RESUMEN
The generation of strong T-cell immunity is one of the main challenges for the development of successful vaccines against cancer and major infectious diseases. Here we have engineered spider silk particles as delivery system for a peptide-based vaccination that leads to effective priming of cytotoxic T-cells. The recombinant spider silk protein eADF4(C16) was fused to the antigenic peptide from ovalbumin, either without linker or with a cathepsin cleavable peptide linker. Particles prepared from the hybrid proteins were taken up by dendritic cells, which are essential for T-cell priming, and successfully activated cytotoxic T-cells, without signs of immunotoxicity or unspecific immunostimulatory activity. Upon subcutaneous injection in mice, the particles were taken up by dendritic cells and accumulated in the lymph nodes, where immune responses are generated. Particles from hybrid proteins containing a cathepsin-cleavable linker induced a strong antigen-specific proliferation of cytotoxic T-cells in vivo, even in the absence of a vaccine adjuvant. We thus demonstrate the efficacy of a new vaccine strategy using a protein-based all-in-one vaccination system, where spider silk particles serve as carriers with an incorporated peptide antigen. Our study further suggests that engineered spider silk-based vaccines are extremely stable, easy to manufacture, and readily customizable.
Asunto(s)
Portadores de Fármacos/química , Nanopartículas/química , Seda/química , Arañas/química , Vacunas de Subunidad/farmacología , Adyuvantes Inmunológicos/farmacología , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Antígenos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Dendríticas/citología , Liberación de Fármacos , Femenino , Humanos , Macrófagos/citología , Ratones Endogámicos C57BL , Ovalbúmina/química , Tamaño de la Partícula , Proteínas Recombinantes/química , Propiedades de Superficie , Linfocitos T Citotóxicos , Distribución TisularRESUMEN
Toll-like receptor (TLR) 7 agonists are effective in topical application for the immunotherapy of skin cancers, but their performance for the systemic treatment of solid tumors is limited by the development of TLR tolerance. In this study, we describe a novel strategy to overcome TLR tolerance and enhance TLR7-dependent antitumor immune responses through reprogramming of TLR signaling pathways. The sensitivity of TLR7 signaling in dendritic cells (DC) was increased by prior stimulation with the dsRNA poly(I:C) that mimics virally induced immune activation. Timing of the stimulations was important, as sequential stimulation with poly(I:C) and the TLR7 agonist R848 interspaced by 24 h induced higher MAPK and NFkB signaling in DC than the simultaneous application of the same ligands. DC activated by sequential poly(I:C)/R848 stimulation efficiently induced Th1 differentiation and primed NK-cell and cytotoxic T-cell responses. We have developed a treatment regimen taking advantage of TLR7 reprogram-ming that cured over 80% of large immunogenic tumors in mice by the action of NK cells and cytotoxic T cells. These results have direct implications for the use of these clinically established ligands in the immunotherapy of cancer.
RESUMEN
Innate immune recognition of RNA is key for the initiation of immunity in response to viral infection. Although the factors controlling the detection of viral RNA by innate immune receptors in host cells are increasingly well understood, little is known about the dynamic changes in signaling after the initial triggering of these receptors. In this study, we report that preconditioning with the synthetic dsRNA polyinosinic-polycytidylic acid [poly(I:C)], a mimetic of viral RNA, rapidly reprograms murine APCs by simultaneously augmenting sensitivity of endosomal TLRs and inhibiting activation of RIG-I-like receptors (RLRs) in an IFN-ß-dependent manner. These changes in receptor sensitivity were also seen in vivo after treatment of mice with poly(I:C). Mechanistically, the increased sensitivity of the TLR pathway was associated with elevated MAPK and NF-κB activity. The RLR response was inhibited downstream of TANK-binding kinase-1, resulting in decreased IFN regulatory factor 3 phosphorylation. Reprogramming of pattern-recognition receptor signaling also occurred after viral infection, because infection of host cells with Sendai virus or their exposure to supernatant from virus-infected cells induced the same changes in TLR and RLR sensitivity as poly(I:C). Thus, innate recognition of viral infection critically modifies responses to pattern-recognition receptor stimulation. These dynamic adaptations to infection may reinforce antiviral immunity and at the same time serve to limit pathological inflammation.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Transducción de Señal/inmunología , Receptores Toll-Like/inmunología , Virosis/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Immunoblotting , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Interferón beta/inmunología , Interferón beta/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/virología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Poli I-C/inmunología , Poli I-C/farmacología , Receptores de Reconocimiento de Patrones/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus Sendai/inmunología , Virus Sendai/fisiología , Transducción de Señal/efectos de los fármacos , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
Early in the course of infection, detection of pathogen-associated molecular patterns by innate immune receptors can shape the subsequent adaptive immune response. Here we investigate the influence of virus-associated innate immune activation on lymphocyte distribution in secondary lymphoid organs. We show for the first time that virus infection of mice induces rapid disruption of the Peyer's patches but not of other secondary lymphoid organs. The observed effect was not dependent on an active infectious process, but due to innate immune activation and could be mimicked by virus-associated molecular patterns such as the synthetic double-stranded RNA poly(I:C). Profound histomorphologic changes in Peyer's patches were associated with depletion of organ cellularity, most prominent among the B-cell subset. We demonstrate that the disruption is entirely dependent on type I interferon (IFN). At the cellular level, we show that virus-associated immune activation by IFN-α blocks B-cell trafficking to the Peyer's patches by downregulating expression of the homing molecule α4ß7-integrin. In summary, our data identify a mechanism that results in type I IFN-dependent rapid but reversible disruption of intestinal lymphoid organs during systemic viral immune activation. We propose that such rerouted lymphocyte trafficking may impact the development of B-cell immunity to systemic viral pathogens.
