29-Color Flow Cytometry: Unraveling Human Liver NK Cell Repertoire Diversity.

Recent studies have demonstrated extraordinary diversity in peripheral blood human natural killer (NK) cells and have suggested environmental control of receptor expression patterns on distinct subsets of NK cells. However, tissue localization may influence NK cell differentiation to an even higher extent and less is known about the receptor repertoire of human tissue-resident NK cells. Advances in single-cell technologies have allowed higher resolution studies of these cells. Here, the power of high-dimensional flow cytometry was harnessed to unravel the complexity of NK cell repertoire diversity in liver since recent studies had indicated high heterogeneity within liver NK cells. A 29-color flow cytometry panel allowing simultaneous measurement of surface tissue-residency markers, activating and inhibitory receptors, differentiation markers, chemokine receptors, and transcription factors was established. This panel was applied to lymphocytes across three tissues (liver, peripheral blood, and tonsil) with different distribution of distinct NK cell subsets. Dimensionality reduction of this data ordered events according to their lineage, rather than tissue of origin. Notably, narrowing the scope of the analysis to the NK cell lineage in liver and peripheral blood separated subsets according to tissue, enabling phenotypic characterization of NK cell subpopulations in individual tissues. Such dimensionality reduction, coupled with a clustering algorithm, identified CD49e as the preferred marker for future studies of liver-resident NK cell subsets. We present a robust approach for diversity profiling of tissue-resident NK cells that can be applied in various homeostatic and pathological conditions such as reproduction, infection, and cancer.