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.
Keywords: ARTC2; NAD-induced cell death; Nanobody; P2X7; T cell preparation; s+16a.