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The Isolation of Mouse Dendritic Cells from Lymphoid Tissues and the Identification of Dendritic Cell Subtypes by Multiparameter Flow Cytometry

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Dendritic Cell Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 595))

Abstract

Dendritic cells (DCs) are powerful initiators of the adaptive immune system and their manipulation is seen as a viable approach in the treatment of many human ailments. Our isolation method purifies DCs from mouse lymphoid organs by efficiently removing them from the tissue using collagenase, selecting the light density fraction of cells and then negatively selecting for DCs using a combination of monoclonal antibodies directed against non-DC lineage cells together with anti-rat immunoglobulin-coated magnetic beads. Remaining contaminating cells can be depleted using a combination of specific biotinylated antibodies and anti-biotin-coated magnetic beads, in the case of natural killer (NK) cells, or by FACS gating, during sorting or analysis, in the case of autofluorescent macrophages. The heterogenous DCs can then be separated into various functionally different populations via immunofluorescent labelling and cell sorting. The study of mouse DC populations may enable us to align them to their human counterparts and subsequently to DC populations produced in vitro.

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References

  1. Jung, S., Unutmaz, D., Wong, P., Sano, G., De los Santos, K., Sparwasser, T., Wu, S., Vuthoori, S., Ko, K., Zavala, F., Pamer, E.G., Littman, D.R. and Lang, R.A. (2002) In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 17, 211–220.

    Article  CAS  PubMed  Google Scholar 

  2. Liu, C.H., Fan, Y.T., Dias, A., Esper, L., Corn, R.A., Bafica, A., Machado, F.S. and Aliberti, J. (2006) Cutting Edge: Dendritic cells are essential for in vivo IL-12 production and development of resistance against Toxoplasma gondii infection in mice. J. Immunol. 177, 31–35.

    CAS  PubMed  Google Scholar 

  3. Ciavarra, R.P., Stephens, A., Nagy, S., Sekellick, M. and Steel, C. (2006) Evolution of immunological paradigms in a virus model: Are dendritic cells critical for antiviral immunity and viral clearance? J. Immunol. 177, 492–500.

    CAS  PubMed  Google Scholar 

  4. Watanabe, N., Wang, Y.H., Lee, H.K., Ito, T., Cao, W. and Liu, Y.J. (2005) Hassall’s corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus. Nature 436, 1181–1185.

    Article  CAS  PubMed  Google Scholar 

  5. Luo, X., Tarbell, K.V., Yang, H., Pothoven, K., Bailey, S.L., Dind, R., Steinman, R.M. and Suthanthiran, M. (2007) Dendritic cells with TGF-β1 differentiate naïve CD4+CD25− T cells into islet-protective Foxp3+ regulatory T cells. Proc. Natl. Acad. Sci. USA 104, 2821–2826.

    Article  CAS  PubMed  Google Scholar 

  6. Yamazaki, S., Iyoda, T., Tarbell, K., Olson, K., Velinzon, K., Inaba, K. and Steinman, R.M. (2003) Direct expansion of functional CD25+CD4+ regulatory T cells by antigen presenting dendritic cells. J. Exp. Med. 198, 235– 247.

    Article  CAS  PubMed  Google Scholar 

  7. Wilson, N.S., El-Sukkari, D., Belz, G.T., Smith, C.M., Steptoe, R.J., Heath, W.R., Shortman, K. and Villadangos, J.A. (2003) Most lymphoid organ dendritic cell types are phenotypically and functionally immature. Blood 102, 2187–2194.

    Article  CAS  PubMed  Google Scholar 

  8. Steinman, R.M. (1991) The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9, 271–296.

    Article  CAS  PubMed  Google Scholar 

  9. Vremec, D and Shortman, K. (1997) Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes on incubation and differences between thymus, spleen and lymph nodes. J. Immunol. 159, 565–573.

    CAS  PubMed  Google Scholar 

  10. Shortman, K. and Caux, C. (1997) Dendritic cell development: multiple pathways to natures adjuvant. Stem Cells 15, 409–419.

    Article  CAS  PubMed  Google Scholar 

  11. Shortman, K. and Liu, Y.J. (2002) Mouse and human dendritic cell subtypes. Natl. Rev. Immunol. 2, 151–161.

    Article  CAS  Google Scholar 

  12. Vremec, D., Pooley, J., Hochrein, H., Wu, L. and Shortman, K. (2000) CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. Immunol. 164, 2978–2986.

