RESUMO
The extent to which unconventional forms of antigen presentation drive T cell immunity is unknown. By convention, CD8 T cells recognize viral peptides, or epitopes, in association with classical major histocompatibility complex (MHC) class I, or MHC-Ia, but immune surveillance can, in some cases, be directed against peptides presented by nonclassical MHC-Ib, in particular the MHC-E proteins (Qa-1 in mice and HLA-E in humans); however, the overall importance of nonclassical responses in antiviral immunity remains unclear. Similarly uncertain is the importance of 'cryptic' viral epitopes, defined as those undetectable by conventional mapping techniques. Here we used an immunopeptidomic approach to search for unconventional epitopes that drive T cell responses in mice infected with influenza virus A/Puerto Rico/8/1934. We identified a nine amino acid epitope, termed M-SL9, that drives a co-immunodominant, cytolytic CD8 T cell response that is unconventional in two major ways: first, it is presented by Qa-1, and second, it has a cryptic origin, mapping to an unannotated alternative reading frame product of the influenza matrix gene segment. Presentation and immunogenicity of M-SL9 are dependent on the second AUG codon of the positive sense matrix RNA segment, suggesting translation initiation by leaky ribosomal scanning. During influenza virus A/Puerto Rico/8/1934 infection, M-SL9-specific T cells exhibit a low level of egress from the lungs and strong differentiation into tissue-resident memory cells. Importantly, we show that M-SL9/Qa-1-specific T cells can be strongly induced by messenger RNA vaccination and that they can mediate antigen-specific cytolysis in vivo. Our results demonstrate that noncanonical translation products can account for an important fraction of the T cell repertoire and add to a growing body of evidence that MHC-E-restricted T cells could have substantial therapeutic value.
Assuntos
Influenza Humana , Humanos , Camundongos , Animais , Epitopos , Linfócitos T Citotóxicos , Linfócitos T CD8-Positivos , Peptídeos , Epitopos de Linfócito TRESUMO
The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.
Assuntos
Anticorpos Neutralizantes/metabolismo , Linfócitos B/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Centro Germinativo/imunologia , SARS-CoV-2/fisiologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas Sintéticas/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Células Cultivadas , Epitopos , Humanos , Ativação Linfocitária , Polissorbatos , RNA Viral/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Esqualeno , Vacinação , Vacinas de mRNARESUMO
SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.
Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , RNA Mensageiro/imunologia , RNA Viral/imunologia , Vacinas Virais/administração & dosagem , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/virologia , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Modelos Animais de Doenças , Furina/genética , Furina/imunologia , Humanos , Imunidade Humoral/efeitos dos fármacos , Imunização/métodos , Imunogenicidade da Vacina , Memória Imunológica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/química , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , RNA Mensageiro/genética , RNA Viral/genética , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Sintéticas , Vacinas Virais/biossíntese , Vacinas Virais/genéticaRESUMO
The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.
