RESUMEN
Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG1, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.
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Anticuerpos Biespecíficos , Inmunoglobulina G , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/genética , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/genética , Medición de Riesgo , Trastuzumab/inmunología , Trastuzumab/genética , Animales , Bevacizumab/inmunología , Bevacizumab/genética , MutaciónRESUMEN
A survey conducted by the Therapeutic Product Immunogenicity (TPI) community within the American Association of Pharmaceutical Scientists (AAPS) posed questions to the participants on their immunogenicity risk assessment strategies prior to clinical development. The survey was conducted in 2 phases spanning 5 years, and queried information about in silico algorithms and in vitro assay formats for immunogenicity risk assessments and how the data were used to inform early developability effort in discovery, chemistry, manufacturing and control (CMC), and non-clinical stages of development. The key findings representing the trends from a majority of the participants included the use of high throughput in silico algorithms, human immune cell-based assays, and proteomics based outputs, as well as specialized assays when therapeutic mechanism of action could impact risk assessment. Additional insights into the CMC-related risks could also be gathered with the same tools to inform future process development and de-risk critical quality attributes with uncertain and unknown risks. The use of the outputs beyond supporting early development activities was also noted with participants utilizing the risk assessments to drive their clinical strategy and streamline bioanalysis.
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Desarrollo de Medicamentos , Humanos , Consenso , Medición de Riesgo/métodosRESUMEN
All nucleated mammalian cells express major histocompatibility complex (MHC) proteins that present peptides on cell surfaces for immune surveillance. These MHC-presented peptides (pMHC) are necessary for directing T-cell responses against cells harboring non-self antigens derived from pathogens or from somatic mutations. Alterations in tumor-specific antigen repertoires - particularly novel MHC presentation of mutation-bearing peptides (neoantigens) - can be potent targets of anti-tumor immune responses. Here we employed an integrated genomic and proteomic antigen discovery strategy aimed at measuring how interferon gamma (IFN-γ) alters antigen presentation, using a human lymphoma cell line, GRANTA-519. IFN-γ treatment resulted in 126 differentially expressed proteins (2% of all quantified proteins), which included components of antigen presentation machinery and interferon signaling pathways, and MHC molecules themselves. In addition, several proteasome subunits were found to be modulated, consistent with previous reports of immunoproteasome induction by IFN-γ exposure. This finding suggests that a modest proteomic response to IFN-γ could create larger alteration to cells' antigen/epitope repertoires. Accordingly, MHC immunoprecipitation followed by mass spectrometric analysis of eluted peptide repertoires revealed exclusive signatures of IFN-γ induction, with 951 unique peptides reproducibly presented by MHC-I and 582 presented by MHC-II. Furthermore, an additional set of pMHCs including several candidate neoantigens, distinguished control and the IFN-γ samples by their altered relative abundances. Accordingly, we developed a classification system to distinguish peptides which are differentially presented due to altered expression from novel peptides resulting from changes in antigen processing. Taken together, these data demonstrate that IFN-γ can re-shape antigen repertoires by identity and by abundance. Extending this approach to models with greater clinical relevance could help develop strategies by which immunopeptide repertoires are intentionally reshaped to improve endogenous or vaccine-induced anti-tumor immune responses and potentially anti-viral immune responses.
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Antígenos de Neoplasias/genética , Antígenos de Neoplasias/aislamiento & purificación , Genómica , Péptidos/inmunología , Complejo de la Endopetidasa Proteasomal , Proteómica , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Epítopos/inmunología , Humanos , Interferón gamma/farmacología , Linfoma , Linfocitos T/inmunologíaRESUMEN
Inflammasomes sense a number of pathogen and host damage signals to initiate a signaling cascade that triggers inflammatory cell death, termed pyroptosis. The inflammatory caspases (1/4/5/11) are the key effectors of this process through cleavage and activation of the pore-forming protein gasdermin D. Caspase-1 also activates proinflammatory interleukins, IL-1ß and IL-18, via proteolysis. However, compared to the well-studied apoptotic caspases, the identity of substrates and therefore biological functions of the inflammatory caspases remain limited. Here, we construct, validate, and apply an antibody toolset for direct detection of neo-C termini generated by inflammatory caspase proteolysis. By combining rabbit immune phage display with a set of degenerate and defined target peptides, we discovered two monoclonal antibodies that bind peptides with a similar degenerate recognition motif as the inflammatory caspases without recognizing the canonical apoptotic caspase recognition motif. Crystal structure analyses revealed the molecular basis of this strong yet paradoxical degenerate mode of peptide recognition. One antibody selectively immunoprecipitated cleaved forms of known and unknown inflammatory caspase substrates, allowing the identification of over 300 putative substrates of the caspase-4 noncanonical inflammasome, including caspase-7. This dataset will provide a path toward developing blood-based biomarkers of inflammasome activation. Overall, our study establishes tools to discover and detect inflammatory caspase substrates and functions, provides a workflow for designing antibody reagents to study cell signaling, and extends the growing evidence of biological cross talk between the apoptotic and inflammatory caspases.
