16-19 NK and NKT cells are components of human and rodent liver lymphoid cell populations and play phase-specific roles in the course of IRI. Deletion of CD39 on NK cells inhibits their activation and protects in partial hepatic IRI by way of diminished interferon-gamma

(IFN-γ) production.10 CD1d knockout (KO) mice, which lack NKT cells, are protected against warm hepatic IRI.14, 20 CD39 is widely expressed within the immune system including on activated B cells,21 skin-specific dendritic cells,7 Selleck GDC-0449 a subset of CD4+ T cells (CD4+CD25+ regulatory T cells),22 and NKT cells.23 Human CD39 transgenic (CD39tg) mice were generated to promote an anticoagulant and antiinflammatory milieu that could alleviate renal IRI.15 Herein we extend these observations using a clinically relevant model of orthotopic liver transplantation characterized by prolonged cold storage of the donor organ. Further, we explore putative mechanisms

of protection through the immune phenotyping of these mice. A2aR, adenosine A2a receptor; ALT, alanine transaminase; ATP, adenosine triphosphate; CD39tg, CD39 transgenic; IFN-γ interferon-gamma; iNKT, invariant NKT; IRI, ischemia-reperfusion Fulvestrant in vitro injury; αGalCer, α-galactosylceramide; NK, natural killer cell; WT, wildtype. C57BL/6 wildtype (WT) mice were purchased from the Walter and Eliza Hall Institute (WEHI, Melbourne, Australia). CD39tg mice overexpressing CD39 under the control of the mouse H2Kb promoter (C57BL/6 background)24 were backcrossed at least 10 generations. Invariant NKT KO mice (Jalpha281−/− on a C57BL/6 background) were a kind gift from Prof. Mark Smyth (Peter MacCallum Cancer Centre, Melbourne, Australia). Experimental mice were housed under specific pathogen-free conditions. All studies were approved by the Animal Ethics Committee of St. Vincent’s Hospital following the Australian Code of Practise for the Care and Use of Animals for Scientific Purposes, 7th ed., 2004. Spleen and thymus were mechanically disrupted and red blood cell lysis buffer (eBioscience, San Diego, CA) was used on spleen preparations. Single cell suspensions were resuspended

in phosphate-buffered saline (PBS) with 2% fetal calf serum (FCS) and counted before flow cytometric analysis. 上海皓元 The livers of anesthetized mice were perfused with PB) by way of the portal vein, harvested, and disrupted through a cell strainer. The resulting cell suspension was washed twice in PBS with 2% FCS and the cell pellet was resuspended in 25 mL of 37% isotonic Percoll (GE Healthcare, Buckinghamshire, UK) gradient and centrifuged. The cell pellet contained liver lymphocytes. Freshly obtained leukocytes were stained with the following antibodies: α-galactosylceramide (α-GalCer)/CD1d tetramer-PE (kind gift from Dr. Dale Godfrey, University of Melbourne, Australia), CD3-PerCPCy5.5, CD4-FITC, CD4-PeCy5, CD4-PerCP-Cy5.5, CD69-FITC, TCRb-PE, TCRb-PeCy5 (BD Biosciences, San Diego, CA), CD4-APC, CD8a-APC (eBioscience).