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Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis

Abstract

Although many aspects of blood production are well understood, the spatial organization of myeloid differentiation in the bone marrow remains unknown. Here we use imaging to track granulocyte/macrophage progenitor (GMP) behaviour in mice during emergency and leukaemic myelopoiesis. In the steady state, we find individual GMPs scattered throughout the bone marrow. During regeneration, we observe expanding GMP patches forming defined GMP clusters, which, in turn, locally differentiate into granulocytes. The timed release of important bone marrow niche signals (SCF, IL-1β, G-CSF, TGFβ and CXCL4) and activation of an inducible Irf8 and β-catenin progenitor self-renewal network control the transient formation of regenerating GMP clusters. In leukaemia, we show that GMP clusters are constantly produced owing to persistent activation of the self-renewal network and a lack of termination cytokines that normally restore haematopoietic stem-cell quiescence. Our results uncover a previously unrecognized dynamic behaviour of GMPs in situ, which tunes emergency myelopoiesis and is hijacked in leukaemia.

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Figure 1: GMP clusters in leukaemic and regenerative myelopoiesis.
Figure 2: GMP clusters are foci of differentiation.
Figure 3: Molecular mechanisms of GMP cluster formation.
Figure 4: Irf8 and β-catenin self-renewal progenitor network.
Figure 5: BM niche controls of GMP cluster formation during regenerative myelopoiesis.
Figure 6: Continuous GMP cluster formation in leukaemic myelopoiesis.

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Acknowledgements

We thank A. Leavitt (UCSF) for providing G-CSF; P. Frenette for advice on imaging approaches and the gift of Clxl4-cre:iDTR mice; M. Kissner and M. Lee for management of our Flow Cytometry core facility; and all members of the Passegué laboratory for critical insights and suggestions. This work was supported by NIH K01DK098315 award to E.M.P.; a Bloodwise and CRUK program grants and Wellcome Trust funding to the Cambridge Stem Cell Institute to B.G.; and NIH R01HL092471, R01HL111266 and P30DK063720 grants, Rita Allen Scholar Award and Leukemia Lymphoma Society Scholar Award to E.P.

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Authors

Contributions

A.H., M.B. and S.L. performed all of the experiments with help from S.Y.Z. for dragon bead assays, Y.-A.K. for β-catenin studies, E.M.P. for IL-1 experiments, F.J.C.-N., X.W. and B.G. for Fluidigm and single-cell RNA-seq analyses, and S.H.C. and S.A. for MLL/AF9 experiments. K.B.-H. initiated the imaging studies. A.H., M.B. and S.L. designed the experiments and interpreted the results. A.H. and E.P. wrote the manuscript.

Corresponding author

Correspondence to Emmanuelle Passegué.

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Extended data figures and tables

Extended Data Figure 1 Imaging GMPs in normal and leukaemic conditions.

a, Gating strategy used to identify GMPs showing representative FACS plots with purified GMPs (purple) stained with immunofluorescence markers, and a representative wild-type GMP (purple circle) on bone section. b, Inducible BA and constitutive jB mouse models of human MPN with representative GMP FACS plots. Dox, doxycycline. c, Representative immunofluorescence staining showing GMPs (purple) in the BM of diseased jB mice. d, Progression of cGMP formation with disease development in BA mice at the indicated weeks after withdrawal of doxycycline. e, Representative examples of loose pGMPs and compact cGMPs in wild-type and BA mice. Solid lines indicate bone surface; dotted lines denote cGMPs and stars indicate pGMPs.

Extended Data Figure 2 GMP cluster features.

a, Representative immunofluorescence staining of GMPs (purple) in spleens from wild-type and diseased BA and jB mice. b, cGMPs in recipient mice developing AML after transplantation of MLL-AF9 (MF9)-transduced LSK-derived cells. Experimental scheme and representative immunofluorescence staining of GMPs (purple) in BM from control and diseased MF9 recipient mice. Three individual recipient mice are shown for MF9. c, Representative immunofluorescence staining showing myeloid progenitors (red) in relation to the indicated stromal features (green) in BM from control and diseased BA mice. d, Representative immunofluorescence staining showing myeloid progenitors (red) in relation to mature lymphoid (green) and myeloid (blue) cells in BM from control and diseased BA mice. i and ii highlight two magnified areas. Orange lines indicate germinal centre; arrowheads denote individual GMPs; stars indicate pGMPs and dotted lines denote cGMPs.

