RESUMO
Treatment with ex vivo-generated regulatory T cells (T-reg) has been regarded as a potentially attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp knock-in (Foxp3gfp.KI) mice and myelin oligodendrocyte glycoprotein (MOG)(35-55)/IA(b) (MHC class II) tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. MOG tetramer-reactive, Foxp3(+) T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. Foxp3(+) T cells isolated from the CNS were effective in suppressing naive MOG-specific T cells, but failed to control CNS-derived encephalitogenic T-eff that secreted interleukin (IL)-6 and tumor necrosis factor (TNF). Our data suggest that in order for CD4(+)Foxp3(+) T-reg to effectively control autoimmune reactions in the target organ, it may also be necessary to control tissue inflammation.
Assuntos
Autoimunidade/imunologia , Encéfalo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição Forkhead/metabolismo , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Imunoterapia/métodos , Camundongos , Modelos Imunológicos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismoRESUMO
Tolerance induction, and thus prevention of autoimmunity, is linked with the amount of self-antigen presented on thymic stroma. We describe that intrathymic (i.t.) delivery of the autoantigen, myelin oligodendrocyte glycoprotein (MOG), via a lentiviral vector (LV), led to tolerance induction and prevented mice from developing fulminant experimental autoimmune encephalomyelitis (EAE). This protective effect was associated with the long-term expression of antigen in transduced stromal cells, which resulted in the negative selection of MOG-specific T cells and the generation of regulatory T cells (Tregs). These selection events were effective at decreasing T-cell proliferative responses and reduced Th1 and Th17 cytokines. In vivo, this translated to a reduction in inflammation and demyelination with minimal, or no axonal loss in the spinal cords of treated animals. Significantly intrathymic delivery of MOG to mice during the priming phase of the disease failed to suppress clinical symptoms despite mice being previously treated with a clearing anti-CD4 antibody. These results indicate that targeting autoantigens to the thymic stroma might offer an alternative means to induce the de novo production of tolerant, antigen-specific T cells; however, methods that control the number and or the activation of residual autoreactive cells in the periphery are required to successfully treat autoimmune neuroinflammation.
Assuntos
Encefalomielite Autoimune Experimental/terapia , Tolerância Imunológica , Glicoproteína Mielina-Oligodendrócito/genética , Glicoproteína Mielina-Oligodendrócito/imunologia , Animais , Progressão da Doença , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Feminino , Vetores Genéticos , Lentivirus/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito/biossíntese , Distribuição Aleatória , Células Th1/imunologia , Células Th17/imunologia , Timo/imunologia , Transdução GenéticaRESUMO
Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Camundongos , Animais , Humanos , Encefalomielite Autoimune Experimental/induzido quimicamente , Emulsões , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito , Camundongos Endogâmicos , Adjuvantes ImunológicosRESUMO
Recent studies suggest that metabolic changes and oxygen deficiency in the central nervous system play an important role in the pathophysiology of multiple sclerosis (MS). In our present study, we investigated the changes in oxygenation and analyzed the vascular perfusion of the spinal cord in a rodent model of MS. We performed multispectral optoacoustic tomography of the lumbar spinal cord before and after an oxygen enhancement challenge in mice with experimental autoimmune encephalomyelitis (EAE), a model for MS. In addition, mice were transcardially perfused with lectin to label the vasculature and their spinal columns were optically cleared, followed by light sheet fluorescence microscopy. To analyze the angioarchitecture of the intact spine, we used VesSAP, a novel deep learning-based framework. In EAE mice, the spinal cord had lower oxygen saturation and hemoglobin concentration compared to healthy mice, indicating compromised perfusion of the spinal cord. Oxygen administration reversed hypoxia in the spinal cord of EAE mice, although the ventral region remained hypoxic. Additionally, despite the increased vascular density, we report a reduction in length and complexity of the perfused vascular network in EAE. Taken together, these findings highlight a new aspect of neuroinflammatory pathology, revealing a significant degree of hypoxia in EAE in vivo that is accompanied by changes in spinal vascular perfusion. The study also introduces optoacoustic imaging as a tractable technique with the potential to further decipher the role of hypoxia in EAE and to monitor it in MS patients.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , Humanos , Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/patologia , Doenças Neuroinflamatórias , Oxigênio/metabolismo , Medula Espinal/metabolismoRESUMO
Autoimmune murine disease models are vital tools for identifying novel targets and finding better treatments for human diseases. Complete Freund's adjuvant is commonly used to induce disease in autoimmune models, and the quality of the adjuvant/autoantigen emulsion is of critical importance in determining reproducibility. We have established an emulsification method using a standard homogenizer and specially designed receptacle. Emulsions are easy to prepare, form stable and uniform water-in-oil particles, are faster to make than the traditional syringe method, use less material and are designed to fill syringes with ease. In the present study, we have validated the emulsions for induction of experimental autoimmune encephalitis, collagen II induced arthritis, antigen induced arthritis, and delayed type hypersensitivity models. These models were induced consistently and reproducibly and, in some cases, the new method outperformed the traditional method. The method described herein is simple, cost-effective and will reduce variability, thereby requiring fewer animals for in vivo research involving animal models of autoimmune disease and in vaccine development.
