Eomes expression identifies the early bone marrow precursor to classical NK cells

Abstract

Natural killer (NK) cells traffic through the blood and mount cytolytic and interferon-γ (IFNγ)-focused responses to intracellular pathogens and tumors. Type 1 innate lymphoid cells (ILC1s) also produce type 1 cytokines but reside in tissues and are not cytotoxic. Whether these differences reflect discrete lineages or distinct states of a common cell type is not understood. Using single-cell RNA sequencing and flow cytometry, we focused on populations of TCF7⁺ cells that contained precursors for NK cells and ILC1s and identified a subset of bone marrow lineage-negative NK receptor-negative cells that expressed the transcription factor Eomes, termed Eomes^hiNK^neg cells. Transfer of Eomes^hiNK^neg cells into Rag2^-/-Il2rg^-/- recipients generated functional NK cells capable of preventing metastatic disease. By contrast, transfer of PLZF⁺ ILC precursors generated a mixture of ILC1s, ILC2s and ILC3s that lacked cytotoxic potential. These findings identified Eomes^hiNK^neg cells as the bone marrow precursor to classical NK cells and demonstrated that the NK and ILC1 lineages diverged early during development.

Extended Data Fig. 1.. Gating strategies for BM precursors.

a, Gating strategy used for identifying NK1.1⁺Eomes-GFP⁺IL-7Rα⁻ NK cells, NK1.1⁺Eomes-GFP⁻IL-7Rα⁺ ILC1s, NK1.1⁻Eomes-GFP⁺Tcf7-mCherry⁺ Eomes^hiNK^neg cells, Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁻CD90⁻ EILPs, and Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁺CD90⁺PD1⁺ ILCPs among CD4⁻CD8⁻CD3ε⁻TCRβ⁻TCRγδ⁻CD19⁻B220⁻Gr1⁻CD11c⁻CD25⁻Ter119⁻(Lin⁻) cells isolated from the BM of Tcf7^mCherry/+Eomes^GFP/+Zbtb16^hCD4/+Rorc^Thy1.1/+ mouse by flow cytometry. b, Gating strategy used for identifying CD244⁺CD27⁺IL-7Rα⁺Flt3^– preNKP and CD244⁺CD27⁺IL-7Rα⁺Flt3^–CD122⁺ rNKP among CD4⁻CD8⁻CD3ε⁻TCRβ⁻TCRγδ⁻CD19⁻B220⁻Gr1⁻CD11c⁻CD25⁻Ter119⁻(Lin⁻) cells isolated from the BM of Tcf7^mCherry/+Eomes^GFP/+Zbtb16^hCD4/+Rorc^Thy1.1/+ mouse by flow cytometry as in a.

Extended Data Fig. 2.. scRNA-seq annotation and data filtering.

a, UMAP embedding of scRNA-seq data shows a combined total of 31,774 cells from the BM of 35 mice, with cells annotated by cluster (color, index number) if the cluster was removed prior to downstream analysis, and colored gray otherwise, i.e., if the cells were maintained for downstream analysis (e.g., Figs. 1–3, 7). b, Dot plot shows expression (dot color, size, as in Fig. 1b) of genes differentially expressed in annotated clusters (x axis) compared to all other clusters (“Other”) in the dataset. FDR-adjusted P < 0.05; abs. log2FC > 0.5. c, d, UMAP embedding as in a colored by the log₁₀ of the total number of unique molecular identifiers (UMIs), indicating unique transcript molecules €, and by the log₁₀ of the total number of unique genes encoded by the transcripts detected (d). e, f, UMAP embedding of filtered scRNA-seq data (as in Fig. 1a) colored by the log₁₀ of the total number of unique molecular identifiers (UMIs), indicating unique transcript molecules €, and by the log₁₀ of the total number of unique genes encoded by the transcripts detected (f). Points in c-f, corresponding to cells, are plotted in ascending order of their deviation from the median color value, such that the extreme values are displayed at the forefront. g, Violin plots show log of size-normalized expression (Methods) of curated genes in the Rorc⁺ APC cluster (Fig. 1a). Horizontal lines in violins denote the 25^th percentile, median, and 75^th percentile of normalized expression, while white diamonds denote the mean.

