Flow Cytometric Techniques for Studying Plasmacytoid Dendritic Cells in Mixed Populations

  • Stacey L. Olshalsky
  • Patricia Fitzgerald-Bocarsly
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 116)


Plasmacytoid dendritic cells (PDCs) are the natural interferon (IFN-α)-producing cells in human peripheral blood that produce vast quantities of IFN-α in response to viral infection and other stimuli. PDCs are a rare cell type, making up less than 0.5% of peripheral blood mononuclear cells. To date, these cells have not been successfully cultured in vitro and are very sensitive to selection via magnetic bead labeling, making them very difficult to study as a purified population. Therefore, our laboratory has developed techniques to study PDCs in mixed populations. Using flow cytometry to label specific cell-surface markers, PDCs can be easily identified from other peripheral blood mononuclear cells or in mononuclear cell suspensions of lymphoid tissue. PDCs can also be permeabilized and stained for intracellular proteins or cytokines. Using surface and intracellular flow cytometry, phenotypic and functional aspects can be combined to accurately study PDCs in a mixed population of cells.

Key Words

Plasmacytoid dendritic cells IFN-α IRF-7 intracellular flow cytometry 


  1. 1.
    Siegal, F., Kadowaki, N., Shodell, M., Fitzgerald-Bocarsly, P., Shah, K., Ho, S., et al. (1999) The nature of the principal type 1 interferon-producing cells in human blood. Science 284, 1835–1837.PubMedCrossRefGoogle Scholar
  2. 2.
    Cella, M., Jarrossay, D., Facchetti, F., Alebardi, O., Nakajima, H., Lanzavecchia, A., and Colonna, M. (1999) Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat. Med. 5, 919–923.PubMedCrossRefGoogle Scholar
  3. 3.
    Grouard, G., Rissoan, M., Filguiera, L., Durand, I., Banchereau, J., and Liu, J. (1997) The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin-3 and CD40 ligand. J. Exp. Med. 185, 1101–1111.PubMedCrossRefGoogle Scholar
  4. 4.
    Fitzgerald-Bocarsly, P. (2002) Natural IFN-alpha producing cells: The plasmacytoid dendritic cells. BioTechniques 33, S16–S29.Google Scholar
  5. 5.
    Sellati, T. J., Waldrop, S. L., Salazar, J. C., Bergstresser, P. R., Picker, L., and Radolf, J. D. (2001) The cutaneous response in humans to Treponema pallidum lipoprotein analogues involves cellular elements of both innate and adaptive immunity. J. Immunol. 166, 4131–4140.PubMedGoogle Scholar
  6. 6.
    Jahnsen, F., Lund-Johansen, F., Dunne, J., Farkas, L., Haye, R., and Brandtzaeg, P. (2000) Experimentally induced recruitment of plasmacytoid (CD123high) dendritic cells in human nasal allergy. J. Immunol. 165, 4062–4068.PubMedGoogle Scholar
  7. 7.
    Feldman, S. B., M. Ferraro, H. M. Zheng, N. Patel, S. Gould-Fogerite, and P. Fitzgerald-Bocarsly. (1994) Viral induction of low frequency interferon-alpha producing cells. Virology 204, 1.PubMedCrossRefGoogle Scholar
  8. 8.
    Svensson, H., Cederblad, B., Lindahl, M., and Alm, G. (1996) Stimulation of natural interferon-α/β-producing cells by Staphylococcus aureus. J. Interferon Cytokine Res. 16, 7–16.PubMedCrossRefGoogle Scholar
  9. 9.
    Remoli, M. E., Giacomini, E., Lutfalla, G., Dondi, E., Orefici, G., Battistini, A., et al. (2002) Selective expression of type I IFN genes in human dendritic cells infected with Mycobacterium tuberculosis. J. Immunol. 169, 366–374.PubMedGoogle Scholar
  10. 10.
    Gibson, S. J., Imbertson, L. M., Wagner, T. L., Testerman, T. L., Reiter, M. J., Miller, R. L., et al. (1995) Cellular requirements for cytokine production in response to the immunomodulators imiquimod and S-27609. J. Interferon Cytokine Res. 15, 537–545.PubMedCrossRefGoogle Scholar