Assuntos
Artrite Experimental , Doenças Autoimunes , Animais , Autoantígenos , Emulsões , Camundongos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
In certain disease context, cells of the monocyte/macrophage lineage are known to exhibit T-cell suppressor function. However, whether naïve monocytes are also able to suppress T-cell responses has not been previously investigated. In this study, we have discovered that CD11b(+)Ly6G(-) mononuclear cells in the blood of naïve mice are potent suppressors of T-cell proliferation in vitro. The suppression of T-cell proliferation requires cell-cell contact and is partially dependent on nitric oxide production. Following the induction of experimental autoimmune encephalomyelitis in mice, the suppressor function of this blood CD11b(+)Ly6G(-) cell population is impaired. Therefore, blood CD11b(+)Ly6G(-) cells appear to be intrinsically suppressive and may have a key role in maintaining immune homoeostasis. Loss of this suppressive function may contribute to development of autoimmunity.
Assuntos
Autoimunidade/imunologia , Monócitos/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Superfície , Antígeno CD11b/imunologia , Comunicação Celular/imunologia , Proliferação de Células , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/citologia , Óxido Nítrico/imunologia , Linfócitos T/citologiaRESUMO
An efficient pathway of cross-presentation common to a range of dendritic cell (DC) populations was identified by targeting Ag to MHC class II molecules. This finding was achieved by conjugating Ag to M1, which is a modified version of the superantigen streptococcal mitogenic exotoxin Z-2 that binds to MHC class II molecules but cannot directly stimulate T cells. M1 conjugates were efficiently presented to CD4(+) and CD8(+) T cells by bone marrow-derived DC and Langerhans cells in vitro. Whereas nonconjugated Ag was preferentially cross-presented by splenic CD8alpha(+) DC in vivo, M1-conjugated Ag was cross-presented by all dendritic subtypes assessed. Potent effector T cell responses with antitumor activity were elicited when M1 conjugates were injected together with an adjuvant. This method of Ag delivery has significant potential in therapeutic applications.
Assuntos
Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/imunologia , Apresentação Cruzada/imunologia , Sistemas de Liberação de Medicamentos/métodos , Exotoxinas/administração & dosagem , Exotoxinas/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Animais , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Toxinas Bacterianas/metabolismo , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Exotoxinas/metabolismo , Ligantes , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais/imunologia , Superantígenos/administração & dosagem , Superantígenos/imunologia , Superantígenos/metabolismoRESUMO
Autoimmune diseases are incurable. We have hypothesized that these diseases can be cured by the transplantation of bone marrow (BM) stem cells that have been genetically engineered to express self-Ag. Here we have tested this hypothesis in experimental autoimmune encephalomyelitis (EAE) induced by the self-Ag myelin oligodendrocyte glycoprotein (MOG). We show that, in mice, transplantation of BM genetically modified to express MOG prevented the induction and progression of EAE, and combined with antecedent corticosteroid treatment, induced long-term remission of established disease. Mice remained resistant to EAE development upon subsequent rechallenge with MOG. Transfer of BM from these mice rendered recipients resistant to EAE. Splenocytes from these mice failed to proliferate or produce IL-17, IFN-gamma, and GM-CSF in response to MOG(35-55) peptide stimulation and they failed to produce MOG autoantibody. Mechanistically, we demonstrated in vivo reduction in development of CD4(+) MOG(35-55)-specific thymocytes, indicative of clonal deletion with no evidence for selection of Ag-specific regulatory T cells. These findings validate our hypothesis that transplantation of genetically modified BM expressing disease-causative self-Ag provides a curative approach by clonal deletion of disease-causative self-reactive T cells.
