Monitoring the Sensitivity of T Cell Populations Towards NAD+ Released During Cell Preparation

  • Björn RissiekEmail author
  • Marco Lukowiak
  • Friedrich Haag
  • Tim Magnus
  • Friedrich Koch-Nolte
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)


Mouse T cells express the toxin-related ecto-ADP-ribosyltransferase ARTC2 that catalyzes the posttranslational ADP-ribosylation of cell surface proteins by transferring the ADP-ribose group of its substrate nicotinamide adenine dinucleotide (NAD+) to arginine residues of its target proteins. One well known target of ARTC2 is the ATP-gated P2X7 ion channel. ADP-ribosylation of P2X7 induces gating of the channel, calcium influx, ecto-domain shedding, phosphatidylserine externalization, and finally cell death. Previous studies have shown that the ARTC2 substrate NAD+ is released during T cell preparation. Since P2X7 is differentially expressed among T cell subpopulations, preparation-related ADP-ribosylation has a strong impact on the vitality of T cells that express high levels of P2X7. With this chapter we provide a protocol to monitor the consequences of preparation-related P2X7 ADP-ribosylation on T cells using regulatory T cells as generic T cell subpopulation known to express high levels of P2X7. However, this protocol could be easily adapted to other T cell populations.

Key words

ARTC2 P2X7 NAD-induced cell death T cell preparation s+16a Nanobody 


  1. 1.
    Adriouch S, Hubert S, Pechberty S et al (2007) NAD+ released during inflammation participates in T cell homeostasis by inducing ART2-mediated death of naive T cells in vivo. J Immunol 179:186–194CrossRefGoogle Scholar
  2. 2.
    Schwarz N, Fliegert R, Adriouch S et al (2009) Activation of the P2X7 ion channel by soluble and covalently bound ligands. Purinergic Signal 5:139–149. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Gu B, Bendall LJ, Wiley JS (1998) Adenosine triphosphate-induced shedding of CD23 and L-selectin (CD62L) from lymphocytes is mediated by the same receptor but different metalloproteases. Blood 92:946–951PubMedGoogle Scholar
  4. 4.
    Moon H, Na H-Y, Chong KH, Kim TJ (2006) P2X7 receptor-dependent ATP-induced shedding of CD27 in mouse lymphocytes. Immunol Lett 102:98–105. CrossRefPubMedGoogle Scholar
  5. 5.
    Menzel S, Rissiek B, Bannas P et al (2015) Nucleotide-Induced Membrane-Proximal Proteolysis Controls the Substrate Specificity of T Cell Ecto-ADP-Ribosyltransferase ARTC2.2. J Immunol 195(5):2057–2066. CrossRefPubMedGoogle Scholar
  6. 6.
    Adriouch S, Ohlrogge W, Haag F et al (2001) Rapid induction of naive T cell apoptosis by ecto-nicotinamide adenine dinucleotide: requirement for mono(ADP-ribosyl)transferase 2 and a downstream effector. J Immunol 167:196–203CrossRefGoogle Scholar
  7. 7.
    Rissiek B, Danquah W, Haag F, Koch-Nolte F (2014) Technical advance: a new cell preparation strategy that greatly improves the yield of vital and functional Tregs and NKT cells. J Leukoc Biol 95:543–549. CrossRefPubMedGoogle Scholar
  8. 8.
    Rissiek B, Haag F, Boyer O et al (2015) ADP-ribosylation of P2X7: a matter of life and death for regulatory T cells and natural killer T cells. Curr Top Microbiol Immunol 384:107–126. CrossRefPubMedGoogle Scholar
  9. 9.
    Hubert S, Rissiek B, Klages K et al (2010) Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment through the ART2-P2X7 pathway. J Exp Med 207:2561–2568. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Lahl K, Loddenkemper C, Drouin C et al (2007) Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease. J Exp Med 204:57–63. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Björn Rissiek
    • 1
    Email author
  • Marco Lukowiak
    • 1
  • Friedrich Haag
    • 2
  • Tim Magnus
    • 1
  • Friedrich Koch-Nolte
    • 2
  1. 1.Department of NeurologyUniversity Medical Center Hamburg-EppendorfHamburgGermany
  2. 2.Institute of ImmunologyUniversity Medical Center Hamburg-EppendorfHamburgGermany

Personalised recommendations