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Picturing Polarized Myeloid Phagocytes and Regulatory Cells by Mass Cytometry

Part of the Methods in Molecular Biology book series (MIMB,volume 1989)

Abstract

The immune monocyte/phagocyte system (MPS) includes numerous cell subsets of the myeloid lineage including monocyte, macrophage, and dendritic cell (DC) populations that are heterogeneous both phenotypically and functionally. Previously, we characterized these diverse MPS phenotypes with multi-parametric mass cytometry (CyTOF). In order to expansively characterize monocytes, macrophages, and dendritic cells, a CyTOF panel was designed to measure 35 identity-, activation-, and polarization-markers. Here we provide a protocol to define a reference map for the myeloid compartment, including sample preparation, to produce reference cell subsets from the monocyte/phagocyte system. In particular, we focused on monocyte-derived macrophages that were further polarized in vitro with cytokine stimulation (i.e., M-CSF, GM-CSF, IL-4, IL-10, IFNγ, and LPS), as well as monocyte-derived DCs, and myeloid-derived suppressor cells (MDSCs), generated in vitro from human bone marrow and/or peripheral blood.

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References

  1. Guilliams M, Ginhoux F, Jakubzick C et al (2014) Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol 14:571–578. https://doi.org/10.1038/nri3712

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ancuta P (2015) A slan-based nomenclature for monocytes? Blood 126:2536–2538. https://doi.org/10.1182/blood-2015-10-675470

    Article  CAS  PubMed  Google Scholar 

  3. Ziegler-Heitbrock L, Ancuta P, Crowe S et al (2010) Nomenclature of monocytes and dendritic cells in blood. Blood 116:e74–e80. https://doi.org/10.1182/blood-2010-02-258558

    Article  CAS  PubMed  Google Scholar 

  4. Bronte V, Brandau S, Chen SH et al (2016) Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nat Commun 7:12150. https://doi.org/10.1038/ncomms12150

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Murray PJ, Allen JE, Biswas SK et al (2014) Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity 41:14–20. https://doi.org/10.1016/j.immuni.2014.06.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Engblom C, Pfirschke C, Pittet MJ (2016) The role of myeloid cells in cancer therapies. Nat Rev Cancer 16:447–462. https://doi.org/10.1038/nrc.2016.54

    Article  CAS  PubMed  Google Scholar 

  7. Ginhoux F, Schultze JL, Murray PJ et al (2016) New insights into the multidimensional concept of macrophage ontogeny, activation and function. Nat Immunol 17:34–40. https://doi.org/10.1038/ni.3324

    Article  CAS  PubMed  Google Scholar 

  8. Greenplate AR, Johnson DB, Roussel M et al (2016) Myelodysplastic syndrome revealed by systems immunology in a melanoma patient undergoing anti-PD-1 therapy. Cancer Immunol Res 4:474–480. https://doi.org/10.1158/2326-6066.CIR-15-0213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bendall SC, Simonds EF, Qiu P et al (2011) Single-cell mass Cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332:687–696. https://doi.org/10.1126/science.1198704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Spitzer MH, Nolan GP (2016) Mass Cytometry: single cells, many features. Cell 165:780–791. https://doi.org/10.1016/j.cell.2016.04.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Saeys Y, Van Gassen S, Lambrecht BN (2016) Computational flow cytometry: helping to make sense of high-dimensional immunology data. Nat Rev Immunol 16:449–462. https://doi.org/10.1038/nri.2016.56

    Article  CAS  PubMed  Google Scholar 

  12. Diggins KE, Greenplate AR, Leelatian N et al (2017) Characterizing cell subsets using marker enrichment modeling. Nat Methods 14:275–278. https://doi.org/10.1038/nmeth.4149

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Amir EAD, Davis KL, Tadmor MD et al (2013) viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol 31:545–552. https://doi.org/10.1038/nbt.2594

    Article  CAS  PubMed Central  Google Scholar 

  14. Qiu P, Simonds EF, Bendall SC et al (2011) Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE. Nat Biotechnol 29:886–891. https://doi.org/10.1038/nbt.1991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Diggins KE, Gandelman JS, Roe CE et al (2018) Generating quantitative cell identity labels with marker enrichment modeling (MEM). Curr Protoc Cytom 83(2018):10.21.1–10.21.28. https://doi.org/10.1002/cpcy.34

    Article  Google Scholar 

  16. Alcántara-Hernández M, Leylek R, Wagar LE et al (2017) High-dimensional phenotypic mapping of human dendritic cells reveals interindividual variation and tissue specialization. Immunity 47(6):1037–1050.e6. https://doi.org/10.1016/j.immuni.2017.11.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. See P, Dutertre CA, Chen J et al (2017) Mapping the human DC lineage through the integration of high-dimensional techniques. Science 356:eaag3009. https://doi.org/10.1126/science.aag3009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Schulz D, Severin Y, Zanotelli VRT et al. (2019) In-Depth Characterization of Monocyte-Derived Macrophages using a Mass Cytometry-Based Phagocytosis Assay. Scientific reports 9:1925 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374473/ PMID: 30760760

  19. Sander J, Schmidt SV, Cirovic B et al (2017) Cellular differentiation of human monocytes is regulated by time-dependent interleukin-4 signaling and the transcriptional regulator NCOR2. Immunity 47:1051–1066.e12. https://doi.org/10.1016/j.immuni.2017.11.024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chevrier S, Levine JH, Zanotelli VRT et al (2017) An immune atlas of clear cell renal cell carcinoma. Cell 169:736–738.e18. https://doi.org/10.1016/j.cell.2017.04.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lavin Y, Kobayashi S, Leader A et al (2017) Innate immune landscape in early lung adenocarcinoma by paired single-cell analyses. Cell 169:750–757.e15. https://doi.org/10.1016/j.cell.2017.04.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Krieg C, Nowicka M, Guglietta S et al (2018) High-dimensional single-cell analysis predicts response to anti-PD-1 immunotherapy. Nat Med 9:2579–2514. https://doi.org/10.1038/nm.4466

    Article  CAS  Google Scholar 

  23. Roussel M, Ferrell PB, Greenplate AR et al (2017) Mass cytometry deep phenotyping of human mononuclear phagocytes and myeloid-derived suppressor cells from human blood and bone marrow. J Leukoc Biol 102:437–447. https://doi.org/10.1189/jlb.5MA1116-457R

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Xue J, Schmidt SV, Sander J et al (2014) Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 40:274–288. https://doi.org/10.1016/j.immuni.2014.01.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Marigo I, Bosio E, Solito S et al (2010) Tumor-induced tolerance and immune suppression depend on the C/EBPbeta transcription factor. Immunity 32:790–802. https://doi.org/10.1016/j.immuni.2010.05.010

    Article  CAS  PubMed  Google Scholar 

  26. Lechner MG, Liebertz DJ, Epstein AL (2010) Characterization of cytokine-induced myeloid-derived suppressor cells from normal human peripheral blood mononuclear cells. J Immunol 185:2273–2284. https://doi.org/10.4049/jimmunol.1000901

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Mikael Roussel or Jonathan M. Irish .

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Roussel, M., Bartkowiak, T., Irish, J.M. (2019). Picturing Polarized Myeloid Phagocytes and Regulatory Cells by Mass Cytometry. In: McGuire, H., Ashhurst, T. (eds) Mass Cytometry. Methods in Molecular Biology, vol 1989. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9454-0_14

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  • DOI: https://doi.org/10.1007/978-1-4939-9454-0_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9453-3

  • Online ISBN: 978-1-4939-9454-0

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