RESUMO
Immunodominance (ID) defines the hierarchical immune response to competing antigens in complex immunogens. Little is known regarding B cell and antibody ID despite its importance in immunity to viruses and other pathogens. We show that B cells and serum antibodies from inbred mice demonstrate a reproducible ID hierarchy to the five major antigenic sites in the influenza A virus hemagglutinin globular domain. The hierarchy changed as the immune response progressed, and it was dependent on antigen formulation and delivery. Passive antibody transfer and sequential infection experiments demonstrated 'original antigenic suppression', a phenomenon in which antibodies suppress memory responses to the priming antigenic site. Our study provides a template for attaining deeper understanding of antibody ID to viruses and other complex immunogens.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Epitopos Imunodominantes/imunologia , Viroses/imunologia , Vírus/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Patrimônio Genético , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Interações Hospedeiro-Patógeno/genética , Imunização , Epitopos Imunodominantes/química , Memória Imunológica , Vírus da Influenza A/imunologia , Linfonodos/imunologia , Camundongos , Modelos Moleculares , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Conformação Proteica , Viroses/genética , Viroses/virologiaRESUMO
The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/biossíntese , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Células HEK293 , Antígenos de Histocompatibilidade Classe I/imunologia , Interações Hospedeiro-Patógeno , Humanos , Vigilância Imunológica , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Melanoma/imunologia , Melanoma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
Antibody responses to influenza vaccines tend to be focused on epitopes encountered during prior influenza exposures, with little production of de novo responses to novel epitopes. To examine the contribution of circulating antibodies to this phenomenon, we passively transferred a hemagglutinin (HA)-specific monoclonal antibody (mAb) into mice before immunizing with whole inactivated virions. The HA mAb inhibited de novo HA-specific antibodies, plasmablasts, germinal center B cells, and memory B cells, while responses to a second antigen in the vaccine, neuraminidase (NA), were uninhibited. The HA mAb potently inhibited de novo antibody responses against epitopes near the HA mAb binding site. The HA mAb also promoted IgG1 class switching, an effect that, unlike the inhibition of HA responses, relied on signaling through Fc-gamma receptors. These studies suggest that circulating antibodies inhibit de novo B cell responses in an antigen-specific manner, which likely contributes to differences in antibody specificities elicited during primary and secondary influenza virus exposures.IMPORTANCEMost humans are exposed to influenza viruses in childhood and then subsequently exposed to antigenically drifted influenza variants later in life. It is unclear if antibodies elicited by earlier influenza virus exposures impact immunity against new influenza virus strains. Here, we used a mouse model to investigate how an anti-hemagglutinin (HA) monoclonal antibody (mAb) affects de novo B cell and antibody responses to the protein targeted by the monoclonal antibody (HA) and a second protein not targeted by the monoclonal antibody [neuraminidase (NA)]. Collectively, our studies suggest that circulating anti-influenza virus antibodies can potently modulate the magnitude and specificity of antibody responses elicited by secondary influenza virus exposures.
RESUMO
Successful direct MHC class I Ag presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides that can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin (Ub)-proteasome system; however, other Ub-like proteins have also been implicated in protein degradation and direct Ag presentation. In this article, we examine the role of neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in direct Ag presentation in mouse cells. NEDD8 is the Ub-like protein with highest similarity to Ub, and fusion of NEDD8 to the N terminus of a target protein can lead to the degradation of target proteins. We find that appending NEDD8 to the N terminus of the model Ag OVA resulted in degradation by both the proteasome and the autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1, resulted in peptide presentation. When directly compared with Ub, NEDD8 fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924 inhibited the presentation of peptides from the defective ribosomal product-derived form of a model Ag. These results demonstrate that NEDD8 activity in the cell is important for direct Ag presentation, but not by directly targeting proteins for degradation.
