RESUMEN
We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes.
Asunto(s)
Venenos de Abeja/enzimología , Membrana Celular/metabolismo , Fosfolipasas A2/química , Animales , Línea Celular Tumoral , Células Dendríticas/metabolismo , Humanos , Fosfolipasas A2/aislamiento & purificación , Fosfolipasas A2/metabolismo , Proteínas Recombinantes de Fusión/químicaRESUMEN
Bee venom phospholipase A2 (bvPLA2) is a small, 15kDa enzyme which hydrolyses many phospholipids through interfacial binding. The mutated bvPLA2H34Q (bvPLA2m), in which histidine-34 is replaced by glutamine, is not catalytically active. This protein has been shown to be a suitable membrane anchor and has been suggested as a suitable tumor-antigen vector for the development of novel dendritic cell-based vaccines. To confirm this feature, in this study the fusion protein PNY, composed of NY-ESO-1(NY(s)) fused to the C-terminus of bvPLA2m, was engineered. bvPLA2m enhanced the binding of NY(s) to the membrane of human monocyte-derived dendritic cells (DCs) and, once taken up by the cells, the antigen fused to the vector was directed to both MHC I and MHC II peptide-loading compartments. bvPLA2m was shown to increase the cross-presentation of the NY(s)-derived, restricted HLA-A*02 peptide, NY-ESO-1157-165(NY157-165), at the T1 cell surface. DCs loaded with the fusion protein induced cross-priming of NY(s)-specific CD8 + T-cells with greater efficiency than DCs loaded with NY(s). Sixty-five percent of these NY(s)-specific CD8+ T-cell lines could also be activated with the DCs pulsed with the peptide, NY157-165. Of these CD8+ T-cell lines, two were able to recognize the human melanoma cell line, SK-MEL-37, in a context of HLA-A*02. Only a small number of bvPLA2m CD8+ T-cell lines were induced, indicating the low immunogenicity of the protein. It was concluded that bvPLA2m can be used as a membrane-binding vector to promote MHC class II peptide presentation and MHC class I peptide cross-presentation. Such a system can, therefore, be tested for the preparation of cell-based vaccines.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Venenos de Abeja/enzimología , Células Dendríticas/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Fosfolipasas A2/metabolismo , HumanosRESUMEN
We describe the creation of cell adhesion mediated by cell surface engineering. The Flt3-ligand was fused to a membrane anchor made of the diphtheria toxin translocation domain. The fusion protein was attached to the surface of a cell by an acid pulse. Contact with another cell expressing the receptor Flt3 lead to its activation. This activity involved direct cell-cell contact. A mean force of 20 nN was needed to separate functionalized cells after 5 min of contact. Overall, we showed that it is possible to promote specific cell-cell adhesion by attaching protein ligands at the surface of cells.
Asunto(s)
Membrana Celular/metabolismo , Toxina Diftérica/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Fusión Celular , Línea Celular , Toxina Diftérica/genética , Ligandos , Proteínas de la Membrana/genética , Ratones , Unión ProteicaRESUMEN
Mycolactone is a polyketide toxin produced by Mycobacterium ulcerans (Mu), the causative agent of the skin disease Buruli ulcer (BU). Surprisingly, infected tissues lack inflammatory infiltrates. Structural similarities between mycolactone and immunosuppressive agents led us to investigate the immunomodulatory properties of mycolactone on dendritic cells (DCs), the key initiators and regulators of immune responses. At noncytotoxic concentrations, phenotypic and functional maturation of both mouse and human DCs was inhibited by mycolactone. Notably, mycolactone blocked the emigration of mouse-skin DCs to draining lymph nodes, as well as their maturation in vivo. In human peripheral blood-derived DCs, mycolactone inhibited the ability to activate allogeneic T cell priming and to produce inflammatory molecules. Interestingly, production of the cytokines interleukin (IL) 12, tumor necrosis factor alpha, and IL-6 was only marginally affected, whereas production of the chemokines macrophage inflammatory protein (MIP) 1alpha, MIP-1beta, regulated on activation, normal T cell expressed and secreted, interferon gamma-inducible protein 10, and monocyte chemoattractant protein 1 was abolished at nanomolar concentrations. Importantly, mycolactone endogenously expressed by Mu mediated similar inhibitory effects on beta-chemokine production by DCs. In accordance with the histopathological features of BUs, our results suggest that bacterial production of mycolactone may limit both the initiation of primary immune responses and the recruitment of inflammatory cells to the infection site. Moreover, they highlight a potential interest in mycolactone as a novel immunosuppressive agent.
Asunto(s)
Toxinas Bacterianas/toxicidad , Células Dendríticas/efectos de los fármacos , Inmunosupresores/toxicidad , Animales , Toxinas Bacterianas/inmunología , Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Células Dendríticas/fisiología , Femenino , Humanos , Inmunosupresores/inmunología , Activación de Linfocitos/efectos de los fármacos , Macrólidos , Ratones , Ratones Endogámicos C57BL , Mycobacterium ulcerans/químicaRESUMEN
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
The authors have investigated a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This method may offer an alternative to gene therapy approaches. It consists in anchoring recombinant cytokines to the cell membrane. Attachment is mediated by the transmembrane domain of diphtheria toxin (T) genetically fused to the cytokine and is triggered by an acid pH pulse. The authors found that the fusion protein T-hIL-2 anchored to the surface of tumor cells retained its IL-2 activity while remaining exposed for several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective antitumor immunity mediated by tumor-specific cytotoxic T lymphocytes. This procedure presents several advantages as compared with the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and the selection of transfected clones, it eliminates the safety problems connected with viral vectors, and it allows the control of the amount of cytokines delivered with the vaccine.