Extended Data Fig. 3.. preNKP, rNKP, and aceNKP markers do not identify a transcriptionally distinct population in scRNA-seq data.

a,b, UMAP embeddings (as in Fig. 1a), with cells colored by normalized expression of pre-NKP and rNKP markers (a), and aceNKP markers (b).

Extended Data Fig. 4.. Eomes^hiNK^neg develop independently of PLZF.

a, Quantification of Eomes^hiNK^neg cells in the BM of WT (n=3) and Zbtb16 +18/32^Δ/Δ (n=4) mice. Data are representative of two independent experiments. Data represent mean ± s.e.m. b, Bar graph showing the relative abundance of BM Eomes⁺NK1.1⁺DX5⁺ NK cells and Eomes⁻NK1.1⁺DX5⁻IL-7Rα⁺ ILC1s in WT (n=3) and Zbtb16 +18/32^Δ/Δ (n=4) mice. Data are representative of two independent experiments. Data represent mean ± s.e.m.

Extended Data Fig. 5.. NK cells emerge post-natally.

, UMAP clustering of high dimensional flow cytometry data and corresponding heatmap displaying relative expression DX5, L-selection, Eomes, KLRG1, CD49a, TRAIL, CD69 and CD200r among CD45⁺CD3ε⁻NK1.1⁺NKp46⁺ liver lymphocytes from 1–6 week-old mice. b, bar graph showing the number of DX5⁺CD49a⁻ NK cells (n=5) or DX5⁻CD49a⁺ ILC1s (n=5) among CD45⁺CD3ε⁻NK1.1⁺NKp46⁺ liver lymphocytes of 1–6 week-old mice. Data are representative of three independent experiments. Data represent mean ± s.e.m. **P<0.01, ***P<0.001, ****P < 0.0001. c, bar graph showing the frequency of DX5⁺CD49a⁻ NK cells (n=4), DX5⁻CD49a⁺ ILC1s (n=4) and DX5^loCD49a⁺ undifferentiated cells (n=4) among CD45⁺CD3ε⁻NK1.1⁺NKp46⁺ liver lymphocytes from 1–6 week-old mice. Data are representative of two independent experiments. Data represent mean. d, UMAP clustering of high dimensional flow cytometry data showing the composition of DX5⁺CD49a⁻ NK cells and DX5⁻CD49a⁺ ILC1s among CD45⁺CD3ε⁻NK1.1⁺NKp46⁺ liver lymphocytes from 1–6 week-old mice.

Extended Data Fig 6.. Instability of Eomes and DX5 in vitro.

a, Representative flow cytormetry plot expression of Eomes-GFP and DX5 on Eomes-GFP⁻NK1.1⁺IL-7Rα⁺ ILC1s, α4β7⁺CD244⁺IL-7Rα⁺CD90⁺PD1⁺ ILCPs, Eomes^hiNK^neg cells or Eomes-GFP⁺NK1.1⁺ NK cells on day 7 of co-culture with OP9 cells with IL2, IL7 and SCF. b, Bar graph showing DX5 expression on single NK cell (n=49) and single ILC1 (n=30) on day 7 of co-culture with OP9 cells with IL2, IL7 and SCF. Data represent mean ± s.e.m. c, Representative flow cytometry plot showing the reconstitution of CD3ε⁻CD19⁻NK1.1⁺ NK cells (top) and NK1.1⁻CD90⁺IL-7Rα⁺CD25⁺IL-33Rα⁺ ILC2s (bottom) in the lung of CD45.2/CD45.2 Rag2^−/−IL2rg^−/− mice at week 2 post-intravenous transfer of equal mixes of CD45.1/CD45.2 Eomes^hiNK^neg cells and CD45.2/CD45.2 Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁺CD90⁺PD1⁺ ILCPs.

