Purification of Human Dendritic Cell Subsets from Peripheral Blood

  • Solana Alculumbre
  • Lucia PattariniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1423)


Blood represents the most accessible source of human dendritic cells (DCs). We present here a method to isolate three DC subtypes, as identified until now, from peripheral blood: plasmacytoid dendritic cells (pDCs), CD141+ myeloid DCs, and CD1c+ myeloid DCs. The method is based on the sequential depletion of non-DCs. First, depletion of granulocytes, erythrocytes, and platelets is obtained by blood centrifugation over a Ficoll gradient. Then, antibodies recognizing non-DCs, combined with magnetic beads, allow enrichment of DCs from peripheral blood mononuclear cells (PBMCs). Finally, enriched DCs are purified and separated into the different subtypes by immunolabeling and fluorescence-activated cell sorting (FACS) using DC-specific surface markers.

DC studies might contribute to the comprehension of human immune processes in physiological and pathological conditions. Human blood DCs targeting might be a useful tool to ameliorate inflammatory diseases and improve vaccination strategies.

Key words

Dendritic cell Human Blood pDC Subtypes 



We are grateful to Vassili Soumelis for his support and suggestions.

We thank Zofia Maciorowski and the Cytometry platform of Curie Institute for help in cell sorting. We thank Cristina Ghirelli and Raphael Zollinger for DC protocol tests and improvement, Carolina Martinez, and Coline Trichot for critical reading and suggestions. S.A. and L.P. were supported by ANR-10-IDEX-0001-02 PSL* and ANR-11-LABX-0043 grants.