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Secuencias de Aminoácidos , Anticuerpos/química , Anticuerpos/metabolismo , Sitios de Unión , Caspasas/metabolismo , Inflamasomas/metabolismo , Secuencia de Aminoácidos , Caspasas/química , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Many cancers evade immune rejection by suppressing major histocompatibility class I (MHC-I) antigen processing and presentation (AgPP). Such cancers do not respond to immune checkpoint inhibitor therapies (ICIT) such as PD-1/PD-L1 [PD-(L)1] blockade. Certain chemotherapeutic drugs augment tumor control by PD-(L)1 inhibitors through potentiation of T-cell priming but whether and how chemotherapy enhances MHC-I-dependent cancer cell recognition by cytotoxic T cells (CTLs) is not entirely clear. We now show that the lysine acetyl transferases p300/CREB binding protein (CBP) control MHC-I AgPPM expression and neoantigen amounts in human cancers. Moreover, we found that two distinct DNA damaging drugs, the platinoid oxaliplatin and the topoisomerase inhibitor mitoxantrone, strongly up-regulate MHC-I AgPP in a manner dependent on activation of nuclear factor kappa B (NF-κB), p300/CBP, and other transcription factors, but independently of autocrine IFNγ signaling. Accordingly, NF-κB and p300 ablations prevent chemotherapy-induced MHC-I AgPP and abrogate rejection of low MHC-I-expressing tumors by reinvigorated CD8+ CTLs. Drugs like oxaliplatin and mitoxantrone may be used to overcome resistance to PD-(L)1 inhibitors in tumors that had "epigenetically down-regulated," but had not permanently lost MHC-I AgPP activity.
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Presentación de Antígeno/inmunología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , FN-kappa B/metabolismo , Neoplasias/tratamiento farmacológico , Factores de Transcripción p300-CBP/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Linfocitos T CD8-positivos , Proliferación Celular , Quimioterapia Combinada , Humanos , Inmunoterapia/métodos , Ratones , FN-kappa B/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Oxaliplatino/farmacología , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Factores de Transcripción p300-CBP/genéticaRESUMEN
Vascular leakage, or edema, is a serious complication of acute allergic reactions. Vascular leakage is triggered by the release of histamine and serotonin from granules within tissue-resident mast cells. Here, we show that expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates mast cell-mediated vascular leakage. In myeloid precursors, the granulocyte-macrophage progenitors (GMPs), loss of NSP4 results in the decrease of cellular levels of histamine, serotonin and heparin/heparan sulfate. Mast cells that are derived from NSP4-deficient GMPs have abnormal secretory granule morphology and a sustained reduction in histamine and serotonin levels. Consequently, in passive cutaneous anaphylaxis and acute arthritis models, mast cell-mediated vascular leakage in the skin and joints is substantially reduced in NSP4-deficient mice. Our findings reveal that NSP4 is required for the proper storage of vasoactive amines in mast cell granules, which impacts mast cell-dependent vascular leakage in mouse models of immune complex-mediated diseases.
