RESUMEN
The use of dendritic cells (DC) for the development of therapeutic cancer vaccines is attractive because of their unique ability to present tumor epitopes via the MHC class I pathway to induce cytotoxic CD8+ T lymphocyte responses. C-Type membrane lectins, DC-SIGN and the mannose receptor (MR), present on the DC surface, recognize oligosaccharides containing mannose and/or fucose and mediate sugar-specific endocytosis of synthetic oligolysine-based glycoclusters. We therefore asked whether a glycotargeting approach could be used to induce uptake and presentation of tumor antigens by DC. To this end, we designed and synthesized glycocluster conjugates containing a CD8+ epitope of the Melan-A/Mart-1 melanoma antigen. These glycocluster-Melan-A conjugates were obtained by coupling glycosynthons: oligosaccharyl-pyroglutamyl-beta-alanine derivatives containing either disaccharides, a dimannoside (Manalpha-6Man) or lactoside, or a Lewis oligosaccharide, to Melan-A 16-40 peptide comprising the 26-35 HLA-A2 restricted T cell epitope, extended with an oligolysine stretch at the C-terminal end. We showed by confocal microscopy and flow cytometry that fluorescent-labeled Melan-A glycoclusters containing either dimannoside or Lewis oligosaccharide were taken up by DC and concentrated in acidic vesicles; conversely lactoside glycopeptides were not at all taken up. Furthermore, using surface plasmon resonance, we showed that dimannoside and Lewis-Melan-A conjugates bind MR and DC-SIGN with high affinity. DC loaded with these conjugates, but not with the lactose-Melan-A conjugate, led to an efficient presentation of the Melan-A epitope eliciting a CD8+ T-lymphocyte response. These data suggest that synthetically designed glycocluster-tumor antigen conjugates may induce antigen cross-presentation by DC and represent a promising tool for the development of tumor vaccines.
Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/síntesis química , Células Dendríticas/inmunología , Glicopéptidos/síntesis química , Antígenos de Neoplasias/metabolismo , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/metabolismo , Endocitosis , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Citometría de Flujo , Colorantes Fluorescentes/química , Glicopéptidos/inmunología , Glicopéptidos/uso terapéutico , Humanos , Lectinas Tipo C/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Melanoma/patología , Microscopía Confocal , Oligosacáridos/química , Polilisina/química , Receptores de Superficie Celular/metabolismo , Resonancia por Plasmón de Superficie , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
We have used bee venom phospholipase A2 as a vector to load human dendritic cells ex vivo with a major histocompatibility complex (MHC) class I-restricted epitope fused to its C-terminus. The fusion protein bound to human monocyte-derived dendritic cells and was internalized into early endosomes. In vitro immunization experiments showed that these dendritic cells were able to generate specific CD8 T cell lines against the epitope carried by the fusion protein. Cross-presentation did not require proteasome, transporter associated with antigen processing, or endosome proteases, but required newly synthesized MHC molecules. Comparison of the antigen presentation pathway observed in this study to that followed by other toxins used as vectors is discussed.
Asunto(s)
Antígenos Virales/inmunología , Reactividad Cruzada , Células Dendríticas/inmunología , Epítopos Inmunodominantes/inmunología , Fosfolipasas A/metabolismo , Fosfoproteínas/inmunología , Proteínas de la Matriz Viral/inmunología , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/fisiología , Venenos de Abeja/enzimología , Brefeldino A/farmacología , Linfocitos T CD8-positivos/inmunología , Membrana Celular/metabolismo , Cloroquina/farmacología , Reactividad Cruzada/efectos de los fármacos , Células Dendríticas/química , Células Dendríticas/fisiología , Endosomas/fisiología , Antígenos de Histocompatibilidad Clase I/análisis , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Epítopos Inmunodominantes/análisis , Epítopos Inmunodominantes/metabolismo , Activación de Linfocitos/inmunología , Fosfolipasas A/análisis , Fosfolipasas A2 , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Inhibidores de Proteasoma , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de la Matriz Viral/análisis , Proteínas de la Matriz Viral/metabolismoRESUMEN