Extended Data Figure 3 Regenerating BM after 5-FU treatment.

a, Gating strategy used to identify the indicated BM populations by flow cytometry in 5-FU-treated wild-type mice. Representative FACS plots are shown at the indicated days after treatment. b, Frequency of BM LSK cells, HSCs, MPP2/MPP3, GMPs and granulocytes at the indicated days after 5-FU treatment. c, Representative immunofluorescence staining showing GMPs (purple) in BM from 5-FU treated mice at the indicated days after treatment. Of note, cGMPs were observed in all investigated bones (that is, femur, tibia, humerus and sternum) at day 12 after 5-FU treatment. Solid lines indicate bone surface; dotted lines denote cGMPs. Data are mean ± s.d. (grey bars, reference range); *P ≤ 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test).

Source data

Extended Data Figure 4 GMP clusters during myeloid regeneration and expansion.

a, Granulocyte depletion in the BM of Ly-6G-treated mice, with experimental scheme (top left), representative FACS plots (top right) and immunofluorescence staining of GMPs (purple) (bottom) at the indicated days after treatment. b, Granulocyte expansion in the BM of G-CSF-treated mice, with experimental scheme (top left), representative FACS plots (top right) and immunofluorescence staining of GMPs (purple) (bottom) at the indicated days after treatment. c, GMP clusters in the BM of HSC-transplanted mice, with experimental scheme (left) and representative immunofluorescence staining of GMPs (purple) at the indicated weeks after transplantation (right). Non-transplanted wild-type BM is shown for comparison; IR, ionizing radiation. Stars indicate pGMPs and dotted lines denote cGMPs.

Extended Data Figure 5 GMP clusters are clonal.

ac, Clonality of regenerative GMP clusters. a, Percentage of CD45.2+ cells in the peripheral blood (PB) pre-treatment and the BM after 5-FU treatment for each of the chimaera mice used at the indicated days after 5-FU treatment. b, Representative immunofluorescence staining showing myeloid progenitors (red) and CD45.2 (green) expression in BM from 5-FU-treated chimaera mice at the indicated days after treatment. c, Experimental scheme (left) and representative immunofluorescence staining (right) showing myeloid progenitors (red) and CD45.2 (green) expression separately in BM from two independent day 12 5-FU-treated chimaera mice. Positive (+) clusters have ≥75% CD45.2+ cells and negative (−) clusters ≤5% CD45.2+ cells. d, Clonality of leukaemic GMP clusters with experimental scheme (left) and representative immunofluorescence staining (right) showing myeloid progenitors (red) and GFP (green) expression from Actb-Gfp cells in BM from diseased BA chimaera mice. Positive (+) clusters have ≥75% GFP+ cells and negative (−) clusters ≤5% GFP+ cells. Dotted lines denote cGMPs.

Extended Data Figure 6 Dynamic proliferation and differentiation in regenerating GMP clusters.

a, Representative FACS plots showing kinetics of BrdU incorporation in LSKs and GMPs from 5-FU-treated wild-type mice at the indicated days after treatment. b, Representative immunofluorescence staining showing myeloid progenitors (red) in relation to proliferating EdU+ (green, top row) and dividing pH3+ (green, bottom row) cells in BM from 5-FU-treated wild-type mice at the indicated days after treatment. c, Representative immunofluorescence staining showing myeloid progenitors (red) in relation to mature lymphoid (green) and myeloid (blue) cells in BM from 5-FU-treated wild-type mice at the indicated days after treatment. Stars indicate pGMPs and dotted lines denote cGMPs.

Extended Data Figure 7 Molecular reprogramming in regenerating and leukaemic GMP clusters.

a, Additional Fluidigm gene expression analyses of regenerating GMPs isolated from 5-FU-treated wild-type mice at the indicated days after treatment (n = 2; 10–16 pools of 100 cells per condition). Results are expressed as fold change compared to levels in untreated (D0) GMPs and presented as boxplots (line: median; box: twenty-fifth and seventy-fifth percentiles; whisker: ninetieth and tenth percentiles). b, Loading association of principal component analyses of Fluidigm gene expression data from regenerating GMPs. c, Representative FACS plots of GFP expression in GMPs of 5-FU-treated Csf1r-Gfp reporter mice at the indicated days after treatment. d, tSNE analyses and loading association of principal component analyses of Fluidigm gene expression data from MPN GMPs isolated from diseased BA, jB and respective age-matched control mice (n = 4; 22–28 pools of 100 cells per condition). e, Principal component analyses of single-cell GMP RNA-seq data showing the distribution of each 5-FU-treatment time point (day 0: 89 cells; day 8: 187 cells; day 10: 89 cells; day 12: 75 cells; day 14: 36 cells) and individual control (94 cells) and BA (BA(1): 68 cells; BA(2): 57 cells; BA(3): 87 cells) mice. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (Student’s t-test).