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Mastocitos/enzimología , Serina Proteasas/metabolismo , Traslado Adoptivo , Animales , Complejo Antígeno-Anticuerpo , Regulación Enzimológica de la Expresión Génica , Histamina/metabolismo , Ratones , Ratones Noqueados , Neutrófilos , Serina Proteasas/genética , Serotonina/metabolismoRESUMEN
Tumor-specific mutations can generate neoantigens that drive CD8 T cell responses against cancer. Next-generation sequencing and computational methods have been successfully applied to identify mutations and predict neoantigens. However, only a small fraction of predicted neoantigens are immunogenic. Currently, predicted peptide binding affinity for MHC-I is often the major criterion for prioritizing neoantigens, although little progress has been made toward understanding the precise functional relationship between affinity and immunogenicity. We therefore systematically assessed the immunogenicity of peptides containing single amino acid mutations in mouse tumor models and divided them into two classes of immunogenic mutations. The first comprises mutations at a nonanchor residue, for which we find that the predicted absolute binding affinity is predictive of immunogenicity. The second involves mutations at an anchor residue; here, predicted relative affinity (compared with the WT counterpart) is a better predictor. Incorporating these features into an immunogenicity model significantly improves neoantigen ranking. Importantly, these properties of neoantigens are also predictive in human datasets, suggesting that they can be used to prioritize neoantigens for individualized neoantigen-specific immunotherapies.
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Antígenos de Neoplasias/inmunología , Mutación , Neoplasias/genética , Neoplasias/inmunología , Aminoácidos/genética , Animales , Afinidad de Anticuerpos , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Antígenos de Histocompatibilidad Clase I/inmunología , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias/patología , Péptidos/genética , Péptidos/inmunología , RNA-Seq , Secuenciación del ExomaRESUMEN
INTRODUCTION: Anti-drug antibody (ADA) responses are becoming an increasing concern as more highly engineered and sophisticated biotherapeutics enter the clinic. An arsenal of tools has been developed to identify potential T cell epitopes that may drive unwanted immunological responses to protein therapeutics; one such tool is termed 'Major Histocompatibility Complex-Associated Peptide Proteomics' (MAPPs). This review highlights the evolution of this MHC II profiling technology, its technological advantages and limitations, and its utility in helping to de-risk the immunogenicity of biotherapeutics. Areas covered: A comprehensive literature review was performed along with discussions with key leaders in the field of MAPPs to summarize the importance of monitoring potential immunogenicity of clinical molecules. Herein we also describe how MAPPs has been applied specifically for monitoring MHC II peptides derived from biotherapeutics. Expert commentary: Given the importance of this growing field we discuss the complementary tools used in conjunction with MAPPs and review case studies where this approach has informed clinical studies and in some cases allowed re-engineering of the biotherapeutic moiety to a less immunogenic format.
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Productos Biológicos/uso terapéutico , Complejo Mayor de Histocompatibilidad , Péptidos/metabolismo , Proteómica , Secuencia de Aminoácidos , Anticuerpos/farmacología , Humanos , Péptidos/químicaRESUMEN
Antibody-drug conjugates (ADC) are designed to selectively bind to tumor antigens via the antibody and release their cytotoxic payload upon internalization. Controllable payload release through judicious design of the linker has been an early technological milestone. Here, we examine the effect of the protease-cleavable valine-citrulline [VC(S)] linker on ADC efficacy. The VC(S) linker was designed to be cleaved by cathepsin B, a lysosomal cysteine protease. Surprisingly, suppression of cathepsin B expression via CRISPR-Cas9 gene deletion or shRNA knockdown had no effect on the efficacy of ADCs with VC(S) linkers armed with a monomethyl auristatin E (MMAE) payload. Mass spectrometry studies of payload release suggested that other cysteine cathepsins can cleave the VC(S) linker. Also, ADCs with a nonprotease-cleavable enantiomer, the VC(R) isomer, mediated effective cell killing with a cysteine-VC(R)-MMAE catabolite generated by lysosomal catabolism. Based on these observations, we altered the payload to a pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) conjugate that requires linker cleavage in order to bind its DNA target. Unlike the VC-MMAE ADCs, the VC(S)-PBD ADC is at least 20-fold more cytotoxic than the VC(R)-PBD ADC. Our findings reveal that the VC(S) linker has multiple paths to produce active catabolites and that antibody and intracellular targets are more critical to ADC efficacy. These results suggest that protease-cleavable linkers are unlikely to increase the therapeutic index of ADCs and that resistance based on linker processing is improbable. Cancer Res; 77(24); 7027-37. ©2017 AACR.
