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. 2011 Nov 17;118(20):5439-47.
doi: 10.1182/blood-2011-04-348912. Epub 2011 Sep 19.

Identification of the Earliest Natural Killer Cell-Committed Progenitor in Murine Bone Marrow

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Free PMC article

Identification of the Earliest Natural Killer Cell-Committed Progenitor in Murine Bone Marrow

John W Fathman et al. Blood. .
Free PMC article

Abstract

Natural killer (NK) cells develop in the bone marrow and are known to gradually acquire the ability to eliminate infected and malignant cells, yet the cellular stages of NK lineage commitment and maturation are incompletely understood. Using 12-color flow cytometry, we identified a novel NK-committed progenitor (pre-NKP) that is a developmental intermediate between the upstream common lymphoid progenitor and the downstream NKP, previously assumed to represent the first stage of NK lineage commitment. Our analysis also refined the purity of NKPs (rNKP) by 6-fold such that 50% of both pre-NKP and rNKP cells gave rise to NKp46+ NK cells at the single-cell level. On transplantation into unconditioned Rag2-/-Il2rγc-/- recipients, both pre-NKPs and rNKPs generated mature NK cells expressing a repertoire of Ly49 family members that degranulated on stimulation ex vivo. Intrathymic injection of these progenitors, however, yielded no NK cells, suggesting a separate origin of thymic NK cells. Unlike the rNKP, the pre-NKP does not express IL-2Rβ (CD122), yet it is lineage committed toward the NK cell fate, adding support to the theory that IL-15 signaling is not required for NK commitment. Taken together, our data provide a high-resolution in vivo analysis of the earliest steps of NK cell commitment and maturation.

Figures

Figure 1
Figure 1
Pre-NKPs and rNKPs can be highly purified from BM using 12-color flow cytometry. (A) Gating strategy for CLP, pre-NKP, and rNKP. (B) Frequency of CLP, pre-NKP, and rNKP in BM isolated from 2 femurs. (C) Absolute number of CLP, pre-NKP, and rNKP from 2 femurs. Shown are the individual data from 10 mice with the mean and standard deviation (B-C).
Figure 2
Figure 2
Pre-NKP and rNKP are lineage restricted and give rise only to NK cells in vitro. (A) Pre-NKP and rNKP lack B-cell potential in vitro. Individual wells with OP9 cells were seeded with 10 indicated progenitor cells and cultured in the presence of SCF, IL-7, Flt3 ligand, and IL-15. Wells were analyzed by flow cytometry 10 days later. No wells were positive for Gr-1+CD11b+ myeloid cells (data not shown). (B) Pre-NKP and rNKP lack in vitro T-cell potential. Flow cytometry analysis of the lineage output of individual wells seeded on OP9-DL1 cells with 10 indicated progenitor cells. No wells analyzed were positive for myeloid cells (data not shown). Data are representative of 1 of 4 experiments. (C) Pre-NKP and rNKP generate NK cells at a high clonal frequency. Number of wells plated with single cell and the number of wells positive for NKp46+ cells after 10 days. Data are representative of 1 of 3 experiments.
Figure 3
Figure 3
Pre-NKP and rNKP are lineage restricted to the NK cell fate in vivo. CD45.1 unfractionated BM, CLP, pre-NKP, and rNKP were transplanted into unconditioned congenic CD45.2 Rag2−/−IL2rγc−/− recipients. Spleens 3 weeks after transplantation were isolated and analyzed by flow cytometry for lineage potential of donor cells. Each progenitor type was injected into 5 individual mice per experiment. Data are representative of 1 of 5 experiments.
Figure 4
Figure 4
Pre-NKP and rNKP in vivo–derived NK cells are phenotypically mature and give rise to NK cells on transplantation faster than CLP or LMPP. (A) CD45.1 unfractionated BM, CLP, pre-NKP, and rNKP were transplanted into unconditioned congenic CD45.2 Rag2−/−IL2rγc−/− recipients. Spleens 3 weeks after transplantation were isolated and analyzed by flow cytometry for mature NK cell markers CD27 and Mac1. Each progenitor type was injected into 5 individual mice per experiment. Data are representative of 1 of 2 experiments. (B) Ly5.2+ progenitors where transplanted into unconditioned Ly5.1+ DKO mice. Mice where bled every week and analyzed by flow cytometry for donor NK cell reconstitution. Pre-NKP gave rise to NK cells as early as 1 week and were exhausted after 3 weeks. CLP gave rise to few, but detectable NK cells at 1 week and also became exhausted by 4 weeks. We observed no NK cells at 1 week in the LMPP transplants, but by 2 weeks they where comparable to both pre-NKP and CLP. Each progenitor type was injected into 5 individual mice per experiment. Data are representative of 1 of 2 experiments. (C) Absolute number of donor-derived NK cells from the spleens of mice transplanted with different progenitors. Data are representative of 1 of 2 experiments.
Figure 5
Figure 5
Pre-NKP–derived NK cells have mature functional phenotype. (A) Pre-NKP–derived splenic NK cells express diverse combinations of Ly49 receptors. Wild-type NK cells and donor NKp46+ cells from pre-NKP transplanted Rag2−/−IL2rγc−/− mice were stained with a panel of anti-Ly49 family antibodies. Data represents 1 of 2 experiments. (B) Pre-NKP–derived NK cells degranulate on activation. Wild-type NK cells and NKp46+ cells derived in vivo from pre-NKP were isolated and stimulated with plate bound anti-NK1.1 for 4 hours in the presence of lysosomal-associated membrane protein 1 (Lamp-1) antibody. Data are representative of 2 independent experiments.
Figure 6
Figure 6
Proposed model for NK bone marrow development. CLP, immature NK cells (iNK), and mature NK cells in the bone marrow, not terminally differentiated splenic NK cells that lack expression of CD27 (mNK).

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