Assuntos
Antígenos de Neoplasias , Neuroblastoma , Proteínas Oncogênicas , Peptídeos , Receptores de Antígenos Quiméricos , Animais , Humanos , Camundongos , África/etnologia , Alelos , Sequência de Aminoácidos , Carcinogênese , Reações Cruzadas , Antígenos HLA-A/química , Antígenos HLA-A/imunologia , Neuroblastoma/genética , Neuroblastoma/imunologia , Neuroblastoma/terapia , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/imunologia , Peptídeos/antagonistas & inibidores , Peptídeos/química , Peptídeos/imunologia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/uso terapêuticoRESUMO
The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos HLA/imunologia , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Oncogênicas/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Antígenos de Neoplasias/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Reações Cruzadas , Apresentação Cruzada , Feminino , Antígenos HLA/metabolismo , Proteínas de Homeodomínio/imunologia , Proteínas de Homeodomínio/metabolismo , Humanos , Interferon gama/imunologia , Camundongos , Neoplasias/metabolismo , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/metabolismo , Linfócitos T/imunologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismoRESUMO
Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Caliciviridae/imunologia , Diferenciação Celular/imunologia , Gastroenterite/imunologia , Norovirus/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Infecções por Caliciviridae/genética , Infecções por Caliciviridae/virologia , Diferenciação Celular/genética , Linhagem Celular , Microambiente Celular/genética , Microambiente Celular/imunologia , Gastroenterite/genética , Gastroenterite/virologia , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Camundongos Endogâmicos C57BL , Norovirus/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
HIV-1-specific CD4+ T cells (TCD4+s) play a critical role in controlling HIV-1 infection. Canonically, TCD4+s are activated by peptides derived from extracellular ("exogenous") Ags displayed in complex with MHC class II (MHC II) molecules on the surfaces of "professional" APCs such as dendritic cells (DCs). In contrast, activated human TCD4+s, which express MHC II, are not typically considered for their APC potential because of their low endocytic capacity and the exogenous Ag systems historically used for assessment. Using primary TCD4+s and monocyte-derived DCs from healthy donors, we show that activated human TCD4+s are highly effective at MHC II-restricted presentation of an immunodominant HIV-1-derived epitope postinfection and subsequent noncanonical processing and presentation of endogenously produced Ag. Our results indicate that, in addition to marshalling HIV-1-specific immune responses during infection, TCD4+s also act as APCs, leading to the activation of HIV-1-specific TCD4+s.
Assuntos
Soropositividade para HIV , HIV-1 , Apresentação de Antígeno , Linfócitos T CD4-Positivos , Células Dendríticas , Epitopos , Antígenos de Histocompatibilidade Classe II , Humanos , Peptídeos , Linfócitos TRESUMO
The source proteins from which CD8+ T cell-activating peptides are derived remain enigmatic. Glycoproteins are particularly challenging in this regard owing to several potential trafficking routes within the cell. By engineering a glycoprotein-derived epitope to contain an N-linked glycosylation site, we determined that optimal CD8+ T cell expansion and function were induced by the peptides that are rapidly produced from the exceedingly minor fraction of protein mislocalized to the cytosol. In contrast, peptides derived from the much larger fraction that undergoes translocation and quality control are produced with delayed kinetics and induce suboptimal CD8+ T cell responses. This dual system of peptide generation enhances CD8+ T cell participation in diversifying both antigenicity and the kinetics of peptide display.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Animais , Linhagem Celular , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Antígenos de Histocompatibilidade Classe I/metabolismo , Cinética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/genéticaRESUMO
Smallpox and monkeypox pose severe threats to human health. Other orthopoxviruses are comparably virulent in their natural hosts, including ectromelia, the cause of mousepox. Disease severity is linked to an array of immunomodulatory proteins including the B22 family, which has homologs in all pathogenic orthopoxviruses but not attenuated vaccine strains. We demonstrate that the ectromelia B22 member, C15, is necessary and sufficient for selective inhibition of CD4+ but not CD8+ T cell activation by immunogenic peptide and superantigen. Inhibition is achieved not by down-regulation of surface MHC- II or co-stimulatory protein surface expression but rather by interference with antigen presentation. The appreciable outcome is interference with CD4+ T cell synapse formation as determined by imaging studies and lipid raft disruption. Consequently, CD4+ T cell activating stimulus shifts to uninfected antigen-presenting cells that have received antigen from infected cells. This work provides insight into the immunomodulatory strategies of orthopoxviruses by elucidating a mechanism for specific targeting of CD4+ T cell activation, reflecting the importance of this cell type in control of the virus.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Proteínas Virais/imunologia , Animais , Ectromelia Infecciosa/metabolismo , Ectromelia Infecciosa/virologia , Feminino , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Virais/metabolismo , VirulênciaRESUMO
Francisella tularensis, the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18. We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response. Using mice deficient in the DNA-sensing inflammasome component AIM2, we demonstrate here that AIM2 is required for sensing F. tularensis. AIM2-deficient mice were extremely susceptible to F. tularensis infection, with greater mortality and bacterial burden than that of wild-type mice. Caspase-1 activation, IL-1beta secretion and cell death were absent in Aim2(-/-) macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA. Our study identifies AIM2 as a crucial sensor of F. tularensis infection and provides genetic proof of its critical role in host innate immunity to intracellular pathogens.
