A 36-antigen spectral flow cytometry panel to unravel human NK cell differentiation and education

Spectral flow cytometry is the latest advance in flow cytometry field. Overcoming the strict association one fluorophore-one photomultiplier, it relies on the measurement of the whole emission spectrum for each fluorophore, thus increasing the resolution and deepening immunophenotyping at single cell level. This technology allows to provide high-throughput characterization of several immune cells, allowing for the discovery of novel populations that can be relevant in the translational setting. Natural Killer (NK) cells are a perfect case in point: historically classified according to the expression of CD56 and CD16, the full exploitation of their cytotoxic features in clinic is limited by the poor knowledge of their differentiation and by the underestimation of their heterogeneity. Given these premises, we designed and optimized a 36-antigen spectral flow cytometry panel for the simultaneous phenotypic and functional characterization of human NK cells, ultimately attempting to bridge and validate the multitude of data developed via high-throughput technologies. The panel presented here, optimized on healthy human samples, allows the discrimination of circulating NK cell subsets from other innate and adaptive immune populations, and the simultaneous evaluation of precursors, enabling the in-depth assessment of their phenotypical and functional development.