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Distinct developmental pathways generate functionally distinct populations of natural killer cells

An Author Correction to this article was published on 10 July 2024

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Abstract

Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H+ NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56dim and CD56bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans.

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Fig. 1: Early NK cell progenitors can be identified in the BM.
Fig. 2: ENKPs are early NK-lineage-biased progenitors.
Fig. 3: ENKPs develop independently of ILCPs.
Fig. 4: ENKP and ILCP give rise to distinct subsets of NK cells.
Fig. 5: ENKP_NK cells respond to MCMV infection.
Fig. 6: ENKP_NK cells and ILCP_NK cells are functionally distinct.

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Data availability

scRNA-seq data are available from the NCBI Gene Expression Omnibus repository (accession number: GSE266109).

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Acknowledgements

We thank V. Lazarevic and C. Spinner for help with acquiring mouse strains and V. Shapiro for critical comments on the paper. We also thank the staff of the NCI CCR Single Cell Analysis Facility for sequencing BM and spleen samples and the staff of the CCR/NCI flow cytometry core for cell sorting. This work was supported by the Division of Intramural Research of the NCI, NIAID and NHLBI, ANR JCJC AAPG2022: ANR-22-CE15-0040-01 (C.H.).

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Authors

Contributions

A.B. and Y.D. conceptualized the study. Y.D., M.L., S.G., V.I.B., L.C., S.C.S. and J.M. were responsible for the methodology. Y.D., M.L., S.G., V.I.B., L.C., A.D., S.D., C.H., S.C.S., J.M., D.P., Y.Z., J.Z., Y.B. and J.C.S. performed the investigations. Y.D. and M.L. produced the visualizations. A.B. supervised the study. Y.D., M.L., V.I.B., L.C. and A.B. wrote the paper.

Corresponding author

Correspondence to Avinash Bhandoola.

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The authors declare no competing interests.

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Nature Immunology thanks Niklas Björkström and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ioana Staicu, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Characterization of early NK cell development in BM.

a, Gating strategy for sorting ALP, EILP, ILCP, ILC2P, CD122+ progenitor, NK cell and ILC1 for scRNA-seq as in Fig. 1a. b, UMAP of scRNA-seq samples grouped by original idents. c, Feature plots of genes critical for identifying UMAP clusters. d, Violin plots of Klrc1, Klrc2 and Klrc3 in ENKP and NK cluster. e, Slingshot pseudotime and heatmap of gene expression ordered by pseudotime. For b - e, same scRNA-seq data as in Fig. 1a.

Extended Data Fig. 2 Characterization of ENKPs.

a, Representative flow plots of ENKPs (left) and quantification of ENKPs and NK cells in the BM of Tcf7-/- mice (n = 3) and Tcf7+/+ mice (n = 5). Data are combined data from 2 independent experiments. b, Representative flow plots of ENKPs (left) and quantification of ENKPs and NK cells in the BM of VAVCreGata3fl/fl mice (n = 5) and Gata3fl/fl mice (n = 5). Data are combined data from 2 independent experiments. c, Histograms of expression of α4β7, Thy1.2, IL7Ra, Kit, CD27, 2B4 and Flt3 by ALP, EILP, ILCP and ENKP in the BM of wild-type C57BL/6 mice. Representative data from 2 independent experiments. d, Quantifications of IL-7RA-YFP+ frequency in ENKPs and NK cells in the BM of IL-7RACreRosa26-YFP reporter mice (n = 5 mice). Representative data from 3 independent experiments. For a, b and d, data are mean ± s.e.m. For a and b, statistical analysis used two-tailed unpaired Student’s t-tests.

Extended Data Fig. 3 Assessment of lineage potential of ENKPs.