Extended Data Fig. 7.. Comparison of scRNA-seq trajectory inference results.

a, PAGA graph shows degree of connectivity (line weight) between clusters (dots) from scRNA-seq data (Fig. 1). Thicker lines represent a stronger connection, while dot size scales with number of cells in cluster. b, UMAP embedding (colored by cluster, as in Fig. 1a) shows RNA velocity streamlines (arrows), indicating cellular state transitions inferred by scVelo from all cells (Methods). c, UMAP embedding (as in Fig. 7d), shows an overlaid grid of average RNA velocity vectors (arrows), as an alternative view of the streamline visualization of inferred cellular state transitions in Fig. 7d. d, UMAP embedding shows RNA velocity streamlines (as in b) indicating cellular state transitions inferred by TopicVelo from all cells (Methods).

Extended Data Fig. 8.. Topic-specific cell weights and streamlines from TopicVelo analysis.

a, UMAP embeddings (as in Fig. 1a) of cells, colored by their weight for each of 11 “topics”, or gene programs, inferred (without supervision or prior knowledge) via a probabilistic topic modeling analysis. Titles indicate post hoc topic annotations, determined by literature-based associations with the genes differentially expressed in each topic (Methods, Supplementary Table 3). b, Close-ups of the UMAP embedding (colored by cluster, as in Fig. 1a) show RNA velocity streamlines (arrows) for curated topic-specific cellular state transitions (titles), inferred by TopicVelo from topic-specific cells (displayed in each close-up) and genes, and then integrated to compute the NK/ILC1-focused transition matrix (Fig. 7d, Extended Data Fig. 6c, Methods).

Extended Data Fig 9.. Eomes expression marks the loss of ILC2/3 potential.

a, Representative flow cytometry plots showing PLZF^neg and PLZF^lo Eomes^hiNK^neg cell-derived NK1.1^–ICOS⁺ ILC2/ILC3s, NK1.1⁺ICOS^–TRAIL⁺KLRG1^– ILC1-like cells and NK1.1⁺ICOS^–TRAIL^–KLRG1⁺ NK-like cells on day 7 of co-culture with OP9 cells with IL-2, IL-7 and SCF. b, Representative flow cytometry plots showing Eomes^neg and Eomes^int ILCP cell-derived NK1.1^–ICOS⁺ ILC2/ILC3s, NK1.1⁺ICOS^–TRAIL⁺KLRG1^– ILC1-like cells and NK1.1⁺ICOS^–TRAIL^–KLRG1⁺ NK-like cells on day 7 of co-culture with OP9 cells with IL-2, IL-7 and SCF. c, Representative flow cytometry plot showing the reconstitution of CD3ε⁻CD19⁻NK1.1⁺ NK cells (top) and NK1.1⁻CD90⁺IL-7Rα⁺CD25⁺IL-33Rα⁺ ILC2s (bottom) in the lung of CD45.2/CD45.2 Rag2^−/−γc^−/− mice at week 2 post-intravenous transfer of equal mixes of CD45.1/CD45.2 PLZF^loEomes^hiNK^neg cells and CD45.2/CD45.2 Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁺CD90⁺PD1⁺ ILCPs.

Extended Data Fig 10.. Model of innate lymphocyte development from BM precursors.

Downstream of CLP, Tcf7-expressing EILP gains the expression of an intermediate level of PLZF and develop into helper ILCs and NK cells. A fraction of EILPs upregulate Eomes expression to become NK/ILC1-restricted PLZF^loEomes^hiNK^neg cells which can either further downregulate PLZF on the way to become NK cells or can lose both PLZF and Eomes expression to become ILC1s. Alternatively, a fraction of PLZF^int EILPs can further upregulate PLZF to become ILCPs. These ILCPs can generate all ILC lineages. However, a small subset of ILCPs upregulate Eomes, lose ILC2/3 potential, and can generate NK cells or ILC1s. Created with BioRender.com.

