3C,D). The peripheral lymphopenia, hypofunction, and poor reconstituting capacities of CD4+ T cells and iNKT cells from CD39tg mice implicated a defect in thymopoiesis. CD39 transgene expression was confirmed in the thymus by histology

and real-time PCR (not shown). Flow cytometric analysis confirmed a thymic maturation blockade at the double positive (DP) stage with an increased proportion of double negative (DN) thymocytes and a significant decrease of DP, CD4 single positive (SP) and CD8 SP thymocyte absolute numbers (Fig. 4A; Table 2). T cell receptor beta (TCR-β) rearrangement and expression Staurosporine datasheet of CD69 are hallmarks of T-cell development, but expression of both was virtually absent in CD39tg DP thymocytes (Fig. 4B). TCR-β expression was significantly decreased in CD4 SP and CD8 SP cells (Fig. 4C). The same immunodeficiency was observed in CD39tg crossed with A2a-receptor KO mice (not shown), suggesting that an

A2a-receptor-independent mechanism was responsible for the thymic maturation blockade. To determine the relative importance of hepatic CD4+ T cell versus iNKT cell deficiencies in protection from IRI, iNKT KO and CD4-depleted WT livers were transplanted into WT recipients. ABT-199 chemical structure CD4 depletion was confirmed by flow cytometric analysis of the spleen of the donor at the time of liver harvesting (Fig. 5A). CD4-depleted (ALT 10,296 ± 1,376) but not iNKT KO donor livers (ALT 26,271 ± 2,231) (Fig. 5B) were protected to the

level observed for CD39tg donor livers. This suggests that the Oxymatrine resident hepatic CD4+ T cells (but not iNKT cells) are predominant mediators of early IRI following prolonged cold preservation and liver transplantation. Herein we have shown that the overexpression of CD39 in the donor mouse is protective against hepatic IRI triggered by extended cold preservation (18 hours) in a model of liver transplantation. Unexpectedly, protection did not appear to be due to elevated levels of CD39 in the liver parenchyma itself, because liver grafts from CD39tg mice reconstituted with a WT immune system prior to transplant were not protected, but rather to a reduction in CD4+ cells in the donor liver. CD39tg mice exhibited a selective CD4+ T-cell panlymphopenia encompassing CD4+ iNKT cells. CD4+ T-cell depletion of WT donor livers, but not the absence of iNKT cells, paralleled the level of hepatic protection observed for CD39tg livers. Together this suggests that the protection imparted by CD39 overexpression is a consequence of depletion of resident hepatic CD4+ T cells. In this study, a clinically relevant model of prolonged cold storage followed by orthotopic liver transplantation was adopted. In clinical transplantation, prolonged cold ischemia and reperfusion activate both the immune response and the interrelated coagulation system.