Assuntos
Apresentação de Antígeno , Complexo de Endopeptidases do Proteassoma , Animais , Linfócitos T CD8-Positivos/metabolismo , Ciclopentanos , Camundongos , Proteína NEDD8/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas , Pirimidinas , Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMO
CD8+ T cell immunosurveillance is based on recognizing oligopeptides presented by MHC class I molecules. Despite decades of study, the importance of protein ubiquitylation to peptide generation remains uncertain. In this study, we examined the ability of MLN7243, a recently described ubiquitin-activating enzyme E1 inhibitor, to block overall cytosolic peptide generation and generation of specific peptides from vaccinia- and influenza A virus-encoded proteins. We show that MLN7243 rapidly inhibits ubiquitylation in a variety of cell lines and can profoundly reduce the generation of cytosolic peptides. Kinetic analysis of specific peptide generation reveals that ubiquitylation of defective ribosomal products is rate limiting in generating class I peptide complexes. More generally, our findings demonstrate that the requirement for ubiquitylation in MHC class I-restricted Ag processing varies with class I allomorph, cell type, source protein, and peptide context. Thus, ubiquitin-dependent and -independent pathways robustly contribute to MHC class I-based immunosurveillance.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/imunologia , Nucleosídeos/farmacologia , Peptídeos/imunologia , Sulfonamidas/farmacologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Citosol/química , Citosol/imunologia , Inibidores Enzimáticos/farmacologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/imunologia , Cinética , Ligantes , Camundongos , Monitorização Imunológica , Peptídeos/metabolismo , Pirazóis , Pirimidinas , Sulfetos , Ubiquitinação , Vaccinia virus/química , Vaccinia virus/imunologiaRESUMO
Influenza A virus gene segment 7 encodes two proteins: the M1 protein translated from unspliced mRNA and the M2 protein produced by mRNA splicing and largely encoded by the M1 +1 reading frame. To better understand the generation of defective ribosomal products relevant to MHC class I Ag presentation, we engineered influenza A virus gene segment 7 to encode the model H-2 K(b) class I peptide ligand SIINFEKL at the M2 protein C terminus. Remarkably, after treating virus-infected cells with the RNA splicing inhibitor spliceostatin A to prevent M2 mRNA generation, K(b)-SIINFEKL complexes were still presented on the cell surface at levels ≤60% of untreated cells. Three key findings indicate that SIINFEKL is produced by cytoplasmic translation of unspliced M1 mRNA initiating at CUG codons within the +1 reading frame: 1) synonymous mutation of CUG codons in the M2-reading frame reduced K(b)-SIINFEKL generation; 2) K(b)-SIINFEKL generation was not affected by drug-mediated inhibition of AUG-initiated M1 synthesis; and 3) K(b)-SIINFEKL was generated in vitro and in vivo from mRNA synthesized in the cytoplasm by vaccinia virus, and hence cannot be spliced. These findings define a viral defective ribosomal product generated by cytoplasmic noncanonical translation and demonstrate the participation of CUG-codon-based translation initiation in pathogen immunosurveillance.
Assuntos
Vírus Defeituosos/genética , Vírus da Influenza A/genética , Peptídeos/genética , Ribossomos/metabolismo , Proteínas da Matriz Viral/genética , Animais , Apresentação de Antígeno/efeitos dos fármacos , Linhagem Celular , Vírus Defeituosos/química , Vírus Defeituosos/efeitos dos fármacos , Vírus Defeituosos/metabolismo , Genes MHC Classe I , Células HeLa , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Biossíntese de Proteínas , Piranos/farmacologia , Splicing de RNA/efeitos dos fármacos , Compostos de Espiro/farmacologiaRESUMO
Green fluorescent protein (GFP) and other fluorescent proteins are essential tools for biological research. When fused to peptides or proteins as a reporter, GFP enables localization and quantitation of gene products in otherwise unmanipulated live cells or organisms. We previously reported that a sizable fraction of nascent GFP is post-translationally converted into a 20-kDa Triton X-100-insoluble proteasome substrate (Qian, S. B., Princiotta, M. F., Bennink, J. R., and Yewdell, J. W. (2006) J. Biol. Chem. 281, 392-400; Dolan, B. P., Li, L., Veltri, C. A., Ireland, C. M., Bennink, J. R., and Yewdell, J. W. (2011) J. Immunol. 186, 2065-2072). Here, we show that a similarly sized fragment is generated by all GFP and red fluorescent protein family members we examined. We demonstrate that fragmentation is a by-product of GFP chromophore rearrangement. A non-rearranging GFP mutant fails to fragment and generates diminished levels of K(b)-SIINFEKL complexes when SIINFEKL is genetically fused to either the C- or N-terminal domains of GFP fusion proteins. Instructively, another fragmenting GFP mutant that cannot create the functional chromophore but still generates fragments also demonstrates diminished K(b)-SIINFEKL generation. However, the mutant and wild-type fragments differ fundamentally in that wild-type fragments are rapidly liberated from the intact molecule and degraded quickly, accounting for increased K(b)-SIINFEKL generation. In the fragmenting mutant, the fragments are generated slowly and remain associated, likely in a native conformation based on their original structural description (Barondeau, D. P., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2006) J. Am. Chem. Soc. 128, 4685-4693). The wild-type GFP fragments represent the first biochemically defined natural defective ribosomal products to contribute peptides for immunosurveillance, enabling quantitation of peptide generation efficiency from this source of defective ribosomal products. More broadly, given the wide use of fluorescent proteins, their ubiquitous and abundant fragmentation must be considered when interpreting experiments using these extremely useful probes.