Figure 1.. Identification of Eomes⁺ cells in the mouse BM.

a, UMAP embedding of scRNA-seq data showing 19,961 CD4⁻CD8⁻CD3ε⁻TCRβ⁻TCRγδ⁻CD19⁻B220⁻Gr1⁻CD11c⁻CD25⁻Ter119⁻(Lin⁻) Tcf7-mCherry⁺ cells and NK1.1⁺ cells sorted from the BM of 19 females and 16 males Eomes^GFP/+Tcf7^mCherry/+Zbtb16^hCD4/+Rorc^Thy1.1/+ mice, annotated by cluster (color, index number). b, Dot plot showing the expression of curated genes that are differentially expressed in a cluster relative to all other cells in clusters as in a. FDR-adjusted P < 0.05; absolute value of the log2-fold change (abs. log2FC) > 0.5. Dot color: row z-scored, cluster average of log- and size-normalized gene counts (normalized expression). Dot size, percent of cells in cluster with positive expression of the gene. c, UMAP embeddings, colored by normalized expression of curated differentially expressed genes as in b.

Figure 2.. Eomes^hiNK^neg cells are distinct from NK cells and ILC1s.

a, Dot plot showing expression of select genes differentially expressed in Eomes⁺Tcf7^int, NK, or ILC1 clusters, relative to the other two clusters in scRNA-seq data. FDR-adjusted P < 0.05; abs. log2FC > 0.5. Dot color, size, as in Fig. 1b. b, Representative flow cytometry plots of Eomes-GFP⁺NK1.1⁻NKp46⁻ cells (Eomes^hiNK^neg) and Eomes-GFP⁺NK1.1⁺NKp46⁺ NK cells in BM CD4⁻CD8⁻CD3ε⁻TCRβ⁻TCRγδ⁻CD19⁻B220⁻Gr1⁻CD11c⁻CD25⁻Ter119⁻(Lin⁻) CD45⁺ cells from Eomes^GFP mice. c, Representative histogram showing the expression of NK associated markers KLRG1, CD11b, DX5, NKG2A/C/E, NKG2D, CD244, CD27, perforin, granzyme B, Ly49H, Ly49C/F/I/H, TCF7, T-bet, L-selectin and IL-7Rα on Eomes^hiNK^neg cells or NK cells from the BM of Eomes^GFP/+Tcf7^mCherry/+ mice. Data are pooled from 2 independent experiments.

Figure 3.. Eomes^hiNK^neg cells are distinct from other progenitors.

a, Gating strategy used for the identification of Eomes^hiNK^neg cells, Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁻CD90⁻ EILPs, Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁺CD90⁺PD1⁺ ILCPs in the BM of Tcf7^mCherry/+Eomes^GFP/+Zbtb16^hCD4/+Rorc^Thy1.1/+ mice by flow cytometry. Gated on Lin⁻ cells as shown in Extended Data 1. b, High-dimensional flow cytometry embedding of BM precursors, shown as UMAP with FlowSOM clustering among cell populations selected as in a. c, Number of EILPs (n=7), ILCPs (n=7), Eomes^hiNK^neg cells (n=7), preNKPs (n=6), rNKPs (n=7) isolated from the BM of 4-week-old Tcf7^mCherry/+Eomes^GFP/+Zbtb16^hCD4/+Rorc^Thy1.1/+ mice. Data are pooled from 7 independent experiments, data represent mean ± s.e.m. d, Heatmap showing relative expression of α4β7, PD1, PLZF, CD90, IL-7Rα, Eomes and CD122 in EILPs, ILCPs and Eomes^hiNK^neg clusters as in b. e, Dot plot showing expression of genes differentially expressed in EILP (cluster 0), ILCP (cluster 1) or Eomes⁺Tcf7^int (cluster 4) relative to the other two clusters in scRNA-seq. FDR-adjusted P < 0.05; abs. log2FC > 0.5. Dot color, size, as in Fig. 1b. f, Bar graph showing the frequency of PLZF-fate mapped YFP⁺ fraction in liver GR1⁺ myeloid cells (n=7), spleen CD3ε⁺Cd1d-tetramer⁺ NKT cells (n=7), spleen CD19⁺ B cells (n=7), spleen CD3ε⁺ T cells (n=7), liver CD3ε⁻NK1.1⁺DX5⁻CD49a⁺ ILC1 (n=7), liver CD3ε⁻NK1.1⁺DX5⁺CD49a⁻ NK cells (n=7), BM Eomes^hiNK^neg cells (n=6) in radiation chimeras reconstituted with YFP⁻ Lin⁻ Sca-1⁺ cKit⁺ BM LSK cells from PLZF^GFP-Cre/+ ROSA26^{fl-STOP-fl-YFP/+} mice. Data are pooled from 4 independent experiments. Data represent mean ± s.e.m.