Assuntos
Autoantígenos/imunologia , Transplante de Medula Óssea , Deleção Clonal/imunologia , Encefalomielite Autoimune Experimental/imunologia , Glicoproteínas/imunologia , Tolerância Imunológica/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Reguladores/imunologia , Corticosteroides/farmacologia , Animais , Autoanticorpos/genética , Autoanticorpos/imunologia , Autoantígenos/genética , Deleção Clonal/efeitos dos fármacos , Deleção Clonal/genética , Citocinas/genética , Citocinas/imunologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/terapia , Feminino , Expressão Gênica/genética , Expressão Gênica/imunologia , Glicoproteínas/genética , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/genética , Imunidade Inata/genética , Imunidade Inata/imunologia , Camundongos , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/genética , Timo/imunologia , Transdução GenéticaRESUMO
Autoreactive myelin-specific CD4(+) T cells play an important role in CNS demyelination observed in MS and EAE. Consequently, it is important to understand the mechanisms of T cell receptor signalling leading to the activation of autoreactive T cells. We have previously generated a chimeric T cell receptor beta-chain (betaIII) displaying increased antigen sensitivity by exchanging most of the transmembrane and the intracellular domain of the TCR-beta chain with the corresponding TCR-gamma sequence. To investigate the effect of this "super-signalling" TCR in an autoimmune setting, we generated MOG(35-55) specific TCR transgenic mice expressing either the wild-type or the chimeric betaIII TCR-beta chain. We found that naïve transgenic T cells expressing the chimeric betaIII chain proliferated more extensively than wild-type cells in response to MOG(35-55)in vitro. Likewise, betaIII T cells skewed into a TH1 phenotype maintained the proliferative advantage over wild-type TH1 T cells at low antigen concentration. However, when skewed into a TH2 phenotype, there was no difference in proliferation between wild-type and betaIII T cells. Blocking of Fas-mediated cell death evenly affected wild-type and betaIII TH1 T cells and resulted in increased proliferation of both subsets, suggesting that betaIII T cells did not show defective Fas-FasL signalling. Finally, we found that betaIII TCR transgenic mice are more susceptible to EAE than wild-type TCR transgenic mice. We conclude that the change in the transmembrane domain of the TCR-beta chain affects TH1 T cells and the susceptibility to EAE, but does not affect TH2 cells. Investigating the molecular interaction within the TCR complex will help us to identify signalling pathways that can be manipulated to stop the progression of MS.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Proliferação de Células/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Suscetibilidade a Doenças/imunologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Proteína Ligante Fas/imunologia , Feminino , Glicoproteínas/farmacologia , Imunização , Interferon gama/biossíntese , Interleucina-17/biossíntese , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/farmacologia , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/química , Proteínas Recombinantes/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Receptor fas/imunologiaRESUMO
BACKGROUND: Fluorescence activated cell sorting (FACS) is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. Beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. As the individual suspension bead passes through the sensing region of the FACS machine, fluorescent signals are acquired and analysed. Currently, antigens are tediously purified and chemically cross-linked to preformed beads. Purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. As an alternative, we genetically engineered Escherichia coli to produce biopolyester (polyhdroxyalkanoate=PHA) granules displaying diagnostically relevant antigens in their native conformation and suitable for FACS analysis. RESULTS: Hybrid genes were constructed, which encode either the mouse interleukin-2 (IL2) or the myelin oligodendrocyte glycoprotein (MOG) fused via an enterokinase site providing linker region to the C terminus of the PHA granule associated protein PhaP, respectively. The hybrid genes were expressed in PHA-accumulating recombinant E. coli. MOG and IL2 fusion proteins were abundantly attached to PHA granules and were identified by MALDI-TOF/MS analysis and N terminal sequencing. A more abundant second fusion protein of either MOG or IL2 resulted from an additional N terminal fusion, which did surprisingly not interfere with attachment to PHA granule. PHA granules displaying either IL2 or MOG were used for FACS using monoclonal anti-IL2 or anti-MOG antibodies conjugated to a fluorescent dye. FACS analysis showed significant and specific binding of respective antibodies. Enterokinase treatment of IL2 displaying PHA granules enabled removal of IL2 as monitored by FACS analysis. Mice were immunized with either MOG or OVA (ovalbumin) and the respective sera were analysed using MOG-displaying PHA granules and FACS analysis showing a specific and sensitive detection of antigen-specific antibodies within a wide dynamic range. CONCLUSION: E. coli can be genetically engineered to produce PHA granules displaying correctly folded eukaryotic proteins and which can be applied as beads in FACS based diagnostics. Since PHA granule formation and protein attachment occurs in one step already inside the bacterial cell, microbial production could be a cheap and efficient alternative to commercial beads.
