Novel role of the ectonucleotidase CD39

Many important processes in our immune system are associated with the release of nucleotides, particularly adenosine triphosphate (ATP), an important biomolecule derived from purine and also known as the energy currency of life. In the extracellular space, however, ATP acts as a potent signaling molecule by triggering purin-ergic receptors. The ectonucleotidase CD39 is a cell surface enzyme that hydrolyses ATP and thus determines the duration and magnitude of nucleotide-induced inflammatory and immune responses. Our intriguing but as yet unpublished observation that CD39 not only exhibits ectonucleotidase activity but may also hydrolyse isoprenoid diphosphates constitutes the basis of the proposed research project. Isoprenoids are evolutionarily highly conserved lipid building blocks that are essential for cell growth and proliferation. During stress-induced dysregulation, isoprenoid diphosphates, which are potentially dangerous molecules, can be specifically detected by a subset of immune cells by use of their gammadelta T lymphocyte receptor. We want to examine this novel isoprenoid diphosphate phosphohydrolase activity of CD39. The planned studies will include the thorough characterization of recombinant and cell-associated CD39 as well as the investigation of isoprenoid diphosphates as novel regulators of CD39 that may competitively inhibit the breakdown of ATP and thus prolong purinergic signaling processes. With the support of our cooperation partner Prof. Huck from the local Institute of Analytical Chemistry we will perform measurements of nucleotides and isoprenoids using highly sensitive analytical methods. The role of CD39 as an isoprenoid diphosphate phosphohydrolase in immune responses will be investigated in human cells as well as in animal models. This novel function of CD39 shall definitively be established in vivo by comparing the efficacy of immunisation in wild type mice and in knock-out mice, in which CD39 has been selectively inactivated by means of genetic manipulation. Likewise, enhanced purinergic signal transduction that results from the competitive inhibition of ATP degradation will be studied in wild type and in knock-out mice lacking individual purinergic receptors. Knock-out mice along with other methods will be provided by our international collaboration partners Prof. Idzko from the University of Freiburg, Germany, and Prof. Simon C. Robson from Harvard University in Boston, USA. The exploration of CD39 as an isoprenoid diphosphate phosphohydrolase represents something like a world première, which will contribute to an improved understanding of inflammation and immunity that may ultimately be harnessed for enhanced vaccination and immunotherapy.

Many important processes in our immune system are associated with the release of nucleotides, particularly adenosine triphosphate (ATP), an important biomolecule derived from purine and also known as the energy currency of life. The ecto-enzyme CD39 has long been known as an ATPase, i.e. a cell surface enzyme that degrades ATP in the extracellular space by hydrolysing its phosphate groups. CD39 thus controls the duration and magnitude of ATP-induced inflammatory and immune responses. In this project, we could ascribe an entirely novel function to CD39. Isoprenoid diphosphates are lipid molecules generated in the mevalonate pathway, which is best known for the synthesis of cholesterol. However, isoprenoid diphosphates also activate T cells, a distinct T cell subset with anti-microbial and anti-tumour properties. Upon activation, these T cells upregulate the levels of CD39, which then not only hydrolyses ATP but also degrades isoprenoid diphosphates and thus terminates the T cell response. Intriguingly, the isoprenoid diphosphate with the longest side chain (GGPP, C20) turned out to be resistant to CD39-mediated hydrolysis and even inhibited ATP hydrolysis by CD39. The resulting modest increase of extracellular ATP activated P2Y11, a member of the P2Y family of G protein-coupled receptors. In our subsequent studies, we found that P2Y11 receptor on the surface of macrophages, another cell type in the immune system, can communicate with two distinct cell surface receptors, the interleukin-1 receptor (IL-1R) and the Toll-like receptor 4 (TLR4). By "talking" to these receptors, P2Y11 induced the release of soluble tumor necrosis factor (TNF) receptors, which are able to neutralise the potentially dangerous cytokine TNF- in the microenvironment. Concomitantly P2Y11 suppressed TNF- secretion induced by microbial products. Our findings thus also establish P2Y11 receptor as a sentinel of TNF- induced inflammation and improve our understanding of P2Y11 receptor signalling in anti-inflammatory macrophages. Collectively, our studies identified a regulatory signalling cascade that controls inflammatory and immune responses. CD39-mediated degradation of isoprenoid diphosphates prevents excessive T cell responses. Subsequent engagement of P2Y11 receptor prevents hyperinflammation and ensures restoration of homeostasis. Agonistic targeting of this pathway may be desirable in infectious diseases such Covid-19. Conversely, antagonistic P2Y11 targeting may be required, for instance, to improve cancer immunotherapy, because ATP, which is already a major constituent of the tumour microenvironment, will increasingly be released during therapy-induced tumour destruction and may then activate anti-inflammatory P2Y11 signalling, thus limiting the efficacy of cancer therapy.