Assuntos
Francisella tularensis/imunologia , Imunidade Inata , Macrófagos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Tularemia/imunologia , Animais , Sinalização do Cálcio/imunologia , Caspase 1/genética , Caspase 1/imunologia , Caspase 1/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA , Francisella tularensis/patogenicidade , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Interleucina-1beta/imunologia , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/imunologia , L-Lactato Desidrogenase/metabolismo , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Proteínas Nucleares/genética , Multimerização Proteica , Tularemia/genética , Tularemia/metabolismoRESUMO
Both immature and mature dendritic cells (DCs) can process and present foreign Ags to CD4 T cells; however, the mechanism by which MHC class II (MHC-II) in mature DCs acquires antigenic peptides remains unknown. To address this, we have studied Ag processing and presentation of two distinct CD4 T cell epitopes of the influenza virus hemagglutinin coat protein by both immature and mature mouse DCs. We find that immature DCs almost exclusively use newly synthesized MHC-II targeted to DM+ late endosomes for presentation to influenza virus-specific CD4 T cells. By contrast, mature DCs exclusively use recycling MHC-II that traffics to both early and late endosomes for antigenic peptide binding. Rab11a knockdown partially inhibits recycling of MHC-II in mature DCs and selectively inhibits presentation of an influenza virus hemagglutinin CD4 T cell epitope generated in early endosomes. These studies highlight a "division of labor" in MHC-II peptide binding, in which immature DCs preferentially present Ags acquired in Rab11a- DM+ late endosomes, whereas mature DCs use recycling MHC-II to present antigenic peptides acquired in both Rab11a+ early endosomes and Rab11a- endosomes for CD4 T cell activation.
Assuntos
Apresentação de Antígeno , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Genes MHC da Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Hibridomas , Ativação Linfocitária , Camundongos , Camundongos Knockout , Orthomyxoviridae/imunologia , Ubiquitina-Proteína Ligases/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
CD4+ T cells play critical roles in defending against poxviruses, both by potentiating cellular and humoral responses and by directly killing infected cells. Despite this central role, the basis for pox-specific CD4+ T cell activation, specifically the origin of the poxvirus-derived peptides (epitopes) that activate CD4+ T cells, remains poorly understood. In addition, because the current licensed poxvirus vaccines can cause serious adverse events and even death, elucidating the requirements for MHC class II (MHC-II) processing and presentation of poxviral Ags could be of great use. To address these questions, we explored the CD4+ T cell immunogenicity of ectromelia, the causative agent of mousepox. Having identified a large panel of novel epitopes via a screen of algorithm-selected synthetic peptides, we observed that immunization of mice with inactivated poxvirus primes a virtually undetectable CD4+ T cell response, even when adjuvanted, and is unable to provide protection against disease after a secondary challenge. We postulated that an important contributor to this outcome is the poor processability of whole virions for MHC-II-restricted presentation. In line with this hypothesis, we observed that whole poxvirions are very inefficiently converted into MHC-II-binding peptides by the APC as compared with subviral material. Thus, stability of the virion structure is a critical consideration in the rational design of a safe alternative to the existing live smallpox vaccine.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Imunogenicidade da Vacina/imunologia , Poxviridae/imunologia , Vacinas de Produtos Inativados/imunologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The notion that peptides bound to MHC class I molecules are derived mainly from newly synthesized proteins that are defective, and are therefore targeted for immediate degradation, has gained wide acceptance. This model, still entirely hypothetical, has strong intuitive appeal and is consistent with some experimental results, but it is strained by other findings, as well as by established and emerging concepts in protein quality control. While not discounting defectiveness as a driving force for the processing of some proteins, we propose that MHC-class-I-restricted epitopes are derived mainly from nascent proteins that are accessed by the degradation machinery prior to any assessment of fitness, and we outline one way in which this could be accomplished.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Modelos Imunológicos , Peptídeos/imunologia , Peptídeos/metabolismo , Animais , Apresentação de Antígeno/imunologia , HumanosRESUMO
