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ILC1s control leukemia stem cell fate and limit development of AML

Nature Immunology volume 23, pages 718–730 (2022)Cite this article

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A Publisher Correction to this article was published on 15 June 2022

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Abstract

Type I innate lymphoid cells (ILC1s) are critical regulators of inflammation and immunity in mammalian tissues. However, their function in cancer is mostly undefined. Here, we show that a high density of ILC1s induces leukemia stem cell (LSC) apoptosis. At a lower density, ILC1s prevent LSCs from differentiating into leukemia progenitors and promote their differentiation into non-leukemic cells, thus blocking the production of terminal myeloid blasts. All of these effects, which require ILC1s to produce interferon-γ after cell–cell contact with LSCs, converge to suppress leukemogenesis in vivo. Conversely, the antileukemia potential of ILC1s wanes when JAK–STAT or PI3K–AKT signaling is inhibited. The relevant antileukemic properties of ILC1s are also functional in healthy individuals and impaired in individuals with acute myeloid leukemia (AML). Collectively, these findings identify ILC1s as anticancer immune cells that might be suitable for AML immunotherapy and provide a potential strategy to treat AML and prevent relapse of the disease.

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Fig. 1: ILC1s induce apoptosis in LSCs.
Fig. 2: ILC1-secreted IFNγ controls the direction of LSC differentiation.
Fig. 3: ILC1s inhibit the differentiation of LSCs into myeloid blasts.
Fig. 4: ILC1s and IFNγ improve survival of leukemic mice.
Fig. 5: ILC1s produce more IFNγ than NK cells when interacting with LSCs.
Fig. 6: ILC1-derived IFNγ inhibits LSC differentiation via JAK–STAT and PI3K–AKT signaling.
Fig. 7: ILC1s are functionally impaired in AML.

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Data availability

Sequencing datasets are accessible from the Gene Expression Omnibus (GEO) under accession number GSE198783. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (CA210087, CA265095 and CA163205 to M.A.C.; NS106170, AI129582, CA247550, CA264512 and CA223400 to J.Y.) and the Leukemia and Lymphoma Society (1364-19 to J.Y.). Research reported in this publication also included work performed in the Hematopoietic Tissue Biorepository Core supported by the National Cancer Institute of the National Institutes of Health under grant number P30CA033572. We appreciate S.T. Wilkinson and L. Sage for editing the manuscript. Images were created with BioRender.com and Adobe Photoshop.

Author information

Author notes

  1. These authors contributed equally: Zhenlong Li, Rui Ma, Shoubao Ma.

Authors and Affiliations

  1. Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center, Los Angeles, CA, USA

    Zhenlong Li, Rui Ma, Shoubao Ma, Lei Tian, Ting Lu, Michael A. Caligiuri & Jianhua Yu

  2. Hematologic Malignancies Research Institute, City of Hope National Medical Center, Los Angeles, CA, USA

    Zhenlong Li, Rui Ma, Shoubao Ma, Lei Tian, Ting Lu, Michael A. Caligiuri & Jianhua Yu

  3. Department of Computational and Quantitative Medicine, City of Hope National Medical Center, Los Angeles, CA, USA

    Jianying Zhang

  4. Division of Hematology, Department of Internal Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA

    Bethany L. Mundy-Bosse

  5. Gehr Family Center for Leukemia Research, Department of Hematological Malignancies Translational Science, City of Hope National Medical Center, Los Angeles, CA, USA

    Bin Zhang & Guido Marcucci

  6. City of Hope Comprehensive Cancer Center, Los Angeles, CA, USA

    Michael A. Caligiuri & Jianhua Yu

  7. Department of Immuno-Oncology, City of Hope, Los Angeles, CA, USA

    Jianhua Yu

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Contributions

J.Y. and M.A.C. conceived and designed the study. Z.L., R.M., S.M., L.T. and T.L developed the methodology. Z.L., R.M., S.M., L.T., T.L., B.L.M.-B., B.Z. and G.M. acquired the data (for example, provided animals, acquired and managed donors, provided facilities and so on). Z.L., J.Y. and J.Z. analyzed and interpreted data (for example, statistical analysis, biostatistics and computational analysis). Z.L., R.M., S.M., J.Y. and M.A.C. wrote, reviewed and/or revised the manuscript. J.Z. provided administrative, technical or material support (for example, reporting or organizing data and constructing databases). J.Y. and M.A.C. supervised the study and acquired funding. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Michael A. Caligiuri or Jianhua Yu.