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Anticuerpos Monoclonales/metabolismo , Antineoplásicos/metabolismo , Catepsina B/fisiología , Inmunoconjugados/metabolismo , Profármacos/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Catepsina B/metabolismo , Línea Celular Tumoral , Células Cultivadas , Citrulina/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Células HEK293 , Humanos , Inmunoconjugados/uso terapéutico , Oligopéptidos , Profármacos/uso terapéutico , Proteolisis , Valina/metabolismoRESUMEN
The common p.D358A variant (rs2228145) in IL-6R is associated with risk for multiple diseases and with increased levels of soluble IL-6R in the periphery and central nervous system (CNS). Here, we show that the p.D358A allele leads to increased proteolysis of membrane bound IL-6R and demonstrate that IL-6R peptides with A358 are more susceptible to cleavage by ADAM10 and ADAM17. IL-6 responsive genes were identified in primary astrocytes and microglia and an IL-6 gene signature was increased in the CNS of late onset Alzheimer's disease subjects in an IL6R allele dependent manner. We conducted a screen to identify variants associated with the age of onset of Alzheimer's disease in APOE É4 carriers. Across five datasets, p.D358A had a meta Pâ=â3 ×10-4 and an odds ratioâ=â1.3, 95% confidence interval 1.12 -1.48. Our study suggests that a common coding region variant of the IL-6 receptor results in neuroinflammatory changes that may influence the age of onset of Alzheimer's disease in APOE É4 carriers.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Polimorfismo de Nucleótido Simple , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Anciano , Anciano de 80 o más Años , Alelos , Animales , Apolipoproteína E4/genética , Astrocitos/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Técnicas de Cocultivo , Estudios de Cohortes , Femenino , Células HEK293 , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Microglía/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Major histocompatibility complex Class I (MHCI) and Class II (MHCII) presented peptides powerfully modulate T cell immunity and play a vital role in generating effective anti-tumor and anti-viral immune responses in mammals. Characterizing these MHCI or MHCII presented peptides can help generate therapeutic treatments, afford information on T cell mediated biomarkers, provide insight into disease progression, and reduce adverse anti-drug side effects from engineered biotherapeutics. Here, we explore the tools and techniques commonly employed to discover both MHCI- and MHCII-presented peptides. We describe complementary strategies that enhance the characterization of these peptides and the informatics tools employed for both predicting and characterizing MHCI- and MHCII-presented epitopes. The evolution of methodologies for isolating MHC-presented peptides is discussed, as are the mass spectrometric workflows that can be employed for their characterization. We provide a perspective on where this field is headed, and how these tools may be applicable to the discovery and monitoring of epitopes in a variety of scenarios.
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Antígenos de Histocompatibilidad Clase II/química , Péptidos/química , Proteómica/métodos , Animales , Epítopos/química , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Linfocitos T/inmunologíaRESUMEN
Manganese plays a central role in cellular detoxification of reactive oxygen species (ROS). Therefore, manganese acquisition is considered to be important for bacterial pathogenesis by counteracting the oxidative burst of phagocytic cells during host infection. However, detailed analysis of the interplay between bacterial manganese acquisition and phagocytic cells and its impact on bacterial pathogenesis has remained elusive for Staphylococcus aureus, a major human pathogen. Here, we show that a mntC mutant, which lacks the functional manganese transporter MntABC, was more sensitive to killing by human neutrophils but not murine macrophages, unless the mntC mutant was pre-exposed to oxidative stress. Notably, the mntC mutant formed strikingly small colonies when recovered from both type of phagocytic cells. We show that this phenotype is a direct consequence of the inability of the mntC mutant to reinitiate growth after exposure to phagocytic oxidative burst. Transcript and quantitative proteomics analyses revealed that the manganese-dependent ribonucleotide reductase complex NrdEF, which is essential for DNA synthesis and repair, was highly induced in the mntC mutant under oxidative stress conditions including after phagocytosis. Since NrdEF proteins are essential for S. aureus viability we hypothesize that cells lacking MntABC might attempt to compensate for the impaired function of NrdEF by increasing their expression. Our data suggest that besides ROS detoxification, functional manganese acquisition is likely crucial for S. aureus pathogenesis by repairing oxidative damages, thereby ensuring efficient bacterial growth after phagocytic oxidative burst, which is an attribute critical for disseminating and establishing infection in the host.