Figure 4.. Eomes^hiNK^neg cells emerge post-natally.

a, Representative flow cytometry plot showing the frequency of Eomes-GFP⁺NK1.1⁻ Eomes^hiNK^neg cells, Eomes-GFP⁺NK1.1⁺ NK cells and Eomes-GFP⁻NK1.1⁺ ILC1s in Lin⁻CD45⁺ lymphocytes isolated from fetal liver at embryonic day 14.5 (E14.5; pooled samples from 9 fetus) and the BM of 4-week-old Tcf7^mCherry/+Eomes^GFP/+ mice. b, Bar graph showing number of Eomes-GFP⁺NK1.1⁻ cells (Eomes^hiNK^neg), Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁺CD90⁺PD1⁺ ILCPs, Eomes-GFP⁺NK1.1⁺ NK cells and Eomes-GFP⁻NK1.1⁺ ILC1s in the E14.5 fetal liver (n=5) or 1–4 week old BM (n=5) of Tcf7^mCherry/+Eomes^GFP/+ mice. Data are representative of two independent experiments. Statistical significance was calculated by two-way ANOVA with Tukey’s multiple comparisons test. Data represent mean ± s.e.m. *P < 0.05. **P < 0.01. ****P < 0.0001.

Figure 5.. Eomes^hiNK^neg cells generate functional NK cells in vitro.

a, Representative flow cytometry plots showing ILCP- and Eomes^hiNK^neg cell-derived NK1.1^–ICOS⁺ ILC2/ILC3s, NK1.1⁺ICOS^–TRAIL⁺KLRG1^– ILC1s and NK1.1⁺ICOS^–TRAIL^–KLRG1⁺ NK cells on day 7 of co-culture with OP9 cells with IL-2, IL-7 and SCF. b, Pie chart showing the average frequency of NK1.1^–ICOS⁺Rorc-Thy1.1^– ILC2s, NK1.1^–Rorc-Thy1.1⁺ ILC3s, NK1.1⁺ICOS^–TRAIL⁺KLRG1^– ILC1s, NK1.1⁺ICOS^–TRAIL^–KLRG1⁺ NK cells and NK1.1⁺ICOS^–TRAIL^–KLRG1^– undifferentiated cells in ILCP or Eomes^hiNK^neg cell-derived progenies at day 7 as in a. Data are representative of three independent experiments. c, Bar graph showing the expression of TRAIL and KLRG1 on NK1.1⁺ICOS^– cells derived from single ILCP (n=37) or Eomes^hiNK^neg cells (n=19) at day 7 as in a. d, Intracellular staining of Tbet and perforin in ILCP- or Eomes^hiNK^neg cell-derived NK1.1⁺ICOS^–Nkp46⁺ cells at day 7 of culture as in a. e, Percentage of efficiency of killing of YAC1 tumor cells cultured at 1:1 ratio with NK1.1⁺ICOS^– cells derived from Eomes-GFP⁻NK1.1⁺IL-7Rα⁺ ILC1s (n=3), α4β7⁺CD244⁺IL-7Rα⁺CD90⁺PD1⁺ ILCPs (n=3), Eomes^hiNK^neg cells (n=3) or Eomes-GFP⁺NK1.1⁺ NK cells (n=3) sorted from the BM of Eomes^GFP mice, co-cultured with OP9 cells with IL-2, IL-7, SCF for 6 days and stimulated with IL-15 overnight. Statistical significance was calculated by two-way ANOVA with Tukey’s multiple comparisons test. Data represent mean ± s.e.m. ***P<0.001, ****P < 0.0001

