1C), suggesting that these receptors were negatively regulated upon cell activation. Expression of P2X7[34],[34] was similar on freshly isolated mDCs from spleen, bone marrow, blood, liver, and kidney (Fig. 2A). Because

CD39 is the key molecule that hydrolyzes ATP and regulates ATP concentration,[34] we considered that the resistance of liver DCs to ATP might be the result of ATP hydrolysis by cell-surface–expressed CD39. However, whereas >95% of mDCs from each tissue expressed CD39 (data not shown), liver mDCs displayed significantly higher levels (mean fluorescence intensity; MFI) than mDCs from lymphoid and other nonlymphoid tissues, including kidney mDCs find more (Fig. 2B). CD39 was not detected on mouse hepatocytes (Fig. 2C). Interestingly, liver mDCs, but not liver pDCs (which represent a comparatively high proportion of liver DCs, compared with spleen DC[35]), expressed greater levels of CD39 than other liver and spleen innate and adaptive immune cells

(Fig. 2D). Liver mDCs also expressed CD73 (Fig. 2E,F), which contributes to adenosine generation. Freshly isolated DCs were cultured in ATP-containing medium, and ATP concentration was determined at various times by luminescence assay. ATP concentration decreased progressively (approximately 80%) over 120 minutes in the presence of liver mDCs from WT B6 mice. Initially (first 30 minutes), liver and spleen mDCs from WT mice hydrolyzed ATP at similar rates, but only liver mDCs continued to reduce ATP levels over the ensuing 120 minutes (Fig. 3A). By contrast, an equivalent number of DCs from CD39−/− mice failed medchemexpress to hydrolyze ATP. NVP-BKM120 As expected, the extent of ATP hydrolysis mediated by liver versus splenic mDCs was consistent with their different levels of CD39 expression (Fig. 2B,C). However, expression levels of other ectoenzymes, such as CD39L1 and CD39L3, were similar on spleen and liver mDCs (Fig. 3C). ATP stimulation (120 minutes) did not alter CD39 expression on spleen or liver mDCs (Fig. 3C). Production of

adenosine (Fig. 3B) also correlated with the differential levels of CD39 and CD73 expression on liver and spleen mDCs (Fig. 2E,F). These data indicate that the superior ability of liver mDCs to hydrolyze ATP results from their comparatively high CD39 expression. To confirm the processing of ATP by CD39 on liver mDCs, we precultured WT or CD39−/− liver mDCs in ATP-containing medium for 3 hours, then applied the cell-free culture supernatant to WT liver mDCs for 18 hours, together with LPS stimulation. As expected, the medium from WT, compared with CD39−/− DCs cultured with ATP, induced less IAb, costimulatory molecule, and B7-H1 expression (Fig. 3D). We assessed CD39 expression on liver DCs freshly isolated from histologically normal surgical resection tissue. Human liver and circulating mDCs were gated on CD45+, lineage (CD3, CD14, CD19, and CD20)−, and BDCA-1+ cells, as previously described.[28, 36] Similarly to mice (Fig.