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Proteínas Bacterianas/genética , Replicación del ADN/genética , Manganeso/metabolismo , Proteínas de Transporte de Membrana/genética , Estrés Oxidativo/genética , Estallido Respiratorio/genética , Staphylococcus aureus/genética , Animales , Regulación Bacteriana de la Expresión Génica/genética , Humanos , Macrófagos/microbiología , Ratones , Neutrófilos/microbiología , Fagocitosis/genética , Proteómica/métodos , Especies Reactivas de Oxígeno/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismoRESUMEN
Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1ß processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1ß maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1ß secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasas/metabolismo , Inflamasomas/metabolismo , Transducción de Señal , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Caspasas Iniciadoras , Línea Celular , Femenino , Bacterias Gramnegativas/inmunología , Humanos , Inflamasomas/efectos de los fármacos , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Mutación/genética , Necrosis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a Fosfato , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Sepsis/microbiología , Transducción de Señal/genética , Análisis de SupervivenciaRESUMEN
T-helper type 17 (TH17) cells that produce the cytokines interleukin-17A (IL-17A) and IL-17F are implicated in the pathogenesis of several autoimmune diseases. The differentiation of TH17 cells is regulated by transcription factors such as RORγt, but post-translational mechanisms preventing the rampant production of pro-inflammatory IL-17A have received less attention. Here we show that the deubiquitylating enzyme DUBA is a negative regulator of IL-17A production in T cells. Mice with DUBA-deficient T cells developed exacerbated inflammation in the small intestine after challenge with anti-CD3 antibodies. DUBA interacted with the ubiquitin ligase UBR5, which suppressed DUBA abundance in naive T cells. DUBA accumulated in activated T cells and stabilized UBR5, which then ubiquitylated RORγt in response to TGF-ß signalling. Our data identify DUBA as a cell-intrinsic suppressor of IL-17 production.
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Interleucina-17/biosíntesis , Biosíntesis de Proteínas , Células Th17/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Estabilidad de Enzimas , Femenino , Inflamación/genética , Inflamación/patología , Intestino Delgado/metabolismo , Intestino Delgado/patología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Transducción de Señal , Especificidad por Sustrato , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/biosíntesis , Proteasas Ubiquitina-Específicas/deficiencia , Proteasas Ubiquitina-Específicas/genética , UbiquitinaciónRESUMEN
Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as 'non-self' neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, â¼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide-MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.
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Exoma/genética , Fenómenos Inmunogenéticos/genética , Espectrometría de Masas , Mutación , Neoplasias/genética , Animales , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Inmunidad Celular/inmunología , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Neoplasias/inmunología , Péptidos/genética , Estructura Terciaria de ProteínaRESUMEN
Proteolysis describes the cleavage of proteins into smaller components, which in vivo occurs typically to either activate or impair the functionality of cellular proteins. Proteolysis can occur during cellular homeostasis or can be induced due to external stress stimuli such as heat, biological or chemical insult, and is mediated by the activity of cellular enzymes, namely, proteases. Proteolytic cleavage of proteins can influence protein activation by exposing an active site or disrupting inhibitor binding. Conversely, proteolytic cleavage of many proteins has also been shown to lead to protein degradation resulting in inactivation of the substrate. Thousands of proteolytic events are known to take place in regulated cellular processes such as apoptosis and pyroptosis, however, their individual contribution to these processes remains poorly understood. Additionally, many cellular homeostatic processes are regulated by proteolytic events, however, in some cases, few proteolytic substrates have been identified. To gain further insight into the mechanism of action of these cellular processes, and to characterize biomarkers of cell death and other pathological indications, it is imperative to utilize a complete arsenal of tools for studying proteolysis events in vivo and in vitro. In this chapter, we focus on alternative methodologies to N-terminomics for profiling substrates of proteolysis and describe an additional suite of tools including orthogonal biophysical separation techniques such as COFRADIC or GASSP, and affinity capture tools that can enrich for newly formed C-termini (C-terminomics) generated as a result of caspase-mediated proteolysis.
