Method of Generating Tolerogenic Maturation-Resistant Dendritic Cells and Testing for Their Immune-Regulatory Functions In Vivo in the Context of Transplantation

  • Sherrie J. Divito
  • Adrian E. MorelliEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1899)


During that past two decades, advances in techniques for generating in vitro immune-suppressive dendritic cells (DCs) have heralded the use of these pro-tolerogenic DCs as therapeutics against transplant rejection and autoimmune diseases. In transplantation, previous dogma assumed that systemically administered therapeutic DCs bearing donor antigens (Ags) control the anti-donor response by directly interacting with anti-donor T cells in vivo. However, recent evidence indicates that the exogenously-administered therapeutic DCs instead function as Ag-transporting cells that transfer donor Ags to recipient’s Ag-presenting cells (APCs) for presentation to T cells. In secondary lymphoid organs, presentation of acquired donor Ags by recipient’s quiescent DCs triggers deficient activation and eventual apoptosis of donor-specific effector T cells, leading to a relative increase in the percentage of donor-specific regulatory T cells. This chapter describes the methodology to generate in vitro immune-suppressive DCs that are resistant to maturation, and to assess in vivo both their survival and their ability to regulate donor-specific T cells in a mouse model.

Key words

Tolerogenic dendritic cells Vitamin D3 Cell therapy Transplantation Mouse 



We thank the comments of the Research Specialist William J. Shufesky (Univ. of Pittsburgh, Pittsburgh, PA, USA).


  1. 1.
    Banchereau J, Briere F, Caux C et al (2000) Immunobiology of dendritic cells. Annu Rev Immunol 18:767–811CrossRefGoogle Scholar
  2. 2.
    Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392:245–252CrossRefGoogle Scholar
  3. 3.
    Cella M, Sallusto F, Lanzavecchia A (1997) Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol 9:10–16CrossRefGoogle Scholar
  4. 4.
    Steinman RM, Hawiger D, Nussenzweig MC (2003) Tolerogenic dendritic cells. Annu Rev Immunol 21:685–711CrossRefGoogle Scholar
  5. 5.
    Steinman RM, Nussenzweig MC (2002) Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc Natl Acad Sci U S A 99:351–358CrossRefGoogle Scholar
  6. 6.
    Schwartz RH (1990) A cell culture model for T lymphocyte clonal anergy. Science 248:1349–1356CrossRefGoogle Scholar
  7. 7.
    Morelli AE, Thomson AW (2007) Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol 7:610–621CrossRefGoogle Scholar
  8. 8.
    Griffin MD, Xing N, Kumar R (2003) Vitamin D and its analogs as regulators of immune activation and antigen presentation. Annu Rev Nutr 23:117–145CrossRefGoogle Scholar
  9. 9.
    Lemire JM (1995) Immunomodulatory actions of 1,25-dihydroxyvitamin D3. J Steroid Biochem Mol Biol 53:599–602CrossRefGoogle Scholar
  10. 10.
    Rigby WF (1988) The immunobiology of vitamin D. Immunol Today 9:54–58CrossRefGoogle Scholar
  11. 11.
    Penna G, Adorini L (2000) 1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cell leading to impaired alloreactive T cell activation. J Immunol 164:2405–2411CrossRefGoogle Scholar
  12. 12.
    Yates SF, Paterson AM, Nolan KF et al (2007) Induction of regulatory T cells and dominant tolerance by dendritic cells incapable of full activation. J Immunol 179:967–976CrossRefGoogle Scholar
  13. 13.
    Piemonti L, Monti P, Sironi M et al (2000) Vitamin D3 affects differentiation, maturation, and function of human monocyte-derived dendritic cells. J Immunol 164:4443–4451CrossRefGoogle Scholar
  14. 14.
    Griffin MD, Lutz W, Phan VA et al (2001) Dendritic cell modulation by 1alpha,25 dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo. Proc Natl Acad Sci U S A 98:6800–6805CrossRefGoogle Scholar
  15. 15.
    Divito SJ, Wang Z, Shufesky WJ et al (2010) Endogenous dendritic cells mediate the effects of intravenously injected therapeutic immunosuppressive dendritic cells in transplantation. Blood 116:2694–2705CrossRefGoogle Scholar
  16. 16.
    Tiffany LJ, Garcia-Ojeda PA, Stein KE (1999) Determination of the IgG2a allotype of CXB recombinant inbred mouse strains by a PCR-based method. Immunogenetics 50:71–73CrossRefGoogle Scholar
  17. 17.
    Wang Z, Larregina AT, Shufesky WJ et al (2006) Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells. Am J Transplant 6:1297–1311CrossRefGoogle Scholar
  18. 18.
    Wang Z, Divito SJ, Shufesky WJ et al (2012) Dendritic cell therapies in transplantation revisited: deletion of recipient DCs deters the effect of therapeutic DCs. Am J Transplant 12:1398–1408CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of DermatologyBrigham and Women’s Hospital and Harvard Medical SchoolBostonUSA
  2. 2.Department of Surgery, T.E. Starzl Transplantation InstituteUniversity of Pittsburgh Medical CenterPittsburghUSA

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