Abstract
On murine T cells, GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) ADP-ribosylates the P2X7 ion channel at arginine 125 in response to nicotinamide adenine dinucleotide (NAD+) released during cell preparation. We have previously shown that chronic gating of P2X7 by ADP-ribosylation reduces the vitality and function of regulatory T cells and natural killer T cells that co-express high levels of ARTC2.2 and P2X7. Here, we evaluated the expression of ARTC2.2 and P2X7 by effector and memory T cells in the liver of naïve mice and after infection with Listeria monocytogenes (Lm). We found that KLRG1−/CD69+ tissue-resident memory T cells (Trm) in the liver of naïve mice and 7 weeks after infection with Lm express high levels of ARTC2.2 and P2X7. Isolation of liver Trm and subsequent incubation at 37°C resulted in cell death of the majority of CD4+ and CD8+ Trm. Injection of the ARTC2.2-blocking nanobody s+16a 30 min prior to organ harvesting effectively prevented ADP-ribosylation of P2X7 during cell preparation and thereby prevented NAD-induced cell death of the isolated Trm upon subsequent incubation at 37°C. Consequently, preserving Trm vitality by s+16a injection enabled a highly sensitive in vitro cytokine expression profile analyses of FACS sorted liver Trm. We conclude that in vivo blockade of ARTC2.2 during cell preparation by nanobody s+16a injection represents a valuable strategy to study the role and function of liver Trm in mice.
Highlights
Mammalian ecto-ADP-ribosyltransferases are a family of toxin-related enzymes that use extracellular (NAD+) to attach an ADP-ribose group to arginine residues of cell surface proteins
A recent study suggests that liver CD8+ Trm are affected by NAD+ released during cell preparation [20], these cells have not yet been fully characterized toward their expression of ARTC2.2 and P2X7
Seven weeks after Listeria monocytogenes (Lm) infection, we found that the majority of CD8+ and CD4+ Trm co-express high levels of ARTC2.2 and P2X7
Summary
Mammalian ecto-ADP-ribosyltransferases (ecto-ARTs) are a family of toxin-related enzymes that use extracellular (NAD+) to attach an ADP-ribose group to arginine residues of cell surface proteins. ARTC2 isoforms are expressed on immune cells. While ARTC2.1 is expressed mainly by innate immune cells such as macrophages, dendritic cells, and microglia, ARTC2.2 is the major ecto-ART expressed by T cells [2,3,4]. The ARTC2 enzymes ADP-ribosylate various target proteins and thereby modulate their function. One well-characterized target of ARTC2.2-mediated ADPribosylation is the adenosine triphosphate (ATP)-gated P2X7 ion channel [5, 6]. Two differentially spliced isoforms of P2X7 are expressed by murine immune cells [7, 8].
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