Molecular mechanisms and function of P2X7 ion channel gating on T cells

• Applicants: Professor Dr. Friedrich Koch-Nolte; Professorin Dr. Eva Tolosa
• Subject Area: Biochemistry ; Immunology
• Term: from 2014 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 263480173

• Final Report Year: 2020

Final Report Abstract

ATP is released from cells during stress, inflammation and tissue damage. The homotrimeric P2X7 ion channel functions as a sensor of extracellular ATP. P2X7 is expressed by T cells, macrophages and other immune cells. ATP-binding induces a reversible conformational shift which allows influx of Ca2+ and Na+ ions and efflux of K+ ions. Activation of P2X7 on T cells induces exposure of phosphatidylserine on the outer leaflet of the plasma membrane and shedding of CD62L. Activation of P2X7 on macrophages induces inflammasome formation and the processing and release of proinflammatory IL-1β. Nanobodies are recombinant single antigen-binding variable domains (VHH) from camelid heavy chain antibodies. In the region corresponding to the hydrophobic interface of the two variable domains of conventional antibodies (VH, VL), nanobodies contain hydrophylic amino acids. This accounts for the excellent solubility and stability of nanobodies, which allows easy reformatting of nanobodies into mono, bi- and multivalent formats. The major goals of this project were to evaluate the mode of action and therapeutic potential of P2X7-specific nanobodies, and to determine the expression, function and experimental modulation of P2X7 on human T cells. Our results indicate that P2X7- blocking and P2X7-potentiating nanobodies, both function by an allosteric mechanism, most likely by hindering and or enhancing ATP-induced conformational changes. For in vitro experiments, we generated recombinant nanobody monomers, dimers, and chimeric mouse IgG or human IgG heavy chain antibodies; for in vivo applications we generated half-life extended dimers after genetic fusion of P2X7-specific nanobody dimers to an albumin-specific nanobody. In mice, a single systemic injection sufficed to completely cover cell surface P2X7 for at least 4 days. Treatment of mice with P2X7-blocking nanobodies showed significant therapeutic effects in experimental models of delayed type hypersensitivity and antibody-induced glomerulonephritis. For human P2X7, treatment with P2X7-blocking nanobodies effectively inhibited ATP- induced release of IL-1ß by LPS-primed monocytes, and ATP-induced shedding of CD62L, externalization of phosphatidylserine and uptake of the DNA-staining dye DAPI by T cells. Regarding the studies on expression of P2X7 on human T lymphocytes, we found that innate-like lymphocytes, namely T cells harbouring a γδ receptor and mucosal-associated invariant T (MAIT) cells, express higher levels of P2X7 on the cell surface, and consequently respond to lower concentrations of ATP than conventional CD4 and CD8 T cells. Our results indicate that it is worthwhile to pursue P2X7 as a target for nanobodybased treatment strategies in inflammatory diseases.

Publications

• 2016 Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation. Sci Transl Med. 8:366ra162
  Danquah W, Meyer-Schwesinger C, Rissiek B, Pinto C, Serracant-Prat A, Amadi M, Iacenda D, Knop JH, Hammel A, Bergmann P, Schwarz N, Assunção J, Rotthier W, Haag F, Tolosa E, Bannas P, Boué-Grabot E, Magnus T, Laeremans T, Stortelers C, Koch-Nolte F
  
• 2018 Modulating ion channel function with antibodies and nanobodies. Curr Opin Immunol. 52:18-26
  Stortelers C, Pinto-Espinoza C, Van Hoorick D, Koch-Nolte F
  
• 2018 TRMmaintenance is regulated by tissue damage via P2RX7. Sci Immunol. 3:eaau1022
  Stark R, Wesselink TH, Behr FM, Kragten NAM, Arens R, Koch-Nolte F, van Gisbergen KPJM, van Lier RAW
  
• 2018. CD39 is upregulated during activation of mouse and human T cells and attenuates the immune response to Listeria monocytogenes. PLOS One 13:e0197151
  Raczkowski F, Rissiek A, Ricklefs I, Heiss K, Schumacher V, Wundenberg K, Haag F, Koch-Nolte F, Tolosa E, Mittrücker HW
  
• 2018. Immunophenotyping of Newly Diagnosed and Recurrent Glioblastoma Defines Distinct Immune Exhaustion Profiles in Peripheral and Tumor-infiltrating Lymphocytes. Clinical Cancer Res. 24(17):4187-4200
  Mohme M, Schliffke S, Maire CL, Rünger A, Glau L, Mende KC, Matschke J, Gehbauer C, Akyüz N, Zapf S, Holz M, Schaper M, Martens T, Schmidt NO, Peine S, Westphal M, Binder M, Tolosa E, Lamszus K
  
• 2018. In Vivo Blockade of Murine ARTC2.2 During Cell Preparation Preserves the Vitality and Function of Liver Tissue-Resident Memory T Cells. Front Immunol. 9:1580
  Rissiek B, Lukowiak M, Raczkowski F, Magnus T, Mittrücker HW, Koch-Nolte F
  
• 2018. Nanobody-Based Biologics for Modulating Purinergic Signaling in Inflammation and Immunity. Front Pharmacol. 9:266
  Menzel S, Schwarz N, Haag F, Koch-Nolte F
  
• 2018. Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody. Elife. 7: pii: e36217
  Kaczmarek-Hajek K, Zhang J, Kopp R, Grosche A, Rissiek B, Saul A, Bruzzone S, Engel T, Jooss T, Krautloher A, Schuster S, Magnus T, Stadelmann C, Sirko S, Koch-Nolte F, Eulenburg V, Nicke A
  
• P2X7 receptor antagonists and agonists WO2013178783, granted in Australia (2018), USA (2018), China (2020)
  Danquah W, Nolte F, Laermans T, Stortelers C
  
• 2019. Generation and function of non-cell-bound CD73. Front Immunol. 10: 1729
  Schneider E, Rissiek A, Winzer R, Puig B, Rissiek B, Haag F, Mittrücker HW, Magnus T, Tolosa E
  
• 2019. Novel biologics targeting the P2X7 ion channel. Curr Opin Pharmacol. 47:110-118
  Koch-Nolte F, Eichhoff A, Pinto-Espinoza C, Schwarz N, Schäfer T, Menzel S, Haag F, Demeules M, Gondé H, Adriouch S
  
• 2019. P2X7 receptor induces mitochondrial failure in monocytes and compromises NLRP3 inflammasome activation during sepsis. Nat Commun. 10:2711
  Martínez-García JJ, Martínez-Banaclocha H, Angosto-Bazarra D, de Torre-Minguela C, Baroja- Mazo A, Alarcón-Vila C, Martínez-Alarcón L, Amores-Iniesta J, Martín-Sánchez F, Ercole GA, Martínez CM, González-Lisorge A, Fernández-Pacheco J, Martínez-Gil P, Adriouch S, Koch-Nolte F, Luján J, Acosta-Villegas F, Parrilla P, García-Palenciano C, Pelegrin P
  
• 2019. Purine Release, Metabolism, and Signaling in the Inflammatory Response. Annu Rev Immunol. 37:325-347
  Linden J, Koch-Nolte F, Dahl G
  
• 2020. The induction and function of the anti-inflammatory fate of TH17 cells. Nat Commun. 11:3334
  Xu H, Agalioti T, Zhao J, Steglich B, Wahib R, Vesely MCA, Bielecki P, Bailis W, Jackson R, Perez D, Izbicki J, Licona-Limón P, Kaartinen V, Geginat J, Esplugues E, Tolosa E, Huber S, Flavell RA, Gagliani N