Multi-methodologic dissection of human Natural Killer cell ontogenesis to study the contribution of unconventional NK cell subsets and other Innate Lymphoid Cells (ILCs)

Natural Killer (NK) cell maturation remains unclear in humans. Although the Bone Marrow (BM) niche is a major site for the initial phases of NK cell ontogenesis, hereupon, committed precursors reach secondary lymphoid tissues to differentiate in CD56brightCD16neg (CD56bright) and eventually CD56dimCD16pos (CD56dim) NK cells. Recently, we described an additional CD56dimCD16neg (unCD56dim) subset, whose role in the ontogenesis is unknown. Herein, we integrated several techniques to dissect NK cell ontogenesis: first we set up an autologous ex-vivo BM-niche by co-culturing BM-derived mesenchymal stromal cells with FACS-sorted NK cell precursors (NKPs), for 28 days. We observed that NKPs differentiate into ILC-resembling cells, and then into CD56bright NK cells and unCD56dim. Through in-silico analysis of single-cell RNA sequencing data, we confirmed CD56bright cells as ancestors for both unCD56dim and CD56dim cells. Lastly, we investigated NK cell immune reconstitution after hematopoietic stem cell transplantation as a model of in vivo NK cell differentiation, by applying single-cell based unbiased pipelines to multi-parametric flow-cytometry data. This allowed us to appreciate fully differentiated unCD56dim NK cells. In conclusion, we demonstrated that ILC-like cells are involved in the initial steps of human NK cell ontogenesis and that unCD56dim cells likely represent a more differentiated NK cell subset.