RESUMO
The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8+ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-gamma secreting CD8+ T cells specific for H-2K(b)-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2(-/-), Tlr4(-/-), Tlr9(-/) (-) or Myd88(-/-) mice generated both specific cytotoxic responses and IFN-gamma secreting CD8+ T cells at levels comparable to WT mice, although the frequency of IFN-gamma+CD4+ cells was diminished in infected Myd88(-/-) mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-gamma, TNF-alpha and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4(-/-) mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Imunidade Inata , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Doença de Chagas/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Trypanosoma cruzi/imunologiaRESUMO
Vaccines have had an unquestionable impact on public health during the last century. The most likely reason for the success of vaccines is the robust protective properties of specific antibodies. However, antibodies exert a strong selective pressure and many microorganisms, such as the obligatory intracellular parasite Trypanosoma cruzi, have been selected to survive in their presence. Although the host develops a strong immune response to T. cruzi, they do not clear the infection and instead progress to the chronic phase of the disease. Parasite persistence during the chronic phase of infection is now considered the main factor contributing to the chronic symptoms of the disease. Based on this finding, containment of parasite growth and survival may be one method to avoid the immunopathology of the chronic phase. In this context, vaccinologists have looked over the past 20 years for other immune effector mechanisms that could eliminate these antibody-resistant pathogens. We and others have tested the hypothesis that non-antibody-mediated cellular immune responses (CD4+ Th1 and CD8+ Tc1 cells) to specific parasite antigens/genes expressed by T. cruzi could indeed be used for the purpose of vaccination. This hypothesis was confirmed in different mouse models, indicating a possible path for vaccine development.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Doença de Chagas/prevenção & controle , Modelos Animais de Doenças , Imunidade Celular , Camundongos , Trypanosoma cruzi/genéticaRESUMO
Immunisation with Amastigote Surface Protein 2 (asp-2) and trans-sialidase (ts) genes induces protective immunity in highly susceptible A/Sn mice, against infection with parasites of the Y strain of Trypanosoma cruzi. Based on immunological and biological strain variations in T. cruzi parasites, our goal was to validate our vaccination results using different parasite strains. Due to the importance of the CD8(+) T cells in protective immunity, we initially determined which strains expressed the immunodominant H-2K(k)-restricted epitope TEWETGQI. We tested eight strains, four of which elicited immune responses to this epitope (Y, G, Colombian and Colombia). We selected the Colombian and Colombia strains for our studies. A/Sn mice were immunised with different regimens using both T. cruzi genes (asp-2 and ts) simultaneously and subsequently challenged with blood trypomastigotes. Immune responses before the challenge were confirmed by the presence of specific antibodies and peptide-specific T cells. Genetic vaccination did not confer protective immunity against acute infection with a lethal dose of the Colombian strain. In contrast, we observed a drastic reduction in parasitemia and a significant increase in survival, following challenge with an otherwise lethal dose of the Colombia strain. In many surviving animals with late-stage chronic infection, we observed alterations in the heart's electrical conductivity, compared to naive mice. In summary, we concluded that immunity against T. cruzi antigens, similar to viruses and bacteria, may be strain-specific and have a negative impact on vaccine development.
Assuntos
Doença de Chagas/prevenção & controle , Glicoproteínas/imunologia , Neuraminidase/imunologia , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Sequência de Bases , Reações Cruzadas , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Camundongos , Dados de Sequência Molecular , Parasitemia/prevenção & controle , Alinhamento de Sequência , Análise de Sobrevida , Linfócitos T/imunologiaRESUMO
Vaccines have had an unquestionable impact on public health during the last century. The most likely reason for the success of vaccines is the robust protective properties of specific antibodies. However, antibodies exert a strong selective pressure and many microorganisms, such as the obligatory intracellular parasite Trypanosoma cruzi, have been selected to survive in their presence. Although the host develops a strong immune response to T. cruzi, they do not clear the infection and instead progress to the chronic phase of the disease. Parasite persistence during the chronic phase of infection is now considered the main factor contributing to the chronic symptoms of the disease. Based on this finding, containment of parasite growth and survival may be one method to avoid the immunopathology of the chronic phase. In this context, vaccinologists have looked over the past 20 years for other immune effector mechanisms that could eliminate these antibody-resistant pathogens. We and others have tested the hypothesis that non-antibody-mediated cellular immune responses (CD4+ Th1 and CD8+ Tc1 cells) to specific parasite antigens/genes expressed by T. cruzi could indeed be used for the purpose of vaccination. This hypothesis was confirmed in different mouse models, indicating a possible path for vaccine development.
