Differentiation of Peripheral Blood Monocytes into Dendritic Cells

David W. O'Neill1, Nina Bhardwaj1

1 New York University School of Medicine, New York, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.4
DOI:  10.1002/0471142735.im22f04s67
Online Posting Date:  July, 2005
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Dendritic cells (DCs) are potent antigen‐presenting cells (APC) that are important in the initiation and control of cellular immune responses. Commonly used in T cell–stimulation experiments, DCs are typically “matured” in vitro with microbial products or proinflammatory cytokines, and then loaded with antigens from any number of sources, including peptides, whole proteins, cell lysates, RNA, microbes, or killed tumor cells. This unit presents a simple and commonly used method for the generation of mature human dendritic cells—differentiating them from peripheral blood monocytes.

Keywords: antigen presenting cells; dendritic; human; monocytes

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Peripheral blood mononuclear cells (PBMC; unit 7.1)
  • RPMI‐10 (see recipe)
  • RPMI 1640 medium (e.g., Invitrogen; no serum or other additives), prewarmed to 37°C
  • PBS/5% albumin (see recipe)
  • 400 IU/µl IL‐4 (see recipe)
  • 100 IU/µl GM‐CSF (see recipe)
  • 100× monocyte conditioned medium (MCM) mimic (see recipe)
  • Heat‐inactivated human AB serum containing 10% (v/v) DMSO
  • Isopropanol
  • Liquid nitrogen
  • 10‐cm tissue culture dishes (BD Falcon)
  • Inverted microscope
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • Tabletop centrifuge
  • 6‐well tissue culture plates (BD Falcon)
  • 1.0 or 1.8‐ml cryovials
  • “Mr. Frosty” Cryo 1°C freezing container (Nalgene)
  • Liquid nitrogen freezer or equivalent
  • Additional reagents and equipment for determining cell viability by trypan blue exclusion ( appendix 3B)
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Literature Cited

Literature Cited
   Albert, M.L., Sauter, B., and Bhardwaj, N. 1998. Dendritic cells acquire antigen from apoptotic cells and induce class I–restricted CTLs. Nature 392:86‐89.
   Ardavin, C., Amigorena, S., and Reis e Sousa, C. 2004. Dendritic cells: Immunobiology and cancer immunotherapy. Immunity 20:17‐23.
   Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran, B., and Palucka, K. 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18:767‐811.
   Banchereau, J., Palucka, A.K., Dhodapkar, M., Burkeholder, S., Taquet, N., Rolland, A., Taquet, S., Coquery, S., Wittkowski, K.M., Bhardwaj, N., Pineiro, L., Steinman, R., and Fay, J. 2001. Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor‐derived dendritic cell vaccine. Cancer Res. 61:6451‐6458.
   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.
   Caux, C., Dezutter‐Dambuyant, C., Schmitt, D., and Banchereau, J. 1992. GM‐CSF and TNF‐alpha cooperate in the generation of dendritic Langerhans cells. Nature 360:258‐261.
   Dhodapkar, M.V., Steinman, R.M., Sapp, M., Desai, H., Fossella, C., Krasovsky, J., Donahoe, S.M., Dunbar, P.R., Cerundolo, V., Nixon, D.F., and Bhardwaj, N. 1999. Rapid generation of broad T‐cell immunity in humans after a single injection of mature dendritic cells. J. Clin. Invest. 104:173‐180.
   Engelmayer, J., Larsson, M., Lee, A., Lee, M., Cox, W.I., Steinman, R.M., and Bhardwaj, N. 2001. Mature dendritic cells infected with canarypox virus elicit strong anti‐human immunodeficiency virus CD8+ and CD4+ T‐cell responses from chronically infected individuals. J. Virol. 75:2142‐2153.
   Fonteneau, J.F., Gilliet, M., Larsson, M., Dasilva, I., Munz, C., Liu, Y.J., and Bhardwaj, N. 2003. Activation of influenza virus‐specific CD4+ and CD8+ T cells: A new role for plasmacytoid dendritic cells in adaptive immunity. Blood 101:3520‐3526.
   Hsu, F.J., Benike, C., Fagnoni, F., Liles, T.M., Czerwinski, D., Taidi, B., Engleman, E.G., and Levy, R. 1996. Vaccination of patients with B‐cell lymphoma using autologous antigen‐ pulsed dendritic cells. Nat. Med. 2:52‐58.
   Jonuleit, H., Kuhn, U., Muller, G., Steinbrink, K., Paragnik, L., Schmitt, E., Knop, J., and Enk, A.H. 1997. Pro‐inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum‐free conditions. Eur. J. Immunol. 27:3135‐3142.
   Lanzavecchia, A. and Sallusto, F. 2001. The instructive role of dendritic cells on T cell responses: Lineages, plasticity and kinetics. Curr. Opin. Immunol. 13:291‐298.
   Larregina, A.T., Morelli, A.E., Tkacheva, O., Erdos, G., Donahue, C., Watkins, S.C., Thomson, A.W., and Falo, L.D. Jr. 2004. Highly efficient expression of transgenic proteins by naked DNA‐transfected dendritic cells through terminal differentiation. Blood 103:811‐819.
   Lechmann, M., Berchtold, S., Hauber, J., and Steinkasserer, A. 2002. CD83 on dendritic cells: More than just a marker for maturation. Trends Immunol. 23:273‐275.
   Lee, A.W., Truong, T., Bickham, K., Fonteneau, J.F., Larsson, M., Da Silva, I., Somersan, S., Thomas, E.K., and Bhardwaj, N. 2002. A clinical grade cocktail of cytokines and PGE(2) results in uniform maturation of human monocyte‐derived dendritic cells: Implications for immunotherapy. Vaccine 20 Suppl 4:A8‐A22.
   Matzinger, P. 2002. The danger model: A renewed sense of self. Science 296:301‐305.
   Nair, S.K., Heiser, A., Boczkowski, D., Majumdar, A., Naoe, M., Lebkowski, J.S., Vieweg, J., and Gilboa, E. 2000. Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells. Nat. Med. 6:1011‐1017.
   O'Neill, D.W., Adams, S., and Bhardwaj, N. 2004. Manipulating dendritic cell biology for the active immunotherapy of cancer. Blood 104:2235‐2246.
   Penna, G., Sozzani, S., and Adorini, L. 2001. Cutting edge: Selective usage of chemokine receptors by plasmacytoid dendritic cells. J. Immunol. 167:1862‐1866.
   Rafiq, K., Bergtold, A., and Clynes, R. 2002. Immune complex‐mediated antigen presentation induces tumor immunity. J. Clin. Invest. 110:71‐79.
   Scandella, E., Men, Y., Gillessen, S., Forster, R., and Groettrup, M. 2002. Prostaglandin E2 is a key factor for CCR7 surface expression and migration of monocyte‐derived dendritic cells. Blood 100:1354‐1361.
   Shortman, K. and Liu, Y.J. 2002. Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2:151‐161.
   Steinman, R.M., Hawiger, D., and Nussenzweig, M.C. 2003. Tolerogenic dendritic cells. Annu. Rev. Immunol. 21:685‐711.
   Timmerman, J.M., Czerwinski, D.K., Davis, T.A., Hsu, F.J., Benike, C., Hao, Z.M., Taidi, B., Rajapaksa, R., Caspar, C.B., Okada, C.Y., van Beckhoven, A., Liles, T.M., Engleman, E.G., and Levy, R. 2002. Idiotype‐pulsed dendritic cell vaccination for B‐cell lymphoma: Clinical and immune responses in 35 patients. Blood 99:1517‐1526.
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