Aminoglycosides have been proposed as therapies for genetic disorders caused by nonsense mutations, because of their capacity to enhance translational read-through of premature termination codons (PTCs), thereby permitting expression of functional full-length protein. However, a potential consequence of this strategy is the development of an autoimmune response to HLA-presented epitopes encoded downstream of the PTC or other stop codons. Using a recombinant virus-expression system in tissue culture and in mice, we demonstrate that gentamicin can induce expression and MHC class I presentation of a model epitope encoded downstream of a PTC at levels sufficient to activate CD8(+) T cells. The degree of read-through-derived peptide presentation varies with the sequence of the stop codon and +1 nucleotide. Additionally, we applied a mass spectrometry exploration of the HLA class I peptide repertoire of gentamicin-treated cells and identified multiple peptides derived from read-through of conventional stop codons. These results substantiate the possibility of self-reactivity to cryptic epitopes revealed by stop codon read-through therapies and potentially other therapeutic approaches involving compounds that alter translational fidelity.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Gentamicinas/farmacologia , Oligopeptídeos/genética , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Regiões 3' não Traduzidas/genética , Animais , Anticorpos Monoclonais , Western Blotting , Linfócitos T CD8-Positivos/imunologia , Códon sem Sentido/genética , Epitopos de Linfócito T/imunologia , Regulação da Expressão Gênica/imunologia , Imunoprecipitação , Luciferases , Camundongos , Oligonucleotídeos/genética , Oligopeptídeos/metabolismo , Elongação Traducional da Cadeia Peptídica/genética , Espectrometria de Massas em TandemRESUMO
The forces that drive conversion of nascent protein to major histocompatibility complex (MHC) class I-restricted peptides remain unknown. We explored the fundamental property of overt hydrophobicity as such a driver. Relocation of a membrane glycoprotein to the cytosol via signal sequence ablation resulted in rapid processing of nascent protein not because of the misfolded luminal domain but because of the unembedded transmembrane (TM) domain, which serves as a dose-dependent degradation motif. Dislocation of the TM domain during the natural process of endoplasmic reticulum-associated degradation (ERAD) similarly accelerated peptide production, but in the context of markedly prolonged processing that included nonnascent species. These insights into intracellular proteolytic pathways and their selective contributions to MHC class I-restricted peptide supply, may point to new approaches in rational vaccine design.
Assuntos
Apresentação de Antígeno/fisiologia , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Antígenos Ly/fisiologia , Células HeLa , Humanos , Immunoblotting , Proteínas de Membrana/fisiologia , Camundongos , Sinais Direcionadores de ProteínasRESUMO
We assessed several routes of immunization with vaccinia virus (VACV) in protecting mice against ectromelia virus (ECTV). By a wide margin, skin scarification provided the greatest protection. Humoral immunity and resident-memory T cells notwithstanding, several approaches revealed that circulating, memory CD8(+) T cells primed via scarification were functionally superior and conferred enhanced virus control. Immunization via the epithelial route warrants further investigation, as it may also provide enhanced defense against other infectious agents.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Epitélio/imunologia , Vaccinia virus/imunologia , Animais , Imunidade Humoral/imunologia , Imunização/métodos , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinação/métodosRESUMO
UNLABELLED: Although the pattern recognition receptor Toll-like receptor 2 (TLR2) is typically thought to recognize bacterial components, it has been described to alter the induction of both innate and adaptive immunity to a number of viruses, including vaccinia virus (VACV). However, many pathogens that reportedly encode TLR2 agonists may actually be artifactually contaminated during preparation, possibly with cellular debris or merely with molecules that sensitize cells to be activated by authentic TLR2 agonists. In both humans and mice, the most relevant natural route of infection with VACV is through intradermal infection of the skin. Therefore, we examined the requirement for TLR2 and its signaling adaptor MyD88 in protective immunity to VACV after intradermal infection. We find that although TLR2 may recognize virus preparations in vitro and have a minor role in preventing dissemination of VACV following systemic infection with large doses of virus, it is wholly disposable in both control of virus replication and induction of adaptive immunity following intradermal infection. In contrast, MyD88 is required for efficient induction of CD4 T cell and B cell responses and for local control of virus replication following intradermal infection. However, even MyD88 is not required to induce local inflammation, inflammatory cytokine production, or recruitment of cells that restrict virus from spreading systemically after peripheral infection. Thus, an effective antiviral response does require MyD88, but TLR2 is not required for control of a peripheral VACV infection. These findings emphasize the importance of studying relevant routes of infection when examining innate sensing mechanisms. IMPORTANCE: Vaccinia virus (VACV) provides the backbone for some of the most widely used and successful viral vaccine vectors and is also related to the human pathogens Cantagalo virus and molluscum contagiosum virus that infect the skin of patients. Therefore, it is vital to understand the mechanisms that induce a strong innate immune response to the virus following dermal infection. Here, we compare the ability of the innate sensing molecule Toll-like receptor 2 (TLR2) and the signaling molecule MyD88 to influence the innate and adaptive immune response to VACV following systemic or dermal infection.