Assuntos
Separação Celular/métodos , Escherichia coli/metabolismo , Citometria de Fluxo/métodos , Interleucina-2/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Poliésteres/química , Engenharia de Proteínas/métodos , Animais , Materiais Biocompatíveis/química , Biopolímeros/química , Escherichia coli/genética , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Interleucina-2/genética , Camundongos , Proteínas da Mielina , Glicoproteína Associada a Mielina/genética , Glicoproteína Mielina-Oligodendrócito , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Many biopharmaceuticals (BPs) are known to be immunogenic in the clinic, which can result in modified pharmacokinetics, reduced efficacy, allergic reactions and anaphylaxis. During recent years, several technologies to predict immunogenicity have been introduced, but the predictive value is still considered low. Thus, there is an unmet medical need for optimization of such technologies. The generation of T cell dependent high affinity anti-drug antibodies plays a key role in clinical immunogenicity. This study aimed at developing and evaluating a novel in vitro T cell:PBMC assay for prediction of the immunogenicity potential of BPs. To this end, we assessed the ability of infliximab (anti-TNF-α), rituximab (anti-CD20), adalimumab (anti-TNF-α) and natalizumab (anti-α4-integrin), all showing immunogenicity in the clinic, to induce a CD4+ T cells response. Keyhole limpet hemocyanin (KLH) and cytomegalovirus pp65 protein (CMV) were included as neo-antigen and recall antigen positive controls, respectively. By analyzing 26 healthy donors having HLA-DRB1 alleles matching the European population, we calculated the frequency of responding donors, the magnitude of the response, and the frequency of BP-specific T cells, as measured by 3[H]-thymidine incorporation and ELISpot IL-2 secretion. KLH and CMV demonstrated a strong T cell response in all the donors analyzed. The frequency of responding donors to the BPs was 4% for infliximab, 8% for adalimumab, 19% for rituximab and 27% for natalizumab, which is compared to and discussed with their respective observed clinical immunogenicity. This study further complements predictive immunogenicity testing by quantifying the in vitro CD4+ T cell responses to different BPs. Even though the data generated using this modified method does not directly translate to the clinical situation, a high sensitivity and immunogenic potential of most BPs is demonstrated.