Assuntos
Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Fragmentos de Peptídeos/imunologia , Apresentação de Antígeno , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/imunologia , Monitorização Imunológica , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Proteína Vermelha FluorescenteRESUMO
Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.
Assuntos
Imunidade Inata , Metionina/metabolismo , Estresse Oxidativo/fisiologia , Aminoacilação de RNA de Transferência/fisiologia , Adenoviridae/fisiologia , Animais , Código Genético , Células HeLa , Humanos , Ligantes , Metionina/genética , Camundongos , Modelos Genéticos , NADPH Oxidases/metabolismo , Orthomyxoviridae/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Especificidade por Substrato , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Aminoacilação de RNA de Transferência/efeitos dos fármacosRESUMO
To better understand the generation of MHC class I-associated peptides, we used a model antigenic protein whose proteasome-mediated degradation is rapidly and reversibly controlled by Shield-1, a cell-permeant drug. When expressed from a stably transfected gene, the efficiency of antigen presentation is ~2%, that is, one cell-surface MHC class I-peptide complex is generated for every 50 folded source proteins degraded upon Shield-1 withdrawal. By contrast, when the same protein is expressed by vaccinia virus, its antigen presentation efficiency is reduced ~10-fold to values similar to those reported for other vaccinia virus-encoded model antigens. Virus infection per se does not modify the efficiency of antigen processing. Rather, the efficiency difference between cellular and virus-encoded antigens is based on whether the antigen is synthesized from transgene- vs. virus-encoded mRNA. Thus, class I antigen-processing machinery can distinguish folded proteins based on the precise details of their synthesis to modulate antigen presentation efficiency.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/metabolismo , Animais , Apresentação de Antígeno/genética , Sequência de Bases , Linhagem Celular , Células HeLa , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Camundongos , Ovalbumina/genética , Ovalbumina/imunologia , Ovalbumina/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Sensitivity is essential in CD8+ T-cell killing of virus-infected cells and tumor cells. Although the affinity of the T-cell receptor (TCR) for antigen is relatively low, the avidity of T cell-antigen-presenting cell interactions is greatly enhanced by increasing the valence of the interaction. It is known that TCRs cluster into protein islands after engaging their cognate antigen (peptides bound to MHC molecules). Here, we show that mouse K(b) class I molecules segregate into preformed, long-lasting (hours) clusters on the antigen-presenting cell surface based on their bound viral peptide. Peptide-specific K(b) clustering occurs when source antigens are expressed by vaccinia or vesicular stomatitis virus, either as proteasome-liberated precursors or free intracellular peptides. By contrast, K(b)-peptide complexes generated by incubating cells with synthetic peptides are extensively intermingled on the cell surface. Peptide-specific complex sorting is first detected in the Golgi complex, and compromised by removing the K(b) cytoplasmic tail. Peptide-specific clustering is associated with increased T-cell sensitivity: on a per-complex basis, endogenous SIINFEKL activates T cells more efficiently than synthetic SIINFEKL, and wild-type K(b) presents endogenous SIINFEKL more efficiently than tailless K(b). We propose that endogenous processing generates peptide-specific clusters of class I molecules to maximize the sensitivity and speed of T-cell immunosurveillance.
Assuntos
Antígenos Virais/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/química , Animais , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Citoplasma/metabolismo , Complexo de Golgi/metabolismo , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Microglobulina beta-2/metabolismoRESUMO
Surprisingly little is known about the interaction of human blood mononuclear cells with viruses. Here, we show that monocytes are the predominant cell type infected when peripheral blood mononuclear cells are exposed to viruses ex vivo. Remarkably, infection with vesicular stomatitis virus, vaccinia virus, and a variety of influenza A viruses (including circulating swine-origin virus) induces monocytes to differentiate within 18 hours into CD16(-)CD83(+) mature dendritic cells with enhanced capacity to activate T cells. Differentiation into dendritic cells does not require cell division and occurs despite the synthesis of viral proteins, which demonstrates that monocytes counteract the capacity of these highly lytic viruses to hijack host cell biosynthetic capacity. Indeed, differentiation requires infectious virus and viral protein synthesis. These findings demonstrate that monocytes are uniquely susceptible to viral infection among blood mononuclear cells, with the likely purpose of generating cells with enhanced capacity to activate innate and acquired antiviral immunity.