a, Flow plots of CD3CD19NKp46+NK1.1+CD49aDX5+EomesGFP+ NK cells and CD3CD19NKp46+NK1.1+CD49a+DX5 EomesGFP- ILC1s sorted from the spleen and liver of EomesGFP reporter mice at day 7 post-culture on OP9 stromal cells with SCF, Flt3L, IL-7 and IL-2. Representative data from 2 independent experiments. b, Representative flow plots of in vitro clonal assay as in Fig. 2c. c, Quantification of NK1.1+EomesGFP+CD220RNK cells at day 7 post-culture of ENKPs (n = 4 wells), ILCPs (n = 4 wells) sorted from the BM (ENKPs and ILCPs) of EomesGFPTcf7YFP reporter mice, or CD3CD19NKp46+NK1.1+CD49aDX5+EomesGFP+ mature NK cells (n = 4 wells) sorted from the spleen of EomesGFP reporter mice on OP9 stromal cells with SCF, Flt3L, IL-7 and IL-2. Representative data from 2 independent experiments. d, Quantification of NK1.1+EomesGFP+CD220RNK cells at day 7 post-culture of ENKPs or ILCPs sorted from the BM of EomesGFPTcf7YFP reporter mice on OP9 stromal cells with SCF, Flt3L, IL-7 and IL-2, comparing with (n = 4 wells) and without Flt3L (n = 4 wells). Representative data from 2 independent experiments. e, Donor chimerism within CD45.2+CD3CD19NK1.1+NKp46+DX5+CD49a NK cells in the liver of CD45.1 NSG mice adoptively co-transferred with CD45.2+ ALP (n = 5), ENKP (n = 5) or ILCP (n = 4) sorted from the BM of wild-type C57BL/6 (CD45.2) mice mixed with an equal number of CD45.1+ ALPs as competitors sorted from the BM of B6-Ly5.2 (CD45.1) mice at week 6 post-transfer. Data are combined from 2 independent experiments. f - h, Representative flow plots of post-sort purity check for ENKP (f), ILCP (g) and ALP (h). i, Quantification of NK cells and ILC1s in the spleen of NSG mice transferred with ENKPs (n = 5) or LinKit+PD-1+α4β7+Flt3Thy1.2+ ILCPs (n = 9) sorted from the BM of IL-7RACreEomesfl/fl mice or IL-7RACreEomes+/+ mice at week 6 post-transfer. Data are combined from 2 independent experiments. For c, d, e, i, data are mean ± s.e.m., and statistical analysis used two-tailed unpaired Student’s t-tests, without any adjustment for multiple comparisons.

Extended Data Fig. 4 ENKP_NK cells and ILCP_NK cells are transcriptionally different.

a, Volcano plot of differentially expressed genes between ENKP_NK cells and ILCP_NK cells. b, Heatmap showing relative expression of genes equally expressed (upper), more highly expressed in ENKP_NK cells (middle) and more highly expressed ILCP_NK (lower). Each column represents one cell. c, Violin plots of genes expressed higher in ENKP_NK (upper) and higher in ILCP_NK (lower) grouped by developmental origins. Data are shown as z-score. d, GO enrichment analysis using genes expressed higher in ENKP_NK cells (upper) or ILCP_NK cells (lower). e, Hierarchically clustered heatmap showing relative gene expression levels of genes differentially expressed between ENKP_NK cells and ILCP_NK cells in ALP_NK cells. Each column represents one cell. Dashed line indicates separation between two major column clusters. Data are shown as z-score. f, UMAP (upper) and stacked bar plot of scRNA-seq of ALP_NK cells, ENKP_NK cells and ILCP_NK cells. g, Volcano plot of differentially expressed genes between cluster_0 and cluster_1 in f. h, Volcano plot of differentially expressed genes between ENKP_NK cells and ILCP_NK cells in cluster_0 (left) and in cluster_1 (right). Arrow highlights the gene “Klra8”. i, Hierarchically clustered Euclidean distance matrix of transcriptomes of scRNA-seq of NK cells and ILC1s in BM and liver (right). j, Heatmap of select genes (see Methods) differentially expressed between NK cells and ILC1s in ENKP_NK cells, ILCP_NK cells and ILC1s in BM (left) and liver (right). For a - h, same scRNA-seq data as in Fig. 4a. For i, j, BM data is from scRNA-seq in Fig. 1a, liver data is from ref. 28, CD45+NK1.1+CD3ε-HobittdTom+ ILC1 and CD45+NK1.1+CD3ε-HobittdTom-CXCR6DX5+ NK cells were sorted from the liver of HobitTom/WT mice at steady-state.