Figure 6.. Eomes^hiNK^neg cells are precursors committed to the NK cell lineage.

a, Representative flow cytometry of DX5⁻CD49a⁺ ILC1s and DX5⁺CD49a⁻KLRG1⁺Ly49H⁺ NK cells within the CD3ε⁻TCRb⁻NK1.1⁺NKp46⁺ cell compartment in the liver of CD45.2/CD45.2 Rag2^−/−IL2rg^−/− mice intravenously transferred with equal mixes of CD45.1/CD45.2 Eomes^hiNK^neg cells and CD45.2/CD45.2 Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁻CD90⁻ EILPs, Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁺CD90⁺PD1⁺ ILCPs, CD244⁺CD27⁺IL7R⁺Flt3^–CD122^– pre-NKPs and CD244⁺CD27⁺IL-7Rα⁺Flt3^–CD122⁺ rNKPs at week 2 post-transfer. b, Frequency of reconstituted DX5⁻CD49a⁺ ILC1s and DX5⁺CD49a⁻ NK cells in the liver of Rag2^−/−IL2rg^−/− mice transferred with EILPs (n=6), ILCPs (n=6), Eomes^hiNK^neg cells (n=8), preNKPs (n=4) and rNKPs (n=4) as in a, at week 2 post-transfer. Data represent mean ± s.e.m. ***P = 0.0008, ****P < 0.0001. Data are representative of five independent experiments. c, Relative contribution of CD45.1/CD45.2 Eomes^hiNK^neg cells and CD45.2/CD45.2 rNKPs to the reconstitution of CD3ε⁻NK1.1⁺NKp46⁺DX5⁺CD49a⁻ NK cells in the liver (n=5), spleen (n=5) and lung (n=3) of Rag2^−/−IL2rg^−/− mice transferred with an equal mix of precursor cells as in a, at week 2 post-transfer. Data are representative of two independent experiments. Data represent mean ± s.e.m. ****P < 0.0001. d, Relative contribution of CD45.1/CD45.2 Eomes^hiNK^neg cells and CD45.2/CD45.2 ILCPs to CD3ε⁻NK1.1⁺NKp46⁺DX5⁺CD49a⁻ NK cells in the liver (n=6), spleen (n=6) and lung (n=6) of Rag2^−/−IL2rg^−/− mice transferred with an equal mix of precursor cells as in a, at week 2 post-transfer. Data are representative of three independent experiments. Data represent mean ± s.e.m. ****P < 0.0001.

Figure 7.. PLZF⁺Eomes⁺ cells have both ILC1 and NK potential.

