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CD66b−CD64dimCD115− cells in the human bone marrow represent neutrophil-committed progenitors

Nature Immunology volume 23, pages 679–691 (2022)Cite this article

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Abstract

Here we report the identification of human CD66b−CD64dimCD115− neutrophil-committed progenitor cells (NCPs) within the SSCloCD45dimCD34+ and CD34dim/− subsets in the bone marrow. NCPs were either CD45RA+ or CD45RA−, and in vitro experiments showed that CD45RA acquisition was not mandatory for their maturation process. NCPs exclusively generated human CD66b+ neutrophils in both in vitro differentiation and in vivo adoptive transfer experiments. Single-cell RNA-sequencing analysis indicated NCPs fell into four clusters, characterized by different maturation stages and distributed along two differentiation routes. One of the clusters was characterized by an interferon-stimulated gene signature, consistent with the reported expansion of peripheral mature neutrophil subsets that express interferon-stimulated genes in diseased individuals. Finally, comparison of transcriptomic and phenotypic profiles indicated NCPs represented earlier neutrophil precursors than the previously described early neutrophil progenitors (eNePs), proNeus and COVID-19 proNeus. Altogether, our data shed light on the very early phases of neutrophil ontogeny.

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Fig. 1: Identification of neutrophil precursors within cGMPs.
Fig. 2: Identification of additional CD34+ and CD34dim/− neutrophil-restricted progenitors.
Fig. 3: NCP1s and NCP2s independently differentiate into neutrophils.
Fig. 4: RNA-seq experiments confirm that NCPs represent very early precursors of neutrophils.
Fig. 5: NCPs stand at earlier stages than human eNePs, proNeu1/2s and COVID-19 proNeus.
Fig. 6: Generation of neutrophils by human NCPs in adoptive transfer experiments.
Fig. 7: scRNA-seq experiments of NCPs and cMoPs reveal that they consist of multiple cell clusters.
Fig. 8: Characterization of the scRNA-seq cell clusters composing NCPs.

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Data availability

Raw datasets have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) and are available under the accession number GSE164687. Bulk RNA-seq datasets of eNePs and HSCs/CMPs were downloaded from GSE157103 and GSE113182, respectively. COVID-19 proNeus and preNeus scRNA-seq datasets were downloaded from https://www.fastgenomics.org. Source data are provided with this paper.

Code availability

R scripts for data processing are available through https://github.com/fbianchetto/Neutrophil-Committed-Progenitors.

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Acknowledgements

This work was supported by grants to M.A.C. (from AIRC grant no. IG-20339; MIUR-PRIN grant no. 20177J4E75_004; and Fondazione Cariverona) and to N.T. (grant no. GR-2016-02361263). We thank P. Mazzi for preparing cytospins, E. Pietronigro for slide acquisition, S. Pasqualato (European Institute of Oncology, Milan, Italy) for providing Tn5 transposase and A. Carraro (Centro Trapianti Fegato, AOUI Verona, Italy) and V. Bergamini (UOC Ostetricia e Ginecologia B, AOUI Verona, Italy) for providing spleen and cord blood samples. We thank G. Zini (University Cattolica, Hematology Department, Rome) for her professional consultancy about NCP morphology. Centro Piattaforme Tecnologiche (CPT) of the University of Verona has been instrumental for the access to the flow cytometry/cell analysis and genomic/transcriptomic platforms.

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Author notes

  1. These authors contributed equally: Federica Calzetti, Giulia Finotti, Nicola Tamassia, Francisco Bianchetto-Aguilera.

Authors and Affiliations

  1. Section of General Pathology, Department of Medicine, University of Verona, Verona, Italy

    Federica Calzetti, Giulia Finotti, Nicola Tamassia, Francisco Bianchetto-Aguilera, Sara Gasperini, Ilaria Signoretto, Patrizia Scapini & Marco A. Cassatella