Extended Data Fig. 5 ENKPs and ILCPs give rise to distinct NK cells.

a, Donor chimerism within CD45.2+Ly49H+ NK cells in the spleen of CD45.1 NSG mice adoptively co-transferred with CD45.2+ ALP (n = 8), ENKP (n = 9) or ILCP (n = 8) sorted from the BM of wild-type C57BL/6 (CD45.2) mice mixed with an equal number of CD45.1+ ALPs as competitors sorted from the BM of B6-Ly5.2 (CD45.1) mice at week 6 post-transfer. Data are combined from 2 independent experiments. b, Representative flow plots (left) and quantification of Ly49H+.frequency in ENKPs (n = 5 mice) and NK cells (n = 5 mice) in the BM of wild-type C57BL/6 mice. Data are combined from 2 independent experiments. c, Quantification of Ly49 receptor positive frequencies in ENKP_NK cells and ILCP_NK cells in the spleen of NSG mice transferred with ENKPs (n = 10) or ILCPs (n = 10) sorted from the BM of Tcf7YFP reporter mice at week 6 post-transfer. Data are combined from 2 independent experiments. d, Representative histogram of TCF1-YFP expression (left) and quantification of TCF1-YFP+ frequency in ENKP_NK cells and ILCP_NK cells in the spleen of NSG mice transferred with ENKPs (n = 10) or ILCPs (n = 10) sorted from the BM of Tcf7YFP reporter mice at week 6 post-transfer. Data are combined from 2 independent experiments. e, Violin plots of Tcf7 from scRNA-seq of ALP_NK cells, ENKP_NK cells and ILCP_NK cells, same scRNA-seq data as in Fig. 4a. f, Quantification of PLZF-YFP+ frequency in total NK cells, Ly49H+ NK cells, Ly49H NK cells in the spleen (n = 7), liver (n = 7), lung (n = 7), mesenteric lymph nodes (mLN) (n = 7) and thymus (n = 7), PLZF-YFP+ frequency in ILC1s in the liver (n = 7) and mLN (n = 7), and PLZF-YFP+ frequency in B cells in the spleen (n = 7), liver (n = 7), lung (n = 7) and mLN (n = 7) from PLZF lineage-tracing chimeras generated in B6-Ly5.2 mice at week 12-16 post-transfer. Data are combined from 2 independent experiments. B cells were used as negative control for PLZF labeling. g, Quantification of Ly49H+ frequency in NK cells in in the spleen (n = 7), liver (n = 7), lung (n = 7), mLN (n = 7) and thymus (n = 7) from PLZF lineage-tracing chimeras generated in B6-Ly5.2 mice at week 12-16 post-transfer. Data are combined from 2 independent experiments. h - i, Quantification of PLZF-YFP+ frequency (h) and Ly49H+ frequency in NK cells (i) in the spleen (n = 7), salivary glands (SG) (n = 7), uterus (n = 6) and BM (n = 6) from PLZF lineage-tracing chimeras generated in NSG mice at week 12-16 post-transfer. Data are combined from 3 independent experiments. For a-d, f - i, data are mean ± s.e.m. For a,c,d, f - i, statistical analysis used two-tailed unpaired Student’s t-tests, without any adjustment for multiple comparisons.

Extended Data Fig. 6 ENKP_NK cells and ILCP_NK cells are similar to human CD56 dim and CD56 bright NK cells, respectively.

a. Feature plots of surface protein expression of CD56 and CD57 in human blood NK measured by CITE-seq. b. UMAP of human blood NK cells analyzed by scRNA-seq. Annotation defined by surface protein expression of CD56 and CD57 measured by CITE-seq. c. Feature plots of KIR2DL1, KIR2DL2 and KIR2DL3 in human blood NK cells analyzed by scRNA-seq. d. Heatmap of Spearman correlation analysis between mouse NK clusters (cluster_0 and cluster_1) (same scRNA-seq data as in Fig. 4a) and human NK clusters (CD56bright NK cells, CD56dimCD57 NK cells and CD56dimCD57+ NK cells). e. Heatmap of Spearman correlation analysis between mouse NK developmental originals (ENKP_NK cells and ILCP_NK cells) and human NK clusters (CD56bright NK cells, CD56dimCD57 NK cells and CD56dimCD57+ NK cells). For a - e, data were integrated from ref. 31,32, same scRNA-seq and CITE-seq data as in Fig. 4d.

