Unveiling the roles of unconventional subsets and other Innate Lymphoid Cells in Natural Killer cell ontogenesis: the development of an autologous Bone Marrow niche mimicking system

Natural Killer (NK) cells are cytotoxic members of the Innate Lymphoid Cells (ILCs) family, capable of killing tumor-transformed and viral infected cells, independently of a prior antigen exposure. Despite an evolving characterization, a firm definition of the maturation intermediates is lacking. In the Adult Bone Marrow (BM), Naïve and NK-committed precursors differentiate upon stimulation in the CD56brightCD16neg (cCD56bright) cytokines-secreting subset that further differentiates in the cytotoxic CD56dimCD16pos (cCD56dim) NK cells. In addition, a third subset of NK cells bearing a CD56dimCD16neg (unCD56dim) phenotype, whose origin is uncertain, has been identified. To study NK cell ontogenesis, we set up an ex-vivo system mimicking the BM-niche by co-culturing autologous BM-derived Mesenchymal Stem Cells with FACS-sorted precursors, cCD56bright or unCD56dim. The results demonstrated that precursors firstly differentiate in ILCs, that give then rise to cCD56bright NK cells that further differentiate in unCD56dim NK cells. Similarly, co-cultured cCD56bright NK cells differentiate in unCD56dim NK cells, while sorted unCD56dim preserve their identity over time, hinting a more differentiated nature. Regardless of the conditions, only meager frequencies of cCD56dim NK cells were obtained, thus suggesting that additional stimuli, likely peripheral, are essential for their ultimate differentiation. In vitro results are supported by the in-silico analysis of single cell RNA sequencing data highlighting that cCD56bright NK cells are the ancestor of both unCD56dim and cCD56dim NK cells. These data led us to hypothesize that precursors, passing through ILCs, can give rise to cCD56bright NK cells that, depending on the microenvironment, further differentiate into cCD56dim or unCD56dim.