Source data

Extended Data Figure 8 Irf8 and Ctnnb1 function in self-renewing GMPs.

a, Representative FACS plots showing GMPs and granulocytes at the steady state in Irf8+/+ and Irf8−/− mice. b, Experimental scheme for BM from Irf8+/+ and Irf8−/− chimaeric mice. c, Representative immunofluorescence staining of donor-derived CD45.2+ (green) myeloid progenitors (red) in BM from 5-FU-treated Irf8+/+ and Irf8−/− chimaeric mice. d, Nuclear β-catenin expression in HSCs, and MPP3 and MPP4 populations from Irf8+/+ and Irf8−/− mice. Results are expressed as the percentage of positive cells. e, f, Experimental scheme for Ctnnb1 control and Ctnnb1 LOF (e) or Ctnnb1 (GOF) (f) mice. Stars indicate pGMPs and dotted lines denote cGMPs. Data are mean ± s.d.

Source data

Extended Data Figure 9 Mechanisms controlling GMP cluster formation during regeneration.

a, ELISA measurement of cytokine levels in BM fluids of 5-FU-treated wild-type mice at the indicated days after treatment (n = 3). b, Quantification of vascular leakage in 5-FU-treated BM at the indicated days after treatment (n = 3). Results are expressed as dragon-green (DG) bead mean fluorescence intensity (MFI) upon masking of laminin+ blood vessels. c, Representative immunofluorescence staining showing GMPs (purple) in BM from 5-FU-treated mice with concomitant daily injections of G-CSF on days 8 to 11. d, Investigation of 5-FU-treated Il1r1+/+ and Il1r1−/− mice at the indicated days after treatment showing representative immunofluorescence staining of GMPs (purple) (left), FACS plots of granulocyte regeneration (middle), and quantification of the indicated BM populations (right). e, Representative immunofluorescence staining of CD150+ megakaryocytes (red) in BM from 5-FU- and Ly-6G-treated mice. f, g, Megakaryocyte depletion studies in diphtheria toxin-injected iDtr (control) and Cxcl4-cre:iDtr (cre) mice showing representative immunofluorescence staining of CD150+ megakaryocytes (red) at the indicated days after 5-FU treatment (f), and representative Ki67 and DAPI staining of HSCs at day 12 (g). Stars indicate pGMPs and dotted lines denote cGMPs. Data are mean ± s.d. (grey bars, reference range); *P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001 (Student’s t-test).

Source data

Extended Data Figure 10 Deregulated GMP cluster formation in leukaemic mice.

a, ELISA measurements of cytokine levels in BM fluids of BA, jB and respective control mice (n = 3). b, qRT–PCR measurement of Cxcl4 expression in BM and megakaryocyte-enriched BSA gradient of control and BA mice (n = 3). c, Quantification of vascular leakage in diseased BA (n = 3) and jB (n = 3) mice. Results are quantified as dragon-green (DG) bead mean fluorescence intensity upon masking of laminin+ blood vessels. d, Representative FACS plots showing granulocyte regeneration in 5-FU-treated control and BA mice at the indicated days after treatment. e, Revised model of emergency myelopoiesis. At the steady state, blood production reflects the differential generation by HSCs of a small number of myeloid-biased MPP2/MPP3 and a large number of lymphoid-biased MPP4 subsets, which both give rise to GMPs and contribute to myeloid output. By contrast, in emergency situations, HSCs are induced to overproduce MPP2/MPP3, and MPP4 are reprogrammed towards almost exclusive myeloid output, in large part due to cytokine stimulations and the triggering of specific regulatory pathways. An important consequence of the activation of this myeloid regeneration axis is the generation of localized pGMP/cGMP differentiation foci in the BM cavity, which drive the granulocyte production. This entire process is tightly regulated by BM niche signals and is transient during emergency myelopoiesis, but is constantly activated in myeloid leukaemia. Important emerging questions are what controls the switch from self-renewing pGMP to differentiating cGMP clusters, if myeloid-reprogrammed MPP4 also generate pGMP/cGMPs and expanded MPP2/MPP3 continue to produce regular GMPs (dotted lines), and whether granulocytes produced through this regeneration axis functionally differ from steady-state granulocytes (heterogeneity). Data are mean ± s.d. *P ≤ 0.05, ***P ≤ 0.001 (Student’s t-test).

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Hérault, A., Binnewies, M., Leong, S. et al. Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis. Nature 544, 53–58 (2017). https://doi.org/10.1038/nature21693

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