The twisted road to become a mature Natural Killer Cell: development of an autologous system mimicking the bone marrow niche to study their ontogenesis

Natural Killer (NK) cells are innate lymphocytes able of killing tumor-transformed and viral infected cells, without a prior antigen sensitization. Although their characterization is deepening, the definition of their maturation steps is still in its infancy. Adult Bone Marrow (BM) is a major site of NK cell ontogenesis. Here, CD34pos Hematopoietic Stem Cells (HSCs) and NK cell precursors (NKPs) in response to different cytokine stimuli, differentiate in the CD56brightCD16neg (cCD56bright) cytokines-secreting NK cell subset that further differentiates later in the cytotoxic CD56dimCD16pos (cCD56dim) NK cells. In addition to these conventional subsets, a third subset of mature NK cells with an unknown origin and bearing a CD56dimCD16neg (unCD56dim) phenotype has been identified. To study NK cell ontogenesis, set up an autologous ex-vivo system mimicking the BM-niche by co-culturing BM-derived Mesenchymal Stem Cells with FACS-sorted HSCs, NKPs, cCD56bright or unCD56dim. The results obtained demonstrated that HSCs and NKPs firstly differentiate in Innate Lymphoid Cells (ILCs), that then gave rise to cCD56bright NK cells that further differentiate in unCD56dim NK cells. Similarly, cultured cCD56bright NK cells differentiate in unCD56dim NK cells, while sorted unCD56dim NK cells remained themself over the time, as typical of more differentiated subsets. In all the co-culture conditions we obtained only reduced frequencies of cCD56dim NK cells, thus suggesting that other stimuli, likely occurring in the periphery, are needed to drive their terminal differentiation. Our in vitro results are also supported by the in silico analysis of publicly available single cell RNA sequencing data demonstrating that cCD56bright NK cells are the precursors of both unCD56dim and cCD56dim NK cells. These data led us to hypothesize that NKPs passing through ILCs, can give rise to cCD56bright NK cells, that further differentiates into cCD56dim or unCD56dim depending on the microenvironment.