Comparative proteome analyses of human natural killer cells from patients after allogeneic stem cell transplantation and healthy individuals challenged with Aspergillus fumigatus.

Natural killer (NK) cells contribute to the innate immune system and are pivotal for the defence against opportunistic pathogens, including fungi. Aspergillus fumigatus (AF), a filamentous mold, can cause invasive pulmonary aspergillosis in immunocompromised patients, e.g. in patients after allogeneic stem cell transplantation (alloSCT). In this pilot study, we challenged NK cell samples from alloSCT recipients collected 90, 120, and 180 days after transplantation and from healthy individuals with AF and characterize the proteome response differences. We identified 2259 differentially abundant proteins between the NK cell proteomes of alloSCT recipients and healthy individuals. Among these, 1118 proteins were differentially abundant at all time points and 1931 proteins specifically at day 180 post-alloSCT. Following stimulation of NK cells with AF, we found a profoundly different early proteome (day 90, n=1652 proteins), while at day 180, only 77 proteins remained significantly differentially abundant. We identified, among others, a major differentially abundant protein cluster related to IL27RA (including OAS, STAT1, and MX). Furthermore, for selected markers [granzyme A (GZMA), Neural Cell Adhesion Molecule 1 (NCAM1/CD56), perforin-1 (PRF1)], we confirmed our proteome data by flow cytometry in NK cells from an independent second patient and healthy individual cohort. In conclusion, we demonstrate the advantage of combining comprehensive proteomic profiling with targeted flow cytometry to investigate NK cell responses to AF. Our data analysis connects STAT1 with IL27RA as well as granzyme, IFNg, and NCAM1 activity, which may be exploited towards future therapeutics warranting confirmation in larger study cohorts.