Assessment of Surface Markers and Functionality of Dendritic Cells (DCs)

Rafael Nuñez1

1 Memorial Sloan‐Kettering Cancer Center, New York, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.17
DOI:  10.1002/0471142956.cy0917s17
Online Posting Date:  August, 2001
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Abstract

Dendritic cells are a complex group of mainly bone marrow derived cells that possess significant ability to take up, process, and present soluble antigens to responder cells in the lymphoid tissues. Characterization of DC surface markers has been a difficult task owing to the lack of appropriate reagents of sufficient specificity. Molecules closely associated with dendritic cells are being identified and investigated and flow cytometric approaches to evaluation of monoclonal antibodies against DC populations are being developed. This informative unit describes assays for determination of the existence of subsets within two DC populations, the kinetics of antigen expression, and the identification of specific markers for DC subsets. A presents an approach for generating dendritic cells and Langerhans cells (a skin‚Äźderived DC) from blood monocytes.

     
 
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Table of Contents

  • Basic Protocol 1: Immunophenotyping of Dendritic Cells
  • Basic Protocol 2: Determination of Antigen Uptake by Dendritic Cells
  • Basic Protocol 3: Assessment of Dendritic Cell Division Under Cytokine Modulation
  • Basic Protocol 4: Assessment of In Vitro Cytotoxicity by Flow Cytometry
  • Basic Protocol 5: Assessment of In Vivo Cytotoxicity by Flow Cytometry
  • Support Protocol 1: Generation of Dendritic Cells (DCs)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunophenotyping of Dendritic Cells

  Materials
  • Dendritic cells (DCs; see protocol 6
  • Complete medium for DC culture (see recipe)
  • Unlabeled monoclonal antibodies (mAbs) and/or FITC‐, PE‐, PerCP‐, and APC‐labeled mAbs: CD1a, CD1b/c, BG6, HP‐F1 (CD85i), BU10, RFD‐1, CMRF‐44, 7H5 (CD85a), ZM3.8 (CD85j), 55K‐2 (fascin), MMR1.16, MMR190.BB3 (CD206), L25, CMRF‐56, RFD‐7, MR15‐2 (CD205), DCGM‐4 (CD207), TPD153, 42D1 (CD85f), DEC‐205 (CD205), MMRI‐4 (CD205), DC‐LAMP (CD208), AZN‐D1 (CD209), AZN‐D2 (CD209), CMRF‐75, CMRF‐82, CD11c, CD80, CD86, and HLA‐DR
  • Phosphate‐buffered saline (PBS; appendix 2A) or stain buffer (Pharmingen)
  • FITC‐labeled anti‐mouse Ig (for the indirect method; Pharmingen)
  • Cellfix (Becton Dickinson)
  • 5‐ml round‐bottom tubes (Falcon)
  • Centrifuge, 4°C
  • Flow cytometer with 488‐nm excitation (e.g., FACSCalibur; Becton Dickinson) and 530 ± 15 nm band‐pass filter for collecting green fluorescence
  • Software for analysis (e.g., CellQuest, Becton Dickinson; FlowJo, Flow Jo; or WinMDI)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 3A)

Basic Protocol 2: Determination of Antigen Uptake by Dendritic Cells

  Materials
  • Dendritic cells (DCs; see protocol 6
  • OVA‐FITC antigen (Molecular Probes); store in aliquots ≤1 year at −20°C
  • FACS buffer (see recipe)
  • Fixative (e.g., Cellfix, Becton Dickinson; or PBS [ appendix 2A] containing 2% formaldehyde)
  • 5‐ml round‐bottom tubes (Falcon)
  • 37°C, 5% CO 2 incubator
  • Flow cytometer (e.g., FACSCalibur; Becton Dickinson)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 3A)

Basic Protocol 3: Assessment of Dendritic Cell Division Under Cytokine Modulation

  Materials
  • Dendritic cells (DCs)
  • Complete medium (see recipe)
  • rhGM‐CSF (Novartis)
  • rhIL‐4 (Genzyme)
  • rhTGF‐β1 (R&D Systems; optional)
  • PBS ( appendix 2A)
  • 5‐(and‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes)
  • 25‐cm2 tissue culture flasks
  • 5‐ml round‐bottom tubes (Falcon)
  • 37°C, 5% CO 2 incubator
  • Flow cytometer (e.g., FACSCalibur, Becton Dickinson)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 3A)

Basic Protocol 4: Assessment of In Vitro Cytotoxicity by Flow Cytometry

  Materials
  • Antigens
  • Target cells
  • PBS ( appendix 2A)
  • LIVE/DEAD Cell‐Mediated Cytotoxicity Kit (Molecular Probes)
  • 3 mM DiOC 18(3) stock solution in DMSO
  • 50 µg/ml propidium iodide (PI) stock solution in PBS
  • Complete medium supplemented with 10% (w/v) FCS (see recipe), ice‐cold
  • Effector cells (purified T or spleen cells) (Kruisbeek, )
  • 37°C, 5% CO 2 incubator
  • 5‐ml round‐bottom tubes (Falcon)
  • Flow cytometer (e.g., FACSCalibur; Becton Dickinson)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 3A)

