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Molekulare Mechanismen und Funktion der Öffnung des P2X7 Ionenkanals in T Zellen

Fachliche Zuordnung Biochemie
Immunologie
Förderung Förderung von 2014 bis 2019
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 263480173
 
Erstellungsjahr 2020

Zusammenfassung der Projektergebnisse

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.

Projektbezogene Publikationen (Auswahl)

 
 

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