Asunto(s)
Inmunidad Innata/inmunología , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/virología , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/virología , Movimiento Celular/inmunología , Células Cultivadas , Femenino , Interferón Tipo I/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ganglios Linfáticos Agregados/patología , ARN Viral/inmunología , Estomatitis Vesicular/patología , Virus de la Estomatitis Vesicular Indiana/genéticaRESUMEN
Basophils are powerful mediators of Th2 immunity and are present in increased numbers during allergic inflammation and helminth infection. Despite their ability to potentiate Th2 immunity the mechanisms regulating basophil development remain largely unknown. We have found a unique role for isotype-switched antibodies in promoting helminth-induced basophil production following infection of mice with Heligmosomoides polygyrus bakeri or Nippostrongylus brasiliensis. H. polygyrus bakeri-induced basophil expansion was found to occur within the bone marrow, and to a lesser extent the spleen, and was IL-3 dependent. IL-3 was largely produced by CD4(+)CD49b(+)NK1.1(-) effector T cells at these sites, and required the IL-4Rα chain. However, antibody-deficient mice exhibited defective basophil mobilization despite intact T-cell IL-3 production, and supplementation of mice with immune serum could promote basophilia independently of required IL-4Rα signaling. Helminth-induced eosinophilia was not affected by the deficiency in isotype-switched antibodies, suggesting a direct effect on basophils rather than through priming of Th2 responses. Although normal type 2 immunity occurred in the basopenic mice following primary infection with H. polygyrus bakeri, parasite rejection following challenge infection was impaired. These data reveal a role for isotype-switched antibodies in promoting basophil expansion and effector function following helminth infection.
Asunto(s)
Anticuerpos Antihelmínticos/inmunología , Basófilos/inmunología , Interleucina-3/metabolismo , Nematospiroides dubius/inmunología , Nippostrongylus/inmunología , Infecciones por Strongylida/inmunología , Animales , Cambio de Clase de Inmunoglobulina/inmunología , Interleucina-3/inmunología , Ratones , Ratones Mutantes , Estadísticas no Paramétricas , Células Th2/inmunologíaRESUMEN
RATIONALE: The incidence of allergic disorders is increasing in developed countries and has been associated with reduced exposure to microbes and alterations in the commensal bacterial flora. OBJECTIVES: To ascertain the relevance of commensal bacteria on the development of an allergic response, we used a model of allergic airway inflammation in germ-free (GF) mice that lack any exposure to pathogenic or nonpathogenic microorganisms. METHODS: Allergic airway inflammation was induced in GF, specific pathogen-free (SPF), or recolonized mice by sensitization and challenge with ovalbumin. The resulting cellular infiltrate and cytokine production were measured. MEASUREMENTS AND MAIN RESULTS: Our results show that the total number of infiltrating lymphocytes and eosinophils were elevated in the airways of allergic GF mice compared with control SPF mice, and that this increase could be reversed by recolonization of GF mice with the complex commensal flora of SPF mice. Exaggerated airway eosinophilia correlated with increased local production of Th2-associated cytokines, elevated IgE production, and an altered number and phenotype of conventional dendritic cells. Regulatory T-cell populations and regulatory cytokine levels were unaltered, but GF mice exhibited an increased number of basophils and decreased numbers of alveolar macrophages and plasmacytoid dendritic cells. CONCLUSIONS: These data demonstrate that the presence of commensal bacteria is critical for ensuring normal cellular maturation, recruitment, and control of allergic airway inflammation.