Assuntos
Diferenciação Celular , Células Dendríticas/fisiologia , Monócitos/fisiologia , Viroses/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/virologia , Humanos , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Influenza Humana/sangue , Influenza Humana/imunologia , Monócitos/imunologia , Monócitos/virologia , Fatores de Tempo , Vacínia/sangue , Vacínia/imunologia , Vaccinia virus/imunologia , Vaccinia virus/fisiologia , Estomatite Vesicular/sangue , Estomatite Vesicular/imunologia , Vesiculovirus/imunologia , Vesiculovirus/fisiologia , Viroses/fisiopatologiaRESUMO
Here, we address the question of why the influenza A virus hemagglutinin (HA) does not escape immunity by hyperglycosylation. Uniquely among dozens of monoclonal antibodies specific for A/Puerto Rico/8/34, escape from H28-A2 neutralization requires substitutions introducing N-linked glycosylation at residue 131 or 144 in the globular domain. This escape decreases viral binding to cellular receptors, which must be compensated for by additional substitutions in HA or neuraminidase that enable viral replication. Sequence analysis of circulating H1 influenza viruses confirms the in vivo relevance of our findings: natural occurrence of glycosylation at residue 131 is always accompanied by a compensatory mutation known to increase HA receptor avidity. In vaccinated mice challenged with WT vs. H28-A2 escape mutants, the selective advantage conferred by glycan-mediated global reduction in antigenicity is trumped by the costs of diminished receptor avidity. These findings show that, although N-linked glycosylation can broadly diminish HA antigenicity, fitness costs restrict its deployment in immune evasion.
Assuntos
Hemaglutininas/química , Sistema Imunitário/fisiologia , Vírus da Influenza A/metabolismo , Animais , Antígenos/química , Cães , Mapeamento de Epitopos , Glicosilação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Conformação Molecular , Mutação , Replicação ViralRESUMO
Antibody responses to influenza vaccines tend to be focused on epitopes encountered during prior influenza exposures, with little production of de novo responses to novel epitopes. To examine the contribution of circulating antibody to this phenomenon, we passively transferred a hemagglutinin (HA)-specific monoclonal antibody (mAb) into mice before immunizing with whole inactivated virions. The HA mAb inhibited de novo HA-specific antibodies, plasmablasts, germinal center B cells, and memory B cells, while responses to a second antigen in the vaccine, neuraminidase (NA), were uninhibited. The HA mAb potently inhibited de novo antibody responses against epitopes near the HA mAb binding site. The HA mAb also promoted IgG1 class switching, an effect that, unlike the inhibition of HA responses, relied on signaling through Fc-gamma receptors. These studies suggest that circulating antibodies inhibit de novo B cell responses in an antigen-specific manner, which likely contributes to differences in antibody specificities elicited during primary and secondary influenza virus exposures.
RESUMO
Antigenic drift, the gradual accumulation of amino acid substitutions in the influenza virus hemagglutinin (HA) receptor protein, enables viral immune evasion. Antibodies (Abs) specific for the drift-resistant HA stem region are a promising universal influenza vaccine target. Although anti-stem Abs are not believed to block viral attachment, here we show that complement component 1q (C1q), a 460-kilodalton protein with six Ab Fc-binding domains, confers attachment inhibition to anti-stem Abs and enhances their fusion and neuraminidase inhibition. As a result, virus neutralization activity in vitro is boosted up to 30-fold, and in vivo protection from influenza PR8 infection in mice is enhanced. These effects reflect increased steric hindrance and not increased Ab avidity. C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the HA, some of which cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-receptor binding domain anti-SARS-CoV-2 spike Abs, an effect dependent on spike density on the virion surface. These findings demonstrate that C1q can greatly expand Ab function and thereby contribute to viral evolution and immune escape.