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Nature Immunology thanks Eric Vivier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: N. Bernard and Z. Fehervari, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 ILC1s are functionally impaired in mice with AML.

a, 0.2×106 LSKs isolated from the liver of healthy mice or MllPTD/WT: Flt3ITD/ITD mice with AML were i.v. injected into immunodeficient Rag2−/−γc−/− mice. The survival of those mice was analyzed by the Kaplan-Meier method and log-rank test (n = 3 individual mice). b, LSCs were isolated from the spleen of MllPTD/WT: Flt3ITD/ITD mice with AML (CD45.2) and then transplanted into lethally irradiated mice (CD45.1). The percentages of LSCs in the liver of the CD45.1 mice were analyzed 9 weeks after LSC transplantation using flow cytometry. c, Gating strategy for flow cytometry analysis of the mouse ILC1s isolated from livers. The mouse ILC1s were defined as CD3−CD19−NK1.1+NKp46+CD49b−CD49a+. d-f, 2×106 C1498 cells were i.v. injected into C57BL/6 J mice. Twenty-one days later, the production of IFN-γ and TNF by ILC1s from the liver (d), bone marrow (e), and spleen (f) of those healthy mice or mice with AML are shown (n = 5 individual mice). g, GSEA plot shows the relative abundance of genes involved in the TNF–NF-κB signaling pathways in liver ILC1s isolated from mice with AML or healthy mice (n = 3 individual mice). Data are representative of two (a, b, d, e, and f) independent experiments. Data (d-f) are shown as mean ± s.d. and are assessed by unpaired two-tailed Student’s t test. NS, not significant.

Source data

Extended Data Fig. 2 Cell purity and gating strategy for flow cytometry analysis.

a,b, Purity of LSCs (a) and ILC1s (b) after cell sorting. c, Gating strategy for flow cytometric analysis of apoptosis of LSCs co-cultured with or without ILC1s, using 7-AAD. CTV: CellTrace™ Violet. d, Gating strategy for flow cytometry analysis of apoptosis of LSCs cocultured with ILC1s using the Violet Live Cell Caspase Probe. e, Gating strategy for flow cytometry analysis of human ILC1s isolated from peripheral blood. Lineage markers: CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD34, CD203c, and FceRI. Human ILC1s were defined as Lin−CD56−CD127+c-Kit−CRTH2−. f, Gating strategy for flow cytometry analysis of human LSCs. Lineage markers: CD2, CD3, CD4, CD8, CD14, CD16, CD19, Mac-1, CD56, and CD235a. Human LSCs were defined as Lin−CD45dimCD34+CD38−.

Extended Data Fig. 3 IFN-γ—but not TNF—induces apoptosis of LSCs.

a, 5,000–10,000 mouse liver ILC1s were sorted and transferred into the top wells of a 96-well Transwell plate. The bottom chambers of the plate were loaded with 10,000–20,000 LSCs from the spleens of MllPTD/WT: Flt3ITD/ITD mice with AML. The cells were then cocultured for 3 days. The percentages of LSCs that were apoptotic were measured by flow cytometry (n = 3 individual mice). b,c, LSCs from the spleen of MllPTD/WT: Flt3ITD/ITD mice with AML were treated with or without the indicated doses of IFN-γ or TNF for 3 days. Representative images (top, 5× magnification, scale bar 200 µm) and flow cytometry plots (bottom) of the percentages of apoptotic cells in LSCs are shown. Data in a are shown as mean ± s.d. and are assessed by one-way ANOVA models. Data in a, b, and c are representative of two independent experiments. NS, not significant.

Source data

Extended Data Fig. 4 ILC1s and IFN-γ transform the differentiation of LSCs.

a, LSCs from the spleen of MllPTD/WT: Flt3ITD/ITD mice with AML were cocultured with or without 0.1 ng/ml, 1 ng/ml, or 10 ng/ml recombinant mouse IFN-γ. The percentages of Lin−Sca-1+c-Kit+, Lin−Sca-1−c-Kit+, and Lin−Sca-1+c-Kit− cells were measured by flow cytometry (n = 4 individual mice). b, ILC1s from healthy mouse liver were sorted and transferred into the top well of a 96-well Transwell plate. The bottom chamber of the plate was loaded with LSCs from the spleen of MllPTD/WT: Flt3ITD/ITD mice with AML, and coincubated for 3 days (n = 3 individual mice). Then the percentages of Lin−Sca-1+c-Kit+, Lin−Sca-1−c-Kit+, and Lin−Sca-1+c-Kit− cells were measured by flow cytometry. All data are representative of three independent experiments, shown as mean ± s.d., and assessed by one-way ANOVA.

