SURFACE AMP DEAMINASE 2 AS A NOVEL REGULATOR MODIFYING THE EXTRACELLULAR ATP-ADENOSINE BALANCE THAT IS DIFFERENTIALLY EXPRESSED IN PATIENTS WITH RHEUMATOID ARTHRITIS

Background: Adenosine and its nucleotides represent crucial immunomodulators in the extracellular environment. ATP and ADP are released from stressed cells in states of inflammation, whereas adenosine serves as a key anti-inflammatory mediator1. The ectonucleotidases CD39 and CD73 are responsible for the sequential catabolism of ATP to adenosine via AMP, thereby promoting an anti-inflammatory milieu induced by the “adenosine halo”. Great importance has been attributed to these enzymes in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) and as targets in cancer therapy2 3. AMPD2 mediates AMP deamination to IMP, thus constituting an ambiguous mediator both enhancing the degradation of inflammatory ATP and reducing the formation of protective adenosine. Here, we postulate that this pathway is also present on the cell surface of immune cells and modified under inflammatory conditions. Objectives: Therefore, we analysed surface AMPD2 expression and its modulation on distinct cell lines and primary immune cells. Methods: Firstly, AMPD2 surface expression was verified by immunoprecipitation from membrane fractions isolated from cell lines (HEK293 and HMEC1) and CD14+ monocytes analysed by western blot and mass spectrometry. In addition, surface biotinylation of the aforementioned cells was performed. Also, AMPD2 surface expression was evaluated by flow cytometry, analysing both cell lines (HEK293, HMEC1, THP1, and Jurkat) and primary human immune cells from healthy donors and patients with RA. Secondly, co-expression of surface AMPD2, CD39 and CD73 on PBMCs was analysed by flow cytometry directly after isolation as well as after a 24h culture period. Moreover, surface expression was assessed after immunostimulation and Golgi transport inhibition. Results: AMPD2 surface expression was confirmed by western blot and mass spectrometry of (i) precipitated AMPD2 from membrane fractions and (ii) biotinylated surface molecules in HEK293 and HMEC1 as well as CD14+ monocytes. Surface expression was reduced after AMPD2 knockdown in HEK293. Flow cytometric analysis further verified AMPD2 surface expression and revealed a significant decrease after Golgi transport inhibition (p Conclusion: We demonstrate AMPD2 surface expression on immune cells for the first time. Hence, we reveal a novel regulator of the extracellular ATP-adenosine balance that is differentially expressed in RA patients compared to healthy controls. The extracellular conversion of AMP into IMP may constitute a shunt-like mechanism adding to the CD39-CD73 system controlling immunomodulation. References: [1]Regateiro FS, Cobbold SP, Waldmann H. CD73 and adenosine generation in the creation of regulatory microenvironments. Clinical and experimental immunology 2013;171(1):1-7. doi: 10.1111/j.1365-2249.2012.04623.x [2]Morandi F, Horenstein AL, Rizzo R, et al. The Role of Extracellular Adenosine Generation in the Development of Autoimmune Diseases. Mediators of inflammation 2018;2018:7019398. doi: 10.1155/2018/7019398 [3]Allard B, Longhi MS, Robson SC, et al. The ectonucleotidases CD39 and CD73: Novel checkpoint inhibitor targets. Immunol Rev 2017;276(1):121-44. doi: 10.1111/imr.12528 Acknowledgments: This project is funded by an unrestricted grant by Horizon Pharma plc. Disclosure of Interests: Lisa Ehlers: None declared, Aditi Kuppe: None declared, Alexandra Damerau: None declared, Marieluise Kirchner: None declared, Cindy Strehl: None declared, Frank Buttgereit Grant/research support from: Amgen, BMS, Celgene, Generic Assays, GSK, Hexal, Horizon, Lilly, medac, Mundipharma, Novartis, Pfizer, Roche, and Sanofi., Timo Gaber: None declared