  2. Centro Piattaforme Tecnologiche, University of Verona, Verona, Italy

    Monica Castellucci

  3. Section of Immunology, Department of Medicine, University of Verona, Verona, Italy

    Stefania Canè, Stefano Ugel & Vincenzo Bronte

  4. Unit of Pathology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy

    Silvia Lonardi & William Vermi

  5. Interdepartmental Laboratory of Medical Research, Research Center LURM, University of Verona, Verona, Italy

    Chiara Cavallini

  6. Section of Internal Medicine B, Department of Medicine, University of Verona and AOUI Verona, Verona, Italy

    Alessandro Matte

  7. Section of Hematology and Bone Marrow Transplant Unit, Department of Medicine, University of Verona, Verona, Italy

    Fabio Benedetti, Massimiliano Bonifacio & Cristina Tecchio

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  1. Federica Calzetti
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Contributions

F.C. and G.F. performed and analyzed flow cytometry, cell sorting and in vitro differentiation experiments. C.C. and A.M. were involved in cell sorting experiments. N.T., M.C. and S.G. prepared samples for RNA-seq and scRNA-seq. N.T. and F.B.-A. analyzed RNA-seq and scRNA-seq data. F.C. and I.S. performed CFU assay. S.L. and W.V. performed and analyzed ICC. F.C., G.F., S.C., S.U., A.M. and V.B. were involved in adoptive transfer experiments. F.B., M.B. and C.T. provided patient samples. F.C., G.F., N.T., P.S. and M.A.C. designed and wrote the manuscript. M.A.C. supervised the project.

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Correspondence to Marco A. Cassatella.

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Nature Immunology thanks Pierre Guermonprez and Hongbo Luo for their contribution to the peer review of this work. Ioana Visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Gating strategy to identify lineage-positive cells and immature SSCloCD45dim myeloid/lymphoid progenitors within BM-LDCs.

a, Flow cytometry workflow for the identification of mature leukocyte populations and SSCloCD45dim immature myeloid/lymphoid progenitors within BM-LDCs. Mature cell populations were identified as: SSChiCD66b+CD16−CD45hi eosinophils (pink); SSChiCD66b+CD16-/+CD45+ neutrophils (green); SSCloCD141+CLEC9A+ cDC1s (magenta); SSCloFcεRI+CD1c− basophils (dark red); SSCloFcεRIdimCD1c+ cDC2s (yellow); SSCloCD45RA+CD33+CD123+CD303+ pDCs (green); SSCloCD3−CD19−CD56+ NK cells (light blue); SSCloCD3−CD19−CD56−CD14+CD16−/CD14+/-CD16+/- total monocytes (light blue); SSCloCD3+CD19− T cells (brown); SSCloCD3−CD19+ B cells (orange). Immature myeloid/lymphoid progenitors were identified as SSCloCD45dim cells (purple). b, Dot plot overlays of lineage-positive/mature cells and SSCloCD45dim immature progenitors.

Extended Data Fig. 2 Biological features of CD34+CD45RA+CD64dimCD115− cells, cMoPs and MDPs prior to, and after treatment with, either SFGc, or SFG.

a, Bar graphs depicting the percentage of PI−, live cells generated by CD34+CD45RA+CD64dimCD115−cells (n = 8), cMoPs (n = 7) and MDPs (n = 4) treated with SFGc for 7 days. Data represent means ± s.e.m. b, Bar graphs showing the fold expansion of CD34+CD45RA+CD64dimCD115−cells (n = 8), cMoPs (n = 6) and MDPs (n = 4) treated with SFGc for 7 days. Data represent means ± s.e.m. Analysis was performed by using Kruskal-Wallis test, Dunn’s post-hoc test. c, Representative histogram overlays showing the expression of CD114 by CD34+CD45RA+CD64dimCD115−cells (green), cMoPs (light blue) and MDP (brown) populations. Gray profile corresponds to isotype control (n = 3). (d) Bar graphs reporting the percentages of neutrophils (green), cDC1s (magenta), pDCs/preDCs/DC5s (orange), monocytes (light blue) and cDC2s (pink) generated by CD34+CD45RA+CD64dimCD115− cells (n = 8), cMoPs (n = 9) and MDPs (n = 6) treated with SFG for 7 days. Data represent means ± s.e.m.