Assuntos
Animais , Camundongos , /imunologia , /imunologia , Doença de Chagas/imunologia , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Doença de Chagas/prevenção & controle , Modelos Animais de Doenças , Imunidade Celular , Trypanosoma cruzi/genéticaRESUMO
Ischemia reperfusion injury (IRI) is a potential contributor for the development of chronic allograft nephropathy. T cells are important mediators of injury, even in the absence of alloantigens. We performed a depletion of TCD4(+)CTLA4(+)Foxp3(+) cells with anti-CD25(PC61), a treatment with anti-GITR (DTA-1) and rat-IgG, followed by 45 min of ischemia and 24/72 h of reperfusion, and then analyzed blood urea, kidney histopathology and gene expression in kidneys by QReal Time PCR. After 24 h of reperfusion, depletion of TCD4(+)CTLA4(+)Foxp3(+) cells reached 30.3%(spleen) and 67.8%(lymph nodes). 72 h after reperfusion depletion reached 43.1%(spleen) and 90.22%(lymph nodes) and depleted animals presented with significantly poorer renal function, while DTA-1(anti-GITR)-treated ones showed a significant protection, all compared to serum urea from control group (IgG: 150.10+/-50.04; PC61: 187.23+/-31.38; DTA-1: 64.53+/-25.65, mg/dL, p<0.05). These data were corroborated by histopathology. We observed an increase of HO-1 expression in animals treated with DTA-1 at 72 h of reperfusion with significant differences. Thus, our results suggest that PC61(anti-CD25) mAb treatment is deleterious, while DTA-1(anti-GITR) mAb treatment presents a protective role in the renal IRI, indicating that some regulatory populations of T cells might have a role in IRI.
Assuntos
Injúria Renal Aguda/imunologia , Traumatismo por Reperfusão/imunologia , Linfócitos T Reguladores/imunologia , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Nitrogênio da Ureia Sanguínea , Citometria de Fluxo , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Heme Oxigenase-1/biossíntese , Heme Oxigenase-1/genética , Rim/imunologia , Rim/lesões , Rim/patologia , Depleção Linfocítica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antígeno Nuclear de Célula em Proliferação/biossíntese , Antígeno Nuclear de Célula em Proliferação/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Interference or competition between CD8(+) T cells restricted by distinct MHC-I molecules can be a powerful means to establish an immunodominant response. However, its importance during infections is still questionable. In this study, we describe that following infection of mice with the human pathogen Trypanosoma cruzi, an immunodominant CD8(+) T cell immune response is developed directed to an H-2K(b)-restricted epitope expressed by members of the trans-sialidase family of surface proteins. To determine whether this immunodominance was exerted over other non-H-2K(b)-restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2K(b)-, H-2K(k)-, or H-2K(d)-restricted CD8(+) T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2K(b)-restricted immunodominant epitope. In contrast, H-2K(k)- or H-2K(d)-restricted immune responses were significantly impaired in heterozygote infected mice when compared with homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi Ags. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8(+) T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Antígenos H-2/imunologia , Epitopos Imunodominantes/imunologia , Trypanosoma cruzi/imunologia , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Antígenos H-2/química , Antígenos H-2/genética , Heterozigoto , Imunização , Camundongos , Camundongos Endogâmicos , Dados de Sequência MolecularRESUMO
Synthetic oligonucleotides (ODNs) containing immunostimulatory CpG motifs (CpG) are a new class of adjuvants suitable for the development of recombinant vaccines. Here we describe that endogenous interferon (IFN) was critical for the adjuvant activity of CpG ODN as genetically deficient mice developed significantly lower IgG antibody titers following immunization with recombinant proteins. In contrast, the absence of endogenous IL-12/IL-23 or IL-4 had little impact on the magnitude of the antibody response but instead caused a dramatic change in the pattern of IgG isotypes. The dependence on IFN-gamma was specific for CpG ODN and it was not observed with other adjuvants tested. IFN-gamma was produced by NK, dendritic cells, CD4+ and CD8+ T cells stimulated in vitro with CpG ODN. Adoptive transfer experiments confirmed that CD4+ or CD8+ T cells were in fact relevant sources of IFN-gamma in vivo. Following CpG ODN injection, splenic dendritic cells from IFN-gamma deficient mice did not up-regulate CD86 or CD40 expression, suggesting a role for these molecules. The importance of CD28 (CD86 ligand) was confirmed using CD28 deficient mice which presented severely impaired immune responses following CpG ODN-assisted immunization.
