RESUMO
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.
Assuntos
Toxinas Bacterianas/toxicidade , Células Dendríticas/efeitos dos fármacos , Imunossupressores/toxicidade , Animais , Toxinas Bacterianas/imunologia , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Células Dendríticas/fisiologia , Feminino , Humanos , Imunossupressores/imunologia , Ativação Linfocitária/efeitos dos fármacos , Macrolídeos , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium ulcerans/químicaRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Toxina Diftérica/metabolismo , Proteínas de Membrana/metabolismo , Animais , Fusão Celular , Linhagem Celular , Toxina Diftérica/genética , Ligantes , Proteínas de Membrana/genética , Camundongos , Ligação ProteicaRESUMO
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.
Assuntos
Venenos de Abelha/enzimologia , Membrana Celular/metabolismo , Fosfolipases A2/química , Animais , Linhagem Celular Tumoral , Células Dendríticas/metabolismo , Humanos , Fosfolipases A2/isolamento & purificação , Fosfolipases A2/metabolismo , Proteínas Recombinantes de Fusão/químicaRESUMO
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.
Assuntos
Antígenos Virais/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Epitopos Imunodominantes/imunologia , Fosfolipases A/metabolismo , Fosfoproteínas/imunologia , Proteínas da Matriz Viral/imunologia , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/fisiologia , Venenos de Abelha/enzimologia , Brefeldina A/farmacologia , Linfócitos T CD8-Positivos/imunologia , Membrana Celular/metabolismo , Cloroquina/farmacologia , Apresentação Cruzada/efeitos dos fármacos , Células Dendríticas/química , Células Dendríticas/fisiologia , Endossomos/fisiologia , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Epitopos Imunodominantes/análise , Epitopos Imunodominantes/metabolismo , Ativação Linfocitária/imunologia , Fosfolipases A/análise , Fosfolipases A2 , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Inibidores de Proteassoma , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Proteínas da Matriz Viral/análise , Proteínas da Matriz Viral/metabolismoRESUMO
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.
Assuntos
Antígenos de Neoplasias/metabolismo , Venenos de Abelha/enzimologia , Células Dendríticas/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Fosfolipases A2/metabolismo , HumanosRESUMO
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.