Source data

Extended Data Fig. 5 ILC1s and IFN-γ do not affect leukemia progenitor cell differentiation into myeloid blasts.

a,b, Mouse LSCs labeled with CTV were cocultured with or without mouse ILC1s in the presence or absence of anti-IFN-γ or anti-TNF antibody. Statistics of absolute cell numbers of Mac-1+ (a) and Gr-1+ (b) cells are shown (n = 3 individual mice). c, Leukemia progenitor cells were sorted from the spleen of MllPTD/WT: Flt3ITD/ITD mice with AML and cocultured with or without WT ILC1s, IFN-γ−/− ILC1s, or IFN-γ. Representative flow cytometry plots (top) and statistics of the percentages (bottom) of Mac-1+ and Gr-1+ cells are shown (n = 4 individual mice). d, A working model of how ILC1s and their secreted IFN-γ regulate differentiation of LSCs. Data in a, b, and c are representative of three independent experiments, shown as mean ± s.d., and assessed by one-way ANOVA models. NS, not significant.

Source data

Extended Data Fig. 6 ILC1s do not induce HSC apoptosis nor impair their differentiation.

a, Wild-type mouse HSCs from bone marrow of mice were cocultured with or without ILC1s. Representative images and statistics of the percentages of apoptotic cells (5× magnification, scale bar 200 µm, n = 5 individual mice). b, HSCs from blood of healthy donors were cocultured with or without ILC1s. Representative images and statistics of the percentages of apoptotic cells (n = 4 individual donors). c-e, Mouse HSCs were cocultured with or without ILC1s, and representative flow cytometry plots (c), statistics of cell numbers (d), and percentages (e) of Lin−Sca-1+c-Kit+ and Lin−Sca-1−c-Kit+ cells (n = 4 individual mice). f, Representative flow cytometry plots and statistics of cell numbers of Mac-1+Gr-1+ cells (n = 4 individual mice). g, Experimental scheme for (h-j). Mouse HSCs from CD45.2 mice were injected into lethally irradiated CD45.1 mice. One day later, ILC1s were injected into those CD45.1 mice. Three weeks later, donor hematopoietic and progenitor cells, myeloid cell subsets, and WBCs were analyzed. h, The cell numbers of donor LSKs, myeloid progenitor cells (L−S−K+, Lin−Sca-1−c-Kit+ cells), early lymphoid-committed precursors (L−S+K−, Lin−Sca-1+c-Kit− cells), short-term hematopoietic stem cells (STHSC, Lin−Sca-1+c-Kit+Flt3−CD150−CD48− cells), long-term hematopoietic stem cells (LTHSC, Lin−Sca-1+c-Kit+Flt3−CD150+CD48− cells), multipotent progenitors 1 and 2 (MPP1, Lin−Sca-1+c-Kit+Flt3−CD150+CD48+ cells; MPP2, Lin−Sca-1+c-Kit+Flt3−CD150−CD48+ cells), Mac-1+Gr-1+ cell subsets, and WBCs derived from CD45.2 mice were analyzed (n = 4 individual mice in no ILC1 group; n = 3 individual mice in ILC1 group). i, Representative flow cytometry plots and statistics of cell numbers of Mac-1+Gr-1+ cells derived from CD45.2 mice (n = 4 individual mice in no ILC1 group; n = 3 individual mice in ILC1 group). j, Statistics of cell numbers of WBCs (n = 4 individual mice in no ILC1 group; n = 3 individual mice in ILC1 group). Data in a, b, d, e, f, h, i, and j are representative of two independent experiments and shown as mean ± s.d.. Statistics are assessed by one-way ANOVA (a and b) or unpaired two-tailed Student’s t test (d, e, h, i, and j). NS, not significant.

Source data

Extended Data Fig. 7 ILC1s control the leukemia burden in mice transplanted with LSCs.

a, LSCs were i.v. co-injected into lethally irradiated (900 cGy) CD45.2 recipient mice on day 0 along with bone marrow cells isolated from IL-15 transgenic mice (CD45.2) as support cells. On day 1, the mice were i.v. injected with WT ILC1s from the liver of C57BL/6 J (CD45.2) mice or i.p. injected daily with recombinant mouse IFN-γ (0.5 μg/mouse/day). Statistics of the numbers of total WBCs at week 5 (n = 5 individual mice in no ILC1 group; n = 4 individual mice in WT ILC1group; n = 6 individual mice in recombinant IFN-γ group). All absolute cell numbers of WBCs were determined by cell counting with the Element HT5 Hematology Analyzer. b, Representative flow cytometry plots of the percentages of CD45.1+ and CD45.2+ cells. c, LSCs were co-injected into lethally irradiated (900 cGy) CD45.1 recipient mice on day 0 along with bone marrow cells isolated from CD45.1 mice (as support cells). Mice were injected with WT ILC1s or IFN-γ−/− ILC1s from the liver of C57BL/6 J (CD45.2) mice on day 1 or injected daily with recombinant mouse IFN-γ (0.5 μg/mouse/day). Statistics of the number of total WBCs at week 3 (n = 7 individual mice). All absolute cell numbers of WBCs were determined by cell counting with the Element HT5 Hematology Analyzer followed by flow cytometry. For box plots, boxplots (a and c) display the median and interquartile range (25th percentile –75th percentile) with whiskers representing the upper- and lower-quartile (1.5× the 75th and 25th percentile values). Data in a and c are representative of two independent experiments and shown as mean ± s.d. and assessed by one-way ANOVA.

