The activation of A2A adenosine receptors (A2AR) at the start of reperfusion following tissue ischemia reduces the ongoing tissue damage that progresses for many hours. Reperfusion injury is manifested by activation of lymphocytes, macrophages, and neutrophils. Through the use of antibodies to selectively deplete lymphocyte subsets, and the use of adoptive transfer of cells into lymphocyte-deficient RAG1-KO mice, CD4+ T lymphocytes have been identified as the key cells that are inhibited by A2AR activation to block the initiation of reperfusion injury in liver, kidney and heart (Yang et al., 2006;Day et al., 2005b;Day et al., 2005a). These effects are accompanied by reductions in tissue production of several cytokines and chemokines, and a marked decrease in neutrophil accumulation between 4 and 12 hours after reperfusion. In the heart we found that there is a small but significant increase in CD3 positive cells that appear in the myocardium within minutes of reperfusion following ischemia, and the accumulation of these cells is largely inhibited by A2A agonists. Since, necrosis during reperfusion injury is reduced to a greater extent by A2AR activation in the liver (> 75%) than in other tissues, we have used liver to study in detail the effects of A2AR activation. Most T lymphocytes are not activated during reperfusion injury via TRC-mediated mechanisms the require peptide antigen presentation via MHC on antigen presenting cells. However, a small subset of T cells, known as invariant NKT cells, can be rapidly activated due to glycolipid presentation on CD1d that is found on antigen presenting cells or hepatocytes. Selective blockade of CD1d-dependent iNKT cell activation reduces liver reperfusion injury to the same extent as A2AR activation (Lappas et al., 2006). In order to further study how NKT cells initiate and trigger reperfusion injury, we have used the synthetic glycolipid, alpha-galactosylceramide (alpha-GalCer) to selectively activate iNKT cells in liver. Although Rag1 KO mice are resistant to reperfusion injury, adoptive transfer of NKT cells into Rag1 KO mice reconstitutes injury. The injection of alpha-GalCer into mice also provoks liver injury and the sequential activation of NKT, NK, and T cells as assessed by intracellular accumulation of gamma-interferon (gamma-INF) and other cytokines based on FACS analysis of cells derived from enzymatically dispersed liver. We have also examined expression of A2AR mRNA in a reporter mouse that expresses eGFP behind the A2AR promoter. In these mice, eGFP expression in increased by 5-10 fold in NKT cell and NK cells within 12 hours after injection of alpha-GalCer. In NKT cells purified by cell sorting, gamma-IFN production in response to alpha-GalCer is markedly inhibited by A2A agonists in vitro. The results indicate that NKT cells are activated by an endogenous glycolipid during reperfusion injury following tissue ischemia via invariant NKT cell receptors. A2A agonists limit liver reperfusion injury by preventing NKT cell activation. The finding that A2AR mRNA is expressed on NK cells is novel. It is not yet know how A2AR activation influences NK cell function, but the results suggest that in addition to NKT cells, NK cells participate in an innate inflammatory cascade that causes reperfusion injury after ischemia. Inhibition of NKT and NK cells by A2AR activation or by depletion or inhibition of the activation of these cells by other means may be useful to lesson tissue injury following ischemia, transplantation or other inflammatory insults.