Source data

Extended Data Fig. 3 Reconstitution of NCPs and mature neutrophils in BMs of allogeneic HSC-transplanted patients.

Flow cytometry strategy for the identification of reconstituting neutrophils (green), eosinophils (pink), NCP1s (orange), NCP2s (green), NCP3s (magenta), NCP4s (turquoise), cMoPs (light blue), MDPs (brown) and pre-monocytes (black) within BM-LDCs of reconstituting BM (at day +21) from a patient undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT) (n = 3).

Extended Data Fig. 4 Functionality of neutrophils derived from NCPs, as well as CD15 expression by both neutrophil and monocyte precursors among BM-LDCs and their mature counterparts.

a, Histogram plot showing the percentages and the type of colonies generated after 14 days of NCP and BM-LDC culture (n = 3). Data represent means ± s.e.m. b, Representative plot showing the time-course of O2− production by neutrophils derived from NCPs cultured for 7 days with SFGc (orange, green, magenta and turquoise) as compared to blood neutrophils (red) (n = 3). c, Representative cytospins showing the phagocytosis of zymosan particles by neutrophils derived from NCPs cultured for 7 days with SFGc (n = 3). d,e, Histogram overlays showing the expression of CD15 by NCPs (orange, green, magenta and turquoise), PMs (purple), SNs (black), cMoPs (light blue), pre-monocytes (blue) and CD14+ monocytes (red) as compared to the fluorescence-negative control (light gray) (n = 5).

Source data

Extended Data Fig. 5 14-color flow cytometry antibody panel to identify NCPs, monocyte/DC progenitors and immature neutrophil populations in human BMs.

a, Flow cytometry workflow (Supplementary Table 6) showing the gating strategy to identify myeloid progenitors within the SSCloCD66b−Lin(CD3/CD19/CD1c/CD141)-CD14−CD16−CD56−CD45dim region of BM-LDCs (dark red): CD34+CD45RA+ cGMPs, CD34+CD45RA+CD123+CD115− CDPs (yellow), CD34+CD45RA+CD123dim/-CD64dimCD115− NCP2s (green), CD34+CD45RA+CD123dim/-CD64+CD115+ cMoPs (light blue), CD34+CD45RA+CD123dim/-CD64−CD115+MDPs (brown), CD34+CD45RA−CD123dim/-CD64dimCD115− NCP1s (orange), CD34dim/-CD45RA+CD123dim/-CD64dimCD115− NCP3s (magenta), CD34dim/-CD45RA+CD123dim/-CD64++CD115+ pre-monocytes (blue), CD34dim/-CD45RA-CD123dim/-CD64dimCD115− NCP4s (turquoise). The same flow cytometry workflow identify SSChiCD66b+CD16−CD45hi eosinophils (black) as well as all the neutrophil precursors within the SSChiCD66b+Lin(CD3/CD19/CD1c/CD141)- region (green): CD66b+CD10−CD11b−CD16−PMs (grey), CD66b+CD10−CD11bdim/+CD16−MYs (light red), CD66b+CD10−CD11b+CD16+ MMs (purple), CD66b+CD10−CD11b+CD16++CD10− BCs (light yellow), and CD66b+CD11b+CD16++CD10+ SNs (pink). b, Dot plot overlays depicting the phenotype variation (black arrows) of the populations composing the neutrophil maturation cascade in terms of SSC parameter and CD66b, CD15 and CD45 marker modulation.

Extended Data Fig. 6 NCP1s and NCP2s derive from phenotypically distinct CD34+ progenitors.