Extended Data Fig. 7 ENKP_NK cells respond to MCMV infection.

a, Gating strategy used for the identification of ENKP_NK cells and ILCP_NK cells in the blood of NSG mice adoptively transferred with ILCPs or ENKPs sorted from the bone marrow of Tcf7YFP reporter mice. At week 5 post-transfer, these NSG mice were infected with MCMV by intraperitoneal injection, and CD45.2+CD3CD19NK1.1+NKp46+DX5+CD49aNK cells from the blood were analyzed for Ly49H expression at day 7 post-infection. b, Quantification of Ly49H+ NK cells and Ly49H NK cells among ENKP_NK cells and ILCP_NK cells in the blood of NSG mice at week 6 post-transfer of ENKPs (n = 5) or ILCPs (n = 5) in the absence of MCMV infection. c, Quantification of Ly49H+ NK cells and Ly49H NK cells among ENKP_NK cells (n = 8 mice) and ILCP_NK cells (n = 8 mice) in the blood of NSG mice established and infected with MCMV as in a. For b and c, 300 ENKPs or ILCPs were transferred into each NSG mice. d, Donor chimerism of ENKP_NK cells and ILCP_NK cells in the spleen of NSG mice co-transferred with ENKPs and ILCPs (ENKPs:ILCPs=1:4, n = 9,) sorted from the BM of wild-type C57BL/6 mice (CD45.2) or B6-Ly5.2 mice (CD45.1). At week 5 post-transfer, these NSG mice were infected with MCMV by intraperitoneal injection, and NK cells from the spleen were analyzed at day 7 post-infection. ENKP_NK cells and ILCP_NK cells were distinguished using congenic markers CD45.1 vs CD45.2. Data are combined from 2 independent experiments. For b - d, data are mean ± s.e.m.

Extended Data Fig. 8 Verification of ENKP_NK and ILCP_NK-enriched cell population.

a, Representative histograms (left) and quantification of frequencies of cells expressing CD226, CXCR3 or Thy1.2 in the spleen of NSG mice transferred with ENKPs (n = 10) or ILCPs (n = 10) sorted from the BM of Tcf7YFP reporter mice at week 6 post-transfer. Data are combined from 2 independent experiments. b, Representative histograms (left) and quantification of TCF1-YFP+ frequency in Ly49H+ and/or Ly49D+ NK cells (n = 5), Ly49HLy49DCD226+Thy1.2+ NK cells (n = 5), Ly49HLy49DCD226+CXCR3+ NK cells (n = 5) in the spleen from Tcf7YFP reporter mice. Data are combined from 2 independent experiments. n = 5. c - d, Quantification of PLZF-YFP+ frequency (c) and numbers (d) in Ly49H+ and/or Ly49D+ NK cells, Ly49HLy49DCD226+Thy1.2+ NK cells, Ly49HLy49DCD226+CXCR3+ NK cells (c,d), and total NK cells and Ly49HLy49D NK cells (d) in the spleen from PLZF lineage-tracing chimeras generated in NSG mice left uninfected (n = 10) or on day 7 post-infection with MCMV (n = 5). Data are combined from 2 independent experiments. e, Quantification of total NK cells, Ly49H+ and/or Ly49D+ NK cells, Ly49HLy49DCD226+Thy1.2+ NK cells, Ly49HLy49DCD226+CXCR3+ NK cells and Ly49HLy49D NK cells in the spleen of wild-type C57BL/6 mice left uninfected (n = 5) or on day 7 post-infection with MCMV (n = 5). Data are combined from 2 independent experiments. f, Quantification of total NK cells, Ly49H+ and/or Ly49D+ NK cells, Ly49HLy49DCD226+Thy1.2+ NK cells, Ly49HLy49DCD226+CXCR3+ NK cells and ILC1s in the spleen of IL-7RACreEomesfl/fl mice (n = 6) and IL-7RACreEomes+/+ mice (n = 5). Dat are combined from 2 independent experiments. g, Quantification of total NK cells, Ly49H+ and/or Ly49D+ NK cells, Ly49HLy49DCD226+Thy1.2+ NK cells and Ly49HLy49DCD226+CXCR3+ NK cells in the spleen of Zbtb16GFPcre/GFPcre mice (n = 6) and Zbtb16+/+ mice (n = 5). Data are combined from 2 independent experiments. h, Quantification of CD19TCRβ+CD1d+ NKT cells in the spleen from Zbtb16GFPcre/GFPcre mice (n = 3) and Zbtb16+/+ mice (n = 3). Data are combined from 2 independent experiments. n = 3. For a - h, data are mean ± s.e.m., and statistical analysis used two-tailed unpaired Student’s t-tests, without any adjustment for multiple comparisons.