Basic Protocol 5: Assessment of In Vivo Cytotoxicity by Flow Cytometry

  Materials
  • Antigen
  • Target cells of interest
  • Virus (optional)
  • PBS ( appendix 2A)
  • LIVE/DEAD Cell‐Mediated Cytotoxicity Kit (Molecular Probes)
  • 3 mM DiOC 18(3) stock solution in DMSO
  • Complete medium (see recipe), 4°C or on ice
  • Mice
  • Lysis solution (Becton Dickinson)
  • Anticoagulant (e.g., citrate, EDTA)
  • Cellfix (Becton Dickinson)
  • 37°C, 5% CO 2 incubator
  • Sterile 1.8‐ml microcentrifuge tubes (Eppendorf)
  • 5‐ml round‐bottom tubes (Falcon)
  • Flow cytometer (e.g., FACSCalibur, Becton Dickinson)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 3A)

Support Protocol 1: Generation of Dendritic Cells (DCs)

  Materials
  • Adherent PBMCs obtained following the protocol for isolation of PBMCs and the supporting protocol for isolation of adherent cells (Kanof et al., )
  • Complete medium for DC culture (see recipe)
  • rhGM‐CSF (Novartis). Store aliquots ≤1 year at −70°C
  • rhIL‐4 (Genzyme). Store aliquots ≤1 year at −70°C
  • rhTGF‐β1 (R & D Systems). Store aliquots ≤1 year at −70°C
  • 25‐cm2 tissue culture flasks (Corning)
  • 37°C, 5% CO 2 incubator
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Banchereau, J. and Steinman, R.M. 1998. Dendritic cells and the control of immunity. Nature 392:245‐252.
   Bender, A., Sapp, M., Schuler, G., Steinman, R.M., and Bhardwaj, N. 1996. Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood. J. Immunol. Methods 196:121‐135.
   Cella, M., Sallusto, F., and Lanzavecchia, A. 1997. Origin, maturation and antigen presenting function of dendritic cells. Curr. Opin. Immunol. 9:10‐16.
   Geissmann, F., Prost, C., Monnet, J.‐P., Dy, M., Brousse, N., and Hermine, O. 1998. Transforming growth factor beta1, in the presence of granulocyte/macrophage colony‐stimulating factor and interleukin 4, induces differentiation of human peripheral blood monocytes into dendritic Langerhans cells. J. Exp. Med. 187:961‐966.
   Hart, D.N.J., Clark, G.J., MacDonald, K., Kato, M., Vuckovic, S., Lopez, A., Wykes, M., and Munster, D. 2001. 7th Leucocyte Differentiation Antigen Workshop, DC section summary(D. Mason, ed.). Leukocyte Typing VII, Oxford University Press, Oxford. In press.
   Hock, B.D, Sterling, G.C, Daniel, P.B. and Hart, D.N. 1994. Characterization of CMRF‐44, a novel monoclonal antibody to an activation antigen expressed by allostimulatory cells within peripheral blood, including dendritic cells. Immunology 83:573‐581.
   Jiang, W., Swiggard, W.J., Heufler, C., Peng, M., Mirza, A., Steinman, R.M., and Nussenzweig, M.C. 1995. The receptor DEC‐205 expressed by dendritic cells and thymic epithelial cells is involved in antigen presentation. Nature 375:151‐155.
   Kamau, S., Hurtado, M., Müller‐Doblies, U., Grimm, F., and Nuñez, R. 2000. Flow cytometric assessment of allopurinol susceptibility in Leishmania infantum promastigotes. Cytometry 40:353‐360.
   Kanof, M.E., Smith, P.D., and Zola, H. 1996. Preparation of human mononuclear cell populations and subpopulations. In Current Protocols in Immunology(J. Cooligan, A. Kruisbeek, D. Margulies, E. Shevach, and W. Strober, eds.) pp.7.1.1‐7.1.7. John Wiley & Sons, New York.
   Katz, S.I., Cooper, K.D., Iijima, M., and Tsuchida, T. 1985. The role of Langerhans cells in antigen presentation. J. Invest. Dermatol. 85:96‐98.
   Kruisbeek, A.M. 2000. Isolation and fractionation of mononuclear cell populations. In Current Protocols in Immunology(J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp.3.1.2‐3.1.5. John Wiley & Sons, New York.
   Nuñez, R. 2001. Flow Cytometry for Research Scientists: Principles and Applications. Horizon Scientific Press, Norfolk, England.
   Nuñez, R. and Filgueira, L. 2001. Flow cytometry assessment of monoclonal antibody (mAb) reactivities against dendritic cells (DC). In Leukocyte Typing VII(D. Mason, ed.). Oxford University Press. Oxford. In press.
   Nuñez, R., Sanchez, M., Filgueira, L., Wild, P., and Nuñez, C. 1998. Characterization of two human dendritic cell lines that express CD1a, take‐up, process and present soluble antigens and induce MLR. Immunol. Lett. 61:33‐43.
   Nuñez, R., Filgueira, L., and Nuñez, C. 2001. Flow cytometric assessment on human dendritic cell lines (HDCL) of monoclonal antibody (mAb) reactivities and determination of surface antigen expression by cytokine modulation. In Leukocyte Typing VII(D. Mason, ed.). Oxford University Press, Oxford. In press.
   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 alpha. J. Exp. Med. 179:1109‐1118.
   Steinman, R.M. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9:271‐296.
Key References
   Geissmann et al., 1998. See above.
  Initial reference for generating LCs from monocytes.
   Hart et al., 2001. See above.
  Describes the new CD markers assigned for dendritic cells, as well as an additional set of markers identified in dendritic cells that did not fulfill the requirements for assignment of a CD.
   Nuñez, 2001. See above.
  Contains a chapter on flow cytometric evaluation of DCs.
   Nuñez and Filgueira, 2001. See above.
  Displays the mAbs used in the DC workshop and the procedures for generating monocyte‐derived DCs and LCs.
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