[This corrects the article on p. 641 in vol. 7, PMID: 28082980.].
- Publikační typ
- tisková chyba MeSH
Deciphering the mechanisms that allow the induction of strong immune responses is crucial to developing efficient vaccines against infectious diseases and cancer. Based on the discovery that the adenylate cyclase from Bordetella pertussis binds to the CD11b/CD18 integrin, we developed a highly efficient detoxified adenylate cyclase-based vector (CyaA) capable of delivering a large variety of Ags to the APC. This vector allows the induction of protective and therapeutic immunity against viral and tumoral challenges as well as against transplanted tumors in the absence of any added adjuvant. Two therapeutic vaccine candidates against human papilloma viruses and melanoma have been developed recently, based on the CyaA vector, and are currently in clinical trials. We took advantage of one of these highly purified vaccines, produced under good manufacturing practice-like conditions, to decipher the mechanisms by which CyaA induces immune responses. In this study, we demonstrate that CyaA binds both human and mouse CD11b(+) dendritic cells (DCs) and induces their maturation, as shown by the upregulation of costimulatory and MHC molecules and the production of proinflammatory cytokines. Importantly, we show that DCs sense CyaA through the TLR4/Toll/IL-1R domain-containing adapter-inducing IFN-β pathway, independent of the presence of LPS. These findings show that CyaA possesses the intrinsic ability to not only target DCs but also to activate them, leading to the induction of strong immune responses. Overall, this study demonstrates that Ag delivery to CD11b(+) DCs in association with TLR4/Toll/IL-1R domain-containing adapter-inducing IFN-β activation is an efficient strategy to promote strong specific CD8(+) T cell responses.
- MeSH
- adaptorové proteiny vezikulární transportní imunologie MeSH
- adenylátcyklasový toxin imunologie MeSH
- antigeny CD11b imunologie MeSH
- antigeny CD80 biosyntéza MeSH
- antigeny CD86 biosyntéza MeSH
- Bordetella pertussis imunologie MeSH
- buněčná diferenciace imunologie MeSH
- cytotoxické T-lymfocyty imunologie MeSH
- dendritické buňky cytologie imunologie MeSH
- interferon beta imunologie MeSH
- interleukin-1beta biosyntéza MeSH
- interleukin-6 biosyntéza MeSH
- kultivované buňky MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- receptor interferonu alfa-beta genetika MeSH
- receptory interleukinu-1 imunologie MeSH
- signální transdukce imunologie MeSH
- TNF-alfa biosyntéza MeSH
- toll-like receptor 4 imunologie MeSH
- tyrosin genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.
- MeSH
- adenylátcyklasový toxin farmakologie MeSH
- buněčné linie MeSH
- draslík metabolismus MeSH
- endocytóza účinky léků MeSH
- iontový transport účinky léků MeSH
- klathrin metabolismus MeSH
- makrofágy cytologie metabolismus MeSH
- membránové mikrodomény metabolismus MeSH
- myši MeSH
- permeabilita buněčné membrány účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The choice of tools that enable efficient targeting of exogenous antigens (Ag) for processing and presentation by professional Ag-presenting cells (APC) remains limited. This represents, indeed, a bottleneck in development of vaccines inducing specific T-cell responses. Here, we describe a novel strategy of Ag delivery into APCs. The Ag of choice is fused to the N- or C-terminus of streptavidin (SA) and tetrameric Ag-SA or SA-Ag fusion proteins are produced in E. coli and purified by 2-Iminobiotin-Agarose affinity chromatography. Alternatively, Ag-SA proteins are purified from urea extracts of E. coli inclusion bodies and refolded in vitro into functional tetramers. Complexes with biotinylated antibodies targeting cell surface receptors are formed and used to deliver the Ags of choice for processing and presentation by APCs and induction of Ag-specific CD4+ and CD8+ T-cell responses in vitro and in vivo.
- MeSH
- antigeny CD11c imunologie metabolismus MeSH
- antigeny chemie genetika imunologie metabolismus MeSH
- biotinylace MeSH
- buněčné linie MeSH
- dendritické buňky imunologie metabolismus MeSH
- kur domácí MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- ovalbumin chemie genetika imunologie metabolismus MeSH
- receptory buněčného povrchu imunologie metabolismus MeSH
- rekombinantní fúzní proteiny chemie genetika imunologie metabolismus MeSH
- rozpustnost MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- streptavidin chemie genetika imunologie metabolismus MeSH
- T-lymfocyty imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The adenylate cyclase toxin-haemolysin of Bordetella (CyaA) targets CD11b(+) myeloid phagocytes and translocates across their cytoplasmic membrane an adenylate cyclase (AC) enzyme that catalyses conversion of cytosolic ATP into cAMP. In parallel, CyaA acts as a cytolysin forming cation-selective pores, which permeabilize cell membrane and eventually provoke cell lysis. Using cytolytic activity, potassium efflux and patch-clamp assays, we show that a combination of substitutions within the pore-forming (E570Q) and acylation-bearing domain (K860R) ablates selectively the cell-permeabilizing activity of CyaA. At the same time, however, the capacity of such mutant CyaA to translocate the AC domain across cytoplasmic membrane into cytosol of macrophage cells and to elevate cellular cAMP concentrations remained intact. Moreover, the combination of E570Q+K860R substitutions suppressed the residual cytolytic activity of the enzymatically inactive CyaA/OVA/AC(-) toxoid on CD11b-expressing monocytes, while leaving unaffected the capacity of the mutant toxoid to deliver in vitro a reporter CD8(+) T cell epitope from ovalbumin (OVA) to the cytosolic pathway of dendritic cells for MHC class I-restricted presentation and induce in vivo an OVA-specific cytotoxic T cell response. CyaA, hence, employs a mechanism of AC enzyme domain translocation across cellular membrane that avoids passage across the cytolytic pore formed by toxin oligomers.
- MeSH
- adenylátcyklasový toxin genetika metabolismus MeSH
- biologické modely MeSH
- buněčná membrána metabolismus MeSH
- cytotoxické T-lymfocyty imunologie MeSH
- dendritické buňky imunologie MeSH
- epitopy T-lymfocytární imunologie MeSH
- kultivované buňky MeSH
- makrofágy imunologie MeSH
- monocyty imunologie MeSH
- mutageneze cílená MeSH
- mutantní proteiny genetika metabolismus MeSH
- myši MeSH
- ovalbumin imunologie MeSH
- substituce aminokyselin MeSH
- transport proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
