Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca entry and, consequently, apoptosis. To date, adenosine 5'-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2. Here we show that 2'-deoxy-ADPR was a significantly better TRPM2 agonist, inducing 10.4-fold higher whole-cell currents at saturation. Mechanistically, this increased activity was caused by a decreased rate of inactivation and higher average open probability. Using high-performance liquid chromatography (HPLC) and mass spectrometry, we detected endogenous 2'-deoxy-ADPR in Jurkat T lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and nicotinamide adenine dinucleotide (NAD)-glycohydrolase CD38 sequentially catalyzed the synthesis of 2'-deoxy-ADPR from nicotinamide mononucleotide (NMN) and 2'-deoxy-ATP in vitro. Thus, 2'-deoxy-ADPR is an endogenous TRPM2 superagonist that may act as a cell signaling molecule.

Immune cells at sites of inflammation are continuously activated by local antigens and cytokines, and regulatory mechanisms must be enacted to control inflammation. The stepwise hydrolysis of extracellular ATP by ectonucleotidases CD39 and CD73 generates adenosine, a potent immune suppressor. Here we report that human effector CD8 T cells contribute to adenosine production by releasing CD73-containing extracellular vesicles upon activation. These extracellular vesicles have AMPase activity, and the resulting adenosine mediates immune suppression independently of regulatory T cells. In addition, we show that extracellular vesicles isolated from the synovial fluid of patients with juvenile idiopathic arthritis contribute to T cell suppression in a CD73-dependent manner. Our results suggest that the generation of adenosine upon T cell activation is an intrinsic mechanism of human effector T cells that complements regulatory T cell-mediated suppression in the inflamed tissue. Finally, our data underscore the role of immune cell-derived extracellular vesicles in the control of immune responses.

Extracellular adenine nucleotides participate in cell-to-cell communication and modulate the immune response. The concerted action of ectonucleotidases CD39 and CD73 plays a major role in the local production of anti-inflammatory adenosine, but both ectonucleotidases are rarely co-expressed by human T cells. The expression of CD39 on T cells increases upon T cell activation and is high at sites of inflammation. CD73, in contrast, disappears from the cellular membrane after activation. The possibility that CD73 could act in trans would resolve the conundrum of both enzymes being co-expressed for the degradation of ATP and the generation of adenosine. An enzymatically active soluble form of CD73 has been reported, and AMPase activity has been detected in body fluids of patients with inflammation and cancer. It is not yet clear how CD73, a glycosylphosphatidylinositol (GPI)-anchored protein, is released from the cell membrane, but plausible mechanisms include cleavage by metalloproteinases and shedding mediated by cell-associated phospholipases. Importantly, like many other GPI-anchored proteins, CD73 at the cell membrane is preferentially localized in detergent-resistant domains or lipid rafts, which often contribute to extracellular vesicles (EVs). Indeed, CD73-containing vesicles of different size and origin and with immunomodulatory function have been found in the tumor microenvironment. The occurrence of CD73 as non-cell-bound molecule widens the range of action of this enzyme at sites of inflammation. In this review, we will discuss the generation of non-cell-bound CD73 and its physiological role in inflammation.