Anales RANF

P4. E CTONUCLEOTIDASES P.18 T CELL-DERIVED EXOSOMAL CD73 MEDIATES IMMUNE SUPPRESSION E. Schneider 1 , R. Winzer 1 , A. Rissiek 1 , R. Fliegert 1 , H.-W. Mittrücker 1 , C. E. Müller 2 , E. Tolosa 1 1 University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2 University of Bonn, Bonn, Germany The ecto-nucleotidases CD39 and CD73 sequentially hydrolyze extracellular ATP to generate adenosine, which in turn engages to P1 receptors on immune cells dampening the inflammatory response. In humans, co-expression of CD39 and CD73 on T cells is a rare event, both ex vivo and after activation. Under steady-state conditions, CD39 is expressed on the membrane of a subset of regulatory CD4 T cells, while CD73 is mostly expressed by naïve CD8 T cells. In vitro activation of T cells induced an increase of CD39 on the cell membrane, while CD73 expression is dramatically reduced. This CD39 + CD73 ― phenotype is observed on T cells at sites of inflammation. We found CD73-specific AMPase activity in cell culture supernatants of activated CD8 T cells as well as in synovial fluid of patients with juvenile idiopathic arthritis, indicating that CD73 is present in a non-cell-bound form. CD73 is a GPI-anchored protein, however, inhibitors of phospholipases and metalloproteases did not prevent activation-induced shedding from the cell surface. We then considered the possibility that the activity is contained in exosomes/extracellular vesicles (EVs). Indeed, differential centrifugation of T cell culture supernatants revealed most AMPase activity in the EVs found in the pellet after ultracentrifugation. The use of specific inhibitors confirmed that the activity is CD73-specific. Furthermore, we could show that these EVs are able to suppress T cell proliferation in vitro . We conclude that CD73- containing EVs released upon T cell activation provide AMPase activity in trans. The occurrence of CD73 as non-cell-bound molecule widens the range of action of this enzyme at sites of inflammation and contributes to the control of inflammatory responses.