Dendritic cells (DC) are essential for the generation of primary adaptive immune responses, but their full immunostimulatory capacities are only reached upon maturation. The authors compared several clinical-grade adjuvants of bacterial origin to determine their ability to induce phenotypic and functional maturation of monocyte-derived DC (Dendritophages, Dphi; IDM, Paris, France) differentiated with granulocyte-macrophage colony-stimulating factor and interleukin-13 in single-use cell processors (VacCell; IDM, Paris, France). Monophosphoryl lipid A, Mycobacterium bovis bacillus Calmette-Guerin, and Ribomunyl (Pierre Fabre Medicament, Boulogne, France) all appeared able to provide the signal necessary to initiate Dphi maturation. However, only Ribomunyl (Pierre Fabre Medicament) (containing membrane and ribosomal fractions from four bacterial strains) allowed the authors to obtain a significant enhancement of allostimulatory abilities and cytokine production by Dphi in the absence of active cellular infection. Addition of interferon-gamma (IFN-gamma) to Ribomunyl resulted in more pronounced upregulation of CD83, major histocompatibility complex class I, and B7 molecules by Dphi. Moreover, the IFN-gamma addition modulated their cytokine secretion, allowing higher levels of bioactive interleukin-12 concomitant with lower levels of interleukin-10. In kinetic studies, Dphi contact with Ribomunyl and IFN-gamma for 6 hours was sufficient to trigger a maturation process that completed spontaneously. Thus, Ribomunyl in association with IFN-gamma represents a suitable agent for the ex vivo production of mature monocyte-derived DC that can be used as cellular vaccines to promote a potent type I immune response.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Células Dendríticas/efectos de los fármacos , Antígenos Bacterianos/farmacología , Células Dendríticas/fisiología , Humanos , Inmunofenotipificación , Interferón gamma/farmacología , Interleucina-10/biosíntesis , Interleucina-12/biosíntesis , Oligodesoxirribonucleótidos/farmacologíaRESUMEN
DC-SIGN, a dendritic cell (DC)-specific lectin, mediates clustering of DCs with T lymphocytes, a crucial event in the initiation of immune responses. DC-SIGN also binds HIV envelope glycoproteins, allowing efficient virus capture by DCs. We show here that DC-SIGN surface levels are upregulated in HIV-1-infected DCs. This process is caused by the viral protein Nef, which acts by inhibiting DC-SIGN endocytosis. Upregulation of DC-SIGN at the cell surface dramatically increases clustering of DCs with T lymphocytes and HIV-1 transmission. These results provide new insights into how HIV-1 spreads from DCs to T lymphocytes and manipulates immune responses. They help explain how Nef may act as a virulence factor in vivo.
Asunto(s)
Moléculas de Adhesión Celular , Células Dendríticas/virología , Productos del Gen nef/fisiología , VIH-1/fisiología , Lectinas Tipo C , Lectinas/metabolismo , Linfocitos/virología , Receptores de Superficie Celular/metabolismo , Transporte Biológico , Células Dendríticas/fisiología , Endocitosis , Células HeLa , Humanos , Linfocitos/fisiología , Regulación hacia Arriba , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Cytotoxic CD8+ T lymphocytes (CTL) directed against the matrix protein pp65 are major effectors in controlling infection against human cytomegalovirus (HCMV), a persistent virus of the Betaherpesvirus family. We previously suggested that cross-presentation of pp65 by nonpermissive dendritic cells (DCs) could overcome viral strategies that interfere with activation of CTL (G. Arrode, C. Boccaccio, J. Lule, S. Allart, N. Moinard, J. Abastado, A. Alam, and C. Davrinche, J. Virol. 74:10018-10024, 2000). It is well established that mature DCs are very potent in initiating T-cell-mediated immunity. Consequently, the DC maturation process is a key step targeted by viruses in order to avoid an immune response. Here, we report that immature DCs maintained in coculture with infected human (MRC5) fibroblasts acquired pp65 from early-infected cells for cross-presentation to specific HLA-A2-restricted CTL. In contrast, coculture of DCs in the presence of late-infected cells decreased their capacity to stimulate CTL. Analyses of DC maturation after either coculture with infected MRC5 cells or incubation with infected-cell-conditioned medium revealed that acquisition of a mature phenotype was a prerequisite for efficient stimulation of CTL and that soluble factors secreted by infected cells were responsible for both up and down regulation of CD83 expression on DCs. We identified transforming growth factor beta1 secreted by late HCMV-infected cells as one of these down regulating mediators. These findings suggest that HCMV has devised another means to compromise immune surveillance mechanisms. Together, our data indicate that recognition of HCMV-infected cells by DCs has to occur early after infection to avoid immune evasion and to allow generation of anti-HCMV CTL.