Assuntos
Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Doadores de Tecidos , Ensaio de Imunoadsorção Enzimática , Antígenos HLA/imunologia , Haplótipos , HumanosRESUMO
Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease model used to investigate mechanisms involved in the activation of self-reactive T cells. Preproenkephalin (PPNK) is the gene that encodes the protein proenkephalin A that has been detected in the brain, adrenal cells and cells of the immune system. In this paper, whether PPNK plays a role in the development of EAE was investigated. PPNK-deficient and wild-type mice were immunized with the MOG(35-55) peptide and the development of EAE observed. Our results show that PPNK-deficient mice developed less severe clinical signs of disease than wild-type mice, and with lower incidence. MOG(35-55)-specific T cells from PPNK-deficient and wild-type mice produced IFNgamma and TNFalpha but no IL-4 or IL-10, indicative of a Th1 phenotype. However, the numbers of MOG(35-55)-specific IFNgamma-producing cells from immunized PPNK-deficient mice were largely reduced at early stages of disease. Interestingly, there was no difference in clinical signs or infiltrating mononuclear cells in the CNS between wild-type and PPNK-deficient mice at the later stage of disease. Our results suggest that PPNK accelerates the generation of autoimmune IFNgamma-producing T cells and MOG(35-55)-induced EAE.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Encefalinas/deficiência , Encefalinas/imunologia , Precursores de Proteínas/deficiência , Precursores de Proteínas/imunologia , Animais , Proliferação de Células , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Encefalinas/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas/imunologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Ativação Linfocitária/imunologia , Camundongos , Camundongos Mutantes , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Precursores de Proteínas/genética , Células Th1/imunologia , Células Th2/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Correct assembly of all TCR complex polypeptides is essential for its cell surface expression and function. The transmembrane region of the TCRalpha chain is highly conserved and to gain insight into the structural and functional role of these residues, single amino acid substitutions were introduced and surface expression and signaling ability studied in T hybridoma cells. Introduction of acid residues within the TCRalpha chain transmembrane region were mostly tolerated, indicating that the net charge within this region of the TCR complex is not crucial to either assembly or signaling. However, mutations of leucine 112 or phenylalanine 127 to aspartic acids (L112D or F127D, respectively) resulted in dramatic loss of surface expression and, therefore, their signaling ability. Intracellular flow cytometry showed that the mutant TCRalpha polypeptides were present at levels comparable to wild-type, indicating that the reduced surface expression was not a consequence of impaired protein survival. The defect was characterized by immunoprecipitation and showed that residues L112 and F127 were involved in early interactions with the CD3 complex. A large proportion of the TCRalpha chain mutants L112D and F127D consisted of immature protein, indicative of a problem during early assembly of the TCR. Our findings provide evidence for the involvement of the conserved L112 and F127 residues of the TCRalpha chain transmembrane region in the assembly process of the TCR complex.
Assuntos
Leucina/fisiologia , Fenilalanina/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/química , Sequência de Aminoácidos , Aminoácidos/genética , Animais , Sequência Conservada , Análise Mutacional de DNA , Humanos , Hibridomas , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Alinhamento de SequênciaRESUMO
A murine model for the assessment of protective immunity to human papillomavirus (HPV) type 16, a virus that does not naturally infect mice, is described. In this system, protection was tested following intranasal challenge of mice with a recombinant vaccinia virus expressing both the selected HPV antigen and a beta-galactosidase (beta-gal) reporter. The extent of viral infectivity was determined by measuring beta-gal positive lung cells using flow cytometry. The efficacy of this model to measure protective immunity was evaluated by priming mice with the beta-gal vaccinia virus then challenging the mice with the same virus. Vaccinia primed mice had negligible numbers of beta-gal positive cells in the lung 5 days following viral challenge indicating protection, whereas around 50% of cells were infected in immunologically naive, challenged mice. The protective efficacy of two dendritic cell vaccines for HPV16 was measured in this model. Both vaccines provided some protection to subsequent viral challenge, compared with their controls. Although this protection model was applied to HPV in this study, it may also have broad application to other viruses that do not infect mice naturally.