Assuntos
Fator 88 de Diferenciação Mieloide/imunologia , Receptor 2 Toll-Like/imunologia , Vaccinia virus/fisiologia , Vacínia/imunologia , Imunidade Adaptativa , Animais , Feminino , Humanos , Imunidade Inata , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Receptor 2 Toll-Like/genética , Vacínia/genética , Vacínia/virologia , Vaccinia virus/genética , Vaccinia virus/imunologiaRESUMO
UNLABELLED: The factors that determine CD4+ T cell (TCD4+) specificities, functional capacity, and memory persistence in response to complex pathogens remain unclear. We explored these parameters in the C57BL/6 mouse through comparison of two highly related (>92% homology) poxviruses: ectromelia virus (ECTV), a natural mouse pathogen, and vaccinia virus (VACV), a heterologous virus that nevertheless elicits potent immune responses. In addition to elucidating several previously unidentified major histocompatibility complex class II (MHC-II)-restricted epitopes, we observed many qualitative and quantitative differences between the TCD4+ repertoires, including responses not elicited by VACV despite complete sequence conservation. In addition, we observed functional heterogeneity between ECTV- and VACV-specific TCD4+ at both a global and individual epitope level, particularly greater expression of the cytolytic marker CD107a from TCD4+ following ECTV infection. Most striking were differences during the late memory phase where, in contrast to ECTV, VACV infection failed to elicit measurable epitope-specific TCD4+ as determined by intracellular cytokine staining. These findings illustrate the strong influence of epitope-extrinsic factors on TCD4+ responses and memory. IMPORTANCE: Much of our understanding concerning host-pathogen relationships in the context of poxvirus infections stems from studies of VACV in mice. However, VACV is not a natural mouse pathogen, and therefore, the relevance of results obtained using this model may be limited. Here, we explored the MHC class II-restricted TCD4+ repertoire induced by mousepox (ECTV) infection and the functional profile of the responding epitope-specific TCD4+, comparing these results to those induced by VACV infection under matched conditions. Despite a high degree of homology between the two viruses, we observed distinct specificity and functional profiles of TCD4+ responses at both acute and memory time points, with VACV-specific TCD4+ memory being notably compromised. These data offer insight into the impact of epitope-extrinsic factors on the resulting TCD4+ responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Vírus da Ectromelia/imunologia , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/virologia , Vaccinia virus/imunologia , Animais , Citocinas/biossíntese , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Memória Imunológica , Camundongos Endogâmicos C57BL , Subpopulações de Linfócitos T/imunologiaRESUMO
CD4(+) T cells are generally regarded as helpers and regulators of the immune response. Although cytolytic CD4(+) T cells have been described, whether those generated during the course of a viral infection play a role in virus control remains unknown. Here we show that during acute infection with ectromelia virus, the mouse homolog of the human virus of smallpox, large numbers of CD4(+) T cells in the draining lymph node and liver of resistant mice have a cytotoxic phenotype. We also show that these cells kill targets in vivo in a perforin-dependent manner and that mice with specific deficiency of perforin in CD4(+) T cells have impaired virus control. Thus, perforin-dependent CD4(+) T-cell killing of infected cells is an important mechanism of antiviral defense.