*Purpose: Hepatic ischemia and reperfusion injury (HIRI) is a significant cause of morbidity and mortality following liver transplantation and major hepatic resections. Double negative T cells (DNT) are unique regulatory T cells which are discovered in recent decades.We have demonstrated that DNT significantly inhibit allo- or auto-immune responses mainly through perforin/granzyme B pathway. However, whether other mechanisms are involved in the immune regulation of DNT and the potential application of DNT in HIRI are still unknow.

*Methods: WT or CD39 deficient DNT were adoptively transferred before surgery, and then the mouse model of partial (70%) warm HIRI was established. In vitro, WT or CD39 deficient DNT were cocultured with neutrophils with or without ATP stimulation. Transcriptome sequencing of ATP treated WT or CD39 deficient DNT were analyzed.

*Results: In this study, we found DNT highly expressed CD39, a cell surface enzyme hydrolyzing extracellular ATP. Adoptively transferring with wild type (WT) but not CD39 deficient DNT significantly inhibited TNF-α and IL-1β secretion of liver infiltrated neutrophils, decreased neutrophil extracellular traps (NETs) generation and increased neutrophil apoptosis in warm HIRI in both C57BL/6 and T cell deficient B6.Rag2/IL2rc KO mice. Which contributed to markedly alleviated liver injury, as shown by lowered levels of serum ALT and reduced hepatocellular necrosis. Mechanistically, compared with WT DNT, CD39 deficient DNT were unable to hydrolyze extracellular cytotoxic ATP and became apoptotic under ATP stimulation. Transcriptome sequencing analysis suggested that ATP treated CD39 KO DNT had reduced expression of cell activation, proliferation and chemotaxis related genes than WT DNT. However, no differences of Prf1 or Gzmb gene expression were observed between groups. Furthermore, the level of extracellular immunosuppressive molecule, adenosine, was significantly higher in the culture of ATP treated WT DNT compared with that of CD39 deficient DNT. ATP-treated WT DNT but not CD39 deficient DNT significantly increased neutrophil apoptosis and inhibited inflammatory mediators secreted by neutrophils in vitro, suggesting that CD39 expressed by DNT might drive a shift from an ATP driven proinflammatory environment to an anti-inflammatory milieu induced by adenosine.

*Conclusions: In conclusion, our study reveals a new intrinsic mechanism that CD39 is crucial for DNT homeostasis and immunosuppressive function. These data support the concept and the feasibility of potentially utilizing this novel cell therapy for the prevention of HIRI during liver transplantation or hepatic surgery.

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