Monitoring the Sensitivity of T Cell Populations Towards NAD+ Released During Cell Preparation.
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.
['Rissiek|Björn|B|', 'Lukowiak|Marco|M|', 'Haag|Friedrich|F|', 'Magnus|Tim|T|', 'Koch-Nolte|Friedrich|F|']
D036002:ADP Ribose Transferases D000246:Adenosine Diphosphate Ribose D000818:Animals D001120:Arginine D051379:Mice D008967:Molecular Biology D009243:NAD D058486:Receptors, Purinergic P2X7 D013601:T-Lymphocytes
ARTC2 P2X7 T cell preparation NAD-induced cell death Nanobody s+16a