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Caspasas/metabolismo , Espectrometría de Masas/métodos , Proteínas/química , Proteolisis , Secuencia de Aminoácidos , Animales , Cromatografía/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Péptidos/química , Péptidos/metabolismo , Proteínas/metabolismo , Proteómica/métodos , Especificidad por SustratoRESUMEN
The detection of microbial pathogens involves the recognition of conserved microbial components by host cell sensors such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs). TLRs are membrane receptors that survey the extracellular environment for microbial infections, whereas NLRs are cytosolic complexes that detect microbial products that reach the cytosol. Upon detection, both sensor classes trigger innate inflammatory responses and allow the engagement of adaptive immunity. Endo-lysosomes are the entry sites for a variety of pathogens, and therefore the sites at which the immune system first senses their presence. Pathogens internalized by endocytosis are well known to activate TLRs 3 and 7-9 that are localized to endocytic compartments and detect ligands present in the endosomal lumen. Internalized pathogens also activate sensors in the cytosol such as NOD1 and NOD2 (ref. 2), indicating that endosomes also provide for the translocation of bacterial components across the endosomal membrane. Despite the fact that NOD2 is well understood to have a key role in regulating innate immune responses and that mutations at the NOD2 locus are a common risk factor in inflammatory bowel disease and possibly other chronic inflammatory states, little is known about how its ligands escape from endosomes. Here we show that two endo-lysosomal peptide transporters, SLC15A3 and SLC15A4, are preferentially expressed by dendritic cells, especially after TLR stimulation. The transporters mediate the egress of bacterially derived components, such as the NOD2 cognate ligand muramyl dipeptide (MDP), and are selectively required for NOD2 responses to endosomally derived MDP. Enhanced expression of the transporters also generates endosomal membrane tubules characteristic of dendritic cells, which further enhanced the NOD2-dependent response to MDP. Finally, sensing required the recruitment of NOD2 and its effector kinase RIPK2 (refs 8, 9) to the endosomal membrane, possibly by forming a complex with SLC15A3 or SLC15A4. Thus, dendritic cell endosomes are specialized platforms for both the lumenal and cytosolic sensing of pathogens.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Endosomas/inmunología , Endosomas/metabolismo , Proteína Adaptadora de Señalización NOD2/inmunología , Proteína Adaptadora de Señalización NOD2/metabolismo , Salmonella typhimurium/inmunología , Acetilmuramil-Alanil-Isoglutamina/inmunología , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Animales , Proteínas Portadoras/metabolismo , Citoplasma/inmunología , Citoplasma/metabolismo , Citoplasma/microbiología , Células Dendríticas/citología , Inmunidad Innata , Inflamación , Enfermedades Inflamatorias del Intestino/genética , Ligandos , Lisosomas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Fagosomas/inmunología , Fagosomas/metabolismoRESUMEN
BACKGROUND: Detailed knowledge on protein repertoire of a pathogen during host infection is needed for both developing a better understanding of the pathogenesis and defining potential therapeutic targets. Such data, however, have been missing for Staphylococcus aureus, a major human pathogen. METHODS: We determined the surface proteome of methicillin-resistant S. aureus (MRSA) clone usa300 derived directly from murine systemic infectiON. RESULTS: The majority of the in vivo-expressed surface-associated proteins were lipoproteins involved in nutrient acquisition, especially uptake of metal ions. Enzyme-linked immunosorbent assay (ELISA) of convalescent human serum samples revealed that proteins that were highly produced during murine experimental infection were also produced during natural human infection. We found that among the 7 highly abundant lipoproteins only MntC, which is the manganese-binding protein of the MntABC system, was essential for MRSA virulence during murine systemic infection. Moreover, we show that MntA and MntB are equally important for MRSA virulence. CONCLUSIONS: Besides providing experimental evidence that MntABC might be a potential therapeutic target for the development of antibiotics, our in vivo proteomics data will serve as a valuable basis for defining potential antigen combinations for multicomponent vaccines.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/metabolismo , Proteómica , Animales , Proteínas Bacterianas/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Riñón/microbiología , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Suero/inmunología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/inmunología , VirulenciaRESUMEN
Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.
Asunto(s)
Proteínas Bacterianas/inmunología , Glicosiltransferasas/inmunología , Interacciones Huésped-Patógeno/inmunología , Staphylococcus aureus Resistente a Meticilina/fisiología , Infecciones Estafilocócicas/inmunología , Staphylococcus epidermidis/fisiología , Factores de Virulencia/inmunología , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana/genética , Adhesión Bacteriana/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catepsina G/genética , Catepsina G/inmunología , Catepsina G/metabolismo , Línea Celular Tumoral , Pared Celular/enzimología , Pared Celular/genética , Pared Celular/inmunología , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Ratones , Secuencias Repetitivas de Aminoácido , Infecciones Estafilocócicas/enzimología , Infecciones Estafilocócicas/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody-peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the ß-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.