophils that are more phenotypically similar to primary human neutrophils than previously characterized wild-type (WT) iNeutrophils. This puts us in a unique position to use iNeutrophils in both therapeutic and mechanistic studies to assess their interactions with human fungal pathogens. This proposal will therefore leverage WT and GATA1-/- iNeutrophils in combination therapy with current antifungals to inhibit the growth of clinically relevant Aspergilli, and to generate host pattern recognition receptor (PRR) knockouts to characterize mechanisms of fungal recognition that drive neutrophil antifungal activity. This proposal seeks to achieve these goals by combining in vitro immunology techniques with iPSC and fungal genetics. Aim 1 will assess the ability of iNeutrophils to independently inhibit the growth of isolates from the two most common causative agents of IPA, A. fumigatus and A. flavus. These findings will then be expanded to assess the ability of iNeutrophils to synergize with current antifungals in vitro to further enhance fungal killing. Aim 2 will then begin to dissect how neutrophils sense pathogenic fungi in their environment by characterizing the receptor network needed to recognize the immunogenic polysaccharide ß(1,3)-glucan in the fungal cell wall. This will be achieved by generating iNeutrophil ß(1,3)-glucan receptor knockouts and by utilizing Aspergillus mutants with altered ß(1,3)-glucan exposure levels. Together, this approach will provide essential training opportunities in stem cell engineering, immunology techniques, and fila...