Assuntos
Células Dendríticas/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Antígenos Virais/imunologia , Linhagem Celular , Proteínas de Ligação a DNA/imunologia , Citometria de Fluxo , Genes Reporter , Humanos , Camundongos , Modelos Animais , Reprodutibilidade dos Testes , Proteínas Virais/imunologia , beta-Galactosidase/genética , beta-Galactosidase/imunologiaRESUMO
In a previous study, we showed that the Ly6G(-)CD11b(+) blood monocytes residing in naïve mice are intrinsically immunosuppressive and that loss of this suppressive function may contribute to the development of autoimmunity in experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis. Here we report that mice treated with a modified superantigen coupled to myelin oligodendrocyte glycoprotein 35-55 (MOG(35-55)) peptide (DM-MOG(35-55)) suppressed the development of EAE. The treatment was associated with impaired MOG(35-55)-specific T cell proliferation and a decrease in IL-17 and IFNγ production in the draining lymph nodes. Analysis of circulating blood immune cells showed that the suppressor function of Ly6G(-)CD11b(+) blood monocytes was reduced in EAE mice, but was restored in mice treated with DM-MOG(35-55). Importantly, adoptive transfer of blood CD11b(+)Ly6G(-) cells isolated from DM-MOG(35-55)-treated mice protected recipient mice from developing EAE. Together, these results show that DM coupled to the auto-antigen MOG(35-55): 1) suppresses EAE via antigen-specific suppression of T cell responses, and 2) re-establishes suppressor function of Ly6G(-)CD11b(+) blood monocytes. Auto-antigens coupled to DM could therefore represent a new therapeutic approach for controlling inappropriate inflammation in autoimmune diseases such as multiple sclerosis by inducing antigen-specific T cell suppression.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Epitopos/imunologia , Monócitos/imunologia , Superantígenos/imunologia , Transferência Adotiva , Animais , Antígenos Ly/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Antígeno CD11b/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/terapia , Exotoxinas/química , Exotoxinas/imunologia , Ativação Linfocitária/imunologia , Camundongos , Monócitos/metabolismo , Glicoproteína Mielina-Oligodendrócito/química , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Subpopulações de Linfócitos T/imunologiaRESUMO
IL-12p40 and macrophages are essential for the induction of disease in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. In this paper, we show that treatment of mice with opsonized erythrocytes, which have been shown to ligate Fcgamma receptors on macrophages and alter their cytokine profile, significantly delayed the onset of experimental autoimmune encephalomyelitis. This protection correlated to the induction of Th2 responses by autoreactive T cells, enhanced basal systemic responses and a significant downregulation of IL-12p40 and nitric oxide synthase-2, but not IFN-gamma expression. IL-4 was essential for the protection by opsonized erythrocytes as the effects of treatment were eliminated in IL-4-deficient mice. Together these studies suggest that the ligation of Fcgamma receptors can modify the development of autoimmune disease by altering macrophage activation and enhancing Th2 responses.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Imunoglobulinas/uso terapêutico , Receptores de IgG/metabolismo , Células Th2/imunologia , Animais , Interleucina-12/metabolismo , Interleucina-4/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Ovinos , Células Th1/imunologiaRESUMO
We used various culture conditions to generate type 1 (Tc1) or type 2 (Tc2) cytotoxic T cells in vitro. T-cell receptor (TCR) transgenic T cells were cultured with antigen and spleen cells, or antigen and dendritic cells (DC), or anti-CD3 and anti-CD28. Tc1 cultures contained interleukin (IL)-2 and IL-6, and Tc2 cultures contained IL-2, IL-6 and IL-4. Tc2 cells generated in each culture condition acquired a CD62L(low) CD44(high) phenotype, had high cytotoxic activity, and secreted IL-4, IL-5 and moderate amounts of interferon-gamma (IFN-gamma). In contrast, the phenotype and function of Tc1 cells varied depending on culture conditions. Tc1 cells from anti-CD3 and anti-CD28 cultures had high cytotoxic activity and were CD62L(low) CD44(high), while Tc1 cells from antigen and spleen cell cultures had low cytotoxic activity and were CD62L(high) CD44(low). Tc1 cells from antigen and DC cultures had an intermediate phenotype. All Tc1 cells secreted high amounts of IFN-gamma, but only Tc1 from anti-CD3 and anti-CD28 cultures had antitumour activity in vivo. Differences were not caused by suboptimal culture conditions, as Tc1 cells divided at a similar rate whether cultured with antigen and spleen cells or with anti-CD3 and anti-CD28. We conclude that IL-4 not only induces 'type 2' cytokine secretion in CD8(+) T cells, but also affects their expression of surface markers and cytotoxic activity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Animais , Antígenos/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Divisão Celular/imunologia , Linhagem Celular Tumoral , Meios de Cultura , Citocinas/imunologia , Citotoxicidade Imunológica/imunologia , Relação Dose-Resposta Imunológica , Epitopos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Baço/citologia , Baço/imunologiaRESUMO