    CAS  PubMed  Google Scholar 

  13. Caminschi, I., Ahmet, F., Heger, K., Brady, J., Nutt, S.L., Vremec, D., Pietersz, S., Lahoud, M.H., Schofield, L., Hansen, D.S., O’Keeffe, M., Smyth, M.J., Bedoui, S., Davey, G.M., Villadangos, J.A., Heath, W.R and Shortman, K. (2007) Putative IKDCs are functionally and developmentally similar to natural killer cells, but not to dendritic cells. J. Exp. Med. 204, 2579–2590.

    Article  CAS  PubMed  Google Scholar 

  14. Vremec, D., O’Keeffe, M., Hochrein, H., Fuchsberger, M., Caminschi, I., Lahoud, M. and Shortman, K. (2007) Production of interferons by dendritic cells, plasmacytoid cells, natural killer cells, and interferon-producing killer dendritic cells. Blood 109, 1165–1173.

    Article  CAS  PubMed  Google Scholar 

  15. Naik, S.H., Metcalf, D., van Nieuwenhuijze, A., Wicks, I., Wu, L., O’Keeffe, M. and Shortman, K. (2006) Intrasplenic steady-state dendritic cell precursors which are distinct from monocytes. Nat. Immunol. 7, 663–671.

    Article  CAS  PubMed  Google Scholar 

  16. O’Keeffe, M., Hochrein, H., Vremec, D., Caminschi, I., Miller, J.L., Anders, E.M., Wu, L., Lahoud, M.H., Henri, S., Scott, B., Hertzog, P., Tatarczuch, L. and Shortman, K. (2002) Mouse plasmacytoid cells: long-lived cells, heterogeneous in surface phenotype and function, that differentiate into CD8+ dendritic cells only after microbial stimulus. J. Exp. Med. 196, 1307–1319.

    Article  PubMed  Google Scholar 

  17. Asselin-Paturel, C., Brizard, G., Pin, J-J., Briere, F. and Trinchieri, G. (2003) Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody. J. Immunol. 171, 6466–6477.

    CAS  PubMed  Google Scholar 

  18. Toyama-Sorimachi, N., Omatsu, Y., Onoda, A., Tsujimura, Y., Iyoda, T., Kikuchi-Maki, A., Sorimachi, H., Dohi, T., Taki, S., Inaba, K, and Karasuyama, H. (2005) Inhibitory NK receptor Ly49Q is expressed on subsets of dendritic cells in a cellular maturation- and cytokine stimulation-dependent manner. J. Immunol. 174, 4621–4629.

    CAS  PubMed  Google Scholar 

  19. Omatsu, Y., Iyoda, T., Kimura, Y., Maki, A., Ishimori, M., Toyama- Sorimachi, N. and Inaba, K. (2005) Development of murine plasmacytoid dendritic cells defined by increased expression of an inhibitory NK receptor, Ly49Q. J. Immunol. 174, 6657–6662.

    CAS  PubMed  Google Scholar 

  20. Naik, S.H. and Shortman, K. (2007) Steady-state and inflammatory dendritic- cell development. Nat. Rev. Immunol. 7, 19–30.

    Article  PubMed  Google Scholar 

  21. Hochrein, H., Shortman, K., Vremec, D., Scott, B., Hertzog, P. and O’Keeffe, M. (2001) Differential production of IL-12, IFN-α and IFN-γ by mouse dendritic cell subsets. J. Immunol. 166, 5448–5455.

    CAS  PubMed  Google Scholar 

  22. Pooley, J.L., Heath, W.R. and Shortman, K. (2001) Cutting edge: intravenous soluble antigen is presented to CD4 T cells by CD8− dendritic cells, but cross- presented to CD8 T cells by CD8+ dendritic cells. J. Immunol. 166, 5327–5330.

    CAS  PubMed  Google Scholar 

  23. Proietto, A.I., O’Keeffe, M., Gartlan, K., Wright, M.D., Shortman, K. Wu, L. and Lahoud, M.H. (2004) Differential production of inflammatory chemokines by murine dendritic cell subsets. Immunobiology 209, 163–172.

    Article  CAS  PubMed  Google Scholar 

  24. Naik, S.H., Proietto, A.I., Wilson, N.S., Dakic, A., Schnorrer, P., Fuchsberger, M., Lahoud, M.H., O’Keeffe, M., Shao, Q.X., Chen, W.F., Villadangos, J.A., Shortman, K. and Wu, L. (2005) Cutting edge: generation of splenic CD8+ and CD8− dendritic cell equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures. J. Immunol. 174, 6592–6597.