Assuntos
Vacinas contra Influenza , Influenza Humana , Camundongos , Animais , Humanos , Hemaglutininas , Complemento C1q , Ligação Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Anticorpos AntiviraisRESUMO
MHC class I molecules function to display peptides generated from cellular and pathogen gene products for immune surveillance by CD8(+) T cells. Cells typically express approximately 100,000 class I molecules, or approximately 1 per 30,000 cellular proteins. Given "one protein, one peptide" representation, immunosurveillance would be heavily biased toward the most abundant cell proteins. Cells use several mechanisms to prevent this, including the predominant use of defective ribosomal products (DRiPs) to generate peptides from nascent proteins and, as we show here, compartmentalization of DRiP peptide generation to prevent competition from abundant cytosolic peptides. This provides an explanation for the exquisite ability of T cells to recognize peptides generated from otherwise undetected gene products.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Antígenos de Histocompatibilidade Classe I , Animais , Apresentação de Antígeno , Ligação Competitiva , Citosol/metabolismo , Citometria de Fluxo/métodos , Genes MHC Classe I , Cinética , Ligantes , Camundongos , Modelos Biológicos , Monitorização Imunológica/métodos , Peptídeos/química , Ligação ProteicaRESUMO
Tissue-resident memory (TRM) CD8+ T cells are largely derived from recently activated effector T cells, but the mechanisms that control the extent of TRM differentiation within tissue microenvironments remain unresolved. Here, using an IFNγ-YFP reporter system to identify CD8+ T cells executing antigen-dependent effector functions, we define the transcriptional consequences and functional mechanisms controlled by TCR-signaling strength that occur within the skin during viral infection to promote TRM differentiation. TCR-signaling both enhances CXCR6-mediated migration and suppresses migration toward sphingosine-1-phosphate, indicating the programming of a 'chemotactic switch' following secondary antigen encounter within non-lymphoid tissues. Blimp1 was identified as the critical target of TCR re-stimulation that is necessary to establish this chemotactic switch and for TRM differentiation to efficiently occur. Collectively, our findings show that access to antigen presentation and strength of TCR-signaling required for Blimp1 expression establishes the chemotactic properties of effector CD8+ T cells to promote residency within non-lymphoid tissues.
Assuntos
Linfócitos T CD8-Positivos , Memória Imunológica , Receptores de Antígenos de Linfócitos T , Pele , Viroses , Linfócitos T CD8-Positivos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Pele/imunologia , Pele/virologia , Viroses/imunologia , Movimento Celular , Feminino , Animais , Camundongos , Camundongos Endogâmicos C57BL , Interferon gama/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Receptores CXCR6/metabolismoRESUMO
The Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that stimulate T- and B-lymphocyte responses after sex-mismatched allogeneic hematopoietic cell transplantation (HCT). A CD8(+) cytotoxic T lymphocyte (CTL) clone that recognizes a novel HLA-B*2705-restricted H-Y antigen encoded by the DDX3Y gene was isolated from a male who had received a hematopoietic cell graft from his human leukocyte antigen (HLA)-identical sister. The antigenic peptide is a decamer that differs from the homologous DDX3X-encoded peptide at 4 positions. Expression of DDX3Y and of the H-Y epitope that it encodes was examined by quantitative polymerase chain reaction (PCR) and by CTL recognition assays. Expression of DDX3Y is detected in all myeloid and lymphoid leukemic cells that carry an intact Y chromosome. Moreover, the DDX3Y-encoded H-Y epitope is presented on the surface of both myeloid and lymphoid leukemic cells from male HLA-B*2705(+) patients. DDX3Y-specific CTLs prevent engraftment of human acute leukemia in nonobese diabetic/severe combined immune deficient mice, demonstrating that the DDX3Y-encoded H-Y antigen is also expressed in leukemic stem cells. These results demonstrate that CD8(+) T-cell responses against DDX3Y have the potential to contribute to graft-versus-leukemia (GVL) activity after female into male allogeneic HCT. This study is registered at http://clinicaltrials.gov as NCT00107354.