a, Representative flow cytometry showing PD1 by PLZF staining (left) and histogram of PLZF expression (right) on Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁺CD90⁺PD1⁺ ILCPs, Eomes^hiNK^neg cells and Tcf7-mCherry⁺α4β7⁺CD244⁺IL-7Rα⁻CD90⁻ EILPs from the BM of Tcf7^mCherry/+Eomes^GFP/+Zbtb16^hCD4/+Rorc^Thy1.1/+ mice. b, Representative flow cytometry plot of PLZF and Eomes expression in ILCPs as in a. c, UMAP embedding of scRNA-seq data as in Fig. 1a, with cells colored by normalized expression of Eomes, Zbtb16, and Pdcd1. d, Close-up of UMAP embedding colored by cluster, as in Fig. 1a, focused on cells with relatively high expression of gene programs relating to NK, ILC1, ILCP, and Eomes⁺Tcf7^int populations (Methods). Arrows, overlaid RNA velocity streamlines indicating the cellular state transitions inferred by TopicVelo from the displayed cells (Methods, Supplementary Fig. 2,3). Arrows are colored gray over very low density UMAP regions and otherwise black. e, Representative flow cytometry plot of PD1 and PLZF expression on PLZF^loEomes^hiNK^neg cells isolated from BM of Tcf7^mCherry/+Eomes^GFP/+Zbtb16^hCD4/+Rorc^Thy1.1/+ mice and cultured on OP9 cells with IL-2, IL-7 and SCF at 24 and 48 hours of co-culture. f, Representative flow cytometry plot (left) and quantification (right) of CD3ε⁻NK1.1⁺DX5⁻CD49a⁺ ILC1s and CD3ε⁻NK1.1⁺DX5⁺CD49a⁻ NK cells in the liver of CD45.2/CD45.2 Rag2^−/−IL2rg^−/− mice transferred with equal mixes of CD45.1/CD45.2 PLZF^loEomes^hiNK^neg or PLZF^negEomes^hiNK^neg cells and CD45.2/CD45.2 ILCPs, at week 2 post transfer (n=3). Data represent mean ± s.e.m. g, Representative flow cytometry plot (left) and quantification (right) of CD3ε⁻NK1.1⁺DX5⁻CD49a⁺ ILC1s and CD3ε⁻NK1.1⁺DX5⁺CD49a⁻ NK cells in the liver of CD45.2/CD45.2 Rag2^−/−IL2rg^−/− mice transferred with equal mixes of CD45.1/CD45.2 Eomes^int ILCPs and CD45.2/CD45.2 Eomes^neg ILCPs at week 2 post transfer (n=3). Data are representative of three independent experiments. Data represent mean ± s.e.m.

Figure 8.. Eomes^hiNK^neg cells give rise to functional NK cells in vivo.

a, Intracellular ex vivo staining of TNF, GM-CSF and IFNγ after PMA and ionomycin stimulation for 4 hours of CD3ε⁻CD19⁻NK1.1⁺NKp46⁺ cells isolated from the liver of Rag2^−/−IL2rg^−/− mice transferred with ILCPs or Eomes^hiNK^neg cells 2 weeks before analysis or liver CD3ε⁻NK1.1⁺DX5⁻CD49a⁺ ILC1 and CD3ε⁻NK1.1⁺DX5⁺CD49a⁻ NK cells isolated from age-matched Eomes^GFP mice as controls. b, Degranulation (measured as percentage of CD107a expression) of CD45.1/CD45.2 CD3ε⁻NK1.1⁺ NK cells isolated from the spleen of CD45.2/CD45.2 Rag2^−/−IL2rg^−/− mice at week 2 post-intravenous injection with CD45.1/CD45.2 Eomes^hiNK^neg cells and 18 hours post-priming with 200 μg of polyinosinic:polycytidylic acid and incubated ex vivo with YAC1 tumor cells at a ratio of 1:1 for 4h. Data are representative of three independent experiments. Data represent mean ± s.e.m. ****P < 0.0001. c, Quantification metastases in the lung right inferior lobe of Rag2^−/−IL2rg^−/− mice at day 12 post-intravenous injection with 50,000 B16-F10 cells in Rag2^−/−IL2rg^−/− mice were intravenously transferred with PBS control (n=5), ~800 ILCPs (n=3) or Eomes^hiNK^neg cells (n=4) 10 days before the transfer of tumor cells. Data are representative of three independent experiments. Statistical significance was calculated by two-way ANOVA with Tukey’s multiple comparisons test. Data represent mean ± s.e.m. ****P < 0.0001.