    CAS  PubMed  Google Scholar 

  25. Carter, R.W., Thompson, C., Reid, D. M., Wong, S.Y.C. and Tough, D.F. (2006) Preferential Induction of CD4+ T cell responses through in vivo targeting of antigen to dendritic cell-associated C-Type lectin-1. J. Immunol. 177, 2276–2284.

    CAS  PubMed  Google Scholar 

  26. Villadangos, J.A. and Heath, W.R. (2005) Life cycle, migration and antigen presenting functions of spleen and lymph node dendritic cells: Limitations of the Langerhans cells paradigm. Semin. Immunol. 17, 262–272.

    Article  CAS  PubMed  Google Scholar 

  27. Belz, G.T., Smith, C.M., Kleinert, L., Reading, P., Brooks, A., Shortman, K., Carbone, F.R. and Heath, W.R. (2004) Distinct migrating and nonmigrating dendritic cell populations are involved in MHC class I-restricted antigen presentation after lung infection with virus. Proc. Natl. Acad. Sci. USA 101, 8670–8675.

    Article  CAS  PubMed  Google Scholar 

  28. Henri, S., Vremec, D., Kamath, A., Waithman, J., Williams, S., Benoist, C., Burnham, K., Saeland, S., Handman, E. and Shortman, K. (2001) The dendritic cell populations of mouse lymph nodes. J. Immunol. 167, 741–748.

    CAS  PubMed  Google Scholar 

  29. Bursch, L.S., Wang, L., Igyarto, B., Kissenpfennig, A., Malissen, B., Kaplan, D.H. and Hogquist, K.A. (2007) Identification of a novel population of langerin+ dendritic cells. J. Exp. Med. 204, 3147–3156.

    Article  CAS  PubMed  Google Scholar 

  30. Tacken, P.J., deVries, J.M., Torensma, R. and Figdor, C.G. (2007) Dendritic cell immunotherapy: from ex vivo loading to in vitro targeting. Nat. Rev. Immunol. 7, 790–802.

    Article  CAS  PubMed  Google Scholar 

  31. Corbett, A.J., Caminschi, I., McKenzie, B.S., Brady, J.L., Wright, M.D., Mottram, P.L., Hogarth, P.M., Hodder, A.N., Zhan, Y., Tarlinton, D.M., Shortman, K. and Lew, A.M. (2005) Antigen delivery via two molecules on the CD8− dendritic cell subset induces humoral immunity in the absence of conventional “danger”. Eur. J. Immunol. 35, 2815–2825.

    Article  CAS  PubMed  Google Scholar 

  32. Bonifaz, L., Bonnyay, D., Mahnke, K., Rivera, M., Nussenzweig, M.C. and Steinman, R.M. (2002) Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. J. Exp. Med. 196, 1627–1638.

    Article  CAS  PubMed  Google Scholar 

  33. Caminschi, I., Proietto, A.I., Ahmet, F., Kitsoulis, S., Teh, J.S., Lo, J.C.Y., Jensen, K., Rizzitelli, A., Wu, L., Vremec, D., Maraskovsky, E., Braley, H., Davey, G., Mottram, P., van de Velde, N., Shizuo, A., Lew, A.M., Wright, M.D., Heath, W.R. Shortman, K. and Lahoud, M.H. (2008) The dendritic cell subtype restricted C-type lectin Clec9A is a target for vaccine enhancement. Blood 112, 3264–3273.

    Google Scholar 

  34. Vandenabeele, S., Hochrein, H., Mavaddat, N., Winkel, K. and Shortman, K. (2001) Human thymus contains 2 distinct dendritic cell populations. Blood 97, 1733–1741.

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

    David Vremec

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  1. David Vremec
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Editors and Affiliations

  1. Quellijnstraat 25A, Amsterdam, 1072 XN, Netherlands

    Shalin H. Naik

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© 2010 Humana Press, a part of Springer Science+Business Media, LLC

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Vremec, D. (2010). The Isolation of Mouse Dendritic Cells from Lymphoid Tissues and the Identification of Dendritic Cell Subtypes by Multiparameter Flow Cytometry. In: Naik, S. (eds) Dendritic Cell Protocols. Methods in Molecular Biology, vol 595. Humana Press. https://doi.org/10.1007/978-1-60761-421-0_14

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  • DOI: https://doi.org/10.1007/978-1-60761-421-0_14

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-420-3

  • Online ISBN: 978-1-60761-421-0

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