Extended Data Fig. 9 Distinguish ENKP_NK cells and ILCP_NK cells by surface markers.

a, Representative flow plots (left) and quantification of frequencies of CD27+CD11b NK cells, CD27+CD11b+ NK cells and CD27+CD11b+ NK cells among ENKP_NK cells and ILCP_NK cells in the spleen of NSG mice transferred with ENKPs (n = 10) or ILCPs (n = 10) sorted from the BM of Tcf7YFP reporter mice at week 6 post-transfer. Data are combined from 2 independent experiments. b, Quantification of PLZF-YFP+ frequency in CD45.2+CD27+CD11b NK cells, CD45.2+CD27+CD11b+ NK cells and CD45.2+CD27+CD11b+ NK cells from PLZF lineage-tracing chimeras generated in NSG mice (n = 6) at week 12-16 post-transfer. Data are combined from 2 independent experiments. c, Representative histograms (left) and quantification of frequencies of cells expressing KLRG1 or CD62L in ENKP_NK cells and ILCP_NK cells in the spleen of NSG mice transferred with ENKPs (n = 10) or ILCPs (n = 10) sorted from the BM of Tcf7YFP reporter mice at week 6 post-transfer. Data are combined from 2 independent experiments. d - e, Quantification of PLZF-YFP+ frequency in CD45.2+CD11bKlRG1 NK cells and CD45.2+CD11b+KlRG1+ NK cells (d), and CD45.2+ total NK cells and CD45.2+Ly49H+CD62LCD27+ NK cells (e) in the spleen of PLZF lineage-tracing chimeras generated in NSG mice (n = 6) at week 12-16 post-transfer. Data are combined from 2 independent experiments. For a - e, data are mean ± s.e.m., and statistical analysis used two-tailed unpaired Student’s t-tests, without any adjustment for multiple comparisons.

Extended Data Fig. 10 Characterization of functions of NK subsets.

a, Representative flow plots (left) and quantification of frequencies of cells expressing IFN-γ, TNF or CD107a in ENKP_NK-enriched cells (n = 4 wells) and ILCP_NK-enriched cells (n = 4 wells) sorted from the spleen of wild-type C57BL/6 mice and simulated in vitro with PMA+ionomycin. Representative data from 2 independent experiments. b, Quantification of specific lysis ratios of Yac-1 cells co-cultured with ENKP_NK-enriched cells (n = 4 wells) and ILCP_NK-enriched cells (n = 4 wells) sorted from the spleen of wild-type C57BL/6 mice. Representative data from 2 independent experiments. c, Histogram of IFN-γ expression in ENKP_NK cells and ILCP_NK cells at day 7 post-culture of ENKPs or ILCPs sorted from the BM of EomesGFPTcf7YFP reporter mice on OP9 stromal cells with SCF, Flt3L, IL-7 and IL-2 and stimulation with IL-12 + IL18 or PMA+ionomycin. Representative histograms from 2 independent experiments. d - e, Representative flow plots of gating (d) and IFN-γ expression (e) in ENKP_NK-enriched cells, ILCP_NK-enriched cells and CD3CD19NK1.1+NKp46+DX5CD226+ ILC1s in the cecum lamina propria of wild-type C57BL/6 mice at day 1, day 3 and day 5 post-infection with Salmonella, or left uninfected. f - g, Representative flow plots of gating (f) and IFN-γ expression (g) of ENKP_NK-enriched cells, ILCP_NK-enriched cells and CD3CD19NK1.1+NKp46+DX5CD226+ ILC1s in the ear skin of wild-type C57BL/6 mice at day 3 and day 7 post-infection with HSV, or left uninfected. For a, b, e, g, ENKP_NK-enriched cells are Ly49H+ and/or Ly49D+ NK cells. For a, b, ILCP_NK-enriched cells are Ly49HLy49DCD226+(Thy1.2+ and/or CXCR3+) NK cells. For e, g, ILCP_NK-enriched cells are Ly49HLy49DCD226+Thy1.2+ NK cells. For a, b, data are mean ± s.e.m, and statistical analysis used two-tailed unpaired Student’s t-tests, without any adjustment for multiple comparisons.

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Ding, Y., Lavaert, M., Grassmann, S. et al. Distinct developmental pathways generate functionally distinct populations of natural killer cells. Nat Immunol 25, 1183–1192 (2024). https://doi.org/10.1038/s41590-024-01865-2

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