Assuntos
Adjuvantes Imunológicos , Ilhas de CpG/imunologia , Interferon gama/imunologia , Proteínas Recombinantes/imunologia , Animais , Antígenos CD28/genética , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Ilhas de CpG/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Epitopos/imunologia , Feminino , Deleção de Genes , Imunoglobulina G/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Baço/citologia , Baço/imunologia , Linfócitos T/imunologiaRESUMO
BACKGROUND: Following infection with viruses, bacteria or protozoan parasites, naïve antigen-specific CD8(+) T cells undergo a process of differentiation and proliferation to generate effector cells. Recent evidences suggest that the timing of generation of specific effector CD8(+) T cells varies widely according to different pathogens. We hypothesized that the timing of increase in the pathogen load could be a critical parameter governing this process. METHODOLOGY/PRINCIPAL FINDINGS: Using increasing doses of the protozoan parasite Trypanosoma cruzi to infect C57BL/6 mice, we observed a significant acceleration in the timing of parasitemia without an increase in mouse susceptibility. In contrast, in CD8 deficient mice, we observed an inverse relationship between the parasite inoculum and the timing of death. These results suggest that in normal mice CD8(+) T cells became protective earlier, following the accelerated development of parasitemia. The evaluation of specific cytotoxic responses in vivo to three distinct epitopes revealed that increasing the parasite inoculum hastened the expansion of specific CD8(+) cytotoxic T cells following infection. The differentiation and expansion of T. cruzi-specific CD8(+) cytotoxic T cells is in fact dependent on parasite multiplication, as radiation-attenuated parasites were unable to activate these cells. We also observed that, in contrast to most pathogens, the activation process of T. cruzi-specific CD8(+) cytotoxic T cells was dependent on MHC class II restricted CD4(+) T cells. CONCLUSIONS/SIGNIFICANCE: Our results are compatible with our initial hypothesis that the timing of increase in the pathogen load can be a critical parameter governing the kinetics of CD4(+) T cell-dependent expansion of pathogen-specific CD8(+) cytotoxic T cells.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Proliferação de Células , Trypanosoma cruzi/fisiologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
One of the most promising vaccine candidates against the erythrocytic forms of malaria is the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)). As part of our studies aimed at the development of a Plasmodium vivax malaria vaccine, we characterized the immunogenic properties of a new bacterial recombinant protein containing the P. vivax MSP1(19) and two helper T-cell epitopes, the synthetic universal pan allelic DR epitope (PADRE) and a new internal MSP1 P. vivax epitope (DYDVVYLKPLAGMYK). We found that the recognition of His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE was as good as the recognition of His6MSP1(19) indicating that the presence of the T-cell epitopes PADRE and DYDVVYLKPLAGMYK did not modify the MSP1(19) epitopes recognized by human IgG. The recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE proved to be highly immunogenic in marmosets (Callithrix jacchus jacchus) when administered in incomplete Freund's adjuvant. However, when administered in other adjuvant formulations such as Quil A, CpG ODN 2006 or MPL/TDM, antibody titers to MSP1(19) were significantly lower. Among these three adjuvants, Quil A proved to be the most efficient one generating antibody titers significantly higher than the others. These results indicated that under the circumstances evaluated, adjuvants were key for the immunogenicity of the recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE.
Assuntos
Anticorpos Antiprotozoários/sangue , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Vacinas Antimaláricas/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium vivax/imunologia , Adjuvantes Imunológicos , Animais , Callithrix , Adjuvante de Freund , Malária/imunologia , Camundongos , Oligodesoxirribonucleotídeos/imunologia , Saponinas de Quilaia , Saponinas/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Sintéticas/imunologiaRESUMO
The pan HLA DR-binding epitope (PADRE) has been proposed as a simple carrier epitope suitable for use in the development of synthetic and recombinant vaccines. Using the mouse model, we evaluated whether PADRE could improve adjuvant-assisted immunizations with a recombinant malarial protein containing the 19kDa C-terminal region of merozoite surface protein 1 (MSP1(19)) that is a Plasmodium vivax vaccine candidate. Initially, the antibody immune response was evaluated in C57BL/6 mice, a mouse strain which develops a strong T cell immune response to PADRE. When administered in distinct adjuvant formulations, antibody titers induced by the recombinant protein His(6)MSP1(19)-PADRE were not significantly different to those generated by complete/incomplete Freund's adjuvant (CFA/IFA) in terms of magnitude, affinity, IgG subclasses and longevity. However, in C57BL/6 mice immunized with the recombinant protein His(6)MSP1(19), strong antibody responses could be generated in the presence of CFA/IFA but not other classes of adjuvants such as CpG ODN 1826 or MPL/TDM. Similarly, in BALB/c mice that do not develop T cells specific for PADRE, the recombinant protein His(6)MSP1(19)-PADRE failed to induce high antibody titers in the presence of adjuvants other than CFA/IFA. Our results indicated that when adjuvants that are not as strong as CFA/IFA are employed, the presence of PADRE greatly improved adjuvant-assisted antibody immune responses induced by a malarial recombinant antigen. Considering the great limitations of adjuvants for human use, our observation may improve the rational design of new vaccine formulations.