  11. 11.
    Kadowaki, N., Antonenko, S., and Liu, Y. J. (2001) Distinct CpG DNA and polyinosinic-polycytidylic acid double-stranded RNA, respectively, stimulate CD11c-type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN. J. Immunol. 166, 2291–2295.PubMedGoogle Scholar
  12. 12.
    Kadowaki, N., Ho, S., Antonenko, S., Malefyt, R. W., Kastelein, R. A., Bazan, F., and Liu, Y. J. (2001) Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J. Exp. Med. 194, 863–869.PubMedCrossRefGoogle Scholar
  13. 13.
    Krug, A., Rothenfusser, S., Hornung, V., Jahrsdorfer, B., Blackwell, S., Ballas, Z. K., et al. (2001) Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur. J. Immunol. 31, 2154–2163.PubMedCrossRefGoogle Scholar
  14. 14.
    Krug, A., Towarowski, A., Britsch, S., Rothenfusser, S., Hornung, V., Bals, R., et al. (2001) Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. Eur. J. Immunol. 31, 3026–3037.PubMedCrossRefGoogle Scholar
  15. 15.
    Megjugorac, N., Young, H., Amrute, S., Olshalsky, S., and Fitzgerald-Bocarsly, P. (2004) Virally stimulated plasmacytoid dendritic cells produce chemokines and induce migration of T and NK cells. J. Leuk. Biol. 75, 504–514.CrossRefGoogle Scholar
  16. 16.
    Penna, G., Sozzani, S., and Adorini, L. (2001) Cutting Edge: Selective usage of chemokine receptors by plasmacytoid dendritic cells. J. Immunol. 167, 1862–1866.PubMedGoogle Scholar
  17. 17.
    Cella, M., Facchetti, F., Lanzavecchia, A., and Colonna, M. (2000) Plasmacytoid dendritic cells activated by influenza virus and CD40L drive a potent TH1 polarization. Nat. Immunol. 1, 305–310.PubMedCrossRefGoogle Scholar
  18. 18.
    Bandyopadhyay, S., Perussia, B., Trinchieri, G., Miller, D. S., and Starr, S. (1986) Requirement for HLA-DR+ accessory cells in natural killing of cytomegalovirus-infected fibroblasts. J. Exp. Med. 164, 180–195.PubMedCrossRefGoogle Scholar
  19. 19.
    Fitzgerald-Bocarsly, P., Feldman, M., Curl, S., Schnell, J., and Denny, T. (1989) Positively selected Leu-11a (CD16+) cells require the presence of accessory cells or factors for the lysis of HSV-infected fibroblasts but not HSV-infected Raji. J. Immunol. 143, 1318–1326.PubMedGoogle Scholar
  20. 20.
    Svensson, H., Johannisson, A., Nikkila, T., Alm, G. V., and Cederblad, B. (1996) The cell surface phenotype of human natural interferon-a producing cells as determined by flow cytometry. Scand. J. Immunol. 44, 164–172.PubMedCrossRefGoogle Scholar
  21. 21.
    Dzionek, A., Fuchs, A., Schmidt, P., Cremer, S., Zysk, M., Miltenyi, S., Buck, D. W., and 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, 6037–6046.PubMedGoogle Scholar
  22. 22.
    Dzionek, A., Sohma, Y., Nagafune, J., Cella, M., Colonna, M., Facchetti, F., et al. (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, 1823–1834.PubMedCrossRefGoogle Scholar
  23. 23.
    Dzionek, A., Inagaki, Y., Okawa, K., Nagafune, J., Rock, J., Sohma, Y., et al. (2002) Plasmacytoid dendritic cells: From specific surface markers to specific cellular functions. Hum. Immunol. 63, 1133–1148.PubMedCrossRefGoogle Scholar
  24. 24.
    Cederblad, B. and Alm, G. (1990) Infrequent but efficient interferon-α-producing human mononuclear leukocytes induced by herpes simplex virus in vitro studies by immunoplaque and limiting dilution assays. J. Interferon Res. 10, 65–73.PubMedCrossRefGoogle Scholar
  25. 25.
    Feldman, S., Stein, D., Amrute, S., Denny, T., Garcia, Z., Kloser, P., et al. (2001) Decreased interferon-α production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors. Clin. Immunol. 101, 201–210.PubMedCrossRefGoogle Scholar
