Advertisement

Modulation of the Immune Response Using Dendritic Cell-Derived Exosomes

  • Nicole R. Bianco
  • Seon-Hee Kim
  • Adrian E. Morelli
  • Paul D. Robbins
Part of the Methods in Molecular Biology™ book series (MIMB, volume 380)

Abstract

Initial studies in our laboratory were focused on the use of dendritic cells (DC) genetically modified to express Th2-derived cytokines (i.e., interleukin [IL]-4 and IL-10) or apoptotic proteins (i.e., Fas Ligand [FasL]) to reduce inflammation in a mouse model of experimentally induced arthritis. Exosomes are nano-sized vesicles (40–100 nm diameter) released by different cell types, including DC, that contain many of the proteins thought to be involved in regulating the immune response. We have demonstrated that exosomes derived from immature DC treated with immunomodulatory cytokines (i.e., IL-10, IL-4) are able to inhibit inflammation in a murine footpad model of delayed-type hypersensitivity (DTH) and reduce the severity of established collagen-induced arthritis (CIA). In fact, the exosomes were as therapeutic as the parental DC. Because purified DC-derived exosomes are very stable vesicles, they may be a better approach for future treatment of arthritis and other autoimmune disorders than the more unstable DC. In this chapter we detail a protocol for preparing the exosomes produced by murine bone marrow-derived DC. We also review methods to assess the purity and concentration of purified exosomes, by using electron microscopy, Western blot analysis, and flow cytometry. Finally, we describe methods to assess the function of exosomes in vitro, using the mixed leukocytes reaction, and in vivo by means of DTH and an experimental model of CIA.

Key Words

Rheumatoid arthritis exosomes autoimmune disease dendritic cells collagen-induced arthritis delayed-type hypersensitivity 

References

  1. 1.
    Pan, B. T. and Johnstone, R. M. (1983) Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33, 967–978.PubMedCrossRefGoogle Scholar
  2. 2.
    Denzer, K., Kleijmeer, M. J., Heijnen, H. R, Stoorvogel, W., and Geuze, H. J. (2000) Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J. Cell Sci. 113, 3365–3374.PubMedGoogle Scholar
  3. 3.
    Thery, C., Zitvogel, L., and Amigorena, S. (2002) Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 2, 569–579.PubMedGoogle Scholar
  4. 4.
    Johnstone, R. M., Adam, M., Hammond, J. R., Orr, L., and Turbide, C. (1987) Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J. Biol. Chem. 262, 9412–9420.PubMedGoogle Scholar
  5. 5.
    Zitvogel, L., Regnault, A., Lozier, A., et al. (1998) Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat. Med. 4, 594–600.PubMedCrossRefGoogle Scholar
  6. 6.
    Chaput, N., Schartz, N. E., Andre, F., et al. (2004) Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection. J. Immunol. 172, 2137–2146.PubMedGoogle Scholar
  7. 7.
    Morse, M. A., Garst, J., Osada, T., et al. (2005) A phase I study of exosome immunotherapy in patients with advanced non-small cell lung cancer. J. Transl. Med. 3, 9.PubMedCrossRefGoogle Scholar
  8. 8.
    Escudier, B., Dorval, T., Chaput, N., et al. (2005) Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial. J. Transl. Med. 3, 10.PubMedCrossRefGoogle Scholar
  9. 9.
    Altieri, S. L., Khan, A. N., and Tomasi, T. B. (2004) Exosomes from plasmacytoma cells as a tumor vaccine. J. Immunother. 27, 282–288.PubMedCrossRefGoogle Scholar
  10. 10.
    Aline, F., Bout, D., Amigorena, S., Roingeard, P., and Dimier-Poisson, I. (2004) Toxoplasma gondii antigen-pulsed-dendritic cell-derived exosomes induce a protective immune response against T. gondii infection. Infect. Immun. 72, 4127–4137.PubMedCrossRefGoogle Scholar
  11. 11.
    Karlsson, M., Lundin, S., Dahlgren, U., Kahu, H., Pettersson, I., and Telemo, E. (2001) “Tolerosomes” are produced by intestinal epithelial cells. Eur. J. Immunol. 31, 2892–2900.PubMedCrossRefGoogle Scholar
  12. 12.
    Peche, H., Heslan, M., Usal, C., Amigorena, S., and Cuturi, M. C. (2003) Presentation of donor major histocompatibility complex antigens by bone marrow dendritic cell-derived exosomes modulates allograft rejection. Transplantation 76, 1503–1510.PubMedCrossRefGoogle Scholar
  13. 13.
    Andreola, G., Rivoltini, L., Castelli, C., et al. (2002) Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles. J. Exp. Med. 195, 1303–1316.PubMedCrossRefGoogle Scholar
  14. 14.
    Martinez-Lorenzo, M. J., Anel, A., Gamen, S., et al. (1999) Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles. J. Immunol. 163, 1274–1281.PubMedGoogle Scholar
  15. 15.
    Abrahams, V. M., Straszewski, S. L., Kamsteeg, M., et al. (2003) Epithelial ovarian cancer cells secrete functional Fas ligand. Cancer Res. 63, 5573–5581.PubMedGoogle Scholar
  16. 16.
    Abusamra, A. J., Zhong, Z., Zheng, X., et al. (2005) Tumor exosomes expressing Fas ligand mediate CD8+ T-cell apoptosis. Blood Cells Mol. Dis. 35, 169–173.PubMedCrossRefGoogle Scholar
  17. 17.
    Frangsmyr, L., Baranov, V., Nagaeva, O., Stendahl, U., Kjellberg, L., and Mincheva-Nilsson, L. (2005) Cytoplasmic microvesicular form of Fas ligand in human early placenta: switching the tissue immune privilege hypothesis from cellular to vesicular level. Mol. Hum. Reprod. 11, 35–41.PubMedCrossRefGoogle Scholar
  18. 18.
    Kim, S. H., Lechman, E. R., Bianco, N., et al. (2005) Exosomes derived from IL-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis. J. Immunol. 174, 6440–6448.PubMedGoogle Scholar
  19. 19.
    Hee Kim, S., Bianco, N., Menon, R., et al. (2005) Exosomes derived from genetically modified DC expressing FasL are anti-inflammatory and immunosuppressive. Mol. Ther. 13, 289–300.CrossRefGoogle Scholar
  20. 20.
    Son, Y. L, Egawa, S., Tatsumi, T., Redlinger, R. E., Jr., Kalinski, P., and Kanto, T. (2002) A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells. J. Immunol. Methods 262, 145–157.PubMedCrossRefGoogle Scholar
  21. 21.
    Thery, C., Boussac, M., Veron, P., et al. (2001) Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J. Immunol. 166, 7309–7318.PubMedGoogle Scholar
  22. 22.
    Wubbolts, R., Leckie, R. S., Veenhuizen, P. T., et al. (2003) Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation. J. Biol. Chem. 278, 10,963–10,972.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Nicole R. Bianco
    • 1
  • Seon-Hee Kim
    • 1
  • Adrian E. Morelli
    • 2
  • Paul D. Robbins
    • 1
  1. 1.Department of Molecular Genetics and BiochemistryUniversity of Pittsburgh School of MedicinePittsburgh
  2. 2.Department of Surgery & Thomas E. Stazl Transplantation InstituteUniversity of Pittsburgh School of MedicinePittsburgh

Personalised recommendations