  1. 1.
    Van Voorhis WC, Hair LS, Steinman RM, Kaplan G (1982) Human dendritic cells. Enrichment and characterization from peripheral blood. J Exp Med 155(4):1172–1187CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Steinman RM, Cohn ZA (1973) Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med 137(5):1142–1162CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Guilliams M, Ginhoux F, Jakubzick C, Naik SH, Onai N, Schraml BU, Segura E, Tussiwand R, Yona S (2014) Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol 14(8):571–578CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Collin M, Bigley V, Haniffa M, Hambleton S (2011) Human dendritic cell deficiency: the missing ID? Nat Rev Immunol 11(9):575–583CrossRefPubMedGoogle Scholar
  5. 5.
    Randolph GJ, Ochando J, Partida-Sanchez S (2008) Migration of dendritic cell subsets and their precursors. Annu Rev Immunol 26:293–316CrossRefPubMedGoogle Scholar
  6. 6.
    Martinez-Cingolani C, Grandclaudon M, Jeanmougin M, Jouve M, Zollinger R, Soumelis V (2014) Human blood BDCA-1 dendritic cells differentiate into Langerhans-like cells with thymic stromal lymphopoietin and TGF-beta. Blood 124(15):2411–2420CrossRefPubMedGoogle Scholar
  7. 7.
    Volpe E, Pattarini L, Martinez-Cingolani C, Meller S, Donnadieu MH, Bogiatzi SI, Fernandez MI, Touzot M, Bichet JC, Reyal F, Paronetto MP, Chiricozzi A, Chimenti S, Nasorri F, Cavani A, Kislat A, Homey B, Soumelis V (2014) Thymic stromal lymphopoietin links keratinocytes and dendritic cell-derived IL-23 in patients with psoriasis. J Allergy Clin Immunol 134(2):373–381CrossRefPubMedGoogle Scholar
  8. 8.
    Dzionek A, Fuchs A, Schmidt P, Cremer S, Zysk M, Miltenyi S, Buck DW, Schmitz J (2000) BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood. J Immunol 165(11):6037–6046CrossRefPubMedGoogle Scholar
  9. 9.
    Ito T, Inaba M, Inaba K, Toki J, Sogo S, Iguchi T, Adachi Y, Yamaguchi K, Amakawa R, Valladeau J, Saeland S, Fukuhara S, Ikehara S (1999) A CD1a+/CD11c + subset of human blood dendritic cells is a direct precursor of Langerhans cells. J Immunol 163(3):1409–1419PubMedGoogle Scholar
  10. 10.
    Bachem A, Guttler S, Hartung E, Ebstein F, Schaefer M, Tannert A, Salama A, Movassaghi K, Opitz C, Mages HW, Henn V, Kloetzel PM, Gurka S, Kroczek RA (2010) Superior antigen cross-presentation and XCR1 expression define human CD11c + CD141+ cells as homologues of mouse CD8+ dendritic cells. J Exp Med 207(6):1273–1281CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Jongbloed SL, Kassianos AJ, McDonald KJ, Clark GJ, Ju X, Angel CE, Chen CJ, Dunbar PR, Wadley RB, Jeet V, Vulink AJ, Hart DN, Radford KJ (2010) Human CD141+ (BDCA-3) + dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens. J Exp Med 207(6):1247–1260CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Merad M, Sathe P, Helft J, Miller J, Mortha A (2013) The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol 31:563–604CrossRefPubMedGoogle Scholar
  13. 13.
    Segura E, Amigorena S (2014) Cross-presentation by human dendritic cell subsets. Immunol Lett 158(1-2):73–78CrossRefPubMedGoogle Scholar
  14. 14.
    Hogg N, Takacs L, Palmer DG, Selvendran Y, Allen C (1986) The p150,95 molecule is a marker of human mononuclear phagocytes: comparison with expression of class II molecules. Eur J Immunol 16(3):240–248CrossRefPubMedGoogle Scholar
  15. 15.
    MacDonald KP, Munster DJ, Clark GJ, Dzionek A, Schmitz J, Hart DN (2002) Characterization of human blood dendritic cell subsets. Blood 100(13):4512–4520CrossRefPubMedGoogle Scholar
  16. 16.
    Ziegler-Heitbrock L, Ancuta P, Crowe S, Dalod M, Grau V, Hart DN, Leenen PJ, Liu YJ, MacPherson G, Randolph GJ, Scherberich J, Schmitz J, Shortman K, Sozzani S, Strobl H, Zembala M, Austyn JM, Lutz MB (2010) Nomenclature of monocytes and dendritic cells in blood. Blood 116(16):e74–80CrossRefPubMedGoogle Scholar
  17. 17.
    Meredith MM, Liu K, Darrasse-Jeze G, Kamphorst AO, Schreiber HA, Guermonprez P, Idoyaga J, Cheong C, Yao KH, Niec RE, Nussenzweig MC (2012) Expression of the zinc finger transcription factor zDC (Zbtb46, Btbd4) defines the classical dendritic cell lineage. J Exp Med 209(6):1153–1165CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Satpathy AT, Kc W, Albring JC, Edelson BT, Kretzer NM, Bhattacharya D, Murphy TL, Murphy KM (2012) Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages. J Exp Med 209(6):1135–1152CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ (1997) The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J Exp Med 185(6):1101–1111CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Liu YJ (2005) IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol 23:275–306CrossRefPubMedGoogle Scholar
  21. 21.
    Gilliet M, Cao W, Liu YJ (2008) Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases. Nat Rev Immunol 8(8):594–606CrossRefPubMedGoogle Scholar
  22. 22.
    Cella M, Jarrossay D, Facchetti F, Alebardi O, Nakajima H, Lanzavecchia A, Colonna M (1999) Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat Med 5(8):919–923CrossRefPubMedGoogle Scholar
  23. 23.
    Siegal FP, Kadowaki N, Shodell M, Fitzgerald-Bocarsly PA, Shah K, Ho S, Antonenko S, Liu YJ (1999) The nature of the principal type 1 interferon-producing cells in human blood. Science 284(5421):1835–1837CrossRefPubMedGoogle Scholar
  24. 24.
    Reizis B, Bunin A, Ghosh HS, Lewis KL, Sisirak V (2011) Plasmacytoid dendritic cells: recent progress and open questions. Annu Rev Immunol 29:163–183CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Rissoan MC, Soumelis V, Kadowaki N, Grouard G, Briere F, de Waal MR, Liu YJ (1999) Reciprocal control of T helper cell and dendritic cell differentiation. Science 283(5405):1183–1186CrossRefPubMedGoogle Scholar
  26. 26.
    Dzionek A, Sohma Y, Nagafune J, Cella M, Colonna M, Facchetti F, Gunther G, Johnston I, Lanzavecchia A, Nagasaka T, Okada T, Vermi W, Winkels G, Yamamoto T, Zysk M, Yamaguchi Y, Schmitz J (2001) BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction. J Exp Med 194(12):1823–1834CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Wu P, Wu J, Liu S, Han X, Lu J, Shi Y, Wang J, Lu L, Cao X (2008) TLR9/TLR7-triggered downregulation of BDCA2 expression on human plasmacytoid dendritic cells from healthy individuals and lupus patients. Clin Immunol 129(1):40–48CrossRefPubMedGoogle Scholar
  28. 28.
    Ghirelli C, Zollinger R, Soumelis V (2010) Systematic cytokine receptor profiling reveals GM-CSF as a novel TLR-independent activator of human plasmacytoid predendritic cells. Blood 115(24):5037–5040CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.INSERM U932 Immunity and CancerParisFrance
  2. 2.Institut Curie, Centre de rechercheParisFrance

Personalised recommendations