a, Flow cytometry strategy for the identification of SSCloCD45dimCD10−CD123dim/-CD34+CD45RA−CD64− subset (CD45RA−CD64− subset, pink) and SSCloCD45dimCD10−CD123dim/-CD38+CD34+CD45RA+CD64−CD115−GMDPs (CD64−GMDPs, dark blue). b,c, Plots showing the differentiation potential of CD45RA−CD64− subset (b) and CD64−GMDPs (c) based on the changes of CD34 and CD45RA, as well as CD64 and CD115, expression by the generated cells during culture with SFGc for 2, 4 or 7 days (n = 3). d Hypothetic model of myeloid cell ontogeny according to our results. The scheme shows that the acquisition of CD45RA represents a very premature event (that occurs prior to that of CD64) along the maturation trajectories of early progenitors of neutrophils, monocytes and DCs, occurring from the multilineage CD34+CD45RA−CD64−CD115−progenitor subset (including HSCs, CMPs, MEPs) into transitional multilineage progenitor pools. The latter pools include GMDPs (also named as neutrophil-, monocyte- and DC-committed progenitors, NMDPs), MDPs and CDPs, that subsequently mature into uni-lineage precursors, including NCP2s/NCP3s and cMoPs/pre-monocytes. The scheme also shows that the acquisition of CD45RA does not occur in those progenitors present within CD34+CD45RA−CD64−CD115− subset that directly upregulates CD64 expression and generates NCP1s. As shown in the scheme, NCP1s directly differentiate into NCP4s, while NCP2s originate NCP4s via NCP3s.

Extended Data Fig. 7 Characterization of NCP transcriptomes as determined by RNA-seq.

a, Developmental path of NCP1s, NCP2s, NCP3s and NCP4s, as well as HSCs/MPPs, CMPs, PMs, MYs, MMs, BCs, SNs and mature neutrophils (PMN), computationally determined from bulk RNA-seq datasets by using the optimal leaf ordering (OLO) algorithm. b, GO terms enriched by genes associated with the ten gene groups (g1-g10) identified by K-means analysis, as shown in Fig. 4d. The top five GO terms with Benjamini-Hochberg-corrected P values <0.05 (one-sided Fisher’s exact test) are shown for every gene group. ‘Gene ratio’ indicate the fraction of DEGs present in the given GO term. (c), Box plots showing the distribution of mRNA expression levels [as log2(FPKM + 1)] for genes associated to cell cycle, AG, SG, GG, SV and GM, as well as ROS biosynthetic process, phagocytosis, and chemotaxis. The box plot shows the median with the lower and upper quartiles representing a 25th to 75th percentile range and whiskers extending to 1.5 × interquartile range (IQR). LOESS fitting of the data with relative confidence interval is represented by a blue line with a shadow area. d, PCA biplots based on the DEGs identified by LRT among bulk RNA-seq of NCP1s (orange), NCP2s (green), NCP3s (magenta) and NCP4s (turquiose). The graph lists the ten most relevant genes contributing to sample variations (indicated by vectors) for both PC1 and PC2, under both positive and negative directions. Vector lengths correlate with the weight of the given gene within the components.

Extended Data Fig. 8 Additional characterization of NCP and cMoP scRNA-seqs.

a, Density plots of NCP1s, NCP2s, NCP3s, NCP4s and cMoPs overlaid on the UMAP of Fig. 7a. Density of cells in each plot is depicted according to the indications of the color bar. b, Hierarchical clustering dendrogram based on the DEGs identified among the neutrophilic and monocytic cell clusters shown in Fig. 7b. The vertical axis of the dendrogram represents the dissimilarity between clusters (that is, Euclidean distance). c, Violin plots showing the mRNA expression levels [as ln (UMI)] of selected genes across the four neutrophil clusters (c1-c4) chosen among the top defining genes indicated in Fig. 8e. Clusters are colored according to Fig. 7bd, Expression patterns of cell-cycle, AG, SG, and GG genes projected on the UMAP plot restricted to neutrophil progenitor clusters (c1-c4). e, Gene Ontology analysis of DEGs for c2-c4. For every cluster (x-axis), the top ten Gene Ontology terms with Benjamini-Hochberg-corrected P values <0.05 (one-sided Fisher’s exact test) are shown. For cluster 1 no enrichment of biological processes GO term was identified. f, Trajectory plots of c1-c4 cells as defined in Fig. 7b. In each plot is depicted the density of cells according to the color bar placed at the right bottom corner of the panel.

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Calzetti, F., Finotti, G., Tamassia, N. et al. CD66b−CD64dimCD115− cells in the human bone marrow represent neutrophil-committed progenitors. Nat Immunol 23, 679–691 (2022). https://doi.org/10.1038/s41590-022-01189-z

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