Springer Protocols: Abstract: The Isolation and Identification of Murine Dendritic Cell Populations from Lymphoid Tissues and Their Production in Culture

The Isolation and Identification of Murine Dendritic Cell Populations from Lymphoid Tissues and Their Production in Culture

By: David Vremec², Ken Shortman²

Abstract

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Dendritic cells (DC) are widely regarded as the most potent cellular inducers of the adaptive immune response; so, immunotherapy through DC manipulation is a promising option in the future fight against many human ailments. We have developed a method of isolating DC from the mouse that involves efficient extraction from tissues, followed by the selection of the lightest density cells, then depletion of non-DC through a cocktail of monoclonal antibodies and anti-immunoglobulin magnetic beads. Finally, purification and segregation into DC subtypes is achieved by immunofluorescent labeling and sorting. This has demonstrated a network of DC populations differing in surface phenotype and function. We can now produce larger numbers of many of these DC subpopulations from their precursors using bone marrow cultures supplemented with fms-like tyrosine kinase 3 ligand (Flt3L). The culture-generated DC can be aligned with the populations directly isolated from tissues. Combining the in vivo and in vitro systems will make study of murine DC and their alignment to their human counterparts an easier break process.

Affiliation(s): (2) The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia

Book Title: Innate Immunity

Series: Methods in Molecular Biology | Volume: 415 | Pub. Date: Nov-19-2007 | Page Range: 163-178 | DOI: 10.1007/978-1-59745-570-1_10

Subject: Immunology

Key Words: Dendritic cell - DC isolation - DC subpopulations - flow cytometry - Flt3 ligand

References

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

2.	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.

3.	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.

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

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

6.	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.

7.	Yasuda, K., Yu, P., Kirschning, C.J., Schlatter, B., Schmitz, F., Heit, A., Bauer, S., Hochrein, H. and Wagner, H. (2005) Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways. J. Immunol. 174, 6129–6136.

8.	Asselin-Paturel, C., Boonstra, A., Dalod, M., Durand, I., Yessaad, N., Dezutter-Dambuyant, C., Vicari, A., O‘Garra, A., Biron, C., Briere, F. and Trinchieri, G. (2001) Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology. Nat. Immunol. 2, 1144–1150.

9.	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.

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

11.	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.

12.	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.

13.	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.

14.	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.

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

16.	Sallusto, F. and Lanzavecchia, A. (1994) Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin-4 and downregulated by tumor necrosis factor-a. J. Exp. Med. 179, 1109–1118.

17.	Brasel, K., De Smedt, T., Smith, J.L. and Maliszewski, C.R. (2000) Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures. Blood 96, 3029–3039.

18.	Gilliet, M., Boonstra, A., Paturel, C., Antonenko, S., Xu, X.L., Trinchieri, G., O‘Garra, A. and Liu, Y.J. (2002) The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT-3-ligand and granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 195, 953–958.

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

20.	Arrighi, J-F., Hauser, C., Chapuis, B., Zubler, R.H. and Kindler, V. (1999) Long-term culture of human CD34⁺ progenitors with FLT3-ligand, thrombopoietin, and stem cell factor induces extensive amplification of a CD34^- CD14^- and a CD34^-CD14⁺ dendritic cell precursor. Blood 93, 2244–2252.

21.	Chen, W., Antonenko, S., Sederstrom, J.M., Liang, X., Chan, A.S.H., Kanzler, H., Blom, B., Blazar, B.R. and Liu, Y-J. (2004) Thrombopoietin cooperates with FLT3-ligand in the generation of plasmacytoid dendritic cell precursors from human hematopoietic progenitors. Blood 103, 2547–2553.

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