Experimental autoimmune encephalomyelitis (EAE) is an animal model commonly used to investigate mechanisms involved in the activation of self-reactive T cells. Whereas auto-reactive T(h)1 cells are believed to be involved in the generation of EAE, T(h)2 cells can induce EAE in immunocompromised hosts. Since the T(h)2 cytokine IL-5 can influence the nature and severity of disease, we investigated the role of IL-5 in the EAE model. Wild-type C57BL/6J and IL-5(-/-) mice were immunized with myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide and the development of EAE observed. Our results show that IL-5(-/-) mice developed EAE with a similar day of onset and comparable severity to wild-type mice. Primed T cells isolated from IL-5(-/-) mice proliferated equally to wild-type cells in response to antigen challenge with MOG(35-55). Antigen-specific T cells from IL-5(-/-) mice produced IFN-gamma and tumor necrosis factor-alpha, but no IL-4 or IL-10, indicating that a predominant T(h)1 environment was induced following immunization. No differences in the types of cells infiltrating into the central nervous system were observed between IL-5(-/-) and wild-type mice. Our results suggest that IL-5 is not directly involved in the initiation or effector phase of MOG(35-55)-induced EAE in immunocompetent C57BL/6J mice.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Interleucina-5/fisiologia , Animais , Diferenciação Celular , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/ultraestrutura , Citocinas/fisiologia , Encefalomielite Autoimune Experimental/genética , Glicoproteínas/metabolismo , Interferon gama/imunologia , Interleucina-5/genética , Ativação Linfocitária , Camundongos , Camundongos Knockout , Modelos Animais , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/metabolismo , Linfócitos T/imunologia , Células Th1/citologia , Células Th2/citologiaRESUMO
A preestablished infection with the parasitic helminth, Schistosoma mansoni, significantly reduced the incidence and delayed the onset of experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice immunized with myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide. The altered disease progression was not solely due to the induction of a strong Th2 response, since intraperitoneal injection of schistosome eggs did not affect disease development. MOG-specific gamma interferon (IFN-gamma), nitric oxide, and tumor necrosis factor alpha production by splenocytes was significantly reduced in schistosome-infected mice compared to uninfected mice. However, similar levels of interleukin-10 (IL-10) were produced in an antigen-specific manner, suggesting that the induction of antigen-specific responses was not inhibited. Analysis of in vivo cytokine production by real-time PCR indicated that IL-12p40, but not IFN-gamma, transcript levels were dramatically reduced in the spinal cords of schistosome-infected, MOG-immunized mice. Furthermore, analysis of the cellular composition of the spinal cords and brains revealed that a preestablished infection with S. mansoni decreased central nervous system (CNS) inflammation, particularly of macrophages and CD4 T cells. These results suggest that schistosomiasis may negatively regulate the onset of EAE by downregulating the production of proinflammatory cytokines and altering CNS inflammation.
Assuntos
Encefalomielite Autoimune Experimental/complicações , Esquistossomose mansoni/complicações , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Inflamação/imunologia , Inflamação/patologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas da Mielina , Glicoproteína Associada a Mielina/administração & dosagem , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Neutrófilos/imunologia , Neutrófilos/patologia , Óxido Nítrico/biossíntese , Esquistossomose mansoni/imunologia , Medula Espinal/imunologia , Medula Espinal/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Self-reactive T cells escape deletion in the thymus and are found in the peripheral repertoire. Because bone-marrow-derived dendritic cells (BM-DC) are potent activators of antigen-specific T cells, these cells could theoretically activate self-reactive T cells leading to autoimmunity. We investigated whether BM-DC could induce the autoimmune disease experimental autoimmune encephalomyelitis (EAE). Our results show that transfer of BM-DC presenting a self-peptide from the myelin oligodendrocyte glycoprotein (MOG35-55) into naive mice induced EAE 7-14 days later. MOG35-55-specific T cells of the Th1 phenotype were present in the lymph nodes and spleens of mice that received live peptide-pulsed BM-DC. Heat-killed or formaldehyde-fixed BM-DC presenting MOG35-55 could induce neither clinical signs of EAE nor a measurable T-cell response in vitro. These data show that live BM-DC presenting a self-antigen can induce the organ-specific autoimmune disorder EAE in a non-transgenic system. Therefore, this new EAE model could be used as a more clinically relevant model for the human disease multiple sclerosis. These findings could also have implications for the use of DC immunotherapy in a clinical setting.