Non-invasive and label-free identification of human natural killer cell subclasses by biophysical single-cell...

Natural killer (NK) cells are indicated as favorite candidates for innovative therapeutic treatment and are divided into two subclasses: immature regulatory NK CD56^bright and mature cytotoxic NK CD56^dim. Therefore, the ability to discriminate CD56^dim from CD56^bright could be very useful because of their higher cytotoxicity. Nowadays, NK cell classification is routinely performed by cytometric analysis based on surface receptor expression. Here, we present an in-flow, label-free and non-invasive biophysical analysis of NK cells through a combination of light scattering and machine learning (ML) for NK cell subclass classification. In this respect, to identify relevant biophysical cell features, we stimulated NK cells with interleukine-15 inducing a subclass transition from CD56^bright to CD56^dim. We trained our ML algorithm with sorted NK cell subclasses (≥86% accuracy). Next, we applied our NK cell classification algorithm to cells stimulated over time, to investigate the transition of CD56^bright to CD56^dim and their biophysical feature changes. Finally, we tested our approach on several proband samples, highlighting the potential of our measurement approach. We show a label-free way for the robust identification of NK cell subclasses based on biophysical features, which can be applied in both cell biology and cell therapy.