Source data

Extended Data Fig. 8 The role of ILC1s and ILC1-derived IFN-γ in controlling LSCs.

a, Healthy mouse liver ILC1s or NK cells were cocultured with or without LSCs in the presence or absence of anti-IL-7Rα neutralizing antibody or isotype IgG control for 12 h along with IL-12 plus IL-15. Representative flow cytometry plots of IFN-γ production by ILC1s (n = 6 individual mice). b, Representative flow cytometry plots of IFN-γ production in healthy liver ILC1s or healthy liver NK cells after treatment with or without IL-7 (100 ng/ml) in the presence of IL-12 plus IL-15 (n = 5 individual mice). c,d, Mouse LSCs were cocultured with or without mouse ILC1s or NK cells for 3 days in the presence or absence of mouse anti-IFN-γ antibody. Representative images (c; 5× magnification, scale bar 200 µm) and statistics of absolute cell numbers (d) are shown (n = 3 individual mice). e,f, Representative flow cytometry plots (e) and statistics of the percentages of apoptotic LSCs (f; n = 3 individual mice). g, To deplete ILC1s or NK cells, WT mice were i.p. injected with IgG control (CTRL), anti-NK1.1, or anti-asialo-GM1 antibody. Three days later, the percentages of NK cells (Lin–NK1.1+NKp46+CD49b+) and ILC1s (Lin–NK1.1+NKp46+CD49a+) in the liver of WT mice were measured by flow cytometry. Data (d and f) are representative of two independent experiments and shown as mean ± s.d. and are assessed by one-way ANOVA models. NS, not significant.

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Extended Data Fig. 9 RNA-seq identifies gene transcriptional changes and signaling pathways in LSCs treated with ILC1s or IFN-γ.

a, Experimental design for RNA sequencing (RNA-Seq). Mouse LSCs were sorted and treated with or without sorted ILC1s or IFNγ for 3 days. LSCs were resorted from cocultured ILC1s or IFN-γ using FACS before RNA-Seq. b, Purity of LSCs (left) and ILC1s (right) after cell sorting. c, A heat map showing differential expression of RNA of 627 genes (n = 3 individual mice) is shown. d-f, Volcano plots showing significantly differentially expressed genes in RNA pools from AML LSCs treated with ILC1s vs. Ctrl (untreated) (d), IFN-γ vs. Ctrl (e), and IFN-γ vs. ILC1s (f) (n = 3 individual mice). g, Hallmark pathway analysis in LSC RNA pools (IFN-γ vs. Ctrl). The left panel shows signaling pathways downregulated in LSCs. The right panel shows signaling pathways upregulated in LSCs (n = 3 individual mice). Genes with an FDR-adjusted P-value < 0.05 and a fold change (FC) > 1.5 or < 0.7 were considered as significantly upregulated and downregulated genes, respectively.

Extended Data Fig. 10 ILC1s or IFN-γ inhibit LSC differentiation via the JAK-STAT and AKT signaling pathways.

a, GSEA plots show enrichment of the indicated target genes in LSCs co-cultured with ILC1s. The x axis shows the rank orders (ILC1s vs. Ctrl) of all the genes. b, GSEA plots show enrichment of the indicated target genes in LSCs treated with IFN-γ. The x axis shows the rank orders (IFN-γ vs. Ctrl) of all the genes. c,d, Heat maps showing differential expression of RNAs of genes downstream of IFN-γ. e-h, Mouse LSCs labeled with CTV were treated with or without the indicated JAK and AKT inhibitors for 30 min and then cocultured with or without WT or IFN-γ−/− ILC1s in the presence of IL-12 and IL-15 for 3 days. Statistics of the percentages of Lin−Sca-1+c-Kit+, Lin−Sca-1−c-Kit+, Lin−Sca-1+c-Kit−, and Lin−Sca-1−c-Kit− cells (n = 3 individual mice). Genes with an FDR-adjusted P-value < 0.05 and a fold change (FC) > 1.5 or < 0.7 were considered to be significantly upregulated or downregulated. Data in e-h are representative of three independent experiments, shown as mean ± s.d., and assessed by one-way ANOVA models. NS, not significant.

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Li, Z., Ma, R., Ma, S. et al. ILC1s control leukemia stem cell fate and limit development of AML. Nat Immunol 23, 718–730 (2022). https://doi.org/10.1038/s41590-022-01198-y

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