Identification of the earliest NK-cell precursor in the mouse BM

Abstract

IntroductionAlthough the developmental stages leading to committed B and T cells have been dissected in great detail, 1 the developmental intermediates downstream of the common lymphoid progenitor (CLP) leading to a committed natural killer (NK)-cell precursor remain poorly defined. The earliest committed NK-cell progenitor (NKP) identified to date is characterized through the expression of the IL-2 receptor ␤ chain (CD122) and the lack of pan-NK-cell surface markers (NK1.1 and CD49b), but this population is likely to be heterogeneous because only a minority of cells give rise to NK cells in in vitro assays. 2,3 The transcription factor inhibitor of DNA binding 2 (ID2) is essential for NK-cell development. In its absence, fetal and adult NK-cell development is blocked at an early stage. [4][5][6][7] Id2 inhibits E proteins in NK cells and is itself regulated by E4BP4, which is also required for the differentiation of NKPs to immature NK cells. 8,9 Using a reporter line in which the green fluorescent protein (GFP) gene has been introduced into the Id2 locus, we report the identification of the earliest committed NK-cell precursor in adult BM. By analogy to B-cell development, we termed these pre-pro NK cells, and they express high levels of ID2, IL-7R␣ (CD127), and lack expression of most, but not all, NK-cell markers. Furthermore, we demonstrate that ID2 gfp expression can be used to isolate the committed NK-cell progenitors within the heterogeneous NKP population. Methods MiceWe generated ID2 gfp reporter mice by inserting an internal ribosome entry site-GFP cassette into the 3Ј untranslated region of the Id2 locus (G.T.B., S.C., and S.L.N. manuscript submitted April 2011). PU.1 gfp and Rag1 gfp mice have been described previously. 10,11 All mice were maintained under specific pathogen-free conditions at the Walter and Eliza Hall Institute according to institute guidelines. Antibodies, cell analysis, and sortingLineage (lin) marker-positive BM cells were depleted and analyzed by flow cytometry as described in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Cell cultureTo determine precursor frequencies, sorted progenitors cells were seeded in limiting dilution on OP9 or OP9-DL1 cells in the presence of 2% IL-7 supernatant and 5 ng/mL FMS-like tyrosine kinase 3 (Flt3) ligand for Band T-cell development, respectively, and on OP9 cells plus 0.5% IL-7 supernatant and 50 ng/mL human IL-15 (R&D Systems) for NK-cell differentiation as described. 12 After 7-10 days, wells were scored for the presence of colonies. Colonies were harvested for flow cytometric analysis, and precursor frequency was calculated according to Poisson statistics with the use of ELDA (extreme limiting dilution analysis) software Version 1.0 (http://bioinf.wehi.edu.au/software/elda/). 13 Results and discussionThe earliest committed NKP identified to date expresses CD122 but not the pan-NK cell-specific markers NK1.1 and CD49b. 2 This population is likely to be heterogene...

PLZF expression maps the early stages of ILC1 lineage development

“…We also examined the Lin − Sca1 + CD117 (cKit) int/− CD135 (Flt3) − IL7Rα + cell, which was proposed to be an early NK precursor, termed pre-pro-NK (20). Strikingly, we found uniform but low expression of GFP contrasting with a high frequency of YFP fate-mapping, suggesting that a large fraction of these cells had, in fact, originated from ILCPs and downregulated PLZF (Fig.…”

“…In addition, after in vivo transfer into Rag −/− Il2rg −/− hosts, both populations generated exclusively CD3e − NK1.1 + cells in the spleen and liver. Another study used an Id2-GFP reporter strain to identify a population of Lin − Id2 high Sca1 + CD117 (cKit) int/-CD135 (Flt3) − IL7Rα + cells, termed pre-pro-NKs (20), that generated CD3e − NK1.1 + cells at a frequency of ∼1 in 2 upon single-cell culture in vitro with OP9 cells and a mixture of IL-7 and IL-15.…”

Innate immunodeficiency following genetic ablation of Mcl1 in natural killer cells

“…Following upregulation of Id2 in pre-pro NK cells 7 , expression of the IL-2/15Rb (CD122) is acquired and NK cells become dependent on IL-15. The near total loss of NK cells following Mcl1 deletion may suggest that their dependency on Mcl-1 for survival is more stringent than that of B and T cells, since specific deletion of Mcl1 using CD19-Cre (B-cell-specific) and Lck-Cre (T-cell-specific) resulted in a less dramatic loss of these cell types in vivo 22 than what we observed for NK cells.…”

“…Natural killer (NK) cells are the most prevalent ILC member (ILC1) being capable of spontaneous cytokine, chemokine and lytic granule production upon activation 3 . NK cell development takes place in the bone marrow of adults and requires the pleiotropic cytokine, interleukin-15 (IL-15) 4,5 with high expression of the IL-15Rb chain (CD122), an essential component of IL-15 signalling 6 induced soon after commitment to the NK cell lineage 7 . IL-15-responsive progenitors need to express dimers of IL-2Rb and the common gamma chain (g c /IL-2Rg/CD132) and receptor proximal kinases (Jak1/3) and Signal Transducer and Activator of Transcription 5 (STAT5A/B) in order to survive, expand and differentiate 8 .…”

Homéostasie des cellulesnatural killer

“…L'utilisation de souris transgéniques dans lesquelles la green fluorescent protein (GFP) est sous contrôle du promoteur du gène codant pour le facteur de transcription Id2 -qui joue un rôle très important dans le développement des cellules NK -a montré que les cellules GFP + Lin -dans ces souris sont d'authentiques précurseurs NK et que leur potentiel est restreint aux cellules NK. Ces cellules qui sont donc Id2 + , baptisées pré-pro-NK, sont par ailleurs Sca1 + CD127 + CD122 - [6]. Leur présence en périphérie n'a pas été étudiée.…”