Assuntos
RNA Helicases DEAD-box/imunologia , Antígeno H-Y , Antígenos de Histocompatibilidade Classe I , Leucemia/patologia , Células-Tronco Neoplásicas/imunologia , Animais , Linfócitos T CD8-Positivos , RNA Helicases DEAD-box/análise , Feminino , Efeito Enxerto vs Leucemia , Antígeno H-Y/análise , Transplante de Células-Tronco Hematopoéticas , Humanos , Masculino , Camundongos , Camundongos SCID , Antígenos de Histocompatibilidade Menor , Linfócitos T Citotóxicos , Transplante HeterólogoRESUMO
Broadly neutralizing antibodies (Abs) that bind the influenza virus hemagglutinin (HA) stem may enable universal influenza vaccination. Here, we show that anti-stem Abs sterically inhibit viral neuraminidase (NA) activity against large substrates, with activity inversely proportional to the length of the fibrous NA stalk that supports the enzymatic domain. By modulating NA stalk length in recombinant IAVs, we show that anti-stem Abs inhibit virus release from infected cells by blocking NA, accounting for their in vitro neutralization activity. NA inhibition contributes to anti-stem Ab protection in influenza-infected mice, likely due at least in part to NA-mediated inhibition of FcγR-dependent activation of innate immune cells by Ab bound to virions. Food and Drug Administration-approved NA inhibitors enhance anti-stem-based Fc-dependent immune cell activation, raising the possibility of therapeutic synergy between NA inhibitors and anti-stem mAb treatment in humans.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A , Neuraminidase , Infecções por Orthomyxoviridae , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Cães , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Imunidade Inata/efeitos dos fármacos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/imunologia , Células Madin Darby de Rim Canino , Camundongos , Neuraminidase/antagonistas & inibidores , Neuraminidase/imunologia , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/enzimologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Domínios Proteicos , Receptores de IgG/imunologia , Receptores de IgG/metabolismoRESUMO
Human antibody-based immunity to influenza A virus is limited by antigenic drift resulting from amino acid substitutions in the hemagglutinin (HA) head domain. Glycan addition can cause large antigenic changes but is limited by fitness costs to viral replication. Here, we report that glycans are added to H1 and H3 HAs at discrete 5-to-7-year intervals, until they reach a functional glycan limit, after which glycans are swapped at approximately 2-fold-longer intervals. Consistent with this pattern, 2009 pandemic H1N1 added a glycan at residue N162 over the 2015-2016 season, an addition that required two epistatic HA head mutations for complete glycosylation. These strains rapidly replaced H1N1 strains globally, by 2017 dominating H3N2 and influenza B virus strains for the season. The pattern of glycan modulation that we outline should aid efforts for tracing the epidemic potential of evolving human IAV strains.IMPORTANCE Frequent mutation of its major antibody target, the glycoprotein hemagglutinin, ensures that the influenza virus is perennially both a rapidly emerging virus and a major threat to public health. One type of mutation escapes immunity by adding a glycan onto an area of hemagglutinin that many antibodies recognize. This study revealed that these glycan changes follow a simple temporal pattern. Every 5 to 7 years, hemagglutinin adds a new glycan, up to a limit. After this limit is reached, no net additions of glycans occur. Instead, glycans are swapped or lost at longer intervals. Eventually, a pandemic replaces the terminally glycosylated hemagglutinin with a minimally glycosylated one from the animal reservoir, restarting the cycle. This pattern suggests the following: (i) some hemagglutinins are evolved for this decades-long process, which is both defined by and limited by successive glycan addition; and (ii) hemagglutinin's antibody dominance and its capacity for mutations are highly adapted features that allow influenza to outpace our antibody-based immunity.
Assuntos
Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/química , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A/genética , MutaçãoRESUMO
Antibodies and cytotoxic T cells represent 2 arms of host defense against pathogens. We hypothesized that vaccines that induce both high-magnitude CD8+ T cell responses and antibody responses might confer enhanced protection against HIV. To test this hypothesis, we immunized 3 groups of nonhuman primates: (a) Group 1, which includes sequential immunization regimen involving heterologous viral vectors (HVVs) comprising vesicular stomatitis virus, vaccinia virus, and adenovirus serotype 5-expressing SIVmac239 Gag; (b) Group 2, which includes immunization with a clade C HIV-1 envelope (Env) gp140 protein adjuvanted with nanoparticles containing a TLR7/8 agonist (3M-052); and (c) Group 3, which includes a combination of both regimens. Immunization with HVVs induced very high-magnitude Gag-specific CD8+ T cell responses in blood and tissue-resident CD8+ memory T cells in vaginal mucosa. Immunization with 3M-052 adjuvanted Env protein induced robust and persistent antibody responses and long-lasting innate responses. Despite similar antibody titers in Groups 2 and 3, there was enhanced protection in the younger animals in Group 3, against intravaginal infection with a heterologous SHIV strain. This protection correlated with the magnitude of the serum and vaginal Env-specific antibody titers on the day of challenge. Thus, vaccination strategies that induce both CD8+ T cell and antibody responses can confer enhanced protection against infection.