  26. 26.
    Soumelis, V., Scott, I., Gheyas, F., Bouhour, D., Cozon, G., Cotte, L., et al. (2001) Depletion of circulating natural type 1 interferon-producing cells in HIV-infected AIDS patients. Blood 98, 906–912.PubMedCrossRefGoogle Scholar
  27. 27.
    Chehimi, J., Campbell, D. E., Azzoni, L., Bacheller, E., Papasavvas, G., Jerandi, K., et al. (2002) Persistent decreases in blood plasmacytoid dendritic cell number and function despite effective highly active antiretroviral therapy and increased blood myeloid dendritic cells in HIV-infected individuals. J. Immunol. 168, 4796–4801.PubMedGoogle Scholar
  28. 28.
    Kadowaki, N., Antonenko, S., Lau, J. Y., and Liu, Y. J. (2000) Natural interferon alpha/beta-producing cells link innate and adaptive immunity. J. Exp. Med. 192, 219–226.PubMedCrossRefGoogle Scholar
  29. 29.
    Ito, T., Amakawa, R., Inaba, M., Ikehara, S., Inaba, K., and Fukuhara, S. (2001) Differential regulation of human blood dendritic cell subsets by IFNs. J. Immunol. 166, 2961–2969.PubMedGoogle Scholar
  30. 30.
    Kohrgruber, N., Halanek, N., Groger, M., Winter, D., Rappersberger, K., Schmitt-Egenolf, M., et al. (1999) Survival, maturation, and function of CD11c-and CD11c+ peripheral blood dendritic cells are differentially regulated by cytokines. J. Immunol. 163, 3250–3259.PubMedGoogle Scholar
  31. 31.
    Howell, D., Feldman, S., Kloser, P., and Fitzgerald-Bocarsly, P. (1994) Decreased frequency of natural interferon producing cells in peripheral blood of patients with the acquired immune deficiency syndrome. Clin. Immunol. Immunopath. 71, 223–230.CrossRefGoogle Scholar
  32. 32.
    Feldman, S. B., Milone, M. C., Kloser, P., and Fitzgerald-Bocarsly, P. (1995) Functional deficiencies in two distinct IFN-α producing cell populations in PBMC from human immunodeficiency virus seropositive patients. J. Leuk. Biol. 57, 214–220.Google Scholar
  33. 33.
    Ferbas, J. J., Toso, J. F., Logar, A. J., Navratil, J. S., and Rinaldo, C. R. (1994) CD4+ blood dendritic cells are potent producers of IFN-α in response to in vitro HIV-1 infection. J. Immunol. 152, 4649–4662.PubMedGoogle Scholar
  34. 34.
    Kerkmann, M., Rothenfusser, S., Hornung, V., Towarowski, A., Wagner, M., Sarris, A., et al. (2003) Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways of type I IFN synthesis in human plasmacytoid dendritic cells. J. Immunol. 170, 4465–4474.PubMedGoogle Scholar
  35. 35.
    Sato, M., Suemori, H., Hata, N., Asagiri, M., Ogasawara, K., Nakao, K., et al. (2000) Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction. Immunity 13, 539–548.PubMedCrossRefGoogle Scholar
  36. 36.
    Barnes, B. J., Lubyova, B., and Pitha, P. (2002) On the role of interferon regulatory factors in host defense. J. Interferon Cytokine Res. 22, 59–71.PubMedCrossRefGoogle Scholar
  37. 37.
    Izaguirre, A., Barnes, B. J., Amrute, S., Yeow, W. S., Megjugorac, N., Dai, J., et al. (2003) Comparative analysis of IRF and IFN-alpha expression in human plasmacytoid and monocyte-derived dendritic cells. J. Leuk. Biol. 74, 1125–1138.CrossRefGoogle Scholar
  38. 38.
    Dai, J. H., Megjugorac, N., Amrute, S., and Fitzgerald-Bocarsly, P. (2004) Regulation of IRF-7 and IFN-alpha production by enveloped virus and LPS in human plasmacytoid dendritic cells. J. Immunol. 173, 1535–1548.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Stacey L. Olshalsky
    • 1
  • Patricia Fitzgerald-Bocarsly
    • 1
  1. 1.Department of Pathology and Laboratory MedicineUniversity of Medicine and Dentistry of New JerseyNewark

Personalised recommendations